DAB-pa, a fluorescence-based assay for measuring and modulating enzymes involved in polyamine biosynthesis and catabolism

Abstract

The invention relates to methods and compositions for measuring the activity and catalytic parameters of the poly amine biosynthetic and catabolic enzymes. The invention also relates to methods for diagnosing, treating, or preventing diseases and disorders, including infectious diseases, cancer, and neurobiological disorders, using the described methods and compositions.

Description

[0001] TITLE OF THE INVENTION DAB-PA, A FLUORESCENCE-BASED ASSAY FOR MEASURING AND MODULATING ENZYMES INVOLVED IN POLYAMINE BIOSYNTHESIS AND CATABOLISM

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to, and benefit of, U.S. Provisional Application No. 63 / 719,499, filed November 12, 2024, and U.S. Provisional Application No. 63 / 575,199, filed April 5, 2024, each of which is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE The present application hereby incorporates by reference the entire contents of the Sequence Listing contained in the XML file named “047162-5374- OOWO_SequenceListing.xml” which was created on April 1, 2025, and is 9,626 bytes in size.

[0005] BACKGROUND OF THE INVENTION

[0006] Polyamines are polycationic aliphatic biogenic molecules containing carbon chains of varying lengths and different number of amino groups. They are found ubiquitously in all eukaryotic and prokaryotic organisms and are essential for cell growth, differentiation, and survival (Handa et al., Front Chem 2018, 6: 10; Cohen et al., 1998, A Guide to the Polyamines). The most common polyamines include putrescine (diamine), spermidine (triamine), and spermine (tetramine) (Cohen et al., 1998). The pathway for the biosynthesis of polyamines has long been considered an attractive target for the development of novel therapies for the treatment of microbial infections, cancer, and neurodegeneration (Gamble et al., Front Oncol 2012, 2: 162; Casero et al., Nat Rev Cancer 2018, 18: 681-695; Jimenez Gutierrez et al., Int J Mol Sci 2023, 24; Hayes, et al., Cancer Immunol Res 2014, 2: 274-285; Wallace et al., Essays Biochem 2009, 46: 1-9). In most organisms, including humans, as well as important human pathogens such as Plasmodium falciparum, the main causative agent of human malaria, and other protozoan parasites such as Leishmania and Trypanosoma species, as well as the majority of fungal pathogens including Candida and Aspergillus species, the polyamine biosynthesis pathway initiates with the decarboxylation of ornithine via ornithine decarboxylase (ODC) to form putrescine (Raina et al., Acta Chem Scand 1968, 22: 2375-2378; Morris et al., Biochem Biophys Res Commun 1965, 20: 697-702). Spermidine synthase (SPDS), a member of the aminopropyl transferase (APT) class of enzymes, then catalyzes the transfer of the propylamine group from decarboxylated S-adenosyl methionine (dc-SAM) to putrescine to form spermidine (Ikeguchi et al., J Biochem 2006, 139: 1-9 ). Spermidine can also accept an aminopropyl group derived from dc-SAM to form spermine, a reaction catalyzed by a second APT enzyme, spermine synthase (SPMS). In the yeast Saccharomyces cerevisiae, SPDS and SPMS activities are catalyzed by the Spe3 and Spe4 enzymes. Genetic studies demonstrated that disruption of the SPE3 gene results in spermidine, spermine, b-alanine, or pantothenic acid auxotrophy, whereas loss of SPE4 results in spermine, b-alanine, or pantothenic auxotrophy (White et al., J Biol Chem 2001, 276: 10794-10800). These findings highlight the importance of polyamine biosynthesis as an attractive target for the development of new antimicrobial drugs. Unlike P. falciparum and S. cerevisiae, other lower eukaryotes, such as Babesia and Eimeria species, lack an ODC enzyme and rely exclusively on the uptake of polyamines for survival (Singh et al., Nat Microbiol 2023, 8: 845-859; Aunin et al., Wellcome Open Res 2021, 6: 225). While polyamines have been implicated in various cellular functions, data available so far suggest that one of the crucial functions of the polyamine biosynthesis pathway is to form spermidine, which serves as a precursor for the synthesis of hypusine, an uncommon but critical amino acid for the activity of the eukaryotic translation factor eIF5A14.

[0007] In addition to their pivotal role in microbial development, pathogenesis, and virulence, polyamines and their metabolic pathways are also central to the pathophysiology of cancer and neurodegenerative diseases. In cancer biology, polyamine biosynthesis is essential due to its promotion of cell proliferation, growth, and survival. Cancer cells often exhibit dysregulated polyamine metabolism, resulting in elevated polyamine levels that drive rapid cell division and tumor progression. Studies have consistently shown that increased polyamine levels and the overexpression of polyamine biosynthesis genes, such as ornithine decarboxylase (0DC1), spermidine synthase (SMR) and spermine synthase (SMS), correlate strongly with poor survival rates in various cancers, including breast cancer, neuroblastoma, and prostate cancer. These elevated levels are associated with more aggressive cancer phenotypes and resistance to apoptosis, leading to a poorer prognosis. Consequently, targeting polyamine biosynthesis or modulating polyamine levels holds promise as a therapeutic approach to slow cancer progression and enhance the efficacy of other treatments (Casero et al., Nat Rev Cancer, 2018, 18(11):681-695; Koomoa et al., Mol Cancer Ther, 2009, 8)7):2067-2075; Gerner & Meyskens, Nat Rev Cancer, 2004, 4(10):781-792).

[0008] Polyamine dysregulation is also significant in neurodegenerative diseases but contributes to disease progression in contrasting ways. In disorders such as Alzheimer’s and Parkinson’s, elevated polyamine levels are linked to neuronal cell death, oxidative stress, and inflammation, all of which exacerbate the degeneration of neurons. Accumulated polyamines disrupt normal cellular processes, including protein synthesis and degradation, which accelerates neurotoxicity and hastens disease progression. Therapeutic approaches that target polyamine metabolism in neurodegeneration seek to mitigate oxidative damage, strengthen cellular resilience, and protect neurons from further decline (Casero et al., Nat Rev Cancer, 2018, 18(11 ):681 -695 ; Koomoa et al., Mol Cancer Ther, 2009, 8)7):2067-2075; Gerner & Meyskens, Nat Rev Cancer, 2004, 4(10):781-792).

[0009] Although ODC and APT enzymes involved in polyamine biosynthesis, as well as enzymes involved in PA catabolism such as deoxyhypusine synthase (DHS), have been known for many years to be attractive targets for the development of new therapeutics, means to inhibit or induce their activity have relied primarily on the use of substrate analogs. One such analog, a-difluoromethylornithine (DFMO; ornithine analog) acts as a competitive inhibitor of ODC. A search for new small molecules or natural products that modulate the activity of these enzymes through alternative mechanisms (e.g., allosteric inhibitors of ODC, SMR, SMS, or DHPS or activators of SSAT) and have specific and potent activity has been hampered by the lack of assays that are amenable to high-throughput screening of chemical libraries. Measurement of the activity and catalytic parameters of APT enzymes has so far relied mainly on the use of radioisotope- based assays, involving radiolabeled substrates, such as radiolabeled [14C]Spermine trihydrochloride and decarboxylated S-adenosyl[methyl-3H]methionine, which can be incorporated into the target substrate by the aminopropyl transferase (Chattopadhyay et al., Proc Natl Acad Sci U S A 2003, 100: 13869-13874; Hamasaki-Katagiri et al., Gene 1997, 187: 35-43). A second method involved dansylation of polyamines synthesized through reactions with dansyl chloride, followed by quantitative analysis using high performance liquid chromatography (HPLC) (Saeki et al., J Chromatogr 1978, 145: 221- 229). A third approach involved capillary electrophoresis with laser-induced fluorescence (CE-LIF) by derivatizing spermidine with 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-F) (Sano et al., J Chromatogr B Analyt Technol Biomed Life Sci 2007, 845: SO- 83). Finally, spermidine synthase activity was assessed using a specific monoclonal antibody against the reaction product, 5 ’-methylthioadenosine (MT A), coupled with a homogeneous time-resolved fluorescence technique (Enomoto et al., Anal Biochem 2006, 351: 229-240). While these four assays are sensitive, they are low-throughput and involve time-consuming procedures.

[0010] Thus, there is a need in the art for improved compositions and methods for measuring the activity and catalytic parameters of polyamine biosynthetic and catabolic enzymes. This invention satisfies this unmet need.

[0011] SUMMARY OF THE INVENTION

[0012] In some embodiments, the invention relates to a method of detecting the presence of at least one polyamine compound in a sample comprising the steps of: a) preparing a solution comprising the sample and 1,2- diacetylbenzene (DAB); b) incubating the solution; c) measuring fluorescence of the solution; and d) comparing the fluorescence of the solution to the fluorescence of a control solution, wherein an increase in fluorescence relative to the control solution indicates the presence of at least one polyamine compound in the sample. In some embodiments, the polyamine is at least one selected from the group consisting of ornithine, putrescine, spermidine, acetyl-spermidine, spermine, acetyl-spermine, and deoxyhypusine. In some embodiments, the solution of step a) further comprises P-mercaptoethanol (0-ME). In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of sodium tetraborate and potassium phosphate.

[0013] In some embodiments, step b) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step c) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step e) comprises 1,2-DAB and 0- ME at about the same concentration as the sample solution of step a), wherein an increase in absorbance at 364 nm in the excitation spectrum relative to the control solution, an increase in emission at 425 nm relative to the control solution, or both indicate the presence of a polyamine. In some embodiments, steps a) through c) are performed in at least one well of a multi-well plate.

[0014] In some embodiments, the invention relates to a method of detecting aminopropyl transferase (APT) activity of a protein comprising the steps of a) preparing a solution comprising the protein, decarb oxy lated-S- adenosylmethionine (dc-SAM), and either putrescine or spermidine; b) incubating the solution; c) adding 1,2-DAB and 0-ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has APT activity.

[0015] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0016] In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of dc-SAM and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has APT activity. In some embodiments, steps a) through e) are performed in at least one well of a multiwell plate.

[0017] In some embodiments, the invention relates to a method of identifying a compound as an inhibitor of a protein with APT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with APT activity, dc-SAM, and either putrescine or spermidine; b) incubating the solution; c) adding to the solution P- E and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0018] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA. In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with APT activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate. In some embodiments, the invention relates to a compound selected from

[0019] each instance of RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl;

[0020] R1, R2, R3, R4, and R are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof; and each instance of L is a divalent linker selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, sulfonic ester, sulfoximine, sulfonamide, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof, and tautomers, conjugates, salts, and solvates thereof.

[0021] In some embodiments, divalent linker L comprises Formula (IV):

[0022] Formula (IV) wherein: n =l-6; o = l-6; p = 1-6; and q = 0-3. In some embodiments, the compound is selected from the group consisting of:

[0023] and tautomers, conjugates, salts, and solvates thereof.

[0024] In some embodiments, the invention relates to a method of detecting ornithine decarboxylase (ODC) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, pyridoxal phosphate

[0025] (PIP), and ornithine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has ODC activity.

[0026] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA). In some embodiments, step b) comprises incubating the In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of PIP and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has ODC activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0027] In some embodiments, the invention relates to a method of identifying a compound as an inhibitor of a protein with ODC activity comprising the steps of: a) preparing a solution comprising the compound, the protein with ODC activity, PIP, and ornithine; b) incubating the solution; c) adding to the solution P-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0028] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA. In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with ODC activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0029] In some embodiments, the invention relates to a method of detecting deoxyhypusine synthase (DHS) activity of a protein comprising the steps of a) preparing a solution comprising the protein, nicotinamide adenine dinucleotide (NAD+), eukaryotic translation initiation factor 5A (eIF-5A), and spermidine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has DHS activity. In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA). In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of NAD+, eIF-5A, and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has DHS activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0030] In some embodiments, the invention relates to a method of identifying a compound as an inhibitor of a protein with DHS activity comprising the steps of: a) preparing a solution comprising the compound, the protein with DHS activity, NAD+, eIF-5A, and spermidine; b) incubating the solution; c) adding to the solution P-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0031] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA. In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with DHS activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0032] In some embodiments, the invention relates to a method of detecting spermidine / spermine N(l)-acetyltransferase (SSAT) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has SSAT activity.

[0033] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA). In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of acetyl -Co A and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has SSAT activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0034] In some embodiments, the invention relates to a method of identifying a compound as an inhibitor of a protein with SSAT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with SSAT activity, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding to the solution -ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0035] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA. In some embodiments, step b) comprises incubating the solution at about 37 °C for about 60 minutes. In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, step d) comprises incubating the solution at about room temperature for about 60 minutes. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. In some embodiments, the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with SSAT activity. In some embodiments, steps a) through e) are performed in at least one well of a multi-well plate.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0038] Figure 1, comprising Figure 1A through Figure 1C depicts representative data demonstrating the application of 1,2-DAB / p-ME based fluorescent assay to monitor aminopropyl transferase (APT) activity. Figure 1A depicts a schematic representation of the APT reaction and the detection of the products, spermidine or spermine, following the conversion of either putrescine or spermidine in the presence of decarboxylated S- adenosyl methionine (dc-SAM), respectively, and subsequent interaction with 1,2- diacetyl benzene (1,2-DAB) to produce fluorescent adducts. Figure IB depicts representative total fluorescence intensities (Xcx= 364 nm and em = 425 nm ) following incubation of 1,2-DAB and P-mercaptoethanol (P-ME) in APT reaction buffer alone or supplemented with either methyl thioadenosine (MTA), dc-SAM, putrescine, spermidine, or spermine after 60 minutes at 22 °C. Figure ID depicts representative total fluorescence intensities (ex= 364 nm and em = 425 nm ) following incubation of 1,2-DAB in APT reaction buffer alone or supplemented with either MTA, dc-SAM, putrescine, spermidine, or spermine after 60 minutes at 22 °C. The data are presented as mean ± S.D.

[0039] Figure 2, comprising Figure 2A through Figure 2D, depicts representative data presenting time dependent and concentration dependent increase in fluorescence intensity following interaction of 1,2-DAB with polyamines. Figure 2A depicts representative total fluorescence (Xex = 364 nm and em = 425 nm) from 1,2-DAB / p-ME- putrescine adducts at different concentrations of putrescine (PUT, 10-100 pM) over a span of 0-160 minutes. Figure 2B depicts representative total fluorescence from 1,2- DAB / p-ME-spermidine adducts at different concentrations of spermidine (SPD, 10-100 pM) over a span of 0-160 minutes. Figure 2C depicts representative total fluorescence from 1,2-DAB / p-ME-spermine adducts at different concentrations of spermine (SPM, 10-100 pM) over a span of 0-160 minutes. Figure 2D. Total fluorescent intensities of adducts formed between 1,2-DAB / p-ME and 100 pM of either putrescine, spermidine, or spermine after 60 minutes of reaction. Significant difference in total fluorescent intensities were observed between PUT-SPD (p = 0.0001, PUT-SPM (p < 0.0001), and SPD-SPM (p = 0.0006). The data are presented from three independent experiments performed in triplicates, and values are mean ± S.D. The statistical significance was calculated using Welch’s t-test.

[0040] Figure 3, comprising Figure 3A through Figure 3F, depicts representative results demonstrating changes in the ratios of putrescine-spermidine and spermidinespermine produce quantitative changes in 1,2-DAB / p-ME fluorescence. Figure 3 A depicts representative results of putrescine, dcSAM, spermidine, and MTA mixed in different ratios as indicated in the (decreasing concentration of putrescine + dcSAM and increasing concentrations of spermidine + MTA). The total PUT + dcSAM + SPD + MTA concentration was maintained at 200 pM. The total fluorescence intensity was measured at 364 nm (excitation) / 425 nm (emission) after 1 hour of incubation with 1,2- DAB / p-ME. Figure 3B depicts a representative graph showing the net fluorescent intensity (FI) calculated from data in Figure 3A. A linear increase in net fluorescence intensity of spermidine with increasing concentration was observed. Figure 3C depicts a representative image of thin-layer chromatography showing the separation of different fractions containing decreasing concentrations of putrescine + dcSAM and increasing concentrations of spermidine + MTA. Figure 3D depicts representative results of spermidine and spermine mixed in different ratios as indicated in the figure (decreasing concentration of spermidine + dcSAM and increasing concentrations of spermine + MT A), and the total SPD + dcSAM +SPM + MTA concentration was maintained at 200 pM. The total fluorescence intensity was measured at 364 nm (excitation) / 425 nm (emission) after 1 hour of incubation with 1 ,2-D AB / 0-ME. Figure 3E depicts a representative graph showing the net fluorescent intensity (FI) calculated from data in Figure 3D. A linear increase in net fluorescence intensity of spermine with increasing concentration was observed. Figure 3F depicts a representative image of thin-layer chromatography showing the separation of different fractions containing decreasing concentrations of spermidine + dcSAM and increasing concentrations of spermine + MTA. The data are from three independent experiments conducted in triplicates, with error bars denoting mean ± S.E.

[0041] Figure 4, comprising Figure 4A and Figure 4B, depicts representative LCMS results of the reaction of putrescine and 1,2-DAB. Figure 4A depicts representative LC-HRMS results of the reaction of putrescine and 1,2-DAB. Figure 4B depicts representative MS / MS fragmentation results of putrescine-DAB adducts.

[0042] Figure 5, comprising Figure 5A and Figure 5B, depicts representative LCMS results of the reaction of spermidine and 1,2-DAB. Figure 5A depicts representative LC-HRMS results of the reaction of spermidine and 1,2-DAB. Figure 5B depicts representative MS / MS fragmentation results of spermidine-DAB adducts.

[0043] Figure 6, comprising Figure 6A and Figure 6B, depicts representative LCMS results of the reaction of spermine and 1,2-DAB. Figure 6A depicts representative LC-HRMS results of the reaction of spermine and 1,2-DAB. Figure 6B depicts representative MS / MS fragmentation results of spermine-DAB adducts.

[0044] Figure 7, comprising Figure 7A and Figure 7B, depicts representative FIA-MS results of the reaction of putrescine and 1,2-DAB. Figure 7A depicts representative FIA-MS full scan results of the reaction of putrescine and 1,2-DAB. Figure 7B depicts representative MS / MS fragmentation results of putrescine-DAB adducts.

[0045] Figure 8, comprising Figure 8A and Figure 8B, depicts representative FIA-MS results of the reaction of spermidine and 1,2-DAB. Figure 8 A depicts representative FIA-MS full scan results of the reaction of spermidine and 1,2-DAB. Figure 8B depicts representative MS / MS fragmentation results of spermidine-DAB adducts. Figure 9, comprising Figure 9A and Figure 9B, depicts representative FIA-MS results of the reaction of spermine and 1,2-DAB. Figure 9A depicts representative FIA-MS full scan results of the reaction of spermine and 1,2-DAB. Figure 9B depicts representative MS / MS fragmentation results of spermine-DAB adducts.

[0046] Figure 10 depicts a proposed mechanism of formation of fluorescent 1,3- dimethyl isoindole adducts formed from the reaction of 1,2-DAB and putrescine, spermidine, or spermine.

[0047] Figure 11, comprising Figure 11A through Figure 1 IF, depicts representative data demonstrating application of the DAB-APT fluorescence assay to determine the activity of S. cerevisiae spermidine synthase Spe3. Figure 11A depicts a schematic representation of the enzymatic reaction catalyzed by Spe3 along with and the anticipated fluorescence signals using the DAB-PA Assay. Figure 1 IB depicts a representative standard curve was generated by mixing different ratios of putrescine, dcSAM, spermidine, and MTA (decreasing concentration of putrescine + dcSAM and increasing concentrations of spermidine + MTA). The total PUT + dcSAM + SPD + MTA concentration was maintained at 1 mM. The total fluorescence intensity was measured at 364 nm (excitation) / 425 nm (emission) after 1 hour of incubation with 1,2- DAB / p-ME. A linear increase in fluorescence is observed with saturation around 0.1 mM of PUT, dcSAM, and 0.4 mM of SPD + MTA. Figure 11C depicts representative results of spermidine synthase assays conducted using affinity-purified recombinant MBP-Spe3 (20 ng / pl) or heat inactivated MBP-Spe3 and 0.5 mM of putrescine and de- SAM as substrate and co-substrate, respectively, at 37 °C for 0-60 minutes. The total fluorescence intensities of the reactions catalyzed by heat inactivated or active MBP-Spe3 at 0 minutes and 60 minutes were measured at 364 nm (excitation) / 425 nm (emission) after 1 hour of incubation with 1,2-DAB / p-ME buffer. Figure 1 ID depicts representative conversion rates of the substrates putrescine and dcSAM by heat inactivated and active MBP-Spe3 as determined by DAB assay shown as percentage of spermidine formed, from data in Figure 11C. Data presented as mean ± S.D from three independent experiments, each conducted in triplicate. Figure 1 IE depicts representative images of thin-layer chromatography showing the reactions performed in Figure 11C, confirming the formation of the product spermidine by active MBP-Spe3. Figure 1 IFdepicts representative concentration of polyamines (substrate putrescine and the product spermidine) in reactions catalyzed by active and heat denatured MBP-Spe3 after 60 minutes of reactions as determined using LC-MS.

[0048] Figure 12, comprising Figure 12A through Figure 12F, depicts representative data demonstrating application of the 1,2-DAB-APT fluorescence assay to determine the activity of S. cerevisiae spermine synthase sped. Figure 12A depicts a schematic representation of the enzymatic reaction catalyzed by Spe4, along with the anticipated fluorescence signals using the DAB-PA Assay. Figure 12B depicts a representative standard curve generated by mixing different ratios of spermidine, dcSAM, spermine, and MTA (decreasing concentration of spermidine + dcSAM and increasing concentrations of spermine + MTA). The total SPD + dcSAM + SPM + MTA concentration was maintained at 1 mM. The total fluorescence intensity was measured at 364 nm (excitation) / 425 nm (emission) after 1 h of incubation with 1,2-DAB / p-ME. A linear increase in fluorescence is observed. Figure 12C depicts representative results of spermine synthase assays conducted using recombinant MBP-Spe4 (20 ng / pl) or heat inactivated MBP-Spe4 and 0.5 mM of spermidine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0-60 minutes. The total fluorescence intensities of the reactions catalyzed by heat inactivated or active MBP-Spe4 at 0 minute and 60 minutes were measured at 364 nm (excitation) / 425 nm (emission) after 1 hour of incubation with 1,2-DAB / p-ME buffer. Figure 12D depicts representative conversion rates of the substrates spermidine and dcSAM by heat inactivated and active MBP-Spe4 as percentage of spermine formed, from Figure 12C. Data presented as mean ± S.D from three independent experiments, each conducted in triplicate. Figure 12E depicts representative images of thin-layer chromatography showing the reactions performed in Figure 12C, confirming the formation of the product spermine by active Spe4. Figure 12F depicts representative concentration of polyamines (substrate spermidine and the product spermine) in reactions catalyzed by active and heat denatured Spe4 after 60 minutes of reaction determined using LC-MS.

[0049] Figure 13, comprising Figure 13 A through Figure 13F, depicts representative data demonstrating the purification of recombinant MBP-tagged SPE3 (Figure 13 A), SPE4 (Figure 13B), PfSPDS (Figure 13C), PfSPDSD127A(Figure 13D), PfSPDSE147A(Figure 13E), and PfSPDSD127A’E147A D196A(Figure 13F).

[0050] Figure 14, comprising Figure 14A through Figure 14D, depicts representative conversion rates of Spe3, Spe4, and PfSPDS enzymes. Figure 14A depicts representative conversion rate of putrescine, in the presence of dcSAM, by heat inactivated and active Spe3 as a function of putrescine consumption rate based on putrescine signal intensities from TLC data in Figure HE. Figure 14B depicts representative conversion rate of spermidine, in the presence of dcSAM, by heat inactivated and active Spe4 as a function of spermidine consumption rate based on spermidine signal intensities from TLC data in Figure 12E. Figure 14C depicts representative conversion rate of putrescine, in the presence of dcSAM, by heat inactivated PfSPDS, active PfSPDS, and triple mutant PfSPDS shown as a function of putrescine consumption rate based on putrescine signal intensities from TLC data in Figure 16D. Figure 14D depicts representative conversion rate of spermidine, in the presence of dcSAM, by heat inactivated and active PfSPDS as a function of spermidine consumption rate based on spermidine signal intensities from TLC data in Figure 16E.

[0051] Figure 15 depicts a representative multiple sequence alignment of P. falciparum spermidine synthase (PfSPDS) with spermidine synthase of E. coli, S. cerevisiae, and H. sapiens. Conserved active site residues are in bold.

[0052] Figure 16, comprising Figure 16A through Figure 16F, depicts representative application of the DAB-APT fluorescence assay to determine the activity of the P. falciparum PfSPDS enzyme. Figure 16A depicts representative results of spermidine synthase assays conducted using affinity-purified recombinant MBP-PfSPDS (20 ng / pl), heat inactivated MBP-PfSPDS, or MBP-triple mutant PfSPDS (MBP- PfSPDSD127A’E147A D196A) and 0.5 mM putrescine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0-60 minutes. The total fluorescence intensities of the reactions at 0 minute and 60 minutes were measured at 364 nm (excitation) / 425 nm (emission) after 1 h of incubation with 1,2-DAB / p-ME buffer. Figure 16B depicts representative conversion rates of putrescine, in the presence of dcSAM, by heat inactivated, active, and triple mutant MBP-PfSPDS (shown as percentage of spermidine formed, from data in Figure 16A. Data presented as mean ± S.D from three independent experiments, each conducted in triplicate. Figure 16C depicts representative results of spermine synthase assays conducted using heat inactivated or active MBP-PfSPDS, with 0.5 mM of spermidine and 0.75 mM of dc-SAM as substrate and co-substrate, respectively, at 37 °C for 0-60 minutes. The total fluorescence intensities of the reactions catalyzed by heat inactivated or active MBP-PfSPDS at 0 minute and 60 minutes were measured at 364 nm (excitation) / 425 nm (emission) after 1 h of incubation with 1,2- DAB / 0-ME buffer. Data presented as mean ± S.D from three independent experiments, each conducted in triplicate. Figure 16D depicts representative images of thin-layer chromatography showing the reactions performed in Figure 16A, confirming the formation of the product spermidine by active MBP-PfSPDS. Figure 16E depicts representative images of thin-layer chromatography showing the reactions performed in Figure 16C, showing the formation of the product spermine by active MBP-PfSPDS at 60 minute. Figure 16F depicts representative concentration of polyamines (substrate putrescine and the products spermidine, and spermine) in a reaction catalyzed by active, heat denatured and the triple mutant PfSPDS after 90 minutes, as determined by LC-MS.

[0053] Figure 17, comprising Figure 17A through Figure 17L, depicts representative data demonstrating application of the DAB-APT fluorescence assay to determine the activities and kinetics of S. cerevisiae spermidine synthase Spe3, spermine synthase Spe4, and P. falciparum spermidine synthase (PfSPDS). Figure 17A depicts representative spermidine synthase assays conducted using affinity -purified recombinant MBP-Spe3 (20 ng / l), heat denatured Spe3 (Spe3_DN) and 100 M of putrescine and dc-SAM as substrate and co-substrate, respectively, at 37 °C for 0-60 min. A parallel reaction at 4 °C (Spe3 4 °C) served as a control, the same reaction was also performed at 4 °C. Purified MBP-Spe4, which lacks the ability to convert putrescine to spermidine, was included as an additional control. The spermidine synthase activity of MBP-Spe3, MBP-Spe4, and respective controls is depicted as net fluorescence intensity over time. Figure 17B depicts representatives spermidine synthase APT activity of yeast SPE3 calculated from Figure 17A and shown as a function of spermidine concentration over time. Figure 17C depicts representative kinetics of the Spe3 spermidine synthase activity as a function of putrescine. Spe3-APT assays were performed with 20 ng / pl of MBP- Spe3 and varying concentrations of either putrescine or dc-SAM at 37 °C for 60 minutes. Figure 17D depicts representative kinetics of the Spe3 spermidine synthase activity as a function of dc-SAM. Spe3-APT assays were performed as in Figure 17C. Figure 17E depicts representative spermine synthase assays mediated by Spe4 performed using affinity-purified recombinant MBP-Spe4 (20 ng / pl) or heat-denatured SPE4 (Spe4_DN), along with 100 pM of spermidine and dc-SAM as substrates at 37 °C for 0-60 minutes. A control reaction was conducted at 4 °C (Spe 4 °C). Spe3, which lack the ability to convert spermidine to spermine was used as an additional control. The spermine synthase activity of MBP-Spe4, MBP-Spe3, and controls is depicted as net fluorescence intensity over time. Figure 17F depicts representative spermine synthase activity of yeast Spe4 calculated from data in Figure 17E and represented as molar amounts of spermidine formed over time. Figure 17G depicts representative kinetics of the Spe4 spermine synthase activity as a function of spermine concentration over time. Spe4-APT assays were performed with 20 ng / l of MBP-Spe3 and varying concentrations of either spermidine or dc-SAM at 37 °C for 60 minutes. Figure 17H depicts representative kinetics of the spe4 spermine synthase activity as a function of dc-SAM concentration. Spe4-APT assays were performed as in Figure 17G. Figure 171 depicts representative spermidine synthase reactions conducted using affinity-purified recombinant MBP- PfSPDS (20 ng / pl) or mutant enzymes (MBP-PfSPDSD127A, MBP-PfSPDSE147A, MBP- PfSPDSD127A’E147A D196A), and 100 pM of putrescine and dc-SAM as substrate and cosubstrate, respectively, at 37 °C for 0-60 minutes. The spermidine synthase activity of MBP-PfSPDS, PfSPDS mutants, and controls is depicted as net fluorescence intensity over time. Figure 17J depicts representative spermidine synthase activity of PfSPDS and mutants calculated from data in Figure 171 and shown as a function of spermidine concentration over time. Figure 17K depicts representative kinetics of PfSPDS spermidine synthase activity as a function of putrescine concentration. PfSPDS-APT assays were conducted with PfSPDS and varying concentrations of putrescine at 37 °C for 60 minutes. Figure 16L depicts representative spermidine synthase activity as a function of dc-SAM concentrations. PfSPDS-APT assays were conducted as in Figure 17K. Vmax and Kmvalues were determined using Michaelis-Menten kinetics in GraphPad Prism. Data are presented as mean ± S.D. from three independent experiments, each conducted in triplicate. Figure 18, comprising Figure 18A through Figure 18F, depicts representative data demonstrating inhibition of S. cerevisiae Spe3 and P. falciparum PfSPDS activity by 4MCHA. Figure 18A depicts a representative dose-dependent decrease in the activity of MBP-Spe3 with increasing concentrations of 4MCHA (0.0195 pM-100 pM). Figure 18B depicts a representative dose-dependent decrease in the activity of MBP-PfSPDS with increasing concentrations of 4MCHA (0.0195 pM-100 pM). The data are from three independent experiments conducted in triplicates, with error bars denoting mean ± S.E. Figure 18C depicts a representative image of thin-layer chromatography of the reactions performed in Figure 18A, showing a dose dependent decrease in the activity of Spe3. Figure 18D depicts a representative thin-layer chromatography of the reactions performed in Figure 18B, showing a dose dependent decrease in the activity of PfSPDS. Figure 18E depicts representative results demonstrating the determination of the inhibition constant (Ki) value for inhibition of S. cerevisiae Spe3 spermidine synthase activity by 4MCHA. Figure 18F depicts representative results demonstrating the determination of the Ki value for inhibition of P. falciparum PfSPDS spermidine synthase activity by 4MCHA. All data are presented as mean ± S.D from three independent experiments, each conducted in triplicate.

[0054] Figure 19 depicts representative fluorescence of ornithine (ORN), putrescine (PUT), spermidine (SPD), and spermine (SPM) in the presence or absence of 1,2-DAB and P-ME.

[0055] Figure 20 depicts a schematic representation of a 1,2-DAB assay for deoxyhypusine synthase (DHS) activity.

[0056] Figure 21 depicts representative images of Coomassie-stained 4-20% SDS-PAGE gels of fractions eluted from amylose affinity columns for MBP-tagged Spe3 and Spe4.

[0057] Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, each of the following terms has the meaning associated with it in this section. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0059] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0060] As used herein, the term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., Ci-6 means one to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups. Examples include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl,” “haloalky 1” and “homoalkyl.”

[0061] The term “haloCi-6 alkyl” as used herein as a group or part of a group refers to a Ci-6 alkyl group as defined herein wherein at least one hydrogen atom is replaced with a halogen. The term “haloCi-6 alkyl” therefore includes monohaloCi-6 alkyl and also polyhaloCi-6 alkyl. There may be one, two, three or more hydrogen atoms replaced with a halogen, so the haloCi-6 alkyl may have one, two, three or more halogens. Examples of such groups include fluoroethyl, fluoromethyl, difluoromethyl, trifluoromethyl, or trifluoroethyl and the like.

[0062] Similarly, the term “haloCi-6 alkoxy” as used herein as a group or part of a group refers to a Ci-6 alkoxy group as defined herein wherein at least one hydrogen atom is replaced with a halogen. The term “haloCi-6 alkoxy” therefore includes monohaloCi- 6 alkoxy and also polyhaloCi-6 alkoxy. There may be one, two, three or more hydrogen atoms replaced with a halogen, so the haloCi-6 alkyl may have one, two, three, or more halogens. Examples of such groups include fluoroethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, trifluoroethoxy, and the like. The term “C3-8 cycloalkyl” as used herein refers to a saturated monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

[0063] As used herein, the term “substituted alkyl” means alkyl, as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, -OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C=N, -C(=O)O(Ci- C4)alkyl, -C(=0)NH2, -SO2NH2, -C(=NH)NH2, and -NO2. In some embodiments, a substituted alkyl contains one or two substituents selected from halogen, -OH, alkoxy, - NH2, trifluoromethyl, -N(CH3)2, and -C(=O)OH. In specific embodiments, the one or two substituents are selected from halogen, alkoxy, and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxy cyclopentyl and 3- chloropropyl.

[0064] As used herein, the term “alkylene” by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified by (-CH2-)n. By way of example only, such groups include, but are not limited to, groups having 24 or fewer carbon atoms such as the structures -CH2CH2- and -CH2CH2CH2CH2-. The term “alkylene,” unless otherwise noted, is also meant to include those groups described below as “heteroalkylene.”

[0065] As used herein, the terms “alkoxy,” “alkylamino” and “alkylthio” are used in their conventional sense, and refer to alkyl groups linked to molecules via an oxygen atom, an amino group, a sulfur atom, respectively. As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1 -propoxy, 2- propoxy (isopropoxy) and the higher homologs and isomers. In certain embodiments, alkoxy refers to (C1-C3) alkoxy, particularly ethoxy and methoxy.

[0066] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. In some embodiments, a halogen is fluorine, chlorine, or bromine. In specific embodiments, a halogen is fluorine or chlorine. As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, Si, P, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0067] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with at least one polyunsaturated ring and having aromatic character, i.e., having (4n + 2) delocalized 71 (pi) electrons, where n is an integer.

[0068] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing at least one ring (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl.

[0069] As used herein, the term “heterocycle” or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system that consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom that affords a stable structure. A heterocycle may be aromatic or non-aromatic in nature. In some embodiments, the heterocycle is a heteroaryl.

[0070] As used herein, the term “heteroaryl” or “heteroaromatic” refers to aryl groups which contain at least one heteroatom selected from N, O, Si, P, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. A polycyclic heteroaryl may include at least one ring that are partially saturated.

[0071] Examples include tetrahydroquinoline, 2,3 -dihydrobenzofuryl, 1-pyrrolyl, 2-pyrrolyl, 3- pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2- phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4- thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4- pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,

[0072] 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0073] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2, 3 -dihydropyran, tetrahydropyran, 1,4-di oxane, 1,3 -di oxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethyleneoxide.

[0074] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly

[0075] 2-pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0076] Examples of polycyclic heterocycles include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7 -benzofuryl), 2,3 -dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2 -benzothiazolyl and 5-benzothiazolyl), purinyl, benzimidazolyl (particularly 2 -benzimidazolyl), benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0077] The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.

[0078] As used herein, the term “amino aryl” refers to an aryl moiety which contains an amino moiety. Such amino moieties may include, but are not limited to primary amines, secondary amines, tertiary amines, masked amines, or protected amines. Such tertiary amines, masked amines, or protected amines may be converted to primary amine or secondary amine moieties. Additionally, the amine moiety may include an amine-like moiety which has similar chemical characteristics as amine moieties, including but not limited to chemical reactivity.

[0079] As used herein, the term “substituted” means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. For aryl, aryl-(Ci-C3)alkyl and heterocyclyl groups, the term “substituted” as applied to the rings of these groups refers to any level of substitution, namely mono-, di-, tri-, tetra-, or pentasubstitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. In some embodiments, the substituents vary in number between one and four. In another embodiment, the substituents vary in number between one and three. In yet another embodiment, the substituents vary in number between one and two. In yet another embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, -OH, Ci-6 alkoxy, halo, amino, acetamido and nitro. In yet another embodiment, the substituents are independently selected from the group consisting of Ci-6 alkyl, Ci-6 alkoxy, halo, acetamido, and nitro. In another embodiment, the substituents are selected from the group consisting of hydrogen, deuterium, Ci-6 alkyl, C2-6 alkenyl, hydroxy, Ci- 6 alkoxy, halogen, haloCi-6 alkyl, haloCi-6 alkoxy, C3-8 cycloalkyl, nitrile, amino, and combinations thereof. As used herein, where a substituent is an alkyl or alkoxy group, the carbon chain may be branched, straight or cyclic, with straight being predominant.

[0080] As used herein, “combinations thereof’ refers to any combination of any two or more of the preceding substituents, without limit. Several references to integers and R, R1, R2, R3, R4, R5, R6, etc. are made in chemical structures and moieties disclosed and described herein. Any description of integers and R, R1, R2, R3, R4, R5, R6, etc. in the specification is applicable to any structure or moiety reciting integers and R, R1, R2, R3, R4, R5, R6, etc. respectively.

[0081] The term “derivative” refers to a small molecule that differs in structure from the reference molecule but retains the essential properties of the reference molecule. A derivative may change its interaction with certain other molecules relative to the reference molecule. A derivative molecule may also include a salt, an adduct, tautomer, isomer, or other variant of the reference molecule.

[0082] The term “tautomers” are constitutional isomers of organic compounds that readily interconvert by a chemical process (tautomerization).

[0083] The term “isomers” or “stereoisomers” refer to compounds, which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0084] The term “solvate” in accordance with this invention should be understood as meaning any form of the active compound in accordance with the invention in which the said compound is bonded by a non-covalent bond to another molecule (normally a polar solvent), including especially hydrates and alcoholates.

[0085] As used herein, the term “specific binding” refers to that binding which occurs between such paired species as enzyme / substrate, receptor / agonist, antibody / antigen, and lectin / carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions. When the interaction of the two species produces a non-covalently bound complex, the binding which occurs is typically electrostatic, hydrogen-bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody / antigen or enzyme / substrate interaction. In particular, the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs. Thus, for example, an antibody preferentially binds to a single epitope and to no other epitope within the family of proteins. “Inhibitors” is used to refer to molecules that inhibit at least one protein having aminopropyl transferase (APT) activity, respectively. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of at least one protein with APT activity. Inhibitors also include naturally occurring and synthetic ligands, antagonists, antibodies, peptides, cyclic peptides, nucleic acids, ribozymes, small organic molecules, and the like.

[0086] The term “inhibit,” as used herein, means to suppress or block an activity or function, for example, about ten percent relative to a control value. For example, the activity is suppressed or blocked by 50% compared to a control value, or by 75%, or by 95%. “Inhibit,” as used herein, also means to reduce the level of a molecule, a reaction, an interaction, and / or a protein’s stability, amount, function, or activity by a measurable amount or to prevent production entirely. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate a protein function or activity, e.g., antagonists.

[0087] The terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of’ includes determining the amount of something present, as well as determining whether it is present or absent.

[0088] “Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity, or amount (which can be an effective amount) of a given substance within a sample.

[0089] “Control” as used herein refers to a predetermined amount of a particular substance that is detectable in a sample. The control is suitable for the use of a method of the invention, for determining the amount of light absorbed or fluorescence emitted by a fluorescent molecule. An established standard curve provides an expected amount of fluorescent molecules for different levels of APT activity. A control may vary depending on the polyamine, protein, or inhibitor being assayed and the nature of the sample (e.g., solvent, buffers, additives, etc.).

[0090] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range, such as from 1 to 6, should be considered to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0091] Description

[0092] The invention relates, in part, to the discovery that the reaction product of aromatic ketones reacting with polyamines results in fluorescent compounds. Thus, in some embodiments, the invention provides novel compounds resulting from the reaction of aromatic ketones and polyamines. In some embodiments, the aromatic ketone is an aromatic diketone. In some embodiments, the aromatic diketone is 1,2-diacetylbenzene (DAB). In some embodiments, the polyamine is selected from the group consisting of spermidine and spermine.

[0093] In some embodiments, the invention relates to methods of detecting polyamines by detecting fluorescence resulting from the reaction of polyamines with aromatic ketones. In some embodiments, the invention provides methods for detecting enzymatic activity that produces polyamines. In some embodiments, the enzymatic activity is aminopropyl transferase (APT) activity. In some embodiments, the invention provides methods for identifying compounds that prevent, inhibit, or reduce APT activity.

[0094] Compounds

[0095] In some embodiments, the invention relates to the reaction products of an aromatic ketone and a polyamine. In some embodiments, the aromatic ketone is a compound of Formula (I): wherein:

[0096] RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0097] R1, R2, R3, R4, and R5are each independently selected from the group consisting ofH, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0098] In some embodiments, the aromatic ketone of Formula (I) is a compound of Formula (II): wherein: each instance of RKis independently selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0099] R1, R2, R3, and R4are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0100] In some embodiments, each instance of RKis the same. In some embodiments, the compound of Formula (II) is 1,2-diacetylbenzne (DAB).

[0101] In some embodiments, the polyamine is a compound of Formula (III):

[0102] H2N'L"NH2

[0103] Formula (III) wherein is a divalent linker selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, carboxylic acid, amide, ester, ether, carbonyl, carbamate, sulfamate, sulfonic ester, sulfoximine, sulfonamide, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof.

[0104] In some embodiments, divalent linker L comprises Formula (IV):

[0105] Formula (IV) wherein: n =1-6; o = l-6; p = 1-6; and q = 0-3.

[0106] In some embodiments, the compound of Formula (III) is selected from the group consisting of

[0107] (spermidine) (spermine)

[0108] In some embodiments, the invention provides compounds resulting from the reaction of an aromatic ketone and a polyamine. In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (I) and a polyamine of Formula (III). In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (II) and a polyamine of Formula (III). In some embodiments, the compounds are the result of the reaction of 1,2-DAB and a polyamine of Formula (III). In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (I) and spermidine. In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (I) and spermine. In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (II) and spermidine. In some embodiments, the compounds are the result of the reaction of an aromatic ketone of Formula (II) and spermine. In some embodiments, the compounds are the result of the reaction of 1,2-DAB and spermidine. In some embodiments, the compounds are the result of the reaction of 1,2-DAB and spermine.

[0109] In some embodiments, the compound is at least one compound selected from the

[0110] wherein each instance of RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl;

[0111] R1, R2, R3, R4, and R5are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof; and each instance of L is a divalent linker selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, carboxylic acid, amide, ester, ether, carbonyl, carbamate, sulfamate, sulfonic ester, sulfoximine, sulfonamide, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof.

[0112] In some embodiments, divalent linker L comprises Formula (IV): p = 1-6; and q = 0-3. In some embodiments, the compound is at least one selected from the group

[0113] and tautomers, conjugates, salts, and solvates thereof.

[0114] Salts

[0115] Certain compounds of the invention can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulfonate and phosphate salts. The term “salts” embraces addition salts of free acids or free bases which are compounds of the invention.

[0116] All such salts are within the scope of this invention, and references to compounds of the invention include the salt forms of the compounds. The salts of the invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

[0117] Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or disalts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4- acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(lS)-camphor-10- sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane- 1 ,2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L- glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2- sulfonic, naphthalene-1 ,5-disulfonic, l-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins. One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Where the compounds of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the compounds of the invention. The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.

[0118] Certain compounds of the invention may form acid addition salts with at least one equivalent of the acid. The invention includes within its scope all possible stoichiometric and non-stoichiometric forms.

[0119] All of these salts may be prepared by conventional means from the corresponding compound according to the invention by reacting, for example, the appropriate acid or base with the compound according to the invention. In some embodiments, salts are in crystalline form, and prepared by crystallization of the salt from a suitable solvent. The person skilled in the art will know how to prepare and select suitable salt forms for example, as described in Handbook of Pharmaceutical Salts: Properties, Selectin and Use by P. H. Stahl and C. G. Wermuth (Wiley-VCH 2002).

[0120] Solvates

[0121] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates.” For example, a complex with water is known as a “hydrate.” In some embodiments, said crystalline form of the compounds of the invention is a cocrystal or coformer. Such a cocrystal or coformer may be prepared using water-soluble molecules such as saccharin, caffeine, nicotinamide, or carboxylic acids. Coformers may be prepared as described in Emami S et al (2018) BioImpacts 8(4), 305-320, the techniques of which are herein incorporated by reference. It will be understood that the invention includes pharmaceutically acceptable derivatives of compounds of Formula (I) and that these are included within the scope of the invention.

[0122] Isotopes

[0123] The subject invention also includes all pharmaceutically acceptable isotopically- labelled compounds which are identical to those recited above but for the fact that at least one atom is replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention comprise isotopes of hydrogen, such as2H (D) and3H (T), carbon, such asnC,13C, and14C, chlorine, such as36C1, fluorine, such as18F, iodine, such as1231 ,123I and131I, nitrogen, such as13N and15N, oxygen, such as15O ,17O, and18O, phosphorus, such as32P, and sulfur, such as34S. Certain isotopically-labelled compounds of Formula (I), for example, those incorporating a radioactive isotope, are useful in drug and / or substrate tissue distribution studies. The compounds of the invention can also have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes, or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase) etc. The radioactive isotopes tritium, i.e.,3H (T), and carbon-14, i.e.,14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, i.e.,2H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo halflife or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such asnC,18F,15O and13N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0124] Isotopically labelled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using appropriate isotopically-labelled reagents in place of the non-labelled reagent previously employed.

[0125] Methods

[0126] In some embodiments, the invention relates to an method of detecting polyamines in a sample. In some embodiments, the method comprises detecting the presence of a polyamine in a sample by reacting the sample with an aromatic ketone to form a fluorescent compound. In some embodiments, detecting the presence of a polyamine includes measuring the amount (absolute or relative) of polyamine in the sample. In some embodiments, the method comprises the steps of a) preparing a solution comprising the sample and a compound of Formula (I); b) incubating the solution; c) measuring the fluorescence of the solution; and d) comparing the fluorescence of the solution to the fluorescence of a comparator or control, wherein an increase in fluorescence relative to the comparator or control indicates the presence of at least one polyamine compound in the sample: wherein:

[0127] RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0128] R1, R2, R3, R4, and R5are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0129] In some embodiments, the aromatic compound of Formula (I) is a compound of Formula (II) wherein: each instance of RKis independently selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0130] R1, R2, R3, and R4are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0131] In some embodiments, the aromatic ketone is 1,2-diacetylbenzene (DAB). In some embodiments, the polyamine to be detected is at least one selected from the group consisting of spermidine and spermine.

[0132] In some embodiments, the solution of step a) further comprises P- mercaptoethanol (P-ME). In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of sodium tetraborate and potassium phosphate.

[0133] In some embodiments, the concentration of 1,2-DAB in the solution of step a) is between about 1 pM and about 100 mM. In some embodiments, the concentration of 1,2- DAB in the solution of step a) is between about 10 pM and about 10 mM. In some embodiments, the concentration of 1,2-DAB in the solution of step a) is between about 100 pM and about 10 mM. In some embodiments, the concentration of 1,2-DAB in the solution of step a) is between about 1 mM and about 10 mM. In some embodiments, the concentration of 1,2-DAB in the solution of step a) is about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0134] In some embodiments, the concentration of P-ME is between about 1 pM and about 100 mM. In some embodiments, the concentration of -ME in the solution of step a) is between about 10 pM and about 10 mM. In some embodiments, the concentration of P-ME in the solution of step a) is between about 100 pM and about 10 mM. In some embodiments, the concentration of P-ME in the solution of step a) is between about 1 mM and about 10 mM. In some embodiments, the concentration of P-ME in the solution of step a) is about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0135] In some embodiments, the concentration of sodium tetraborate in the solution of step a) is between about 10 pM and about 1 M. In some embodiments, the concentration of sodium tetraborate in the solution of step a) is between about 100 pM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step a) is between about 1 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step a) is between about 10 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step a) is about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0136] In some embodiments, the concentration of potassium phosphate in the solution of step a) is between about 1 pM and about 100 mM. In some embodiments, the concentration of potassium phosphate in the solution of step a) is between about 10 pM and about 10 mM. In some embodiments, the concentration of potassium phosphate in the solution of step a) is between about 100 pM and about 1 mM. In some embodiments, the concentration of potassium phosphate in the solution of step a) is between about 200 pM and about 1 mM. In some embodiments, the concentration of potassium phosphate in the solution of step a) is about 1 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about I .9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0137] In some embodiments, the pH of the solution of step a) is between about 7 and about 12. In some embodiments, the pH of the solution is between about 8 and about 11. In some embodiments, the pH of the solution is between about 9 and about 10. In some embodiments, the pH of the solution is about 7, about 8, about 9, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10, about

[0138] I I, or about 12.

[0139] In some embodiments, step b) comprises incubating the solution at a temperature between about 4 °C and about 50 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 10 °C and about 37 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about

[0140] 20 °C and about 25 °C. In some embodiments, step b) comprises incubating the solution at a temperature of about 4 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about

[0141] 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 45 °C, or about 50 °C. In some embodiments, step b) comprises incubating the solution at about room temperature.

[0142] In some embodiments, step b) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step b) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step b) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step b) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours.

[0143] In some embodiments, step c) comprises analyzing the solution with a spectrophotometer. In some embodiments, step c) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength. In some embodiments, step c) comprises analyzing the solution with a spectrophotometer to obtain an emission spectrum for at least one second wavelength. In some embodiments, step c) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength and an emission spectrum for at least one second wavelength.

[0144] In some embodiments, the excitation spectrum wavelength is between about 350 nm and about 370 nm. In some embodiments, the excitation spectrum wavelength is between about 355 nm and about 369 nm. In some embodiments, the excitation spectrum wavelength is between about 360 nm and about 368 nm. In some embodiments, the excitation spectrum wavelength is between about 361 nm and about 367 nm. In some embodiments, the excitation spectrum wavelength is between about 362 nm and about 366 nm. In some embodiments, the excitation spectrum wavelength is between about 363 nm and about 365 nm. In some embodiments, the excitation spectrum wavelength is about 350 nm, about 355 nm, about 360 nm, about 361 nm, about 362 nm, about 363 nm, about 364 nm, about 365 nm, about 366 nm, about 367 nm, about 368 nm, about 369 nm, or about 370 nm.

[0145] In some embodiments, the emission spectrum wavelength is between about 400 nm and about 450 nm. In some embodiments, the emission spectrum wavelength is between about 410 nm and about 440 nm. In some embodiments, the emission spectrum wavelength is between about 420 nm and about 430 nm. In some embodiments, the emission spectrum wavelength is about 400 nm, about 410 nm, about 420 nm, about 421 nm, about 422 nm, about 423 nm, about 424 nm, about 425 nm, about 426 nm, about 427 nm, about 428 nm, about 429 nm, about 430 nm, about 440 nm, or about 450 nm.

[0146] In some embodiments, the control solution of step e) comprises 1,2-DAB and P- ME. In some embodiments. In some embodiments, the control solution of step e) comprises 1,2-DAB and -ME at about the same concentration as the solution of step a). In some embodiments, an increase in absorbance in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in absorbance at 364 nm in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in emission in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in emission at 425 nm in the emission spectrum indicates the presence of a polyamine. In some embodiments, both an increase in absorbance in the excitation spectrum and an increase in emission in the emission spectrum indicates the presence of a polyamine. In some embodiments, both an increase in absorbance at 364 nm in the excitation spectrum and an increase in emission at 425 nm in the emission spectrum indicate the presence of a polyamine.

[0147] In some embodiments, the method is performed in a multi-well plate. In some embodiments, steps a) through c) are all performed in a single well of a multi-well plate. In some embodiments, the method is performed in a high-throughput manner, wherein multiple samples are analyzed in multiple wells of a single multi-well plate.

[0148] In some embodiments, the invention provides methods of detecting polyamine synthesis or catabolism activity of a protein in a sample. In some embodiments, the polyamine synthesis activity is an aminopropyl transferase (APT) activity. In some embodiments, the polyamine synthesis activity is an ornithine decarboxylase (ODC) activity. In some embodiments, the polyamine catabolism activity is a deoxyhypusine synthase (DHS) activity. In some embodiments, the polyamine catabolism activity is a spermidine / spermine N(l)-acetyltransferase (SSAT) activity. In some embodiments, the method comprise: a) preparing a solution comprising the sample of interest; pyridoxal phosphate (PLP), decarboxylated-S-adenosylmethionine (dc-SAM), nicotinamide adenine dinucleotide (NAD+), eukaryotic translation initiation factor 5A (eIF-5A), and / or acetyl-CoA; and ornithine, putrescine, or spermidine; b) incubating the solution; and c) detecting the presence of at least one polyamine in the solution according to a method of the invention, wherein an increase in fluorescence in the solution relative to the control solution indicates that the protein has polyamine synthesis activity.

[0149] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA). In some embodiments, step b) comprises incubating the solution at a temperature between about 15 °C and about 50 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 20 °C and about 45 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 25 °C and about 40 °C. In some embodiments, step b) comprises incubating the solution at a temperature of about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C.

[0150] In some embodiments, step b) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step b) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step b) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step b) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours.

[0151] In some embodiments, the sample solution is being compared to a control solution comprising all the components of the sample solution except one. In some embodiments, the control solution comprises all the components of the sample solution except PLP, dc- SAM, NAD+, eIF-5A, acetyl-CoA, and / or 1,2-DAB.

[0152] In some embodiments, the method comprises the steps of: a) preparing a solution comprising the protein; PLP, dc-SA, NAD+, eIF-5A, and / or acetyl-CoA; and ornithine, putrescine, or spermidine; b) incubating the solution; c) adding a compound of Formula (I) and P-ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has polyamine synthesis activity: wherein:

[0153] RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0154] R1, R2, R3, R4, and R5are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0155] In some embodiments, the aromatic compound of Formula (I) is a compound of Formula (II): wherein: each instance of RKis independently selected from the group consisting of Ci-C6alkyl and Ci-Ce haloalkyl; and

[0156] R1, R2, R3, and R4are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0157] In some embodiments, the aromatic ketone is 1,2-diacetylbenzene (DAB).

[0158] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA). In some embodiments, step b) comprises incubating the solution at a temperature between about 15 °C and about 50 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 20 °C and about 45 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 25 °C and about 40 °C. In some embodiments, step b) comprises incubating the solution at a temperature of about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C.

[0159] In some embodiments, step b) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step b) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step b) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step b) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours.

[0160] In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 10 pM and about 1 M. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 100 pM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 1 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 10 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0161] In some embodiments, step d) comprises incubating the solution at a temperature between about 4 °C and about 50 °C. In some embodiments, step d) comprises incubating the solution at a temperature between about 10 °C and about 37 °C. In some embodiments, step d) comprises incubating the solution at a temperature between about

[0162] 20 °C and about 25 °C. In some embodiments, step d) comprises incubating the solution at a temperature of about 4 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about

[0163] 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 45 °C, or about 50 °C. In some embodiments, step d) comprises incubating the solution at about room temperature.

[0164] In some embodiments, step d) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step d) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step d) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step d) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an emission spectrum for at least one second wavelength. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength and an emission spectrum for at least one second wavelength.

[0165] In some embodiments, the excitation spectrum wavelength is between about 350 nm and about 370 nm. In some embodiments, the excitation spectrum wavelength is between about 355 nm and about 369 nm. In some embodiments, the excitation spectrum wavelength is between about 360 nm and about 368 nm. In some embodiments, the excitation spectrum wavelength is between about 361 nm and about 367 nm. In some embodiments, the excitation spectrum wavelength is between about 362 nm and about 366 nm. In some embodiments, the excitation spectrum wavelength is between about 363 nm and about 365 nm. In some embodiments, the excitation spectrum wavelength is about 350 nm, about 355 nm, about 360 nm, about 361 nm, about 362 nm, about 363 nm, about 364 nm, about 365 nm, about 366 nm, about 367 nm, about 368 nm, about 369 nm, or about 370 nm.

[0166] In some embodiments, the emission spectrum wavelength is between about 400 nm and about 450 nm. In some embodiments, the emission spectrum wavelength is between about 410 nm and about 440 nm. In some embodiments, the emission spectrum wavelength is between about 420 nm and about 430 nm. In some embodiments, the emission spectrum wavelength is about 400 nm, about 410 nm, about 420 nm, about 421 nm, about 422 nm, about 423 nm, about 424 nm, about 425 nm, about 426 nm, about 427 nm, about 428 nm, about 429 nm, about 430 nm, about 440 nm, or about 450 nm.

[0167] In some embodiments, the control solution of step f) comprises all the components of the solution of step e) except one. In some embodiments, the control solution of step f) comprises all the components of the solution of step f) except PIP, dc- SAM, NAD+, eIF-5A, acetyl-CoA, and / or 1,2-DAB. In some embodiments, an increase in absorbance in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in absorbance at 364 nm in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in emission in the excitation spectrum indicates the presence of a polyamine. In some embodiments, an increase in emission at 425 nm in the emission spectrum indicates the presence of a polyamine. In some embodiments, both an increase in absorbance in the excitation spectrum and an increase in emission in the emission spectrum indicates the presence of a polyamine. In some embodiments, both an increase in absorbance at 364 nm in the excitation spectrum and an increase in emission at 425 nm in the emission spectrum indicate the presence of a polyamine.

[0168] In some embodiments, the method is performed in a multi-well plate. In some embodiments, steps a) through e) are all performed in a single well of a multi-well plate. In some embodiments, the method is performed in a high-throughput manner, wherein multiple samples are analyzed in multiple wells of a single multi-well plate.

[0169] In some embodiments, the invention provides methods of identifying a compound as an inhibitor of a protein with polyamine biosynthesis or catabolism activity. In some embodiments, the polyamine synthesis activity is APT activity. In some embodiments, the protein with APT activity is a spermidine synthase (SRM) or a spermine synthase (SMS). In some embodiments, the SRM is human SRM. In some embodiments, the SRM is an SRM from a human pathogen. In some embodiments, the SMS is human SMS. In some embodiments, the SMS is an SMS from a human pathogen. In some embodiments, the polyamine synthesis activity is ornithine decarboxylase activity. In some embodiments, the protein with ornithine decarboxylase activity is an ornithine decarboxylase (ODC). In some embodiments, the ODC is human ODC. In some embodiments, the ODC is an ODC from a human pathogen. In some embodiments, the polyamine catabolism activity is deoxyhypusine synthase activity. In some embodiments, the protein with deoxyhypusine synthase activity is a deoxyhypusine synthase (DHS). In some embodiments, the DHS is human DHS. In some embodiments, the polyamine catabolism activity is a spermidine / spermine N(l)-acetyltransferase activity. In some embodiments, the protein with polyamine catabolism activity is a spermidine / spermine N(l)-acetyltransferase (SSAT). In some embodiments, the SSAT is human SSAT. In some embodiments, the method comprises the steps of detecting polyamine biosynthesis activity of a sample, wherein the sample comprises the protein with polyamine biosynthesis activity, a polyamine precursor, and the compound of interest, and wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein with polyamine biosynthesis activity. In some embodiments, a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity.

[0170] In some embodiments, the method comprises the steps of detecting polyamine catabolism activity of a sample, wherein the sample comprises the protein with polyamine catabolism activity, a polyamine precursor, and the compound of interest, and wherein an alteration in fluorescence relative to the control solution indicates the compound is a modulator of the protein with polyamine catabolism activity. In some embodiments, an increase in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein with deoxy hypusine synthase activity. In some embodiments, a decrease in fluorescence relative to the control solution indicates the compound is an activator of the protein with spermidine / spermine N(l)-acetyltransferase activity.

[0171] In some embodiments, the method comprises the steps of a) preparing a solution comprising the compound, the protein with polyamine biosynthesis activity; pyridoxal phosphate (PLP), decarboxylated-S-adenosylmethionine (dc-SAM), nicotinamide adenine dinucleotide (NAD+), eukaryotic translation initiation factor 5A (eIF-5A), and / or acetyl-CoA; and ornithine, putrescine, or spermidine; b) incubating the solution; c) adding to the solution a compound of Formula (I) and |3-ME; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution;

[0172] Formula (I) wherein:

[0173] RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0174] R1, R2, R3, R4, and R5are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0175] In some embodiments, a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein. In some embodiments, a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of a protein with APT activity or ornithine decarboxylase activity. In some embodiments, an increase in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein. In some embodiments, an increase in fluorescence relative to the control solution indicates the compound is an inhibitor of a protein with deoxyhypusine synthase activity.

[0176] In some embodiments, the aromatic compound of Formula (I) is a compound of Formula (II)

[0177] Formula (II) wherein: each instance of RKis independently selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl; and

[0178] R1, R2, R3, and R4are each independently selected from the group consisting ofH, D, halogen, Ci-Ce alkylene, Ci-Ce haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof.

[0179] In some embodiments, the aromatic ketone is 1,2-diacetylbenzene (DAB).

[0180] In some embodiments, the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0181] In some embodiments, step b) comprises incubating the solution at a temperature between about 15 °C and about 50 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 20 °C and about 45 °C. In some embodiments, step b) comprises incubating the solution at a temperature between about 25 °C and about 40 °C. In some embodiments, step b) comprises incubating the solution at a temperature of about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50 °C.

[0182] In some embodiments, step b) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step b) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step b) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step b) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours.

[0183] In some embodiments, step c) further comprises adding sodium tetraborate to the solution. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 10 pM and about 1 M. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 100 pM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 1 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is between about 10 mM and about 100 mM. In some embodiments, the concentration of sodium tetraborate in the solution of step c) is about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 100 pM, about 200 pM, about 300 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0184] In some embodiments, step d) comprises incubating the solution at a temperature between about 4 °C and about 50 °C. In some embodiments, step d) comprises incubating the solution at a temperature between about 10 °C and about 37 °C. In some embodiments, step d) comprises incubating the solution at a temperature between about

[0185] 20 °C and about 25 °C. In some embodiments, step d) comprises incubating the solution at a temperature of about 4 °C, about 5 °C, about 10 °C, about 15 °C, about 20 °C, about

[0186] 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 45 °C, or about 50 °C. In some embodiments, step d) comprises incubating the solution at about room temperature.

[0187] In some embodiments, step d) comprises incubating the solution for between about 1 minute and about 6 hours. In some embodiments, step d) comprises incubating the solution for between about 10 minutes and about 3 hours. In some embodiments, step d) comprises incubating the solution for between about 30 minutes and about 2 hours. In some embodiments, step d) comprises incubating the solution for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, or about 6 hours.

[0188] In some embodiments, step e) comprises analyzing the solution with a spectrophotometer. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an emission spectrum for at least one second wavelength. In some embodiments, step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum for at least one first wavelength and an emission spectrum for at least one second wavelength.

[0189] In some embodiments, the excitation spectrum wavelength is between about 350 nm and about 370 nm. In some embodiments, the excitation spectrum wavelength is between about 355 nm and about 369 nm. In some embodiments, the excitation spectrum wavelength is between about 360 nm and about 368 nm. In some embodiments, the excitation spectrum wavelength is between about 361 nm and about 367 nm. In some embodiments, the excitation spectrum wavelength is between about 362 nm and about 366 nm. In some embodiments, the excitation spectrum wavelength is between about 363 nm and about 365 nm. In some embodiments, the excitation spectrum wavelength is about 350 nm, about 355 nm, about 360 nm, about 361 nm, about 362 nm, about 363 nm, about 364 nm, about 365 nm, about 366 nm, about 367 nm, about 368 nm, about 369 nm, or about 370 nm.

[0190] In some embodiments, the emission spectrum wavelength is between about 400 nm and about 450 nm. In some embodiments, the emission spectrum wavelength is between about 410 nm and about 440 nm. In some embodiments, the emission spectrum wavelength is between about 420 nm and about 430 nm. In some embodiments, the emission spectrum wavelength is about 400 nm, about 410 nm, about 420 nm, about 421 nm, about 422 nm, about 423 nm, about 424 nm, about 425 nm, about 426 nm, about 427 nm, about 428 nm, about 429 nm, about 430 nm, about 440 nm, or about 450 nm.

[0191] In some embodiments, the control solution of step f) comprises all the components of the solution of step e) except one. In some embodiments, the control solution of step f) comprises all the components of the solution of step f) except PIP, dc- SAM, NAD+, eTF-5A, acetyl-CoA, and / or 1 ,2-DAB. In some embodiments, a decrease in absorbance in the excitation spectrum indicates the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity. In some embodiments, an increase in absorbance in the excitation spectrum indicates the compound is an inhibitor of the protein with deoxy hypusine synthase activity. In some embodiments, a decrease in absorbance at 364 nm in the excitation spectrum indicates the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity. In some embodiments, an increase in absorbance at 364 nm in the excitation spectrum indicates the compound is an inhibitor of the protein with deoxyhypusine synthase activity. In some embodiments, a decrease in emission in the excitation spectrum indicates the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity. In some embodiments, an increase in emission in the excitation spectrum indicates the compound is an inhibitor of the protein with deoxhyhypusine synthase activity. In some embodiments, a decrease in emission at 425 nm in the emission spectrum indicates the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity. In some embodiments, an increase in emission at 425 nm in the emission spectrum indicates the compound is an inhibitor of the protein with deoxyhypusine activity. In some embodiments, both a decrease in absorbance in the excitation spectrum and a decrease in emission in the emission spectrum indicates the compound is an inhibitor of a protein with APT activity or ornithine decarboxylase activity. In some embodiments, both an increase in absorbance in the excitation spectrum and an increase in emission in the emission spectrum indicates the compound is an inhibitor of a protein with deoxyhypusine synthase activity. In some embodiments, both a decrease in absorbance at 364 nm in the excitation spectrum and a decrease in emission at 425 nm in the emission spectrum indicate the compound is an inhibitor of the protein with APT activity or ornithine decarboxylase activity. In some embodiments, both an increase in absorbance at 364 nm in the excitation spectrum and an increase in emission at 425 nm in the emission spectrum indicate the compound is an inhibitor of the protein with deoxyhypusine synthase activity.

[0192] In some embodiments, the method is performed in a multi-well plate. In some embodiments, steps a) through e) are all performed in a single well of a multi-well plate. In some embodiments, the method is performed in a high-throughput manner, wherein multiple samples are analyzed in multiple wells of a single multi-well plate.

[0193] In some embodiments, the identified compound is an inhibitor of polyamine synthesis activity. In some embodiments, the identified compound is an inhibitor of polyamine synthesis activity involved in the development or maintenance of a microbial infection, cancer, or neurodegeneration. In some embodiments, the identified compound is a target for the treatment of a microbial infection, cancer, or neurodegeneration.

[0194] Suitable test compounds include, but are not limited to, a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a nucleic acid, an antisense nucleic acid, an shRNA, a ribozyme, and a small molecule chemical compound.

[0195] In various embodiments, the test compound concentration in the screening assay can be fixed or varied. A single test compound, or a plurality of test compounds, can be tested at one time. The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam et al., 1997, Anti cancer Drug Des. 12:45).

[0196] Examples of methods for the synthesis of molecular libraries can be found in the art, for example, in: DeWitt et al., 1993, Proc. Natl. Acad. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261: 1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.

[0197] Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladner supra).

[0198] In situations where “high-throughput” modalities are preferred, it is typical that new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.

[0199] In one embodiment, high throughput screening methods involve providing a library containing a large number of test compounds potentially having the desired activity. Such “combinatorial chemical libraries” are then screened in at least one assay, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used.

[0200] EMBODIMENTS

[0201] Embodiment l is a method of detecting the presence of at least one polyamine compound in a sample comprising the steps of: a) preparing a solution comprising the sample and 1,2- diacetylbenzene (DAB); b) incubating the solution; c) measuring fluorescence of the solution; and d) comparing the fluorescence of the solution to the fluorescence of a control solution, wherein an increase in fluorescence relative to the control solution indicates the presence of at least one polyamine compound in the sample.

[0202] Embodiment 2 is the method of embodiment 1, wherein the polyamine is at least one selected from the group consisting of ornithine, putrescine, spermidine, acetyl-spermidine, spermine, acetyl-spermine, and deoxyhypusine.

[0203] Embodiment 3 is the method of embodiment 1 or 2, wherein the solution of step a) further comprises P-mercaptoethanol (P-ME). Embodiment 4 is the method of any one of embodiments 1-3, wherein the solution of step a) further comprises at least one selected from the group consisting of sodium tetraborate and potassium phosphate.

[0204] Embodiment 5 is the method of any one of embodiments 1-4, wherein step b) comprises incubating the solution at about room temperature for about 60 minutes.

[0205] Embodiment 6 is the method of any one of embodiments 1-5, wherein step c) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0206] Embodiment 7 is the method of any one of embodiments 1-6, wherein the control solution of step d) comprises 1,2-DAB and P-ME at about the same concentration as the sample solution of step a), wherein an increase in absorbance at 364 nm in the excitation spectrum relative to the control solution, an increase in emission at 425 nm relative to the control solution, or both indicate the presence of a polyamine.

[0207] Embodiment 8 is the method of any one of embodiments 1-7, wherein steps a) through c) are performed in at least one well of a multi-well plate.

[0208] Embodiment 9 is a method of detecting aminopropyl transferase (APT) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, decarboxylated-S- adenosylmethionine (dc-SAM), and either putrescine or spermidine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has APT activity.

[0209] Embodiment 10 is the method of embodiment 9, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0210] Embodiment 11 is the method of embodiment 9 or 10, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0211] Embodiment 12 is the method of any one of embodiments 9-12, wherein step c) further comprises adding sodium tetraborate to the solution.

[0212] Embodiment 13 is the method of any one of embodiments 9-, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0213] Embodiment 14 is the method of any one of embodiments 9-13, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0214] Embodiment 15 is the method of any one of embodiments 9-14, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of dc-SAM and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has APT activity.

[0215] Embodiment 16 is the method of any one of embodiments 9-15, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0216] Embodiment 17 is a method of identifying a compound as an inhibitor of a protein with APT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with APT activity, dc-SAM, and either putrescine or spermidine; b) incubating the solution; c) adding to the solution P-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0217] Embodiment 18 is the method of embodiment 17, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA.

[0218] Embodiment 19 is the method of embodiment 17 or 18, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0219] Embodiment 20 is the method of any one of embodiments 17-19, wherein step c) further comprises adding sodium tetraborate to the solution.

[0220] Embodiment 21 is the method of any one of embodiments 17-20, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0221] Embodiment 22 is the method of any one of embodiments 17-22, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0222] Embodiment 23 is the method of any one of embodiments 17-22, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with APT activity.

[0223] Embodiment 24 is the method of any one of embodiments 17-23, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0224] Embodiment 25 is a compound selected from the group consisting of

[0225] wherein each instance of RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl;

[0226] R1, R2, R3, R4, and R are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Cs haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof; and each instance of L is a divalent linker selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, sulfonic ester, sulfoximine, sulfonamide, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof, and tautomers, conjugates, salts, and solvates thereof.

[0227] Embodiment 26 is the compound of embodiment 25, wherein divalent linker L comprises Formula (IV):

[0228] Formula (IV) wherein: n =1-6; o = l-6; p = 1-6; and q = 0-3.

[0229] Embodiment 27 is the compound of embodiment 25 or 26, wherein the compound is selected from the group consisting of:

[0230] and tautomers, conjugates, salts, and solvates thereof.

[0231] Embodiment 28 is a method of detecting ornithine decarboxylase (ODC) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, pyridoxal phosphate (PEP), and ornithine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has ODC activity.

[0232] Embodiment 29 is the method of embodiment 28, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0233] Embodiment 30 is the method of embodiment 28 or 29, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0234] Embodiment 31 is the method of any one of embodiments 28-30, wherein step c) further comprises adding sodium tetraborate to the solution.

[0235] Embodiment 32 is the method of any one of embodiments 28-31, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0236] Embodiment 33 is the method of any one of embodiments 28-32, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0237] Embodiment 34 is the method of any one of embodiments 28-33, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of PIP and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has ODC activity.

[0238] Embodiment 35 is the method of any one of embodiments 28-34, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0239] Embodiment 36 is a method of identifying a compound as an inhibitor of a protein with ODC activity comprising the steps of: a) preparing a solution comprising the compound, the protein with ODC activity, PIP, and ornithine; b) incubating the solution; c) adding to the solution P-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0240] Embodiment 37 is the method of embodiment 36, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA.

[0241] Embodiment 38 is the method of embodiment 36 or 37, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0242] Embodiment 39 is the method of any one of embodiments 36-38, wherein step c) further comprises adding sodium tetraborate to the solution.

[0243] Embodiment 40 is the method of any one of embodiments 36-39, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0244] Embodiment 41 is the method of any one of embodiments 36-40, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0245] Embodiment 42 is the method of any one of embodiments 36-41, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with ODC activity.

[0246] Embodiment 43 is the method of any one of embodiments 36-42, wherein steps a) through e) are performed in at least one well of a multi-well plate. Embodiment 44 is a method of detecting deoxyhypusine synthase (DHS) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, nicotinamide adenine dinucleotide (NAD+), eukaryotic translation initiation factor 5A (eIF-5A), and spermidine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has DHS activity.

[0247] Embodiment 45 is the method of embodiment 44, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0248] Embodiment 46 is the method of embodiment 44 or 45, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0249] Embodiment 47 is the method of any one of embodiments 44-46, wherein step c) further comprises adding sodium tetraborate to the solution.

[0250] Embodiment 48 is the method of any one of embodiments 44-47, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0251] Embodiment 49 is the method of any one of embodiments 44-48 method of embodiment 44, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm. Embodiment 50 is the method of any one of embodiments 44-49, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of NAD+, eIF-5A, and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has DHS activity.

[0252] Embodiment 51 is the method of any one of embodiments 44-50, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0253] Embodiment 52 is a method of identifying a compound as an inhibitor of a protein with DHS activity comprising the steps of: a) preparing a solution comprising the compound, the protein with DHS activity, NAD+, eIF-5A, and spermidine; b) incubating the solution; c) adding to the solution -ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0254] Embodiment 53 is the method of embodiment 52, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA.

[0255] Embodiment 54 is the method of embodiment 52 or 53, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0256] Embodiment 55 is the method of any one of embodiments 52-54, wherein step c) further comprises adding sodium tetraborate to the solution.

[0257] Embodiment 56 is the method of any one of embodiments 52-55, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0258] Embodiment 57is the method of any one of embodiments 52-56, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0259] Embodiment 58 is the method of any one of embodiments 52-57, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with DHS activity.

[0260] Embodiment 59 is the method of any one of embodiments 52-58, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0261] Embodiment 60 is a method of detecting spermidine / spermine N(l)- acetyltransferase (SSAT) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has SSAT activity.

[0262] Embodiment 61 is the method of embodiment 60, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

[0263] Embodiment 62 is the method of embodiment 60 or 61, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes. Embodiment 63 is the method of any one of embodiments 60-62, wherein step c) further comprises adding sodium tetraborate to the solution.

[0264] Embodiment 64 is the method of any one of embodiments 60-63, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0265] Embodiment 65 is the method of any one of embodiments 60-64, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0266] Embodiment 66 is the method of any one of embodiments 60-65, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of acetyl-CoA and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has SSAT activity.

[0267] Embodiment 67 is the method of any one of embodiments 60-66, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0268] Embodiment 68 is a method of identifying a compound as an inhibitor of a protein with SSAT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with SSAT activity, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding to the solution [3-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

[0269] Embodiment 69 is the method of embodiment 68, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and BSA.

[0270] Embodiment 70 is the method of embodiment 68 or 69, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

[0271] Embodiment 71 is the method of any one of embodiments 68-70, wherein step c) further comprises adding sodium tetraborate to the solution.

[0272] Embodiment 72 is the method of any one of embodiments 68-71, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

[0273] Embodiment 73 is the method of any one of embodiments 68-72, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

[0274] Embodiment 74 is the method of any one of embodiments 68-73, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with SSAT activity.

[0275] Embodiment 75 is the method of any one of embodiments 68-74, wherein steps a) through e) are performed in at least one well of a multi-well plate.

[0276] EXPERIMENTAL EXAMPLES

[0277] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0278] Example 1 : 1,2-D AB-APT, a Fluorescence-Based Assay for Measuring Polyamine Biosynthesis Aminopropyl Transferase-Mediated Catalysis.

[0279] Polyamines are polycationic molecules that are crucial in a wide array of cellular functions. Their biosynthesis is mediated by aminopropyl transferases (APTs), promising targets in antimicrobial, antineoplastic and anti-neurodegenerative therapies. A major limitation, however, is the lack of high-throughput assays to measure their activity. The first fluorescence-based assay, 1,2-D AB-APT, for measurement of APT activity using 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with increasing carbon chain length has been developed. The assay has been validated using APT enzymes from S. cerevisiae and P. falciparum and is suitable for high-throughput screening of large chemical libraries. Given the importance of APTs in infectious diseases, cancer and neurobiology, the 1,2-D AB-PA Assay reported herein has broad applications for advancing research and drug discovery efforts. Here, the development of a simple and easy to use assay (DAB-APT) for the measurement of APT enzyme activity is reported. The assay, which can be used in 96- and 384-well formats uses the primary amine reactive 1,2-diacetyl benzene, which forms fluorescent conjugates with putrescine, spermidine and spermine with fluorescence intensity increasing with the length of each of these polyamines. The 1,2-D AB-PA Assay was evaluated and validated using the Spe3 and Spe4 enzymes of S. cerevisiae, and the SPDS enzyme of P. falciparum and shown to be suitable for determining their biochemical activity, catalytic properties, and inhibition by known inhibitors, setting the stage for future chemical screens to identify new drugs with antimicrobial activity as well as other with applications in other fields such as cancer and neurodegeneration.

[0280] The materials and methods are now described: Materials

[0281] S. cerevisiae SPE3 and SPE4, and P. falciparum SPDS were codon- optimized for expression in E. coli, chemically synthesized and cloned into pMAL-c4x-l- H(RBS) plasmid by GenScript. Putrescine (P5780-5G), spermidine (S0266-1G), spermine (S4264-1G), 5-Deoxy-5-methylthioadenosine (260585), 1,2-Diacetylbenzene (DAB) (242039) were purchased from Millipore Sigma. Decarboxylated S-adenosyl methionine was purchased from BOC Sciences, USA, and 2-Mercaptoethanol (1610710) was purchased from Bio-Rad, USA.

[0282] Expression and purification of MBP-tagged SPE3, SPE4 and PfSPDS The SPE3-pMAL-c4x-l-H(RBS), SPE4-pMAL-c4x-l-H(RBS) and PfSPDS-pMAL-c4x-l-H(RBS) plasmids were transformed into Rosetta (DE3) E. coli cells (Fisher Scientific, 713973). Two clones from each were selected and tested for expression of MBP-tagged Spe3, Spe4, and PfSPDS. Briefly, each clone was inoculated into 5 mb of Luria broth 24 containing ampicillin (50 pg / ml) and allowed to grow overnight at 37 °C incubator shaker (200 rpm). The following day, secondary cultures were initiated using the primary cultures and grown to 0.6 OD600. Following this, 0.5 mM isopropyl -thiogalactopyranoside (IPTG) was added and the cultures were shifted from 37 °C to 16 °C and allowed to grow for 16 hours. The cultures were collected by centrifugation at 5000 g for 5 minutes. The pellets were resuspended in IX Laemmli sample buffer (1610737EDU; Bio-Rad), boiled at 95 °C for 10 min and centrifuged at 10,000 g for 5 minutes. The supernatants from uninduced and induced samples were run on 4-20% SDS-PAGE (4561096; Bio-Rad) to check protein expression using Coomassie staining.

[0283] For the purification of recombinant MBP-tagged Spe3, MBP-Spe4, MBP- PfSPDS, 500 mL culture for each protein was grown in LB medium containing ampicillin (50 pg / ml) and induced with 0.5 mM IPTG. The bacterial pellets were harvested ~12 hours after induction and resuspended in lysis buffer (25 mM Tris-HCl pH 8.0, 500mM NaCl, 0.5% glycerol, and 50 mM L-arginine, DNase 250 UL / pL, protease inhibitor cocktail, 0.002% 3-((3-cholamidopropyl) dimethylammonuium-1- propane sulfonate (CHAPS) and disrupted by sonication on ice (Omni Sonic Ruptor 400 Ultrasonic Homogenizer) by 15 sec burst at 70% amplitude, 5 times, with 30 sec cooling intervals. The bacterial lysates were centrifuged at 16,000 x g for 20 min and the supernatants containing the recombinant MBP-Spe3, MBP-Spe4, and MBP-PfSPDS were collected. Next, three columns containing 500 pL of amylose magnetic beads (NEB, E8021L) each were packed and equilibrated with column buffer (200 mM NaCl, 20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT). The supernatants were incubated with the amylose resin for 2.5 hour at 4 °C with end-to-end shaking. Following this, the columns were washed with 10 volumes of the column buffer and the protein was eluted using 10 mM maltose (M75- 100; Fisher Scientific). The purified recombinant MBP-Spe3, MBP-Spe4, and MBP- PfSPDS were run on SDS-PAGE and stained with Coomassie blue to check the purity. The protein concentration was determined using nanodrop (Biotek SynergyMX with take3 plate; TAKE3-SN).

[0284] Fluorescence based APT assay.

[0285] The APT enzyme reaction and subsequent detection of the products (spermidine and spermine) using 1,2-DAB / p-ME was performed in the following sequential steps. In the first step, recombinant Spe3, Spe4, and PfSPDS were used in the aminopropyl transferase activity assay. For determining the spermidine synthase activity of recombinant Spe3 and PfSPDS, the enzyme reactions were set up containing either 0.1 mM or 0.5 mM dc-SAM (BOC Biosciences), 0.1 mM or 0.5 mM putrescine, 1 mM EDTA, ImM DTT, 1 pg BSA, 1 pg of either Spe3 or PfSPDS, and 50 mM potassium phosphate buffer (pH 7.5), in 50 pL total volume. The enzyme reaction was incubated at 37 °C for 60 minutes. For determination of spermine synthase activity of Spe4, the enzyme reactions were set up containing 0.75 mM dc-SAM, 0.5 mM spermidine, ImM EDTA, 1 mM DTT, 1 pg BSA, Ipg Spe4, and 50 mM potassium phosphate buffer (pH

[0286] 7.5), in 50 pL total volume. The enzyme reaction was incubated at 37 °C for 60 minutes.

[0287] Following incubation, enzyme activities were terminated by the addition of detection buffer. 35 pL of the above enzyme reactions were mixed with 85 pL detection buffer (1.75 mM b-mercaptoethanol, 64.5 mM sodium tetraborate buffer (pH

[0288] 9.6), 0.22 mM potassium phosphate buffer, 1.48 mM 1,2-diacetyl benzene) in a 96-well black clear bottom plate (265301, ThermoFisher Scientific) and incubated at 22 °C (room temperature) for 60 minutes. Following this, total fluorescence intensities (lex = 364 nm and lem =425 nm) were measured using a plate reader (Biotek Synergy Hl, Agilent) and data was analyzed in GraphPad prism.

[0289] The dual activity of PfSPDS was also tested using putrescine as a substrate. Briefly, the enzyme reactions were set up using 1 pg active or heat inactivated PfSPDS, 0.5 mM putrescine, 1 mM dc-SAM, , 1 mM EDTA, 1 mM DTT, 1 pg BSA, and 50 mM potassium phosphate buffer (pH 7.5), in 50 pl total volume. The enzyme reactions were incubated at 37 °C for 90 minutes. The reactions were stopped by heat inactivation at 85 °C for 15 minutes. The reactions were precipitated with 3 volumes of cold acetonitrile and the samples were subjected to LC-MS analysis to identify the substrate (putrescine) and products (spermidine and spermine).

[0290] Polyamine Analysis by Thin Layer Chromatography

[0291] Spermidine formation from putrescine and dcSAM substrates after spermidine synthase reaction was confirmed by thin layer chromatography (TLC) on Silica 60 plates (Merck; 500 pm) using a solvent system consisting of n-butanol: acetic acid: pyridine: water (3:3:2: 1, v / v / v / v). Polyamines were visualized with ninhydrin spray followed by incubation at 110 °C for 5 minutes.

[0292] Analysis of Polyamine-DAB Adducts using Flow Injection Analysis (FIA)-MS and MS / MS Fragmentation

[0293] FIA-MS: 2 pL of each sample was injected for analysis by F1A-MS using a Sciex 4000 QTRAP mass spectrometer, operated in ESI positive mode with QI MS full scan data acquisition from m / z 50-2000. The gradient mobile phase was delivered at a flow rate of 0.2 mL / min, with 80% methanol and 20% water as the mobile solvent. The MS / MS data was obtained by using Product Ion MS / MS scan type mode to obtain the fragmentation products of specified masses.

[0294] For LCMS, the system utilized a binary solvent system with buffer A as 100% water, 0.1% formic acid and buffer B asl00% acetonitrile, 0.1% formic acid. Trapping was performed at 5 pl / min, 97% buffer A for 3 min using a Waters Symmetry® C18 180pm x 20mm trap column. Samples were separated using an ACQUITY UPLC PST (BEH) Cl 8 nanoACQUITY Column 1 .7 pm, 75 pm x 250 mm (37°C) and eluted at 300 nL / min with the following gradient: 3% buffer B at initial conditions; 5% B at 1 minute; 30% B at 10 minutes; 50% B at 20 minutes; 95% B at 25-35 minutes; return to initial conditions at 40-55 minutes. MS was acquired in an Orbitrap in profile mode over the 50-700 m / z range using wide quadrupole isolation, 1 microscan, 120,000 resolution, AGC target of 4E5, and a maximum injection time of 60 ms. Data dependent MS / MS were collected in top speed mode with a 3 second cycle time on species with an intensity threshold of 5E4, charge states 2-8, and peptide monoisotopic precursor selection preferred. Dynamic exclusion was set to 30 seconds. MS / MS were acquired in the Orbitrap in centroid mode using quadrupole isolation (window 1.6 m / z), HCD activation with a collision energy of 28%, 1 microscan, 60,000 resolution, AGC target of 1E5, maximum injection time of 100 ms.

[0295] The concentrations of putrescine, spermidine, and spermine are measured by API 4000 QTrap® mass spectrometer (Applied Biosystems Sciex, Toronto, Canada) coupled with Agilent HP 1200 HPLC system (Agilent Technologies, Santa Clara, CA). A targeted Multiple Reaction Monitoring (MRM) method was utilized to quantify the level of putrescine, spermidine, and spermine. The standards were prepared at the concentration range of 25-2,500 ng / mL, quality controls (QCs) were prepared at concentrations of 100, 500 and 2,000 ng / mL. Analyst 1.7 software was used for data acquisition and analysis (Applied Biosystems Sciex, Toronto, Canada). An Agilent Eclipse XDB-C18 column (3.5 microns, 2.1 x 100 mm) coupled with Agilent C18 guard column was utilized for the liquid chromatography separation at 50 °C. The gradient started with 98% of 0.1% formic acid in water (A) and 2% of 0.1% formic acid in acetonitrile (B), maintained for 0.2 minutes and increased to 30% B in 4.3 minutes, further increased to 50% B in 1.5 minutes, and 85% in 0.5 minute, and maintained at 85% B for 1.5 minutes, followed by return to 2% B in 0.5 minutes and equilibration for 3.5 minutes before next injection.

[0296] An API 4000 Qtrap mass spectrometer was operated using an electrospray ionization (ESI) source in positive ion mode. MRM transitions monitored for putrescine, spermidine, and spermine were 89.1 / 72.0, 146.2 / 72.0, and 203.2 / 129.1, respectively. For de-clustering, potentials were 48 eV, 35 eV, and 60 eV, entrance potentials were 5 eV, 5 eV, and 9 eV, collision cell exit potentials were 10 eV each, and collision energy were 14 eV, 20 eV, and 17 eV, respectively. The ion spray voltage was 5,500 eV, source temperature was 400 °C and ion source gases 1 and 2 pressures were both 50 psi, curtain gas and CAD gas was set as 15 psi and high mode.

[0297] Data analysis to determine Z’ score.

[0298] The signal to background (S / B) ratios for ySpe3 and ySpe4 were calculated using the following equation:

[0299] FI : Fluorescent intensity

[0300] The coefficient of variation (CV) were calculated using the following formula:

[0301] Coefficient of variation CV) = SD of FI / verage of FI x 100

[0302] The Z’ factor was calculated using the following equation:

[0303] The results are now described:

[0304] Putrescine, spermidine and spermine interact with 1,2-DAB to form fluorescent adducts with increasing fluorescence intensity.

[0305] Previous studies by Medici et al. (Medici et al., Advanced Synthesis & Catalysis 2011, 353: 2369-2376) and Choi et al. (Choi et al., J Biol Chem 2020; 295: 9211-9222) have demonstrated the interaction of 1,2-diacetylbenzene (DAB) with the primary amines of molecules such as tyramine, GABA, and ethanolamine, but not those primary amines attached to oc-carboxylated compounds such as serine), leading to the formation of fluorescent adducts. These fluorescent adducts could be detected using a spectrophotometer with excitation and emission spectra at wavelengths of 364 nm and 425 nm, respectively. Therefore, whether a 1,2-DAB-mediated fluorescence interaction with the polyamines, putrescine, spermidine and spermine could be used to measure the activity of aminopropyl transferase (APT) enzymes, which catalyze the conversion of putrescine to spermidine (SPDS activity) or spermidine to spermine (SPMS activity) was examined. First, the fluorescence intensity following incubation of 1,2-DAB with these polyamines as well as co-substrate, dc-SAM, or reaction product methylthioadenosine (MT A) in the absence or presence of 2 beta mercaptoethanol (0-ME) was compared (Figure 1A-C). The reaction of spermidine with 1,2-DAB / p-ME yielded ~4-fold higher fluorescence than that of putrescine with 1,2-DAB / p-ME (Figure IB). On the other hand, the reaction of spermine with 1,2-DAB / p-ME yielded ~1.5-fold higher fluorescence than that of spermine with 1,2-DAB / p-ME (Figure IB). The fluorescence intensities of dc- SAM and 1,2-DAB, with or without P-ME were similar to that observed with buffer control (Figure IB and Figure 1C). While the overall fluorescence signals were reduced when the polyamines reacted with a detection buffer lacking P-ME, the fold difference in the fluorescence signals between putrescine-spermidine (~3-fold) and spermidinespermine (~E 5-fold) remained unchanged (Figure 1C).

[0306] The total fluorescence (364 / 425 nm) at different concentrations (10- lOOpM) of putrescine, spermidine, and spermine over time was then measured (Figure 2A-C). All the tested concentrations of putrescine, spermidine, and spermine showed a linear increase in the total fluorescence intensity upon reaction with 1,2-DAB / p-ME during the first 60 minutes after which the fluorescence signal reached a plateau. At 60 minutes, the ratio of fluorescent intensity between spermidine and putrescine was ~4 fold, whereas that between spermine and spermidine was ~1.5-fold (Figure 2D).

[0307] To mimic APT activities from fluorescence emission data and estimate the conversion rates in enzyme reactions, standard curves were generated using fixed ratios of the substrate and product (putrescine and spermidine or spermidine and spermine) (Figure 3). To test whether the formation of spermidine in Spe3 mediated reaction, and spermine in Spe4 mediated reaction depicts a linear trend, two different standard curves were generated, one for each enzyme by setting up mock reactions. The first set of mock reactions were conducted for Spe3 by decreasing the concentrations of putrescine (substrate) and dc-SAM (co-substrate) and increasing the concentrations of spermidine (product) and MTA (by product) in the range of 0-100 pM as shown in Figure 3 A. Net fluorescence intensities of increasing spermidine in the mixed putrescine / spermidine reactions were calculated after background correction with the fluorescence value of 0 pM spermidine + 0 pM MTA + 100 pM putrescine + 100 pM dc-SAM from data in Figure 3 A. A linear increase in fluorescence is observed with increasing concentration of spermidine (Figure 3B). To confirm whether the increase in fluorescence observed in Figure 3A and Figure 3B is indeed due to increase in spermidine concentration, the samples were analyzed by running on a thin-layer chromatography plate (500 pm) and stained with 0.2 % ninhydrin (Figure 3C). The second set of mock reactions were conducted for Spe4 by decreasing the concentrations of spermidine (substrate) and dc- SAM (co-substrate) and increasing the concentrations of spermine (product) and MTA (by product) in the range of 0-100 pM (Figure 3D). Net fluorescence intensities of increasing spermine in the mixed spermidine / spermine reactions were calculated after background correction with fluorescence value of the 0 pM spermine + 0 pM MTA + 100 pM spermidine + 100 pM dc-SAM from data in Figure 3D. A linear increase in fluorescence is observed with increasing concentration of spermine (Figure 3E). The reactions performed in Figure 2D were also analyzed by running on a thin-layer chromatography plate (500 pm) and stained with 0.2 % ninhydrin (Figure 3F). These data demonstrate a direct correlation between fluorescence intensity and the rate of conversion of the substrate to the product (Figure 3).

[0308] The ability to differentiate between putrescine, spermidine and spermine using 1,2-DAB, makes this assay suitable for measuring APT enzyme activities of spermidine synthases and spermine synthases.

[0309] Structures of fluorescent adducts were determined by mass spectrometry analysis

[0310] Putrescine, spermidine, and spermine were incubated in the presence of 1,2-DAB in a buffer solution, and the resulting fluorescent complexes were analyzed using flow injection analysis mass spectrometry (FIA-MS) and liquid chromatography high resolution mass spectrometry (LC-HRMS), both with MS / MS fragmentation. Primary [M+H]+ions were identified from full scan data, and their respective MS / MS fragmentation patterns were obtained (Figure 4 through Figure 9). For all three polyamines, the primary mass ion corresponded to a substituted 1,3-dimethyl isoindole. Similar fluorescent substituted 2H-isoindoles and 1,3-dimethyl isoindoles have been shown to be produced by the combination of phthalaldehyde or 1,2-DAB and various nucleophilic reagents, including tyramine, GABA, ethanolamine, and mercaptoethanol (Choi, J. Y., et al., 2020, Journal of Biological Chemistry, 295:9211-9222; Maslivetc, V. A., et al., 2022, RSC Advances, 12:6947-6950). The structure of the observed polyamine- DAB adduct depended upon the structure of the polyamine used (Figure 4 through Figure 9). Putrescine reacted with 1,2-DAB to yield a fluorescent cyclic putrescine-DAB adduct (FIRMS calculated for C14H19N2 [M + H]+= 215.1543, observed [M + H]+= 215.1545). spermidine and spermine also provided fluorescent cyclic adducts spermidine-DAB adduct (HRMS calculated for C17H26N3 [M + H]+= 272.2121, observed [M + H]+= 272.2124) and spermine-DAB adduct (HRMS calculated for C20H33N4 [M + H]+= 329.2700, observed for [M + H]+= 329.2701). However, oxidized versions, spermidine- DAB adduct N-Oxide (HRMS calculated for C17H26N3O1 [M + H]+= 288.2070, observed for [M + H]+= 288.2072) and spermine-DAB adduct N-Oxide (HRMS calculated for C20H33N4O1 [M + H]+= 345.2649, observed for [M + H]+= 345.2651), were also observed. The oxidation was determined to have occurred on the primary amine via analysis of the MS / MS fragmentation pattern. It is worth noting that the oxidized version of the putrescine-DAB adduct, which does not possess a primary amine, was not observed.

[0311] The formation of the polyamine-DAB adducts can be rationalized using a simple, well-precedented arrow-pushing mechanism (Figure 10) (Maslivetc, V. A., et al., 2022, RSC Advances, 12:6947-6950). Adduct formation is initiated by the reaction of a primary amine and a ketone to provide a Schiff base intermediate. The mechanism is the same for spermidine and spermine, with the assumption that only a primary amine can form the Schiff base. Remarkably, these Schiff base intermediates were also observed in the LC-HRMS data: Putrescine-DAB Imine Intermediate (HRMS calculated for C14H21N2O1 [M + H]+= 233.1648, observed for [M + H]+= 233.1646) and Spermidine- DAB Imine Intermediate (HRMS calculated for C17H28N3O1 [M + H]+= 290.2227, observed for [M + H]+= 290.2227) (Figure 4A and Figure 5A). Cyclization via intramolecular reaction between the nucleophilic enamine nitrogen and remaining ketone gives an amino alcohol intermediate, which eliminates to form a bicyclic intermediate. An intramolecular Michael-type cyclization between the remaining putrescine amine and the electrophilic methylene provides the putrescine-DAB adduct. The spermidine-DAB and spermine-DAB adducts are formed as a result of cyclization with their secondary amines.

[0312] Activity of yeast APT enzymes, Spe3 and Spe4, using the 1,2-D AB-APT fluorescence assay

[0313] To validate the use of the 1,2-DAB-PA Assay for measurement of APT enzyme activity, the activity of the yeast Spe3 (Figure 11 A) and Spe4 (Figure 12A) was examined, which have been well-characterized at the genetic level and shown to catalyze either putrescine to spermidine or spermidine to spermine, respectively (Hamasaki- Katagiri, N., et al., 1997, Gene, 187:35-43; Hamasaki-Katagiri, et al., 1998, Gene, 210: 195-201). Standard curves were generated using reactions containing either decreasing concentrations of putrescine, dc-SAM (range: 0 mM to 0.5 mM), and increasing concentrations of spermidine, MTA (range: 0 mM to 0.5 mM) (Figure 1 IB), or decreasing concentrations of spermidine, dc-SAM (range: 0 mM to 0.5 mM), and increasing concentrations of spermine, MTA (range: 0 mM to 0.5 mM) (Figure 12B). These standard curves were used for determination of the conversion rates (spermidine or spermine formation rates) of Spe3 and Spe4 enzymes. Spe3 and Spe4 were expressed in E. coli as N-terminal fusion proteins with the maltose binding protein (MBP) (Figure 13 A and Figure 13B), affinity purified, and used in enzyme reactions in the presence of either putrescine (0.5 mM) or spermidine (0.5 mM) and co-substrate dc-SAM (0.5 mM). The reactions were performed with either active or heat inactivated enzymes (Spe3 and Spe4) and the reactions were stopped by the addition of the detection buffer consisting of 1,2- DAB / p-ME in sodium tetraborate buffer (pH 9.6) followed by incubated at 22 °C (room temperature) for 60 minutes. Fluorescence was measured using a plate reader with excitation and emission at wavelengths of 364 nm and 425 nm, respectively. The active yeast Spe3 catalyzed the conversion of putrescine and dc-SAM to form spermidine over time (Figure 11C through Figure 1 IE), with complete conversion to spermidine achieved in ~40 minutes, whereas the heat inactivated Spe3 failed to convert putrescine into spermidine (Figure 11C). The active yeast Spe4 catalyzed the conversion of spermidine and dc-SAM to form spermine (Figure 12C through Figure 12E), with complete conversion to spermine achieved in ~60 minutes. To validate the products of the reactions catalyzed by Spe3 and Spe4, reactions mixtures were separated by thin-layer chromatography (TLC) (Figure 1 IE and Figure 12E), and the levels, and conversion rates, of putrescine and spermidine were quantified using ImageJ (Figure 14A and Figure 14B). The levels of spermidine determined using 1,2-DAB-PA Assay were similar to those determined using quantification of TLC blots (Figure 1 ID, Figure 1 IE, and Figure 14A), whereas the levels of spermine determined from 1,2-DAB-PA Assay were slightly lower in comparison to those determined using TLC blots (Figure 12D, Figure 12E, and Figure 14B). Further validation of the products of the reactions was achieved using LC- MS analysis (Figure 1 IF and Figure 12F). Together, these data demonstrate that the 1,2- DAB-PA Assay is a reliable assay for measuring the activity of spermidine and spermine synthases.

[0314] Dual activity of P. falciparum spermidine synthase (PfSPDS) using the L2-D AB-APT fluorescence assay

[0315] The use of the 1,2-DAB-PA Assay was further examined by measuring the activity of the spermidine synthase PfSPDS of P. falciparum. This enzyme has been shown to have a primary function as a spermidine synthase but can also convert spermidine to spermine, with the spermine synthase activity accounting for only a small but significant amount of spermine found in P. falciparum-infected erythrocytes (Haider, N., et al., 2005, Molecular and Biochemical Parasitology, 142:224-236; Assaraf, Y. G., et al., 1984, The Biochemical Journal, 222:815-819; Das Gupta, R., et al., 2005, Antimicrobial Agents and Chemotherapeutics, 49:2857-2864; Assaraf, Y. G., et al., 1987, The Biochemical Journal, 242:221-226). The catalytic site residues are conserved between PfSPDS and APT enzymes from other organisms like E. coll. S. cercvisiae, and H. sapiens (Figure 15). PfSPDS, as well as the triple mutant PfSPDSD127A,E147A’D196A,with the three key catalytic residues, D127, E147 and D196 mutated to alanine, were expressed in E. coli as N-terminal fusion proteins with the maltose binding protein (MBP) (Figure 13C and Figure 13F), affinity purified and used in enzyme reactions in the presence of putrescine (0.5 mM) and the co-substrate dc-SAM (0.5 mM). The reactions were performed with active, heat inactivated, and triple mutant PfSPDS recombinant proteins, and the reactions were stopped by the addition of the detection buffer consisting of 1,2-DAB / p-ME in sodium tetraborate buffer (pH 9.6) and incubated at 22 °C (room temperature) for 60 minutes. Fluorescence was measured using a plate reader with excitation and emission spectra at wavelengths of 364 nm and 425 nm, respectively. The active PfSPDS catalyzed the conversion of putrescine and dc-SAM to form spermidine (Figure 16A, Figure 16B, and Figure 16D) over time, with complete conversion to spermidine achieved in ~40 minutes, whereas the heat inactivated, and triple mutant PfSPDS failed to convert putrescine into spermidine (Figure 16A, Figure 16B, and Figure 16D). These reaction mixtures were further separated by thin-layer chromatography (TLC) to visualize and quantify the substrate and product of the reactions (Figure 16D) and estimate the rate of catalysis (PfSPDS-mediated conversion rate) (Figure 14C). This data shows that the levels of spermidine determined using the 1,2-DAB-PA Assay were similar to those determined by TLC. The spermine synthase activity of PfSPDS was also tested by conducting enzyme reactions with PfSPDS as well as a heat inactivated enzyme (negative control) in the presence of spermidine and the co-substrate dc-SAM. Unlike with the yeast Spe4 enzyme, the 1,2-DAB-PA Assay using active PfSPDS showed little to no significant increase in fluorescence over that of the spermidine substrate, suggesting that the spermine synthase activity of PfSPDS is either weak or absent. No conversion of spermidine to spermine in reactions containing the heat inactivated PfSPDS enzyme using the 1,2-DAB-PA Assay (Figure 16C and Figure 16E). Interestingly, when the same enzyme reaction mixtures were analyzed by TLC (Figure 16E) and mass spectrometry (Figure 16F), a small amount of spermine was detected after a 60 min enzyme incubation, with an estimated -20% of the spermidine substrate converted into spermine (Figure 16E, Figure 16F, and Figure 14D).

[0316] Kinetics of Spe3, Spe4, and PfSPDS using the L2-DAB-APT fluorescence assay

[0317] The 1,2-DAB-APT fluorescence assay for detection of APT activity provides an attractive platform for screening of novel compound libraries in a high- throughput format. Since high throughput screens are performed using substrate concentrations near the Km values of the substrates, the kinetic constants were determined for the yeast Spe3, Spe4, and PfSPDS. Firstly, it was tested whether the 1,2-DAB-PA Assay can be used with lower substrate concentrations, i.e., 100 pM instead of 500 pM. A time-dependent increase in fluorescence was detected when Spe3, but not Spe4, was incubated with putrescine and dc-SAM, consistent with an increase in spermidine formation (Figure 17A). As a control, no fluorescence signals could be detected using heat-denatured Spe3 (Spe3_DN) or an Spe3 reaction conducted at 4 °C (Figure 17A). The molar amounts of spermidine formed in the Spe3 catalyzed reaction were calculated (Figure 17B) from the net fluorescence data in Figure 17A, using the standard curves shown in Figure 3B. For Spe3, the Km(38.6 pM) and Fmax (4.2 nmol / pg / min) (n=3) for putrescine as well as the Km (36.1 pM) and Fmax (4.5 nmol / pg / min) (n=3) for dc-SAM were determined (Figure 17C and Figure 17D) using Michaelis-Menten kinetics. Similarly, when yeast Spe4 was used in the APT reaction with spermidine and dc-SAM as substrate and co-substrate (Figure 17E), respectively, a time-dependent increase in fluorescence resulting from the formation of spermine was detected using active but not heat-inactivated enzyme (Figure 17E). The molar amounts of spermine formed in the Spe4 reaction (Figure 17F) were calculated from the net fluorescence data in Figure 17E, using the standard curves shown in Figure 3E. Characterization of the kinetic parameters of Spe4 using the 1,2-DAB-PA Assay yielded a K of 40.4 pM and a Fmax of 1.1 nmol / pg / min for spermidine, and a Km of 6.7 pM and a Fmax of 8.6 nmol / pg / min for dc- SAM (Figure 17G and Figure 17H). In the case of PfSPDS, a time-dependent increase in fluorescence was only observed with active PfSPDS, but not with the mutant enzymes (PfSPDSD127A, PfSPDSE147Aand PfSPDSD127A’E147A’D196A) (Figure 171). The molar amounts of spermidine formed in the PfSPDS-catalyzed reaction were calculated (Figure 17J) from the net fluorescence data in Figure 171. For PfSPDS, the Km (29.13 pM) and Fmax (5.26 nmol / pg / min) (n=3) for putrescine as well as the Km (26.46 pM) and Fmax (5.358 nmol / pg / min) (n=3) for dc-SAM were determined (Figure 17K and Figure 17L) using Michaelis-Menten kinetics.

[0318] The 1,2-DAB-PA Assay is amenable to HTS screening.

[0319] The adaptability of 1,2-DAB / p-ME-APT assay to a 96-well plate format for conducting inhibitor screening in HTS platforms was evaluated using trans-4- methylcyclohexylamine (4MCHA), a known potent inhibitor of spermidine synthase. 4MCHA has been demonstrated to compete with putrescine binding sites on spermidine synthase enzymes (Dufe, V. T., et al., 2007, Journal of Molecular Biology, 373: 167-177). The putrescine to spermidine APT activity of S. cerevisiae Spe3 and PfSPDS was determined in 96-well format in a 100 pl volume in the absence or presence of increasing concentrations of 4MCHA (0-100 pM) and spermidine formation was determined by measuring fluorescence intensity using the 1,2-DAB-PA Assay (Figure 18A and Figure 18B). Samples from the same enzyme inhibition reaction mixtures analyzed by the 1,2- DAB-PA Assay (Figure 18A and Figure 18B) were further separated by TLC to visualize the substrate and product of the reactions (Figure 18C and Figure 18D). The inhibition constants for the compound were determined to be Kt = 6.4 pM for Spe 3 and Ki = 2.5 pM for PfSPDS enzymes (Figure 18E and Figure 18F). The kinetic parameters Kmand Emax of the inhibition curves obtained with different concentration of 4MCHA indicates a competitive inhibition by 4MCHA, which supports the previous findings where 4MCHA has been demonstrated to compete with putrescine for binding to the enzyme (Dufe, V. T., et al., 2007, Journal of Molecular Biology, 373: 167-177). The amenability of the 1,2- DAB-PA Assay to HTS was determined for the yeast Spe3 enzyme by calculating the signal to background (S / B) and Z’ score as detailed in the Methods. From these analyses, the S / B (~4) and Z’ score (0.83), suggesting that the assay is suitability for high- throughput screening, and may be used to screen chemical libraries to search for novel compounds for various indications.

[0320] Discussion

[0321] These results provide a novel fluorescence assay utilizing 1,2- diacetylbenzene (DAB) and P-mercaptoethanol (P-ME) for the measurement of aminopropyltransferase (APT) activity. This assay builds upon previous successful applications of 1,2-DAB, notably in measuring the activity of phosphatidylserine decarboxylase enzymes, where 1,2-DAB interacts specifically with ethanolamine but not serine (Choi, J. Y., et al., 2020, Journal of Biological Chemistry, 295:9211-9222). 1,2- DAB interacts with the primary amines of polyamines - putrescine, spermidine, and spermine, to form fluorescent adducts, likely through a chemical process similar to that observed with ethanolamine (Choi, J. Y ., et al., 2020, Journal of Biological Chemistry, 295:9211-9222). Although all three polyamines carry two primary amine groups available for reaction with 1,2-DAB, differential fluorescence signals result from reaction with putrescine, spermidine, and spermine (Figure IB). The higher fluorescence signals observed with spermine (tetramine) versus spermidine (triamine) and putrescine (diamine) are likely due to the increased chain length of the polyamines, which separates the two primary amine groups and reduces potential steric hindrance, thereby enhancing fluorescence.

[0322] The addition of P-ME further enhanced the assay's sensitivity, enabling the detection of subtle changes in APT enzyme activity. This suggests a potential role for P- ME in facilitating the interaction between 1,2-DAB and polyamines, thereby amplifying the fluorescence signal. This underscore the importance of optimizing assay conditions to maximize both sensitivity and specificity, which are essential for accurate enzyme activity measurements.

[0323] To validate the 1,2-DAB-based assay for APT enzyme activity, an optimized thin-layer chromatography (TLC) assay and mass spectrometry were utilized alongside the fluorescence assay. The 1,2-DAB assay confirmed that in S. cerevisiae, Spe3 catalyzes the conversion of putrescine to spermidine but lacks spermine synthase activity, while Spe4 catalyzes the conversion of spermidine to spermine but lacks spermidine synthase activity. While PfSPDS was clearly shown to convert putrescine to spermidine, in the presence of spermidine as a substrate the 1,2-DAB-PA Assay failed to detect a significant change in fluorescence above that of spermidine alone (Figure 16C). However, TLC and mass spectrometry analyses demonstrated that PfSPDS does indeed convert spermidine to spermine, but this activity is very weak, with only ~ 20% of the substrate being converted into the product after a 90-minute reaction (Figure 16E and Figure 16F). These findings suggest that while the 1,2-DAB-PA Assay can reliably be used to examine the activity of both spermidine and spermine synthases, the fact that the fluorescence of spermine is only 1.5-fold higher than that of spermidine indicates that only highly active enzymes, capable of converting more than 25% of spermidine to spermine, would be suitable for detection using this assay. On the other hand, because the fluorescence difference between putrescine and spermidine is more than threefold, the assay can reliably detect all spermidine synthases, even those with weak activity. Using FIA-MS and LC-HRMS, both with MS / MS fragmentation, the structure of fluorescent adducts formed between putrescine, spermidine, spermine, and 1,2-DAB are likely substituted 1,3-dimethyl isoindoles. Similar structures have been reported in the literature (Choi, J. Y., et al., 2020, Journal of Biological Chemistry, 295:9211-9222). Interestingly, the resulting adduct structures varied depending on the polyamine involved. A fluorescent cyclic 1,2-DAB-polyamine adduct was observed for putrescine ([M + H]+= 215.1545), spermidine ([M + H]+= 272.2124), and spermine ([M + H]+= 329.2701). For spermidine and spermine, oxidized cyclic adducts ([M + H]+= 288.2072 and 345.2651, respectively) were also observed. The formation of these adducts can be explained through a well-precedented mechanistic pathway involving Schiff base formation, cyclization, and an intramolecular Michael-type reaction.

[0324] The stability of the 1,2-DAB-polyamine adducts follows a continuum (DAB-putrescine < 1,2-DAB-spermidine < 1,2-DAB-spermine), which correlates with the observed fluorescence intensities. The MS / MS fragmentation patterns confirmed that the more stable cyclic structures are preferentially observed, aligning with the fluorescence data. Every step in the polyamine-DAB adduct-forming mechanism is reversible. The putrescine-DAB adduct can occur as one cyclic isomer, an 8-membered ring. The spermine-DAB adduct can theoretically occur as a 7-, 12-, or 16-membered ring, depending on which amine participates in the final cyclization step. However, only the 7-membered ring is observed via MS / MS, which is an indication of its relative stability. Because unsymmetrical spermidine has two different primary amine ends, the spermidine-DAB adduct can occur as a 7-, 8-, or 12-membered ring. As for spermine, only the 7-membered ring is observed via MS / MS. This was surprising, since the 8- membered ring of the putrescine-DAB adduct is observed via MS / MS. Because of the relative stability and the reversibility of formation of the polyamine-DAB adducts, the 7- membered spermine-DAB adduct is more fluorescent than the 8-membered putrescine- DAB adduct. Similarly, because either primary amine end of non-symmetrical spermidine is equally likely to form the initial Schiff base, it is possible to rationalize the observed intermediate level of fluorescence intensity of the spermidine-DAB adduct as resulting from, relative to spermine, less of the stable 7-membered ring being formed. However, the order of observed fluorescence intensities may also be due to the number of amine functional groups available for reaction: 4 for spermine, 3 for spermidine and 2 for putrescine.

[0325] Attempts to purify the fluorescent adduct from the reaction of putrescine with 1,2-DAB or to synthesize it via a more controlled sequence of reactions are ongoing. Experiments using Nuclear Magnetic Resonance to verify the polyamine-DAB adduct structures are similarly ongoing. The likely cyclic isoindole products are stable enough for robust fluorescence assays and for analysis by MS, but they are minor components of the total assay reaction products and are likely unstable toward isolation. Further investigations using a combination of advanced analytical techniques and potentially novel synthetic approaches to provide additional characterization of the adducts are ongoing.

[0326] Importantly, this assay exhibited suitability for inhibitor screening in high- throughput platforms. By utilizing known inhibitors, such as trans-4- methylcyclohexylamine (4MCHA), their inhibitory potency against APT enzymes was successfully determined (Figure 18). The competitive inhibition observed with 4MCHA underscores the potential of this assay in identifying novel inhibitors targeting polyamine metabolism, with implications for therapeutic intervention in diseases such as cancer and parasitic infections.

[0327] In conclusion, this study presents a robust and versatile fluorescence assay for accurately measuring APT enzyme activity. Despite the challenges in synthesizing the adducts, the assay's sensitivity, specificity, and suitability for high-throughput screening make it a valuable tool for drug discovery and polyamine metabolism research. These findings will pave the way for the development of novel therapeutics targeting polyamine metabolism pathways.

[0328] Example 2: Identification of Anti-Cancer Treatments

[0329] Heightened levels of polyamine biosynthetic enzymes (PABEs) are present in multiple cancers, including neuroblastoma, hepatocarcinoma, and colorectal, prostate, lung, breast, and gastric cancers. Additionally, high expression of PABEs such as spermidine synthase (SRM) and spermine synthase (SMS) is with correlated with decreased survival in humans. Previously putrescine was examined as a starting point for polyamine biosynthesis with the 1,2-DAB assay, however polyamine synthesis can be tracked back further, to the conversion of ornithine to putrescine by ornithine decarboxylase (ODC). Elevated ODC expression, like expression of other PABEs, is also correlated with decreased survival in cancer patients. ODC is a transcriptional target of the MYC oncogene in both normal and neoplastic cells. MYC-driven tumors rely on increased biomass creation (specifically proteins) to support proliferation, and MYC targets include ribosome components, tRNAs and initiation and elongation factors, including eukaryotic initiation factor 5A isoform 1 (eIF5A).

[0330] In view of this, and the successful development of an assay for detecting the presence of poly amines and inhibitors of aminopropyl transferases, the next question was to determine if the assay could be expanded to ornithine decarboxylase activity in order to cover the entire polyamine biosynthesis pathway.

[0331] To this end, the 1,2-DAB assay was performed with ornithine, putrescine, spermidine, and spermine to see if ornithine would form a fluorescent adduct like the other polyamines, and if so, to see if the fluorescence intensity would be distinct from the other polyamines. In a fortuitous turn of events, it was observed that ornithine and 1,2- DAB do form a fluorescent adduct, and the resulting fluorescence intensity was lower than that of putrescine (Figure 19). Putrescine-DAB adducts were seen to be ~3-times more fluorescent than ornithine-DAB adducts, with spermidine-DAB adducts and spermine-DAB adducts being ~9- and ~13.5-times more fluorescent.

[0332] As the 1,2-DAB assay is able to differentiate ornithine, putrescine, spermidine, and spermine by fluorescence intensity, it is possible to detect enzymatic activity of all of ODC, SRM, and SMS. In view of this, the 1,2-DAB assay can be used for high-throughput identification of inhibitors of any of the enzymes involved in polyamine biosynthesis, including those which can potentially be used in the treatment of cancer.

[0333] Additionally, the versatility of the assay lends itself to identifying additional anticancer treatments through a different mechanism, hypusination of eIF5A. One of the polyamines that can be detected with this assay is spermidine, which is the substrate for deoxyhypusine synthase (DHS). After deoxyhypusine is generated deoxyhypusine hydroxylase (DOHH) then forms the active hypusinated form of eIF5A. Hypusinated eIF5A facilitates the translation of polyproline tracks and prevents ribosome stalling on specific mRNAs in addition to promoting the translation necessary for cell growth in both normal and tumor tissue. This absolute requirement of spermidine in hypusination of eIF5A may represent its essential requirement for cell proliferation. Furthermore, oncogene-driven upregulation of hypusinated eIF5A could potentially lead to a skewing of protein translation towards a hypusine-dependent translatome (Flynn, A. T., et al., 2018, Medical Sciences, 6:E41). eIF5A has also recently been implicated in enhancing nonsense-mediated mRNA decay (Nakanishi, S., et al., 2016, Amino Acids, 48:2353-2362; Hoque, M., et la., 2017, Translation, 5:el366294; Mathews, M. B., et al., 2015, Biochimica et Biophysica Acta - Gene Regulatory Mechanisms, 1849:638-844). The polyamine putrescine may also contribute to protein synthesis via its effects on mTOR complex 1 (mTORCl) and the eukaryotic translation initiation factor 4E (eIF4FE) cap-binding translation initiation complex (Flynn, A. T., et al., 2018, Medical Sciences, 6:E41).

[0334] As with monitoring the conversion of spermidine into spermine by SMS, the 1,2-DAB assay allows for monitoring of deoxyhypusine formation by DHS. The reaction of deoxyhypusinylated eIF5A (EIF5A-DH) with 1,2-DAB results in low fluorescence, while the spermidine-DAB adduct is highly fluorescent (Figure 20. As such, DHS activity can be monitored by decrease in fluorescence, and inhibitors of DHS may be identified as compounds which maintain fluorescence. Similarly, the activity of spermidine / spermine N(l)-acetyltransferase (SSAT), which N(l) acetylates spermine or spermidine, can be monitored. As acetylated spermidine is less fluorescent than spermidine, and acetylated spermine is less fluorescent than spermine, compounds which increase SSAT activity can be identified as compounds which result in a greater decrease in fluorescence than a control reaction without the compounds.

[0335] Example 3: Detailed DAB-PA Assay Protocol

[0336] Polyamines are a class of small organic molecules distinguished by their polycationic nature, possessing multiple amino groups which impart unique chemical properties crucial for cellular function (Schibalski, R. S., et al., 2024, Am J Physiol Cell Physiol, 327(2):C341-C356). Evolutionarily, polyamines are conserved across a wide range of organisms, from bacteria to humans, underscoring their fundamental biological importance (Michael, A. J., 2018, J Biol Chem, 293(48): 18693-18701). The three most studied polyamines are putrescine, spermidine, and spermine; each exhibit specific structural features that influence their biological functions. Putrescine, a di-amine, consists of a four-carbon chain with two amino groups. Spermidine, synthesized from putrescine, features an additional aminopropyl group, while spermine, the most complex, includes two aminobutyl groups, resulting in a highly branched, positively charged structure (Bowman, W. H., et al., 1973, J Biol Chem, 248(7):2480-2486; Dudley, H. W., et al., 1926, Biochem J, 20(5): 1082-1094). Polyamines are essential molecules that regulate diverse functions across different organisms. In bacteria and archaea, polyamines stabilize nucleic acids and cellular structures, facilitating growth and stress adaptation (Michael, A. J., 2018, J Biol Chem, 293(48): 18693-18701; Terui, Y., et al., 2005, Biochem J, 388(Pt 2):427-433). In eukaryotes, including plants, fungi, and animals, polyamines are crucial for regulating cell proliferation, differentiation, and apoptosis by stabilizing chromatin, influencing gene expression, and modulating ribosomal function. They also play significant roles in stress responses, helping organisms manage environmental challenges (Rhee, H. J., et al., 2007, J Coll Mol Med, 11(4):685-703; Solmi, L., et al., 2023, Sci Rep, 13 ( 1):4279) During development, polyamines contribute to cellular differentiation and tissue formation, while in animals, they offer neuroprotection and regulate immune responses (Blazquez, 2024, M. A., Annu Rev Plant Biol, 75(1):95-117; Valdes-Santiago, L., et al., 2013, Front Chem, 1 :42; Tang, G., et al., 2021, Front Microbiol, 12:765398; Pegg, A. E., 2016, J Biol Chem, 291(29): 14904- 14912; Seiler, N., et al., 2005, J Cell Mol Med, 9(3):623-642; Brooks, W. H., 2013, Front Immunol, 4:91). Additionally, polyamines are involved in reproduction, influencing processes such as spermatogenesis in animals and seed germination in plants (Lefevre, P. L., et al., 2011, Endocr Rev, 32(5):694-712). This broad spectrum of functions highlights the evolutionary conservation and critical importance of polyamines in maintaining cellular and organismal homeostasis.

[0337] Polyamine biosynthesis involves a series of enzymatic reactions that convert the amino acids into polyamines, with ornithine and methionine as primary precursors. The pathway initiates with the decarboxylation of ornithine-by-ornithine decarboxylase (ODC), producing putrescine (Raina, A., et al., 1968, Acta Chem Scand, 22(7):2375-2378; Morris, D. R., et al., 1965, Biochem Biophys Res Commun, 20(6):697- 702). Putrescine serves as the substrate for the synthesis of spermidine and spermine. Spermidine is generated through the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine, a reaction catalyzed by spermidine synthase, an aminopropyl transferase (APT) enzyme (Ikeguchi, Y., et al., 2006, J Biochem, 139(1): 1-9). The subsequent formation of spermine involves the addition of an aminobutyl group, also derived from dcSAM, facilitated by another APT enzyme, spermine synthase (Figure 1A) (Pegg, A. E., et al., 2010, Cell Mol Life Sci, 67(1): 113-121).

[0338] The polyamine biosynthetic pathway represents an attractive target for therapeutic intervention due to its crucial role in regulating cell growth, differentiation, and apoptosis. Dysregulation of this pathway is linked to various diseases, including cancer, neurodegenerative disorders, and infectious diseases, making it an attractive candidate for drug development (Casero R. A., et al., 2018, Nat Rev Cancer, 18(11):681- 695; Han, X., et al., 2024, Proc Natl Acad Sci USA, 121(13):e2319429121; Murray- Stewart, T. R., et al., 2016, Biochem J, 473(19):2937-2953; Cervelli, M., et al., 2022, Biomedicines, 10(7); Cason, A. L., et al., 2003, Eur J Hum Genet, 11912): 937-944; Morrison, L. D., et al., 1995, Neurosci Lett, 197(l):5-8; Brooks, W. H., 2024, J Alzheimers Dis, 98(3):837-857; Huang, M., et al., 2020, Infect Drug Resist, 13:4335- 4346; Firpo, M. R , et al., 2021, ACS Infect Dis, 7(6): 1423-1432; Kaiser, A., 2023, Biomolecules, 13(5)). Specifically, APT enzymes, involved in spermidine and spermine formation, represent promising targets for antimicrobial therapies due to the essential role of polyamines in the survival and virulence of many pathogens. In P. falciparum, the causative agent of malaria, polyamine biosynthesis is critical for the parasite's rapid growth and replication within human erythrocytes. Inhibiting the APT enzyme PfSPDS could disrupt polyamine production, leading to impaired parasite development and survival, making it an attractive target for antimalarial therapy. Similarly, polyamine biosynthesis is crucial for bacterial pathogens like . coli, H. pylori, P. aeruginosa, S. aureus and S. ihyphimurium where polyamines contribute to bacterial colonization, biofilm formation, microbial carcinogenesis and protection from oxidative and acid stress (Shah, P., et al., 2008, Mol Microbiol, 68(1):4- 16; Zhang, H., et al., 2017, Biochem Biophys Res Commun, 490(3):861-867; Thongbhubate, K., et al., 2021, J Bacteriol, 203(10); Tofalo, R., et al., 2019, Front Nutr, 6: 16; Mendez, R., et al., 2020, Carcinogenesis, 41 (5): 561 -570). Targeting APT enzymes in these bacteria could pave the way for the development of novel antibiotics.

[0339] The search for novel small molecules or natural products targeting APT enzymes has been hindered by the lack of assays suitable for high-throughput screening (HTS) of chemical libraries. To date, four primary methods have been employed to measure APT enzyme activity in vitro. The classical method involves radioisotope-based assays, using radioactive substrates such as [14C]-Putrescine and [14C]-Spermidine to measure APT activity (Zappia, V., et al., 1980, J Biol Chem, 255(15):7276-7280; Haider, N., et al., 2005, Mol Biochem Parasitol, 142(2):224-236). A second approach utilizes dansylation of polyamines synthesized during APT reactions, followed by quantification using high-performance liquid chromatography (HPLC) (Saeki, Y., et al., 1978, J Chromatogr, 145(2):221-229). A third approach employs capillary electrophoresis with laser-induced fluorescence (CE-LIF), where spermidine is derivatized with 7-fluoro-4- nitrobenzo-2-oxa-l,3-diazole (NBD-F) (Sano, M., et al., 2007, J Chromatogr B Analyt Technol Biomed Life Sci, 845(l):80-83). The fourth approach uses a specific monoclonal antibody against the byproduct, 5 ’-methylthioadenosine (MTA), coupled with a homogeneous time-resolved fluorescence technique (Enomoto, K., et al., 2006, Anal Biochem, 351(2):229-240). Although these assays are sensitive and specific for measuring APT activity, they are low-throughput and involve labor-intensive procedures. The DAB-PA Assay described here presents a breakthrough, offering a HTS platform for large chemical libraries, facilitating the search for novel APT enzyme inhibitors for therapeutic applications.

[0340] Previous studies by Medici et al. and Choi et al. demonstrated that 1,2- DAB reacts specifically with the primary amines of various molecules such as gamma- aminobutyric acid (GABA), tyramine, and ethanolamine, but does not react with the primary amines that are attached to a-carboxylated compounds such as serine (Medici, R., et al., 2011, Adv Synth Catal, 353(13):2369-2376; Choi, J. Y , et al., 2020, J Biol Chem, 295(27):9211-9222). This reactions forms fluorescent adducts that can be easily detected using a plate reader, with excitation and emission wavelengths of 364 nm, and 425 nm, respectively. DAB-mediated adduct formation with polyamines - putrescine, spermidine, and spermine - can be employed to measure the activity of APT enzymes from various organisms (Singh, P., et al., 2024, J Biol Chem, 107832). The DAB-PA Assay generates differential fluorescence between the substrate and product of the reaction. In spermidine synthase-catalyzed reactions, the fluorescence signal for the product spermidine is approximately 4-fold higher than for the substrate putrescine. Similarly, in spermine synthase-catalyzed reactions, the product spermidine shows about a 1.5-fold increase in fluorescence compared to the substrate spermidine (Figure 1A and Figure 10).

[0341] To carry out the DAB-PA Assay, recombinant APT enzymes must first be expressed and purified. Exemplary enzymes include spermidine synthase and spermine synthase enzymes expressed as recombinant fusion proteins with an N-terminal maltose- binding protein (MBP) tag. To generate a standard curve for the APT reactions, appropriate substrates (putrescine or spermidine for spermidine synthase and spermine synthase reactions, respectively) and co-substrates (e.g., decarboxylated S- adenosylmethionine (dcSAM)) are required. Additionally, the reaction products, (e.g., spermidine and spermine), as well as the byproducts (e.g., methylthioadenosine (MTA)), and 1,2-diacetyl benzene (1,2-DAB) for fluorescence detection are required. All required components, including putrescine, spermidine, spermine, dcSAM, MTA, 1,2-DAB, and buffer reagents, are commercially available and listed in the key resources table.

[0342] Table 1: Example Reagent Sources

[0343]

[0344] Preparation of reagents and standard curves for APT reactions

[0345] Transformation of pMAL-Spe3, pMAL-Spe4, and pMAL-PfSPDS plasmids: i. The plasmids (pMAL-Spe3, pMAL-Spe4, and pMAL-PfSPDS) were purified and resuspended in sterile distilled water to a final concentration of 0.1 mg / mL. ii. Rosetta 2 (DE3) competent E. coli cells were thawed on ice for 15-20 minutes. Approximately 40 pl of cells were aliquoted into three pre-chilled microcentrifuge tubes. iii. To each tube, 100 ng of either pMAL-Spe3, pMAL-Spe4, or pMAL- PfSPDS plasmid was added, and the cells were incubated on ice for 20 minutes. iv. The cells underwent a heat shock at 42 °C for exactly 30 seconds, followed by immediate placement on ice for 2 minutes. Afterward, 1 mL of pre-warmed SOC medium was added to each tube. v. The tubes were incubated on a shaker at 37 °C for 1 hour. vi. After incubation, 100 pL of the transformed cell suspension from each tube was spread onto LB-ampicillin agar plates. The plates were incubated overnight at 37 °C. vii. The next morning, the plates were removed from the incubator and stored at 4 °C for further use.

[0346] Making buffers for protein purification

[0347] I) Bacterial lysis buffer: i) 25 mM Tris-HCl (pH 8.0); ii) 500 mM NaCl; iii) 0.5 % Glycerol; iv) IX Protease inhibitor cocktail; v) 0.002% CHAPS; vi) 250 U / mL Benzonase

[0348] II) Binding buffer: i) 25 mM Tris-HCl (pH 8.0); ii) 500 mM NaCl; iii) 0.5 % Glycerol; iv) 1 mM DTT; v) 0.5 mM EDTA

[0349] III) Elution buffer: i) 25 mM Tris-HCl (pH 8.0); ii) 500 mM NaCl; iii) 0.5% Glycerol; iv) ImM DTT; v) 0.5 mM EDTA; vi) 10 mM Maltose

[0350] Purification of MBP-Spe3, MBP-Spe4, and MBP-PfSPDS proteins

[0351] Note: LB broth used for growing bacteria must be autoclaved before use, and ampicillin should be freshly prepared from powder and filter-sterilized prior to adding it to the LB broth.

[0352] 1. Inoculation and Primary Culture: Pick a single bacterial colony from the LB plates (one each for MBP-Spe3, MBP-Spe4, and MBP-PfSPDS as per step 5.a.vii.) and inoculate 20 mL LB broth containing 200 pg / mL ampicillin. Grow these primary cultures overnight (12-16 hours) at 37 °C in an incubator shaker at 180-200 rpm.

[0353] 2. Secondary Culture: The next morning, set up 1 -liter secondary cultures for each protein by inoculating 10 mL of the primary culture from step 5.c.i. into fresh LB broth.

[0354] 3. Growth and Induction: Grow the secondary cultures at 37 °C and 200 rpm until the optical density at 600 nm (OD600) reaches approximately 0.6. Remove 1 mL from each culture as uninduced control samples. To the remaining cultures, add isopropyl P-d-1 -thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Continue incubating the cultures, along with the uninduced controls, overnight at 16 °C with 200 rpm shaking. 4. Harvesting Cells: Harvest the cells from both the induced cultures and uninduced controls by centrifugation at 5000 x g for 30 minutes at 4 °C. Discard the supernatant.

[0355] 5. Validation of Protein Induction: To verify protein induction, take 1 mL from the induced cultures and uninduced controls, centrifuge at 6000 x g for 10 minutes, and discard the supernatant. Resuspend the cell pellets in IX Laemmli buffer, mix, and boil at 95 °C for 10 minutes. Run 5 pL of each sample on SDS-PAGE gels, and check protein induction using Coomassie staining.

[0356] 6. Cell Lysis: Resuspend the remaining induced cell pellets in 20 mL of lysis buffer (section 5.b.i.) and incubate on ice for 30 minutes.

[0357] 7. Sonication: Sonicate the resuspended cells at 10% amplitude, using a 5- second ON and 5-second OFF cycle for a total of 10 minutes, with breaks every 2.5 minutes.

[0358] 8. Supernatant Collection: After sonication, centrifuge the cell lysates at 12,000 x g for 45 minutes at 4 °C. Collect the supernatant, discarding the cell debris. For protein expression analysis, take 50 pL of the supernatant from each protein, add 15 pL of 4X Laemmli buffer, boil at 95 °C for 10 minutes, and run on SDS-PAGE gels to confirm protein expression using Coomassie staining.

[0359] 9. Amylose Resin Preparation: Pack six poly-prep chromatography columns with 2 mL of amylose resin (50% slurry), two for each of MBP-Spe3, MBP- Spe4, and MBP-PfSPDS. Wash the columns with 10 column volumes (CV) of chilled binding buffer.

[0360] 9. Protein Binding to the amylose resin: After washing, close the bottom of the columns with snap-off tips. Add the supernatants containing the proteins to their respective columns. Cap the tops of the columns and place them on a nutator shaker for 3 hours at 4 °C to allow the proteins to bind to the amylose resin.

[0361] 10. Protein Elution: After the binding step, place the columns on a QIArack, remove the top caps and bottom snap-off tips, and allow the unbound supernatant to flow through by gravity. Wash the columns three times with 20 C.V. of chilled binding buffer. 11. Elution of Proteins: Elute the MBP-Spe3, MBP-Spe4, and MBP- PfSPDS proteins by adding 10 m of elution buffer to each column and collecting 10 fractions of 1 m each.

[0362] 12. Protein Confirmation: Confirm the presence of the correct-sized

[0363] 5 proteins in the collected fractions by running 5-10 pL of each fraction on SDS-PAGE gels, followed by Coomassie staining (Figure 21).

[0364] 13. Protein Pooling and Concentration: Pool the fractions containing the proteins of interest. Perform buffer exchange (to remove maltose) using binding buffer and Amicon filter tubes (10 kDa cut-off) according to the manufacturer’s instructions.

[0365] 10 After buffer exchange, measure the protein concentration using a Nanodrop, and store the concentrated proteins in 100 pL aliquots at -80 °C for use in enzyme reactions.

[0366] The purification process should yield 2-4 mg of MBP-Spe3, MBP-Spe4, and MBP-PfSPDS per liter of bacterial culture.

[0367] 15 Preparation of Standard Curves for Spermidine and Spermine Synthase Reactions

[0368] I) Detection buffer recipe: i) lOOmM Sodium tetraborate buffer (pH 9.6) - 6.5 mL (final 65 mM); ii) ImM Potassium phosphate buffer (pH 7.4) - 2.2 mb (final 0.22 mM); iii) 72 mM beta-mercaptoethanol - 243 pL (final 1.75 mM); iv) 15 mM Triton X-100 - 830 pL (1.25 mM); v) 61 mM DAB - 242.5 pL (1.48 mM)

[0369] 20 II) Set up the standards (120 pL total volume, Table 2): i) In a 96 Well

[0370] Black plate with a clear bottom, add 35 pl of each of the six samples to their respective well in triplicate; ii) Add 85 pL of detection buffer (recipe in section 6.a) to each well and incubate for 60 minutes at room temperature in the dark. (Note: Include three additional well with 120 pL of detection buffer alone to serve as background

[0371] 25 fluorescence controls.); iii) Using a plate reader, read the plates with excitation at 364 nm excitation and emission at 425 nm (X) (Figure 3).

[0372] Table 2: Spermine Standard Solution

[0373]

[0374] III) Set up the following standards (120 pL total volume, Table 3): i)

[0375] In a 96-well black plate with a clear bottom, add 35 pL of each of the six samples to their respective wells in triplicate, iii) Add 85 pL of detection buffer (recipe in section 6.a) to each well, and incubate for 60 minutes at room temperature in the dark. (Note-. In three additional wells, add 120 pL of detection buffer alone to serve as background fluorescence controls.) iv) Measure the fluorescence using a plate reader with excitation at 364 nm and emission at 425 nm ( ) (Figure 3).

[0376] Table 3: Spermidine Standard Solutions

[0377] Setting up Spermidine and Spermine Synthase Reactions and Detection Using the DAB- PA Assay

[0378] 5 Assessing the Activity of Spermidine Synthase (MBP-Spe3, MBP-

[0379] PfSPDS) and Spermine Synthase (MBP-Spe4)

[0380] The activity of freshly purified enzymes can be evaluated using the DAB- PA Assay and thin-layer chromatography (TLC) analysis (see section 7) of the reaction products. This is done by conducting enzyme reactions at 37 °C for 60 minutes, using

[0381] 10 putrescine and dcSAM as substrates for MBP-Spe3 and MBP-PfSPDS, or spermidine and dcSAM as substrates for MBP-Spe4. Reactions are terminated by heat denaturation at 80 °C for 15 minutes. The resulting reaction mixtures can be analyzed using the DAB-PA Assay and simultaneously run on TLC plates, along with appropriate controls. TLC plates are then stained with ninhydrin (as detailed in section 7.c) for further analysis.

[0382] 15 I) Aminopropyl transferase (APT) enzyme reaction buffer: i) 50 mM potassium phosphate buffer (pH 7); ii) 1 mM DTT; iii) ImM EDTA; iv) 0.01% bovine serum albumin.

[0383] II) Preparation of solutions: i) 10 mM Putrescine (PUT) - Dissolve 1.61 mg of PUT powder in 1 mL water; ii) 10 mM Spermidine (SPD) - Take 3 pL of 6.7 M SPD stock (liquid) and add to 997 pL water; iii) 10 mM Spermine (SPM) - Dissolve 20.23 mg of SPM powder in 1 mL water; iv) 10 mM Decarboxylated S-adenosyl methionine (dcSAM) - Dissolve 10 mg of dcSAM in 2.07 mL water (Note: make small aliquots of 10 mM dcSAM and store in -20 °C.); v) lOmM Methylthioadenosine (MTA) - Dissolve 3 mg of MTA in 1 mL of ethanol.

[0384] III) Setting up spermidine synthase reaction:

[0385] For setting up 100 pL of spermidine synthase reaction for DAB-PA Assay, add the components listed below in the sequential order: i) APT enzyme buffer - 79 pL ii) 1 mM putrescine - 10 pL (100 pM final) iii) 1 mM dcSAM - 10 pL (100 pM final) iv) Spe3 or PfSPDS enzyme - 1 pL (1 pg)

[0386] For setting up 100 pL of spermidine synthase reaction for TLC analysis, add the components listed below in the sequential order: i) APT enzyme buffer - 86 pL ii) 10 mM putrescine - 5 pL (500 pM final) iii) 10 mM dcSAM - 5 pL (500 pM final) iv) Spe3 or PfSPDS enzyme - 4 pL (4 pg)

[0387] IV) Setting up spermine synthase reaction

[0388] For setting up 100 pL of spermine synthase reaction for DAB-PA Assay, add the components listed below in the sequential order: i) APT enzyme buffer - 79 pL ii) 1 mM spermidine - 10 pL (100 pM final) iii) 1 mM dcSAM - 10 pL (100 pM final) iv) Spe4 - 1 pL (1 pg)

[0389] For setting up 100 pL of spermine synthase reaction for TLC analysis, add the components listed below in the sequential order: i) APT enzyme buffer - 86 pL ii) 10 mM spermidine - 5 pL (500 pM final) iii) 10 mM dcSAM - 5 pL (500 pM final) iv) Spe4 - 4 pL (4 pg)

[0390] To measure the activity of spermidine synthase and spermine synthase reactions, follow these steps using a 96-well plate format:

[0391] 1. Add 35 pL of either the spermidine synthase or spermine synthase reaction mixtures to separate wells of a 96-well plate in triplicate.

[0392] 2. Include control reactions (35 pL per well, in triplicate) for enzyme- only, substrate-only, and product-only conditions. (Note'. If generating standard curves alongside the enzyme reactions, add 35 pL of each standard to separate wells in triplicate.)

[0393] 3. Add 85 pL of detection buffer to each well and incubate the plate in the dark at room temperature for 60 minutes.

[0394] 4. Measure fluorescence using a plate reader with excitation at 364 nm and emission at 425 nm (X).

[0395] Calculating kinetic parameters of APT enzymes using DAB-PA Assay

[0396] The DAB-PA Assay can also be used to determine the kinetic parameters (Am and Fmax) of substrates for spermidine or spermine synthase enzymes. Following is an example setup for calculating the kinetic parameters of the Spe3 enzyme (determining Kmand Fmax for putrescine). Table 4: Spe3 Experimental Enzyme Reaction Setups

[0397] Table 5: Spe3 Control Enzyme Pre-Reaction Setups:

[0398] Table 6: Spe3 Experimental Enzyme Post-Reaction Setups

[0399] 1) Incubate all enzyme reactions and controls at 37 °C for 60 minutes. After incubation, transfer 35 LLL of each reaction and control (in triplicate) into a clearbottom 96-well plate. Add 85 pL of detection buffer to each well, then incubate the plate in the dark at room temperature for 60 minutes. Measure the fluorescence using a plate reader with excitation at 364 nm and emission at 435 nm.

[0400] 2) To determine the Km and Vmax for dcSAM with the Spe3 enzyme, maintain a fixed concentration of putrescine at 120 pM, while varying the concentration of dcSAM from 0 to 120 pM. Include controls with varying concentrations of dcSAM alone (0-120 pM) and spermidine alone (0-120 pM).

[0401] 3) Perform data analysis (described below).

[0402] Validation of APT reactions using thin-layer chromatography (TLC) and Mass spectrometry

[0403] I) Preparation of solvent solution for running TLC: i) n-butanol - 50 mL; ii) Acetic acid - 50 mL; iii) Pyridine - 33.3 mL; iv) Water - 16.7 mb.

[0404] II) Preparation of ninhydrin solution for staining TLC plates: i) Ninhydrin - 0.2 g; ii) Ethanol - 99.5 mL; iii) Acetic acid - 0.5 mL.

[0405] III) Step by step procedure for running APT reactions on TLC plates i) Inside a chemical hood, place Whatman blotting papers inside the TLC tank to cover the inner edges of the tank. Put 100 mL of methanol in the tank and subsequently place a TLC plate inside it. Cover the tank and allow the methanol to run through the plate. ii) Once the methanol has run to the top of the plate, take out the TLC plate and let it dry inside the chemical hood. iii) After the plate is dry, place it on a white light transilluminator. iv) Spot 20 pL of the APT enzyme reactions in horizontal lanes (~0.8 cm to 1 cm in width) along with standards (500 pM each of putrescine, dcSAM, spermidine and spermine) on the TLC plate. v) Let the plate dry at room temperature for 20 minutes, then carefully place it into the TLC tank containing the solvent solution, ensuring no solvent splashes on the plate. vi) Allow the solvent to move up the plate for ~ 4 hours, until the solvent front reaches about 3 / 4thup the plate. Afterward, remove the plate from the tank and allow it to dry in the chemical hood for 8-10 hours. vii) Once completely dry, spray the plate with ninhydrin solution and let it stand for 5 minutes in the chemical hood. Thereafter, place the TLC plate in an oven preheated to 110 °C and dry for 4-6 minutes until the color develops (Figure 1 IE and Figure 12E). viii) Capture an image of the plate for qualitative or quantitative analysis. Quantitative analysis can be performed using ImageJ software, with the percentage of enzyme catalysis determined by the intensity of the substrate band’s disappearance or the product band’s formation on the TLC plate.

[0406] Validation of the enzyme reactions by LC-MS

[0407] While TLC confirmation of product formation in the APT reactions is typically sufficient, LC-MS can be used as an additional method to confirm product formation if needed.

[0408] The products of APT reactions, namely spermidine (from spermidine synthase reactions) and spermine (from spermine synthase reactions), can be confirmed using LC-MS. Quantification of substrates (putrescine or spermidine) and products (spermidine or spermine) can be performed using an API 4000 QTrap® mass spectrometer (Applied Biosystems Sciex, Toronto, Canada) coupled with an Agilent HPI1200 HPLC system (Agilent Technologies, Santa Clara, CA). A targeted Multiple Reaction Monitoring (MRM) method can be applied for polyamine quantification. Polyamine standards (putrescine, spermidine, and spermine) should be prepared in a concentration range of 25-2500 ng / mL, and quality controls (QCs) should be prepared at 100, 500, and 2000 ng / mL. For liquid chromatography separation, an Agilent Cl 8 guard column should be used at a column temperature of 50 °C.

[0409] 5 The gradient elution should be set as in Table 7.

[0410] Table 7: LCMS Gradient

[0411] The mass spectrometer (i.e., an API 4000 QTrap mass spectrometer)

[0412] 10 should be operated with an electrospray ionization (ESI) source in positive ion mode. Data acquisition and analysis can be performed using Analyst 1.7 software (Applied Biosystems Sciex, Toronto, Canada).

[0413] Quantification and Statistical analysis

[0414] 15 Plotting the data to create a standard curve i) Subtract the average value of the blank wells (containing detection buffer only) from all sample readings to correct for background fluorescence. ii) Plot the corrected fluorescence values against the concentration of the poly amines (PUT + de SAM + SPD + MTA for spermidine synthase reactions, or SPD +

[0415] 20 dcSAM + SPM + MTA for spermine synthase reactions) in GraphPad Prism. iii) Use GraphPad Prism to determine the linear fit of the data. This will allow calculation of the concentration of the product (spermidine or spermine) based on the equation generated by the software. Calculating the conversion rate of spermidine synthase and spermine synthase catalyzed reactions i) To calculate the percent conversion of substrate (putrescine, 100 pM) into the product (spermidine), first calculate net fluorescence readings by subtracting the fluorescence value of the substrate alone (100 pM putrescine after 60 minutes in DAB reaction buffer) from total fluorescence. ii) The fluorescence value of the product alone (100 pM spermidine after 60 minutes in DAB reaction buffer) is set as 100% for reference. iii) Calculate the percent conversion of putrescine to spermidine by active Spe3 and control (heat inactivated Spe3) over time (0, 20, 40, 60 minutes) (Figure 1 ID) using the formula below:

[0416] Net fluorescence intensity from enzyme reaction at x minute

[0417] - * 100 Net fluorescence intensity of 100 pM spermidine control at 60 minute.

[0418] Calculating the kinetic parameters of APT enzymes (example: Spe3) i) Subtract the average fluorescence value of the blank wells (containing detection buffer only) from all readings to corrects for background fluorescence. ii) Calculate the average fluorescence readings of the putrescine + dcSAM controls at each concentration, ranging from 0 pM to 120 pM putrescine (dcSAM is at a fixed concentration of 120 pM). iii) From the Spe3-catalyzed reactions, where dcSAM is held constant at 20 pM and putrescine varies (0 - 120 pM), subtract the reading of the corresponding control reading (putrescine + dcSAM without enzyme) to normalize the values. (Example: For Spe3 reaction performed with 10 pM of putrescine and 120 pM dcSAM, subtract the fluorescence readings of the 10 pM putrescine + 120 pM dcSAM control (no enzyme) from the reaction reading.) iv) Calculate the moles of spermidine formed at different substrate concentrations using the following formula:

[0419] FI at X pM of spermidine control.

[0420] * FI in Spe3 reaction at X pM of putrescine substrate v) The specific activity of Spe3 at different substrate concentrations can be calculated using the formula: nmoles of spermidine formed Specific Activity = - - - — - pg of enzyme used / minute vi) Enter the specific activity values into GraphPad prism in an XY table format, with the substrate (putrescine) concentration on the X-axis and specific activity (spermidine formed) on the Y-axis. vii) In GraphPad prism, analyze the data and generate a graph using nonlinear fitting with allosteric sigmoidal curve (Figure 17C).

[0421] Summary

[0422] The DAB-PA Assay offers a robust and straightforward fluorescencebased approach for the detailed biochemical characterization of APT enzymes (Singh, P., et al., J Biol Chem, 2024, 300(11): 107832). This assay is particularly well-suited for high-throughput screening of chemical libraries, making it invaluable for identifying novel inhibitors across various therapeutics areas, including cancer, neuroprotection, and infectious diseases. By facilitating the discovery of targeted therapies, the DAB-PA Assay paves the way for combating a wide range of pathogens and diseases. For protozoan targets, the assay could help identify inhibitors that disrupt polyamine metabolism in Plasmodium species, potentially leading to novel treatments for malaria. It may also aid in developing therapeutic strategies against Toxoplasma gondii, Trypanosoma species, and Leishmania species by disrupting polyamine synthesis in these parasites. In fungal pathogens, the DAB-PA Assay can be used to identify specific inhibitors that impair polyamine metabolism in important human pathogens, such as Candida albicans, Candida auris, Aspergillus fumigatus, Histoplasma capsulatum, Cryptosporidium parvum and Mucorales, further enhancing our arsenal of antifungal therapies. Beyond infectious diseases, the DAB-PA Assay holds considerable promise in oncology and neurodegenerative conditions. Dysregulation of polyamine biosynthesis is a hallmark of many cancers, and inhibiting APT enzymes may hinder tumor growth and promote apoptosis in cancer cells, potentially leading to novel anticancer drugs (Casero, R. A., et al., Nat Rev Cancer, 2018, 18(11 ):681 -695; Murray- Stewart, T. R., Biochem J, 2016, 473(19):2937-2953; Novita Sari, I., Cancer Lett, 20121, 519:91-104). In neurodegenerative diseases, where polyamine levels are often disrupted, APT inhibitors could restore balance and provide neuroprotective benefits (Morrison, L. D., et al., Neurosci Lett, 1995, 197(l):5-8; Minois, N., et al., Aging, 2011, 3(8):716-732). Overall, the DAB-PA Assay represents a promising platform that could transform therapeutic strategies against a wide array of protozoan, fungal, and chronic diseases, ultimately enhancing our capabilities to combat some of the most challenging health threats.

[0423] Example 4: Sequences

[0424] EcSpeE 61 (SEQ ID NO: 1) FIYHEMMTHVPLLAHGHAKHVLIIGGGDGAMLREVTRHKNVESITMVEID AGWSFCRQYLPNHNAGSYDDPRFKLVIDDGVNFVNQTSQTFDVIISDCT

[0425] ScSpe3 72 (SEQ ID NO:2) FAYQEMIAHLALNSHPNPKKVLVIGGGDGGVLREWKHDSVEEAWLCDID EAVIRLSKEYLPEMAASYSHPKVKTHIGDGFQFLRDYQNTFDVIITDSS

[0426] PfSPDS 100 (SEQ ID NO:3)

[0427] FAYHEMMTHVPMTVSKEPKNVLVVGGGDGGIIRELCKYKSVENIDICEID ETVIEVSKI YFKNISC GYEDKRVNVFIED ASKFLENVTNT YD VIIVD S S

[0428] HsSPDS 77 (SEQ ID NO:4)

[0429] F S YQEMIANLPLC SHPN PRE VLI IGGGDGG VL E WK HP S VES VVQCEID ED VIQ VSKKFLPGMAIGYS S SKLTLHVGDGFEFMKQNQD AFD VIITD S S

[0430] EcSpeE (SEQ ID NO: 5) MAEKKQWHETLHDQFGQYFAVDNVLYFIEKTDHQDLIIFENAAFGRVMALDGV VQTTERDEFIYHEMMTHVPLLAHGHAKHVLIIGGGDGAMLREVTRHKNVESITM VEIDAGVVSFCRQYLPNHNAGSYDDPRFKLVIDDGVNFVNQTSQTFDVIISDCT DPIGPGESLFT S AF YEGCKRCLNPGGIF VAQNGVCFLQQEE AID SHRKL SHYF SD V GFYQAAIPTYYGGIMTFAWATDNDALRHLSTEIIQARFLASGLKCRYYNPAVHT AAFALPQYLQDALASQPS

[0431] ScSpe3 (SEQ ID N0:6)

[0432] MAQEITHPTIVDGWFREISDTMWPGQAMTLKVEKVLHHEKSKYQDVLIFKSTTY GNVLVLDNVIQATERDEFAYQEMIAHLALNSHPNPKKVLVIGGGDGGVLREVVK HDSVEEAWLCDIDEAVIRLSKEYLPEMAASYSHPKVKTHIGDGFQFLRDYQNTF DVIITDSSDPEGPAETLFQKEYFQLLNSALTEKGVITTQAESMWIHLPIIKDLKKAC SEVFPVAEYSFVTIPTYPTGTIGFMVCSKDKTCNVKKPLREISDEKEAELYRYYNK KIHEASFVLPTWAAKELN

[0433] PfSPDS (SEQ ID N0 7)

[0434] MDKLISNNKLKLSVVLLGGLCSLAYYHLKNKFHLSQFCFSKKWFSEFSIMWPGQ AFSLKIKKILYETKSKYQNVLVFESTTYGKVLVLDGVIQLTEKDEFAYHEMMTH VPMTVSKEPKNVLVVGGGDGGIIRELCKYKSVENIDICEIDETVIEVSKIYFKNISC

[0435] GYEDKRVNVFIEDASKFLENVTNTYDVIIVDSSDPIGPAETLFNQNFYEKIYNALK PNGYCVAQCESLWIHVGTIKNMIGYAKKLFKKVEYANISIPTYPCGCIGILCCSKT DT-GLTKPNKKLESKEFADLKYYNYENHSAAFKLPAFLLKEIENI

[0436] HsSPDS (SEQ ID N0:8)

[0437] MEPGPDGPAASGPAAIREGWFRETCSLWPGQALSLQVEQLLHHRRSRYQDILVF RSKT YGNVL VLDGVIQC TERDEF S YQEMIANLPLC SHPNPRKVLIIGGGDGGVLR EVVKHPS VES VVQCEIDEDVIQVSKKFLPGMAIGYS S SKLTLHVGDGFEFMKQN QDAFDVIITDSSDPMGPAESLFKESYYQLMKTALKEDGVLCCQGECQWLHLDLI KEMRQFCQSLFPVVAYAYCTIPTYPSGQIGFMLCSKNPSTNFQEPVQPLTQQQVA QMQLKYYNSDVHRAAFVLPEFARKALNDVS The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting the presence of at least one polyamine compound in a sample comprising the steps of: a) preparing a solution comprising the sample and 1,2- diacetylbenzene (DAB); b) incubating the solution; c) measuring fluorescence of the solution; and d) comparing the fluorescence of the solution to the fluorescence of a control solution, wherein an increase in fluorescence relative to the control solution indicates the presence of at least one polyamine compound in the sample.

2. The method of claim 1, wherein the polyamine is at least one selected from the group consisting of ornithine, putrescine, spermidine, acetyl-spermidine, spermine, acetyl-spermine, and deoxyhypusine.

3. The method of claim 1, wherein the solution of step a) further comprises -mercaptoethanol (P-ME).

4. The method of claim 1, wherein the solution of step a) further comprises at least one selected from the group consisting of sodium tetraborate and potassium phosphate.

5. The method of claim 1, wherein step b) comprises incubating the solution at about room temperature for about 60 minutes.

6. The method of claim 1, wherein step c) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelengthand an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

7. The method of claim 6, wherein the control solution of step e) comprises 1,2-DAB and P-ME at about the same concentration as the sample solution of step a), wherein an increase in absorbance at 364 nm in the excitation spectrum relative to the control solution, an increase in emission at 425 nm relative to the control solution, or both indicate the presence of a polyamine.

8. The method of any one of claims 1-7, wherein steps a) through c) are performed in at least one well of a multi-well plate.

9. A method of detecting aminopropyl transferase (APT) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, decarboxylated-S- adenosylmethionine (dc-SAM), and either putrescine or spermidine; b) incubating the solution; c) adding 1,2-DAB and P-ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has APT activity.

10. The method of claim 9, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).11 . The method of claim 9, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

12. The method of claim 9, wherein step c) further comprises adding sodium tetraborate to the solution.

13. The method of claim 9, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

14. The method of claim 9, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

15. The method of claim 14, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of dc-SAM and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has APT activity.

16. The method of any one of claims 9-15, wherein steps a) through e) are performed in at least one well of a multi-well plate.

17. A method of identifying a compound as an inhibitor of a protein with APT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with APT activity, dc-SAM, and either putrescine or spermidine; b) incubating the solution; c) adding to the solution - E and 1,2-DAB; d) incubating the solution;e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

18. The method of claim 17, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and B SA.

19. The method of claim 17, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

20. The method of claim 17, wherein step c) further comprises adding sodium tetraborate to the solution.

21. The method of claim 17, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

22. The method of claim 17, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

23. The method of claim 22, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with APT activity.

24. The method of any one of claims 17-23, wherein steps a) through e) are performed in at least one well of a multi-well plate.

25. A compound selected from the group consisting of:wherein each instance of RKis selected from the group consisting of Ci-Ce alkyl and Ci-Ce haloalkyl;R1, R2, R3, R4, and R5are each independently selected from the group consisting of H, D, halogen, Ci-Ce alkylene, Ci-Cs haloalkylene, Ci-Ce heteroalkylene, amide, ester, carbonyl, and combinations thereof; and each instance of L is a divalent linker selected from the group consisting of alkylene, cycloalkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, silyl, amine, amide, ester, ether, carbonyl, carbamate, sulfamate, sulfonic ester, sulfoximine, sulfonamide, thioether, thioester, disulfide, hydrazine, urea, thiourea, phosphate, phosphonate ester, poly(alkyl ether), heteroatom, and combinations thereof, and tautomers, conjugates, salts, and solvates thereof.

26. The compound of claim 25, wherein divalent linker L comprisesFormula (IV):Formula (IV) wherein: n =1-6; o = l-6; p = 1-6; and q = 0-3.

27. The compound of claim 25, wherein the compound is selected from the group consisting of:and tautomers, conjugates, salts, and solvates thereof.

28. A method of detecting ornithine decarboxylase (ODC) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, pyridoxal phosphate (PIP), and ornithine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has ODC activity.

29. The method of claim 28, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenedi aminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

30. The method of claim 28, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

31. The method of claim 28, wherein step c) further comprises addingsodium tetraborate to the solution.

32. The method of claim 28, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

33. The method of claim 28, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

34. The method of claim 28, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of PIP and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has ODC activity.

35. The method of any one of claims 28-34, wherein steps a) through e) are performed in at least one well of a multi-well plate.

36. A method of identifying a compound as an inhibitor of a protein with ODC activity comprising the steps of: a) preparing a solution comprising the compound, the protein with ODC activity, PIP, and ornithine; b) incubating the solution; c) adding to the solution 0-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solutionindicates the compound is an inhibitor of the protein.

37. The method of claim 36, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and B SA.

38. The method of claim 36, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

39. The method of claim 36, wherein step c) further comprises adding sodium tetraborate to the solution.

40. The method of claim 36, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

41. The method of claim 36, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

42. The method of claim 36, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with ODC activity.

43. The method of any one of claims 36-42, wherein steps a) through e) are performed in at least one well of a multi-well plate.

44. A method of detecting deoxyhypusine synthase (DHS) activity of aprotein comprising the steps of: a) preparing a solution comprising the protein, nicotinamide adenine dinucleotide (NAD+), eukaryotic translation initiation factor 5A (eIF-5A), and spermidine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has DHS activity.

45. The method of claim 44, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

46. The method of claim 44, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

47. The method of claim 44, wherein step c) further comprises adding sodium tetraborate to the solution.

48. The method of claim 44, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

49. The method of claim 44, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, andthe emission spectrum wavelength is about 425 nm.

50. The method of claim 44, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of NAD+, eIF-5A, and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has DHS activity.

51. The method of any one of claims 44-50, wherein steps a) through e) are performed in at least one well of a multi-well plate.

52. A method of identifying a compound as an inhibitor of a protein with DHS activity comprising the steps of: a) preparing a solution comprising the compound, the protein with DHS activity, NAD+, eIF-5A, and spermidine; b) incubating the solution; c) adding to the solution P-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

53. The method of claim 52, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and B SA.

54. The method of claim 52, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

55. The method of claim 52, wherein step c) further comprises adding sodium tetraborate to the solution.

56. The method of claim 52, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

57. The method of claim 52, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

58. The method of claim 52, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with DHS activity.

59. The method of any one of claims 52-58, wherein steps a) through e) are performed in at least one well of a multi-well plate.

60. A method of detecting spermidine / spermine N(l)-acetyltransf erase (SSAT) activity of a protein comprising the steps of: a) preparing a solution comprising the protein, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding 1,2-DAB and -ME to the solution; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence ofa control solution; wherein an increase in fluorescence relative to the control solution indicates that the protein has SSAT activity.

61. The method of claim 60, wherein the solution of step a) further comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), potassium phosphate, dithiothreitol (DTT), and bovine serum albumin (BSA).

62. The method of claim 60, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

63. The method of claim 60, wherein step c) further comprises adding sodium tetraborate to the solution.

64. The method of claim 60, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

65. The method of claim 60, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelength and an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

66. The method of claim 60, wherein the control solution of step f) comprises all components of the solution of step d) except one selected from the group consisting of acetyl-CoA and 1,2-DAB, and wherein an increase in absorbance at 364 nm in the excitation spectrum compared to the control solution, an increase in the fluorescence at 425 nm compared to the control solution, or both indicate the protein has SSAT activity.

67. The method of any one of claims 60-66, wherein steps a) through e) are performed in at least one well of a multi-well plate.

68. A method of identifying a compound as an inhibitor of a protein with SSAT activity comprising the steps of: a) preparing a solution comprising the compound, the protein with SSAT activity, acetyl-CoA, and spermidine or spermine; b) incubating the solution; c) adding to the solution [3-ME and 1,2-DAB; d) incubating the solution; e) measuring fluorescence of the solution; and f) comparing the fluorescence of the solution to the fluorescence of a control solution; wherein a decrease in fluorescence relative to the control solution indicates the compound is an inhibitor of the protein.

69. The method of claim 68, wherein the solution of step a) further comprises at least one selected from the group consisting of EDTA, potassium phosphate, DTT, and B SA.

70. The method of claim 68, wherein step b) comprises incubating the solution at about 37 °C for about 60 minutes.

71. The method of claim 68, wherein step c) further comprises adding sodium tetraborate to the solution.

72. The method of claim 68, wherein step d) comprises incubating the solution at about room temperature for about 60 minutes.

73. The method of claim 68, wherein step e) comprises analyzing the solution with a spectrophotometer to obtain an excitation spectrum at a first wavelengthand an emission spectrum at a second wavelength, wherein the excitation spectrum wavelength is about 364 nm, and the emission spectrum wavelength is about 425 nm.

74. The method of claim 68, wherein the control solution of step f) comprises all components of the solution of step d) except the compound of interest, and wherein a decrease in absorbance at 364 nm in the excitation spectrum compared to the control solution, a decrease in the fluorescence at 425 nm compared to the control solution, or both indicate the compound is an inhibitor of the protein with SSAT activity.

75. The method of any one of claims 68-74, wherein steps a) through e) are performed in at least one well of a multi-well plate.

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