Engineering premarineosin production in streptomyces and derivatization of the premarineosin scaffold for drug development

Abstract

Provided herein are semi-synthetic derivatives of premari neosin and pharmaceutically acceptable salts thereof. Also provided are methods for producing a semi-synthetic derivative of premarineosin or a pharmaceutically acceptable salt thereof, and methods for inhibiting Plasmodium parasites (such as Plasmodium falciparum parasites) and / or treating or preventing malaria infections with the compounds and salts. Further provided are engineered Streptomyces bacteria having increased expression of a PmaD protein or a PmaD protein and a PmaG protein, cultures thereof, and plasmids comprising a nucleotide sequence encoding a PmaD protein or a PmaD protein and a PmaG protein, and their use in engineering bacteria and / or producing premarineosin.

Description

Docket No. 30275 / 70841ENGINEERING PREMARI NEOSIN PRODUCTION IN STREPTOMYCES AND DERIVATIZATION OF THE PREMARINEOSIN SCAFFOLD FOR DRUG DEVELOPMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is an International Application which claims priority to U.S.S.N. 63 / 811,085, filed May 23, 2025, and U.S.S.N. 63 / 747,216, filed January 20, 2025, the respective disclosures of which are each incorporated herein by reference in their entireties.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant no. R35 GM118101 and grant no. 5F31 DA055451 -03, awarded by the National Institutes of Health, and grant no. DGE 2241144, awarded by the National Science Foundation. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety is a sequence listing in computer-readable form submitted concurrently herewith and identified as follows: Filename: 70841_SeqListing.xml; Size: 15,024 bytes; Created: January 9, 2026.FIELD

[0004] The disclosure relates to methods of increasing premarineosin production in Streptomyces bacteria, including engineering increased expression of biosynthetic gene cluster regulators in the Streptomyces bacteria and optimizing culture conditions.BACKGROUND

[0005] Known for their striking red hue - which is hailed as responsible for the historical "bloody bread” phenomenon and the pinkish residue found around some bathroom drains - the prodiginines are a class of microbial natural products marked by a core tripyrrole moiety.

[0006] Cyclic prodiginines are particularly notable prodiginines, as their constrained conformation encourages the binding of charged ions, leading to enhanced bioactivity in comparison to their straight-chained counterparts. One pair of cyclic prodiginines - the diastereomers marineosin A and B, discovered from the marine-derived Streptomyces sp. CNQ617 - are known for their unique spiroaminal structure, which confers potent cytotoxic activity. The Reynolds lab pioneered the interpretation of the late-stage chemistry of the marineosin pathway: the bright red 23-hydroxyundecylprodiginine (23-HUP) undergoes an oxidative bicyclization at the C8 double bond catalyzed by a putative Rieske oxygenase (MarG) to form premarineosin, which then undergoes a MarA-catalyzed reduction of the 06-07 double bond to yield marineosin11.

[0007] Despite the growing preventative and curative efforts, malaria has remained a major global health crisis. In 2023 alone, malaria was responsible for an estimated 263 million cases and 597,000 deaths. To complicate this issue, the causative Plasmodium parasites are becoming more resistant to availableDocket No. 30275 / 70841therapeutics, making it difficult to treat and prevent disease progression. Hence, discovering new antimalarial drug candidates is crucial to mitigating this crisis. One promising contender is the natural product premarineosin A, which is an intermediate of the marineosin biosynthetic pathway that exhibits potent and selective antimalarial activity against both nonresistant and resistant Plasmodium strains. However, its complex chemical structure and low production titers have prevented further exploration. To improve access to this promising metabolite, a biosynthetic gene cluster that encoding the biosynthetic genes for (-)-premarineosin A production in Streptomyces eitanensis was identified. Metabolic engineering approaches increased production from trace levels to nearly 35 mg / L, which enabled semi-synthetic and biocatalytic derivatization of the (-)-premarineosin A scaffold. Late-stage C-H bromination of the (-)-premarineosin A B-ring resulted in a very potent antimalarial compound with low cytotoxicity. These findings report progress toward the sustainable access of (-)-premarineosin A and the first structure-activity relationship study for this unique scaffold in the search for improved antimalarial therapeutics.

[0008] Of particular interest is the marineosin precursor - premarineosin - as it exhibits single digit nanomolar antimalarial activity in vitro and is notably less cytotoxic than marineosin. As nearly half of the world population is actively at risk for malaria infection - and the causative parasites are becoming increasingly more resistant -finding new treatments that match or exceed the efficacy of the current antimalarial arsenal is paramount.

[0009] Disclosed herein are materials and methods for enhancing premarineosin production in Streptomyces bacteria. Aside from their visible cultural presence, prodiginines have been explored for their anticancer, antibacterial, antifungal, antimalarial, and immunosuppressant4properties. For example, the prototypical prodiginine, prodigiosin, is revered for its dual anticancer and antibiotic activity, and a synthetic prodiginine derivative (Obatoclax) yielded positive results as an experimental cancer treatment in phase I and II clinical trials. Hence, the discovery of novel prodiginine analogs is a promising route toward the identification of effective drug candidates.SUMMARY

[0010] One aspect of the disclosure provides a compound, or pharmaceutically acceptable salt thereof, having a structure of formula I:wherein X1is N or N+-O-; each RNis independently H, OH, Ci-ealkyl, C^alkenyl, or C(O)-Ci-3alkyl; R1is halo, H, OH, OHO, C(O)-Ci-3alkyl, C(O)-Ci-3haloalkyl, C1-6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2; R2Docket No. 30275 / 70841and R3are independently H, OH, halo, C^alkenyl, or NO2; and each R4is independently OH or Ci-shaloalkyl, with the proviso that at least one of R1, R2, and R3is not H. In some cases, R1is H, OH, OHO, C(O)-Ci-3alkyl, Ci.6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2. In some cases, R1is halo. In some cases, R1is F. In some cases, R1is Cl, Br, or I. In some cases, R1is H. In some cases, R1is C(O)CF3. In some cases, R1is OHO or C(O)CH3. In some cases, R1is Ci-ealkyl optionally substituted with 1 to 3 R4. In some cases, R1is unsubstituted Ci-ealkyl. In some cases, R1is Ci-ealkyl substituted with 1-3 R4. In some cases, each R4isindependently OH or CFs. In some cases, R1

[0011] Another aspect of the disclosure provides a compound, or pharmaceutically acceptable salt thereof, having a structure of formula II:wherein each of X1and X2is independently N or N+-Q-; each RNis independently H, OH, Ci-ealkyl, C2-6 alkenyl, or C(O)-Ci-3alkyl; L is a linker; and each of R2, R2', R3, and R3' is independently H or halo.

[0012] In various embodiments of formula I or formula II, X1is N. In some cases, X2is N. In some cases, at least one RNis H. In some cases, each RNis H. In some cases, R2is H. In some cases, R2is halo. In some cases, R2is Br. In some cases, R3is H. In some cases, R3is halo. In some cases, R3is Br. In some cases, R2' is H. In some cases, R2' is halo. In some cases, R2' is Br. In some cases, R3' is H. In some cases, R3' is halo. In some cases, R3' is Br. In some cases, L is Ci-sal ky lene or Ci-shaloalky lene. In some cases, L is,

[0013] Another aspect of the disclosure relates to a compound or salt having a structure as shown in Table A.

[0014] Still another aspect of the disclosure relates to a pharmaceutical composition comprising a compound or salt disclosed herein, and a pharmaceutically-acceptable excipient.

[0015] Yet another aspect of the disclosure relates to a method for producing a semi-synthetic derivative of premarineosin or a pharmaceutically acceptable salt thereof, comprising contacting premarineosin or a derivative thereof with an acid catalyst, a halogenase, or both, under conditions suitable for carrying out an electrophilic aromatic substitution reaction on one or more pyrrole rings of the premarineosin or derivative thereof. In some cases, the electrophilic aromatic substitution reaction comprises an acylation reaction, a condensation reaction, aDocket No. 30275 / 70841halogenation reaction, or a combination thereof. In some cases, the method comprises contacting premarineosin or a derivative thereof with an acid catalyst. In some cases, the acid catalyst comprises trifluoroacetic acid.

[0016] In some cases, the acylation or condensation reaction further comprises contacting the premarineosin or derivative thereof with a ketone or aldehyde reagent. In some cases, the ketone or aldehyde reagent is acetone, trifluoroacetone, or formaldehyde. In some cases, the reaction is an acylation reaction. In some cases, the acylation reaction is stopped before complete consumption of the premarineosin or derivative thereof. In some cases, the method further comprises a reduction step. In some cases, the reduction step comprises reducing a carbonyl group of the semi-synthetic derivative of premarineosin or salt thereof. In some cases, the reaction is a condensation reaction.

[0017] In some cases, the reaction is a halogenation reaction. In some cases, the method comprises contacting the premarineosin or derivative thereof with a halogenase. In some cases, the method comprises contacting the premarineosin or derivative thereof with an indole halogenase. In some cases, the indole halogenase is a flavin-dependent halogenase (“FDH”). In some cases, the flavin-dependent halogenase (“FDH”) is FDH 4V or FDH D3.

[0018] In some cases, the semi-synthetic derivative of premarineosin or pharmaceutically acceptable salt thereof is a compound or salt disclosed herein.

[0019] Another aspect of the disclosure relates to a method of inhibiting a Plasmodium falciparum parasite, comprising contacting the parasite with a compound, salt, or pharmaceutical composition disclosed herein. In some cases, the parasite is a drug-resistant or drug-sensitive Plasmodium falciparum parasite. In some cases, the parasite is a Dd2 or 3D7 Plasmodium falciparum parasite.

[0020] Yet another aspect of the disclosure relates to a method of treating or preventing malaria in a patient, comprising administering to the patient a therapeutically effective amount of a compound, salt, or pharmaceutical composition disclosed herein. In some cases, malaria is caused by a drug-resistant or drug-sensitive Plasmodium. In some cases, malaria is caused by a Dd2 or 3D7 Plasmodium falciparum parasite.

[0021] In still another aspect of the disclosure, provided is an engineered Streptomyces bacteria having increased expression of a PmaD protein, wherein the engineered Streptomyces bacteria produces high levels of premarinosin. In another aspect of the disclosure, provided is an engineered Streptomyces bacteria having increased expression of a PmaD protein and a PmaG protein, wherein the engineered Streptomyces bacteria produces high levels of premarineosin.

[0022] In some cases, the engineered Streptomyces bacteria expresses a PmaD protein comprising: I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, ii) the amino acid sequence of SEQ ID NO: 1, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 3, or, iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 3. In various embodiments, the engineered StreptomycesDocket No. 30275 / 70841bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the nucleotide sequence of SEQ ID NO: 3, operably linked to a constitutive promoter.

[0023] In some cases, the engineered Streptomyces bacteria expresses a PmaG protein comprising: I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, II) the amino acid sequence of SEQ ID NO: 2, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 4, or, iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 4. In various embodiments, the engineered Streptomyces bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4, operably linked to a constitutive promoter.

[0024] In some cases, the PmaD protein comprises the amino acid sequence of SEQ ID NO: 1, and the PmaG protein comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the PmaD protein is encoded by the nucleotide sequence of SEQ ID NO: 3, and the PmaG protein is encoded by the nucleotide sequence of SEQ ID NO: 4.

[0025] In another aspect of the disclosure, provided is a culture comprising the engineered Streptomyces bacteria disclosed herein. In some cases, the bacteria is Streptomyces eitanensis, Streptomyces stelliscabiei, Streptomyces griseiscabiei, Streptomyces scabiei, Streptomyces neyagawensis, Streptomyces sp. CNQ617, Streptomyces smyrnaeus, Streptomyces sp. RK75, Streptomyces spirodelae, Streptomyces diacarni, Streptomyces tubbatahanensis, Streptomyces sp. JV178, Streptomyces NPDC008159, Streptomyces scabiei, Streptomyces sp. 11x1, Streptomyces sp. MNU76, or Streptomyces roseoverticillatus

[0026] In yet another aspect of the disclosure, provided is a plasmid comprising a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter. In some cases, the plasmid comprises I) a nucleotide sequence encoding a PmaD protein and II) a nucleotide sequence encoding a PmaG protein, wherein the nucleotide sequences are operably linked to a constitutive promoter. In some cases, the nucleotide sequence encoding the PmaD protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the sequence of SEQ ID NO: 3. In some cases, the PmaD protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1. In some cases, the nucleotide sequence encoding the PmaG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4. In some cases, the PmaG protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the constitutive promoter is KasO*p. In some cases, the constitutive promoter is SEQ ID NO: 5.

[0027] In still another aspect of the disclosure, provided is a method of engineering a bacteria comprising contacting a Streptomyces bacteria with the plasmids disclosed herein.Docket No. 30275 / 70841

[0028] In another aspect of the disclosure, provided is a method of producing premarineosin comprising culturing the engineered Streptomyces bacteria disclosed herein. In come cases, the culture comprises soybean meal as the complex protein source in the media. In some cases, the method further comprises extraction and isolation of premarineosin using 25% methanol in dichloromethane. In some cases, the method further comprises extraction and isolation of premarineosin using acetone.

[0029] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description, taken in conjunction with the drawings. The description hereafter includes specific embodiments with the understanding that the disclosure is illustrative, and is not intended to limit the invention to the specific embodiments described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows overproduction of (-)-premarineosin A in S. eitanensis. FIG. 1 A provides a comparison of the S. eitanensis (-)-premarineosin A BGC and the Streptomyces sp. CNQ617 marineosin BGC using clinker.29The (-)-premarineosin A BGC is lacking marA and marE (box). FIG. 1 B provides a schematic of late-stage biosynthetic steps in the (-)-premarineosin A pathway. FIG. 1 C shows the crystal structure of (-)-premarineosin A. FIG. 1 D shows (l)-premarineosin A and 23-HUP production (mg / L), quantified by AUG (HPLC) against standard curves of known concentrations of each compound. Dots represent three independent culture replicates. Error bars indicate standard deviation (SD). ND: the measured production was below the limit of detection. FIG. 1 E provides (-)-premarineosin A production (mg) per gram of dry cell weight (DCW, g) over time by wildtype and engineered Se_V, Se_D and Se_DG strains.

[0031] FIG. 2 demonstrates the effect of crude complex protein source on yield of premarineosin in 1mL extracts from 50mL cultures of PmaDG. The soybean-meal-based media resulted in significantly higher production (p<0.05) than the other tested media. Error bars represent standard error.

[0032] FIG. 3 shows the crystal structure of the dimethyl-bridged premarineosin A dimer.

[0033] FIG. 4 demonstrates biocatalytic bromination of (-)-premarineosin A. FIG. 4 A provides in-silico docking of (-)-premari neosin A (dark grey) docked in the AlphaFold model of D3 displaying flexibility in the large open active site pocket (Left). FIG. 4 B shows an overlay of (-)-premarineosin A docked in D3 with the crystal structure of RebH (PBD ID: 2OA1) showing the sterically hindering helix that prevents binding in RebH, as opposed to the large, open active site cavity of D3 which accommodates (-)-premarineosin A. FIG. 4 C shows schemes for the biocatalysis (top) and semi-synthesis (bottom) of brominated premarineosin A analogs. FIG. 4 D shows HPLC traces of D3 and no enzyme control reactions. A peak corresponding to the molecular weight of 1 -bromo premarineosin A appears post-reaction. FIG. 4 E shows HPLC traces of the NBS reaction. A peak corresponding to the molecular weight of 12-bromo premarineosin A appears post-reaction.

[0034] FIG. 5 shows the selectivity index and activity of (-)-premarineosin A and analogs. Compounds were tested against P. falciparum 3D7 (chloroquine-sensitive) and Dd2 (chloroquine-resistant) for antimalarial activityDocket No. 30275 / 70841and the HEK293 embryonic kidney and MOLT4 T-cell leukemia cell lines for cytotoxicity to mammalian cells. Selectivity Index (SI) = Mammalian Cell Line IC50 / P. falciparum IC50.DETAILED DESCRIPTION

[0035] Provided herein are compounds which are semi-synthetic derivatives of premarineosin and pharmaceutically acceptable salts thereof, and pharmaceutical compositions comprising the semi-synthetic derivatives of premarineosin and salts thereof. Also provided are methods of producing semi-synthetic derivatives of premarineosin and pharmaceutically acceptable salts thereof. Further provided are methods of inhibiting a Plasmodium falciparum parasite using the compounds and salts disclosed herein, and methods of treating or preventing malaria in a patient using the compounds and salts disclosed herein. Also provided are engineered Streptomyces bacteria having increased expression of a PmaD protein, and engineered Streptomyces bacteria having increased expression of a PmaD protein and a PmaG protein, wherein the engineered Streptomyces bacteria produces high levels of premarineosin. Also provided are plasmids comprising a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter, and plasmids comprising a nucleotide sequence encoding a PmaD protein and a nucleotide sequence encoding a PmaG protein, wherein the nucleotide sequences are operably linked to a constitutive promoter. Also disclosed are materials and methods for sustainable overproduction of premarineosin and premarineosin derivatives. For example, premarineosin overproduction can be achieved by media and culture optimization, increased expression of target regulatory genes, or a combination thereof.

[0036] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0038] The term "a” and "an” as used herein mean "one or more” and include the plural unless the context is appropriate.

[0039] As used herein, the term "bacteria” includes both singular and plural forms, such as a bacterium, a single bacterial cell and bacteria (plural), and genetically modified (recombinant) bacterial cells, bacteria and bacterial strains thereof.

[0040] As used herein, the term "operably linked” refers to a functional linkage between one or more nucleic acid sequences, such as a linkage between a regulatory or promoter sequence and a coding region sequence in which regulatory or promoter and the coding sequence are positioned and oriented for transcription of the coding region sequence to be positively or negatively regulated by the linked regulatory sequence.Docket No. 30275 / 70841Compounds of the Disclosure

[0041] The prodiginines are a class of microbial natural products distinguished by a core tripyrrole moiety and a striking red color.1Prodiginines have exhibited remarkable anticancer2-4, antibacterial5’6, antifungal6, antimalarial7-11, and immunosuppressant3properties. Cyclic prodiginines are particularly notable, as their constrained conformations encourage the binding of charged ions, leading to enhanced bioactivity compared to their straight-chained counterparts.4’12 13For instance, a cyclic prodiginine isolated from Streptomyces CNQ-617, marineosin A, incorporates a unique spiroaminal structure that confers potent cytotoxicity.14In a key late-stage step in marineosin A biosynthesis, the bright red 23-hydroxyundecylprodiginine (23-HUP) undergoes an oxidative bicyclization at the C8-C9 double bond catalyzed by a Rieske oxygenase (MarG) to form premarineosin A.11Finally, premarineosin A is converted to marineosin A by a MarA-catalyzed reduction of the C6-C7 double bond.11 14Premarineosin A is of particular interest as it exhibits single-digit nanomolar antimalarial activity in vitro and is notably less cytotoxic than marineosin A.11

[0042] Due to emerging resistance, developing new antimalarial drugs that match or exceed the efficacy of current therapeutics is paramount.15 16Various prodiginine analogs have been generated to improve their antiplasmodial and cytotoxic properties.7-10’17 18Most often, total synthesis has been employed to derivatize linear prodiginines, with modifications occurring early in the route through the functionalization of simple precursors.9Due to the synthetic rigor of the spirocyclic core, premarineosin A is significantly less amenable to total synthesis, as evidenced by all prior efforts resulting in low overall yields.1920Design of premarineosin A analogs has been attempted via bioconversion of 23-HUP, but is limited by low conversion rates.7Heterologous expression of the marineosin biosynthetic cluster in Streptomyces venezuelae disrupted in marA has enabled the isolation of 0.5 mg of premarineosin A.11However, no production was observed in cultures >100 mL, limiting structural characterization, derivatization, and drug development efforts.

[0043] Provided herein are compounds and pharmaceutically acceptable salt thereof having a structure of formula I:whereinX1is N or N+-O-;each RNis independently H, OH, Ci-ealkyl, C^alkenyl, or C(O)-Ci-3alkyl;R1is halo, H, OH, OHO, C(O)-Ci-3alkyl, C(O)-Ci-3haloalkyl, Ci-ealkyl optionally substituted with 1-3 R4, C2-ealkenyl, or NO2;Docket No. 30275 / 70841R2and R3are independently H, OH, halo, C^alkenyl, or NO2; andeach R4is independently OH or Ci-shaloalkyl,with the proviso that at least one of R1, R2, and R3is not H. In some cases, X1is N or N+-Q-; each RNis independently H, OH, Ci-ealkyl, C^alkenyl, or C(O)-Ci-3alkyl; R1is halo, H, OH, OHO, C(O)-Ci-3alkyl, Ci-ealkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2; R2and R3are independently H, OH, halo, C^alkenyl, or NO2; and each R4is independently OH or Ci-shaloalkyl, with the proviso that at least one of R1, R2, and R3is not H. In some cases, X1is N or N+-O-; each RNis independently H, OH, Ci-ealkyl, C^alkenyl, or C(O)-Ci-salkyl; R1is H, OH, OHO, C(O)-Ci-3alkyl, Ci-ealkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2; R2and R3are independently H, OH, halo, C^alkenyl, or NO2; and each R4is independently OH or Ci-shaloalkyl, with the proviso that at least one of R1, R2, and R3is not H.

[0044] Also provided are compounds and pharmaceutically acceptable salts thereof having a structure of formula II:whereineach of X1and X2is independently N or N+-Q-;each RNis independently H, OH, Ci-ealkyl, C2-6 alkenyl, or C(O)-Ci-salkyl;L is a linker; andeach of R2, R2', R3, and R3' is independently H or halo.

[0045] In some cases, X1is N. In some cases, X1is N+-O-. In some cases, X2is N. In some cases, X2is N+-O-. In some cases, X1and X2are N. In some cases, X1and X2are N+-O-. In some cases, X1is N and X2is N+-O-. In some cases, X1is N+-O- and X2is N.

[0046] In some cases, at least one RNis H. In some cases, each RNis H. In some cases, at least one RNis OH. In some cases, each RNis OH. In some cases, at least one RNis Ci-ealkyl . In some cases, each RNis Ci. ealkyl. In some cases, at least one RNis C2-6 alkenyl. In some cases, each RNis C2-6 alkenyl. In some cases, at least one RNis Csalkenyl. In some cases, each RNis Csalkenyl. In some cases, at least one RNisone RNis C(O)-Ci-salkyl. In some cases, each RNis C(O)-Ci-salkyl.

[0047] In some cases, R1is halo, H, OH, OHO, C(O)-Ci-salkyl, C(O)-Ci-3haloalkyl, C1-6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2. In some cases, R1is halo, H, OH, OHO, C(O)-Ci-3alkyl, C1-6 alkylDocket No. 30275 / 70841optionally substituted with 1-3 R4, C^alkenyl, or NO2. In some cases, R1is H, OH, CHO, C(O)-Ci-3alkyl, C1-6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2. In some cases, R1is H, CHO, C(O)-Ci-3alkyl, or Ci. ealkyl optionally substituted with 1-3 R4. In some cases, R1is halo. In some cases, R1is F. In some cases, R1is Cl, Br, or I. In some cases, R1is Cl. In some cases, R1is Br. In some cases, R1is I. In some cases, R1is H. In some cases, R1is OH. In some cases, R1is CHO. In some cases, R1is C(O)-Ci-3alkyl. In some cases, R1is C(O)CH3. In some cases, R1is CHO or C(O)-Ci-3alkyl. In some cases, R1is CHO or C(O)CH3. In some cases, R1is C(O)-Ci-3haloalkyl . In some cases, R1is C(O)CF3. In some cases, R1is C^alkyl optionally substituted with 1-3 R4. In some cases, R1is unsubstituted C^alkyl. In some cases, R1is C^alkyl substituted with 1-3 R4. In some cases, R1is C^alkyl substituted with 1 R4. In some cases, R1is C^alkyl substituted with 2 R4. In some cases, R1is Ci-ealkyl substituted with 3 R4. In some cases, at least one R4is OH. In some cases, each R4is OH. In some cases, at least one R4is Ci-3haloalkyl. In some cases, each R4is Ci-shaloalkyl. In some cases, atleast one R4is CF3. In some cases, each R4is CF3. In some cases, R1isIn somecases, R1In some cases, R1isIn some cases, R1is C^alkenyl. In some cases, R1somecases,some cases, R1is NO2.

[0048] In some cases, R2is H. In some cases, R2is halo. In some cases, R2is Cl or Br. In some cases, R2is Cl. In some cases, R2is Br. In some cases, R2' is H. In some cases, R2' is halo. In some cases, R2' is Cl or Br. In some cases, R2' is Cl. In some cases, R2' is Br. In some cases, R3is H. In some cases, R3is halo. In some cases, R3is Cl or Br. In some cases, R3is Cl. In some cases, R3is Br. In some cases, R3' is H. In some cases, R3' is halo. In some cases, R3' is Cl or Br. In some cases, R3' is Cl. In some cases, R3' is Br. In some cases, the compound or salt has a structure of formula I and R1and R3are halo. In some cases, the compound or salt has a structure of formula I and R1and R3are Br.

[0049] In some cases, L is optionally substituted Ci-ealkylene. In some cases, L isthat is optionally further substituted, e.g., with one or more OH, halo, Ci-salkyl, Ci-shydroxyalkyl, Ci-shaloalkyl, oxo, amino, and the like. In some cases, L is Ci-3alkylene or Ci-shaloalkylene. In some cases, L is C salkylene. In some cases, L isDocket No. 30275 / 70841

[0050] Specific compounds contemplated include those listed in Table A, and pharmaceutically acceptable salts thereof:Table ADocket No. 30275 / 70841Docket No. 30275 / 70841"&>Docket No. 30275 / 70841>"

[0051] In some cases, the compound or salt is selected from compound A1, A2, A3, A4, A5, A6, A7, A8, A9, and pharmaceutically acceptable salts thereof. In some cases, the compound or salt is selected from compound A10, A11, A12, A13, and pharmaceutically acceptable salts thereof. In some cases, the compound or salt is selected from compound A10, A11, A12, A14, and pharmaceutically acceptable salts thereof. In some cases, the compound or salt is selected from compound A14, A15, A16, A17, and pharmaceutically acceptable salts thereof.

[0052] As used herein, the term "alkyl” refers to straight chained and branched saturated hydrocarbon groups containing one to six carbon atoms. The term Cnmeans the alkyl group has “n” carbon atoms. For example, Ce alkyl refers to an alkyl group that has 6 carbon atoms. “Ci-Cealkyl” refers to an alkyl group having a number of carbon atoms encompassing the entire range (e.g., 1 to 6 carbon atoms), as well as all subgroups (e.g., 1-6, 2-6, 1-5, 3-6, 1, 2, 3, 4, 5, and 6 carbon atoms). Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl (1,1 -dimethylethyl), and 3-methylpentyl. Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group.

[0053] The term "alkylene” used herein refers to an alkyl group having a substituent. For example, an alkylene group can be -CH2CH2- or -CH2-. The term Cnmeans the alkylene group has “n” carbon atoms. For example, C1-3 alkylene refers to an alkylene group having a number of carbon atoms encompassing the entire range of 1 to 3, as well as all subgroups, as previously described for "alkyl” groups. Unless otherwise indicated, an alkylene group can be an unsubstituted alkylene group or a substituted alkylene group.

[0054] The term "alkenyl” used herein refers to an unsaturated aliphatic group analogous in length and possible substitution to an alkyl group described above, but that contains at least one double bond. For example, the term "alkenyl” includes straight chain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl), and branched alkenyl groups (e.g., prenyl and reverse prenyl). A straight chain or branched alkenyl group can have six or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term "C^al keny I” includes chains having a number of carbon atoms encompassing the entire range (e.g., 2 to 6 carbon atoms), as well as all subgroups (e.g., 2-6, 2-5, 2-4, 3-6, 2, 3, 4, 5, and 6 carbon atoms). Unless otherwise indicated, an alkenyl group can be an unsubstituted alkenyl group or a substituted alkenyl group.

[0055] As used herein, the term “haloalkyl” refers to an alkyl group substituted with one or more halogen substituents. Haloalkyl is alternatively referred to as "alkylene-halo.” For example, Ci-ehaloalkyl refers to a C1-6Docket No. 30275 / 70841alkyl group substituted with one or more halogen atoms, e.g., 1, 2, 3, 4, 5, or 6 halogen atoms. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, and trichloromethyl groups. Similarly, haloalkoxy refers to an alkoxy group substituted with one or more halogen atoms e.g., 1, 2, 3, 4, 5, or 6 halogen atoms.

[0056] The term “haloal kylene” used herein refers to a haloalkyl group having two or more further substituents. For example, a haloalky lene group can be -C(CH3)(CF3)-. The term Cnmeans the haloalky lene group has “n” carbon atoms. For example, Ci-shaloal kylene refers to a haloalky lene group having a number of carbon atoms encompassing the entire range of 1 to 3, as well as all subgroups, as previously described for "haloalkyl” groups.

[0057] As used herein, the term "halo” or "halogen” refers to fluorine, chlorine, bromine, or iodine.

[0058] Compounds of the present disclosure can exist in particular geometric or stereoisomeric forms having one or more asymmetric carbon atoms. The present disclosure contemplates such forms, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the disclosed compounds. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are intended for inclusion herein.

[0059] As used herein, the term "pharmaceutically acceptable” means that the referenced substance, such as a compound of the present disclosure, or a formulation containing the compound, or a particular excipient, are safe and suitable for administration to a patient or subject. The term "pharmaceutically acceptable excipient” refers to a medium that does not interfere with the effectiveness of the biological activity of the active ingredient(s) and is not toxic to the host to which it is administered.

[0060] The compounds disclosed herein can be as a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-Docket No. 30275 / 70841hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such salts include, but are not limited to, alkali metal, alkaline earth metal, aluminum salts, ammonium, N+(Ci-4alkyl)4 salts, and salts of organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.Pharmaceutical Formulations, Dosing, and Routes of Administration

[0061] Further provided are pharmaceutical formulations (alternatively referred to as compositions throughout herein) comprising a compound as described herein or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0062] The compounds described herein can be administered to a subject in a therapeutically effective amount, alone or as part of a pharmaceutically acceptable composition or formulation. In addition, the compounds can be administered all at once, multiple times, or delivered substantially uniformly over a period of time. It is also noted that the dose of the compound can be varied over time.

[0063] A particular administration regimen for a particular subject will depend, in part, upon the compound, the amount of compound administered, the route of administration, and the cause and extent of any side effects. The amount of compound administered to a subject (e.g., a mammal, such as a human) in accordance with the disclosure should be sufficient to affect the desired response over a reasonable time frame. Dosage typically depends upon the route, timing, and frequency of administration. Accordingly, the clinician titers the dosage and modifies the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art.

[0064] Purely by way of illustration, the method comprises administering, for example, from about 0.1 mg / kg up to about 100 mg / kg of compound or more, depending on the factors mentioned above. In other embodiments, the dosage ranges from 1 mg / kg up to about 100 mg / kg; or 5 mg / kg up to about 100 mg / kg; or 10 mg / kg up to about 100 mg / kg. Some conditions require prolonged treatment, which may or may not entail administering lowerDocket No. 30275 / 70841doses of compound over multiple administrations. If desired, a dose of the compound is administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. The treatment period will depend on the particular condition and type of pain, and may last one day to several months.

[0065] Suitable methods of administering a physiological ly-acceptable composition, such as a pharmaceutical composition comprising the compounds disclosed herein are well known in the art. Although more than one route can be used to administer a compound, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition comprising the compound is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and / or introduced into circulation. For example, in certain circumstances, it will be desirable to deliver a pharmaceutical composition comprising the agent orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems, or by implantation devices. If desired, the compound is administered regionally via intrathecal administration, intracerebral (intra-parenchymal) administration, intracerebroventricular administration, or intraarterial or intravenous administration feeding the region of interest. Alternatively, the composition is administered locally via implantation of a membrane, sponge, or another appropriate material onto which the desired compound has been absorbed or encapsulated. Where an implantation device is used, the device is, in one aspect, implanted into any suitable tissue or organ, and delivery of the desired compound is, for example, via diffusion, timed-release bolus, or continuous administration.

[0066] To facilitate administration, the compound is, in various aspects, formulated into a physiologically-acceptable composition comprising a carrier (e.g., vehicle, adjuvant, or diluent). The particular carrier employed is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. Physiologically- acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (for example, see U.S. Patent No. 5,466,468). Injectable formulations are further described in, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). A pharmaceutical composition comprising the compound is, in one aspect, placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.Docket No. 30275 / 70841

[0067] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0068] These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Microorganism contamination can be prevented by adding various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0069] Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, mannitol, and silicic acid; (b) binders, as for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as for example, glycerol; (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (a) solution retarders, as for example, paraffin; (f) absorption accelerators, as for example, quaternary ammonium compounds; (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate; (h) adsorbents, as for example, kaolin and bentonite; and (I) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, and tablets, the dosage forms may also comprise buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols, and the like.

[0070] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. The solid dosage forms may also contain opacifying agents. Further, the solid dosage forms may be embedding compositions, such that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.Examples of embedding compositions that can be used are polymeric substances and waxes. The active compound can also be in micro-encapsulated form, optionally with one or more excipients.

[0071] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as forDocket No. 30275 / 70841example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame seed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[0072] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, in addition to the active compound, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances, and the like.

[0073] Compositions for rectal administration are preferably suppositories, which can be prepared by mixing the compounds of the disclosure with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary room temperature, but liquid at body temperature, and therefore, melt in the rectum or vaginal cavity and release the active component.

[0074] The compositions used in the methods of the disclosure may be formulated in micelles or liposomes. Such formulations include sterically stabilized micelles or liposomes and sterically stabilized mixed micelles or liposomes. Such formulations can facilitate intracellular delivery, since lipid bilayers of liposomes and micelles are known to fuse with the plasma membrane of cells and deliver entrapped contents into the intracellular compartment.

[0075] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

[0076] The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration. The optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990) Mack Publishing Co., Easton, PA, pages 1435-1712, incorporated herein by reference. Such formulations may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in animals or human clinical trials.Docket No. 30275 / 70841

[0077] The precise dosage to be employed depends upon several factors including the host, whether in veterinary medicine or human medicine, the nature and severity of the condition, e.g., disease or disorder, being treated, the mode of administration and the particular active substance employed. The compounds may be administered by any conventional route, in particular enterally, and, in one aspect, orally in the form of tablets or capsules. Administered compounds can be in the free form or pharmaceutically acceptable salt form as appropriate, for use as a pharmaceutical, particularly for use in the prophylactic or curative treatment of a disease of interest. These measures will slow the rate of progress of the disease state and assist the body in reversing the process direction in a natural manner.

[0078] It will be appreciated that the pharmaceutical compositions and treatment methods of the invention are useful in fields of human medicine and veterinary medicine. Thus, the subject to be treated is in one aspect a mammal. In another aspect, the mammal is a human.

[0079] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of "administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique {e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the "administering” of compositions includes both methods practiced on the human body and also the foregoing activities.Methods of Synthesizing the Compounds of the Disclosure

[0080] The compounds disclosed herein can be described as semi-synthetic derivatives of premarineosin. Thus, the compounds disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A) and salts thereof can be synthesized using a semi-synthesis strategy. Semisynthesis is a type of chemical synthesis that uses chemical compounds isolated from natural sources (such as microbial cell cultures or plant material) as the starting materials to produce novel compounds with distinct chemical and medicinal properties.

[0081] Thus, the compounds disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A) and salts thereof can be synthesized via a process starting with overexpression of premarineosin in an engineered bacteria. Exemplary methods of premarineosin overexpression and production are described herein below.

[0082] Provided herein are methods for producing a semi-synthetic derivative of premarineosin or a pharmaceutically acceptable salt thereof, comprising contacting premarineosin or a derivative thereof with an acid catalyst, a halogenase, or both, under conditions suitable for carrying out an electrophilic aromatic substitution reaction on one or more pyrrole rings of the premarineosin or derivative thereof. In some cases, the semi-synthetic derivative of premarineosin or pharmaceutically acceptable salt thereof is a compound or salt disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A and salts thereof).Docket No. 30275 / 70841

[0083] In some cases, the electrophilic aromatic substitution reaction comprises an acylation reaction, a condensation reaction, a halogenation reaction, or a combination thereof. In some cases, the electrophilic aromatic substitution reaction is an acylation reaction or a condensation reaction. In some cases, the electrophilic aromatic substitution reaction is an acylation reaction. In some cases, the electrophilic aromatic substitution reaction is a condensation reaction. In some cases, the acylation reaction or condensation reaction comprises contacting premarineosin or a derivative thereof with an acid catalyst. In some cases, the acid catalyst comprises trifluoroacetic acid. In some cases, the acylation reaction or condensation reaction further comprises contacting the premarineosin or derivative thereof with a ketone or aldehyde reagent. In some cases, the ketone or aldehyde reagent is acetone, trifluoroacetone, or formaldehyde. In some cases, the ketone or aldehyde reagent is acetone. In some cases, the ketone or aldehyde reagent is trifluoroacetone. In some cases, the ketone or aldehyde reagent is formaldehyde. In some cases, the the acylation reaction is stopped before complete consumption of the premarineosin or derivative thereof. In some cases, the acylation reaction further comprises a reduction step. In some cases, the reduction step comprises contacting the premarineosin or derivative thereof with a suitable reducing agent, (e.g., sodium borohydride). In some cases, the reduction step comprises reducing a carbonyl group of the semi-synthetic derivative of premarineosin or salt thereof.

[0084] A schematic representation of the acylation reactions and condensation reactions disclosed herein is shown in Scheme 1, below.Scheme 1

[0085] Compounds of Formula (II) can be synthesized using the procedure shown in Scheme 1. For example, premarineosin or a derivative thereof a can be subjected to a condensation reaction i by dissolving a in a suitable solvent and adding an acid catalyst (e.g., trifluoroacetic acid) and a carbonyl-containing substrate (such as a ketone or aldehyde having the general structure of L=O). Allowing the reaction to go to completion followed by quenching (e.g., with a base such as aqueous NaOH) and optional further derivatization yields compounds of Formula (II).Docket No. 30275 / 70841

[0086] Compounds of Formula (I) can be synthesized using the procedure shown in Scheme 1. For example, premarineosin or a derivative thereof a can be subjected to an acylation reaction ii by dissolving a in a suitable solvent and adding an acid catalyst (e.g., trifluoroacetic acid) and an aldehyde reagent having the general structure HC(O)Rc, where Rcis H (i.e., the reagent is formaldehyde) or Rcis a suitable alkyl or haloalkyl group. The reaction is quenched before completion (e.g., with a base such as aqueous NaOH). Optional further derivatization yields compounds of Formula (I).

[0087] In some cases, the electrophilic aromatic substitution reaction comprises a halogenation reaction. In some cases, the halogenation reaction comprises contacting the premarineosin or derivative thereof with a halogenase, e.g., an indole halogenase. In some cases, the indole halogenase is a flavin-dependent halogenase (“FDH”). In some cases, the flavin-dependent halogenase (“FDH”) is FDH 4V or FDH D3.

[0088] Compounds of Formula (I) and (II) wherein one or more of R2, R2', R3, and R3' is halo can also be synthesized using the procedure shown in Scheme 1. For example, a premarineosin derivative b or c wherein each of R2, R2', R3, and R3' is H can be produced via an acylation or condensation reaction as described above. To produce a halogenated compound, the premarineosin derivative b or c is then subjected to a further derivatization step comprising contacting the premarineosin or derivative thereof with a halogenase. For example, the premarineosin or derivative thereof can be dissolved in a suitable aqueous buffer solution comprising a suitable halogenase (e.g., FDH D3) and a suitable halide salt (e.g., NaBr). Incubation for a suitable time under suitable temperature conditions yields a halogenated derivative having a structure of Formula (I) or (II) as appropriate, wherein one or more of R2, R2', R3, and R3' is halo.Methods of Use

[0089] The compounds disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A) and salts thereof can inhibit parasites of the Plasmodium group, such as inhibiting the growth and / or activity of the parasites of the Plasmodium group. Plasmodium is a genus of unicellular eukaryotes that are obligate parasites of vertebrates and insects. The life cycles of Plasmodium species involve development in a bloodfeeding insect host which then injects parasites into a vertebrate host during a blood meal. Parasites grow within a vertebrate body tissue (often the liver) before entering the bloodstream to infect red blood cells. The ensuing destruction of host red blood cells can result in malaria. During this infection, some parasites are picked up by a blood-feeding insect (mosquitoes in majority cases), continuing the life cycle. In some cases, the compounds disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A) and salts thereof can inhibit a Plasmodium falciparum parasite. It should be understood that the methods disclosed herein as inhibiting Plasmodium falciparum parasites can also inhibit other Plasmodium parasites capable of causing malaria in a patient.

[0090] Provided herein are methods of inhibiting a Plasmodium parasite (such as a Plasmodium falciparum parasite), comprising contacting the parasite with compounds disclosed herein (e.g., compounds of Formula I, Formula II, or as shown in Table A) or salts thereof. In some cases, the compounds disclosed herein (e.g.,Docket No. 30275 / 70841compounds of Formula I, Formula II, and as shown in Table A) and salts thereof decrease the amount of a Plasmodium parasite (e.g., a Plasmodium falciparum parasite) in a patient, thereby inhibiting the Plasmodium parasite. In some cases, the parasite is a drug-resistant or drug-sensitive Plasmodium parasite, such as a drugresistant or drug-sensitive Plasmodium falciparum parasite. In some cases, the parasite is a Dd2 or 3D7 Plasmodium falciparum parasite.

[0091] Specifically contemplated are methods of using a therapeutically effective amount of a compound disclosed herein for use as a therapeutic in a subject. As used herein, the term "therapeutically effective amount” means an amount of a compound or combination of therapeutically active compounds (e.g., compounds of Formula I, Formula II, or as shown in Table A or salts thereof) that ameliorates, attenuates or eliminates one or more symptoms of a particular disease or condition (e.g., malaria), or prevents or delays the onset of one of more symptoms of a particular disease or condition.

[0092] The therapeutically effective amount can vary depending upon the intended application, or the subject and disease condition being treated, e.g., the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the weight and age of the patient, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., inhibiting the growth or activity of a Plasmodium parasite (e.g., a Plasmodium falciparum parasite to treat or prevent malaria). The specific dose will vary depending on, for example, the particular compounds chosen, the species of subject and their age / existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0093] As used herein, the term "patient” refers to animals, such as dogs, cats, cows, horses, and sheep (e.g., non-human animals) and humans. Particular patients or subjects are mammals (e.g., humans). The term "patient” includes males and females.

[0094] Provided herein are methods of treating or preventing malaria in a patient, comprising administering to the patient a therapeutically effective amount of a compound disclosed herein (e.g., compounds of Formula I, Formula II, and as shown in Table A) or a pharmaceutically acceptable salt thereof. In some cases, the malaria is caused by a drug-resistant or a drug-sensitive parasite, such as a drug-resistant or drug-sensitive Plasmodium. In some cases, the malaria infection is caused by a Dd2 or 3D7 Plasmodium falciparum parasite.

[0095] As used herein, the terms "treatment" or "treating" a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition before or after it has occurred. The terms "prevention" or "preventing" a disease or disorder specifically refers to a method of reducing, delaying or ameliorating such a condition before it has occurred. Treatment may be directed at one or more effects or symptoms of a disease and / or the underlying pathology. Treatment is aimed to obtain beneficial or desired results including, but not limited to, therapeutic benefit and / or a prophylactic benefit. By "therapeutic benefit” is meant eradication or amelioration of theDocket No. 30275 / 70841underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder. For prophylactic benefit, the pharmaceutical compounds and / or compositions can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The treatment can be any reduction and can be, but is not limited to, the complete ablation of the disease or the symptoms of the disease. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

[0096] As used herein, the term "therapeutic effect" refers to a therapeutic benefit and / or a prophylactic benefit as described herein. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.Engineered Bacteria

[0097] Described herein are bacteria of the Streptomyces genus engineered to express one or more proteins that induce increased production of premarineosin. In various embodiments, the Streptomyces bacteria are engineered to have increased expression of PmaD, PmaG, or a combination thereof, and in some cases the Streptomyces bacteria are engineered to overexpress PmaD, PmaG, or a combination thereof.

[0098] As used herein, the terms "engineered” or "recombinant” can generally refer to a non-naturally occurring organism, nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein) (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0099] Engineering of a bacteria involves the transfer of a nucleotide into the bacterial cell. The nucleotide can be a recombinant nucleotide, wherein the transfer of the recombinant nucleotide into the bacterial cell results in the expression of a homologous nucleotide or a heterologous nucleotide by the bacterial cell. Transferring a recombinant nucleotide into the bacterial cell can result in the integration of the nucleotide into the bacterial genome, or the stable expression of the nucleotide outside of the bacterial genome. T ransferring a nucleotide into the bacterial cell can also delete a homologous nucleotide from the bacterial genome. The introduction of theDocket No. 30275 / 70841nucleotide into the bacterial cell can be accomplished by any method known in the art, including, but not limited to, transformation, transfection, transduction, conjugation, and the like.

[0100] The introduction of nucleotide into a host cell may, for instance, but not limited thereto, be affected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). Specific transformation protocols are known in the art for various types of host cells (see, e.g., for Streptomyces conjugation, Matsushima and Baltz, 1996, Microbiology 142: 261-267; see also Bierman et al., 1992, Gene 116(1): 43-49).

[0101] The engineered Streptomyces disclosed herein are genetically modified to have increased expression of at least one protein involved in the regulation of a premarineosin biosynthetic gene cluster (BGC). As used herein, "increased expression” means that the engineered Streptomyces has an improved or increased expression of a premarineosin BGC regulator relative to a control cell (e.g., an unmodified bacteria). The term "overexpress” or "overexpression” refers to expression of a protein that is improved or increased compared to the expression level of the protein in wild type bacteria or naturally occurring bacteria.

[0102] As used herein "increased expression” is used to refer to an increased expression of a gene, or gene product thereof (e.g., the encoded protein) in a sample as compared to the expression of said gene or gene product in a suitable control, such as a naturally occurring or wild type cell or bacteria. The term "increased expression” preferably refers to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%, 1010%, 1020%, 1030%, 1040%, 1050%, 1060%, 1070%, 1080%, 1090%, 1100%, 1110%, 1120%, 1130%, 1140%, 1150%, 1160%, 1170%, 1180%, 1190%, 1200%, 1210%, 1220%, 1230%, 1240%, 1250%, 1260%, 1270%, 1280%, 1290%, 1300%, 1310%, 1320%, 1330%, 1340%, 1350%, 1360%, 1370%, 1380%, 1390%, 1400%, 1410%, 1420%, 1430%, 1440%, 1450%, 1460%, 1470%, 1480%, 1490%, or / to 1500% or more increased expression relative to a suitable control. For example, "increased expression” refers to about 10% to about 100% increase in expression, or about 50% to about 200% increase in expression, or about 100% to about 250% increase in expression, or about 250% to 500% increase in expression or about 300% to 700% increase in expression, or about 500% to about 1000% increase in expression or about 50% to about 250% increase in expression or about 100% to 500% increase in expression or about 100% to about 1000% increase in expression.

[0103] As used herein, "increased expression” includes when a nucleotide sequence or protein is native to the host cell, whose expression is quantitatively altered or whose expression is directed from a genomic locationDocket No. 30275 / 70841different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter. "Increased expression” also includes when a nucleotide sequence or protein that is not native to the host cell is expressed in the host cell.

[0104] In the context of gene expression, "expression” means the transcription of a specific gene or specific genes or a specific nucleotide construct; the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA construct.

[0105] Protein variants may be defined by their sequence identity when compared to a reference protein. Sequence identity usually is provided as "% sequence identity” or "% identity”. To determine the percent identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

[0106] After aligning the two sequences, in a second step, an identity value shall be determined from the alignment. Therefore, according to the present invention the following calculation of percent identity applies: % identity = (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give "% identity”.

[0107] For calculating the percent identity of two DNA sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For DNA sequences encoding for a protein the pairwise alignment shall be made over the complete length of the coding region from start to stop codon excluding introns. For non-protein-coding DNA sequences the pairwise alignment shall be made over the complete length of the sequence of this invention, so the complete sequence of this invention is compared to another sequence, or regions out of another sequence. Moreover, the preferred alignment program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).

[0108] In various embodiments, the engineered Streptomyces bacteria disclosed herein produces significantly more premarineosin than a control Streptomyces bacteria, such as a naturally occurring or wild type bacteria. The naturally occurring or wild type bacteria produces an "endogenous” level of premarineosin. In someDocket No. 30275 / 70841embodiments, the engineered Streptomyces bacteria produces about 10-fold to about 250-fold more premarineosin than a control Streptomyces bacteria. In various embodiments, the engineered Streptomyces bacteria produces about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 150-fold, about 200-fold, or about 250-fold more premarineosin than a control Streptomyces bacteria.Increased expression of Biosynthetic Gene Cluster regulators

[0109] PmaD is a putative transcriptional activator of the pathway to upregulate the transcription of the entire premarineosin BGC. PmaD is a homolog (40% protein identity, 52% similarity) of the RedD transcriptional activator from the undecylprodigiosin (UDP) pathway in Streptomyces coelicolor A3(2).23Overexpression of redD in both its native host and Streptomyces lividans enables overproduction of UDP23-25.

[0110] In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a PmaD protein, as compared to endogenous PmaD protein levels. In some embodiments, an engineered Streptomyces bacteria expresses a PmaD protein, wherein the amino acid sequence of the PmaD protein is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or wherein the amino acid sequence of the PmaD protein is SEQ ID NO: 1. In some embodiments, an engineered Streptomyces bacteria expresses a PmaD protein, wherein the amino acid sequence is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or wherein the amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0111] In some embodiments, an engineered Streptomyces comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the nucleotide sequence of SEQ ID NO: 3.

[0112] PmaG is a putative Rieske oxygenase which catalyzes the bicyclization of 23-HUP into premarineosin, the last step of (-)-premarineosin A biosynthesis. In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a PmaG protein, as compared to endogenous PmaG protein levels. In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a PmaD protein, and exhibits increased expression of a PmaG protein when compared to endogenous PmaG protein expression in a naturally occurring or wild type bacteria, or is higher than a production level previously reported in the art.

[0113] In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of the PmaG protein, wherein the amino acid sequence of the PmaG protein is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or wherein the amino acid sequence of the PmaG protein is SEQ ID NO: 2. In some embodiments, an engineered Streptomyces bacteria has increased expression of the PmaG protein, wherein the amino acid sequence is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or wherein the amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 4.Docket No. 30275 / 70841

[0114] In some embodiments, an engineered Streptomyces comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4.

[0115] The present disclosure further contemplates a culture comprising any of the Streptomyces bacteria disclosed herein. Exemplary Streptomyces bacteria include, but are not limited to, Streptomyces eitanensis, Streptomyces stelliscabiei, Streptomyces griseiscabiei, Streptomyces scabiei, Streptomyces neyagawensis, Streptomyces sp. CNQ617, Streptomyces smyrnaeus, Streptomyces sp. RK75, Streptomyces spirodelae, Streptomyces diacarni, Streptomyces tubbatahanensis, Streptomyces sp. JV178, Streptomyces NPDC008159, Streptomyces scabiei, Streptomyces sp. 11x1, Streptomyces sp. MNU76, and Streptomyces roseoverticillatus.

[0116] In some embodiments, the engineered Streptomyces described above produce high levels of premarineosin. As used herein, "high levels” refer to premarineosin titers higher than those previously reported in the art by wild-type or naturally occurring Streptomyces strains. To date, the highest titers of premarineosin have been produced in a heterologous MarA deletion mutant which prevented premarineosin from converting to marineosin, but such methods were only able to deliver 5mg / L of premarineosin11, with no production in larger-scale (>100 mL) cultures. In some embodiments, the engineered Streptomyces bacteria described herein produce at least about 10mg / L, 20mg / L, 30mg / L, 40mg / L, or 50mg / L of premarineosin.Plasmids

[0117] The term "plasmid”, "vector”, or "construct” as used herein refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in a microorganism such as a bacteria, or integrates into the host chromosome or a microorganism such as a bacteria. The plasmid can be part of an expression system. The plasmid is useful for creating an engineered bacterial cell, for example, that expresses increased levels of PmaD, PmaG, or a combination thereof, when compared to a non-engineered control cell.

[0118] The terms "expression” or "express” refers to the production of mRNA from the polynucleotide sequence of a gene or portion of a gene. The production of any polypeptide which is encoded by the mRNA, gene, or portion of the gene is also included within the scope of the terms. The term "encoding” refers to the property of polynucleotide sequences to behave as templates for the production of other macromolecules such as mRNA and proteins.

[0119] When used herein the term "coding sequence" refers to a nucleotide sequence which directly encodes the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG, CTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence. The start codon can also be named herein as "translational start signal” or "translational start site”. The stop codon can also be named herein as "translational stop signal” or "translational stop site”.Docket No. 30275 / 70841

[0120] As used interchangeably herein, "operably linked” and "operatively linked” in the context of recombinant or engineered polynucleotide molecules (e.g., DNA and RNA) vectors, and the like refers to the regulatory and other sequences useful for expression, stabilization, replication, and the like of the coding and transcribed noncoding sequences of a nucleic acid that are placed in the nucleic acid molecule in the appropriate positions and / or orientation relative to the coding sequence so as to positively or negatively affect expression or another characteristic of the coding sequence or transcribed non-coding sequence. This same term can be applied to the position and / or orientation of coding sequences, non-coding and / or transcription control elements (e.g., promoters, enhancers, and termination elements), and / or selectable markers in an expression vector. "Operably linked” can also refer to an indirect attachment (i.e., not a direct fusion) of two or more polynucleotide sequences or polypeptides to each other via a linking molecule (also referred to herein as a linker).

[0121] Further disclosed herein are polynucleotides encoding the amino acid sequences disclosed herein, and plasmids comprising the polynucleotides of the disclosure. In various embodiments, a plasmid of the disclosure comprises a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter. In some embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter, wherein the nucleotide sequence encoding the PmaD protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the sequence of SEQ ID NO: 3. In some embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter, wherein the PmaD protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1.

[0122] As used herein, "promoter” includes all sequences capable of driving transcription of a coding or a noncoding sequence. In particular, the term "promoter” as used herein refers to a DNA sequence generally described as the 5' regulator region of a gene, located proximal to the start codon. The transcription of an adjacent coding sequence(s) is initiated at the promoter region. The term "promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. As used herein, the term "constitutive promoter” describes sequences capable of continuously driving transcription of a coding or non-coding sequence. Any plasmids disclosed herein can comprise any constitutive promoter. In some embodiments, the plasmid's constitutive promoter is KasO*p. In some embodiments, the promoter comprises SEQ ID NO: 5.

[0123] In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a PmaG protein operably linked to a constitutive promoter. In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a PmaD protein and a nucleotide sequence encoding a PmaG protein, wherein both sequences are operably linked to a constitutive promoter. The disclosed plasmids can comprise any constitutive promoter. In some embodiments, the nucleotide sequence encoding the PmaG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4. In some embodiments, the nucleotide sequence encodes a PmaG protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQDocket No. 30275 / 70841ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the plasmid's constitutive promoter is KasO*p. In some embodiments, the promoter comprises SEQ ID NO: 5.

[0124] Further contemplated herein is a method of engineering a Streptomyces bacteria, wherein the method comprises contacting the Streptomyces bacteria with any of the plasmids disclosed herein.Optimizing Culture Conditions

[0125] Further contemplated herein is a method of producing premarineosin, wherein the method comprises culturing any of the Streptomyces bacteria disclosed herein, and wherein in some embodiments the Streptomyces culture yields high levels (or increased levels) of premarineosin. High levels or increased levels are determined by comparing with a control level, such as a production level by a wild type bacteria or naturally occurring bacteria, alternatively the production level is higher than a known standard level of production.

[0126] Disclosed herein are methods of optimizing bacterial culture conditions to improve premarineosin yields, wherein the method comprises culturing any of the engineered Streptomyces bacteria disclosed herein in media supplemented with a complex protein. As used herein, "media” refers to a liquid or solid preparation to support growth of bacteria in culture. In some embodiments, engineered Streptomyces bacteria are cultured in TSB media, T2YE media, YEME media, R2YE media, or any other media acceptable for bacterial culture. In some embodiments, engineered Streptomyces bacteria are cultured in media comprising production base media (such as a base media comprising 10g glucose, 5g bacto peptone, 5g yeast extract, and 2g CaCOs per liter, pH=7.0) supplemented with a complex protein. In various embodiments, the complex protein is soybean meal, corn gluten, or peanut meal. In some embodiments, the engineered Streptomyces bacteria are cultured in GMSYE media comprising production media base supplemented with glucose, maltodextrin, and soybean meal. In various embodiments, the engineered Streptomyces bacteria are cultured in GSSY media comprising production media base supplemented with glucose, soluble starch, and soybean meal. In some embodiments, the engineered Streptomyces bacteria are cultured in GICYE media comprising production media base supplemented with glucose, inulin, and corn gluten. In various embodiments, the engineered Streptomyces bacteria are cultured in GSPYE media comprising production media base supplemented with glucose, soluble starch, and peanut meal. In some embodiments, the engineered Streptomyces bacteria are cultured in GISYE media comprising production media base supplemented with glucose, inulin, and soybean meal. In various aspects, the GISYE media comprises 10 g / L of soybean meal.

[0127] In some embodiments, the engineered Streptomyces bacteria are cultured in a small-scale culture. In some aspects, a small-scale culture comprises a culture of about 1mL to about 100 mL. In various aspects, a small-scale culture comprises a culture of about 1mL, about 10mL, about 20mL, about 30mL, about 40mL, about 50mL, about 60mL, about 70mL, about 80mL, about 90mL, or about 100mL. In some embodiments the engineered Streptomyces bacteria are cultured in a large-scale culture. In some aspects, a large-scale culture comprises a culture of at least about 0.5L. In various aspects, a large-scale culture comprises a culture of at least about 0.2L, at least about 0.3L, at least about 0.4L, at least about 0.5L, at least about 0.6L, at least about 0.7L,Docket No. 30275 / 70841at least about 0.8L, at least about 0.9L, at least about 1.0L, at least about 1.1 L, at least about 1.2L, at least about 1 ,3L, at least about 1 ,4L, at least about 1 ,5L, at least about 1 ,6L, at least about 1 ,7L, at least about 1 ,8L, at least about 1 ,9L, or at least about 2L. In some aspects, a large-scale culture comprises a culture of at least about 2L. In some embodiments, the engineered Streptomyces bacteria are cultured for two or more days prior to premarineosin extraction. In various embodiments, the engineered Streptomyces bacteria are cultured for three, four, or five or more days prior to premarineosin extraction. In some embodiments, the engineered Streptomyces bacteria are cultured for five or more days prior to premarineosin extraction.

[0128] In some embodiments, a method of producing premarineosin comprises culturing an engineered Streptomyces eitanensis bacteria in media, wherein the S. eitanensis bacteria is engineered to express a plasmid encoding PmaD, and wherein the culture media is supplemented with a complex protein such as soybean meal. In various embodiments, a method of producing premarineosin comprises culturing an engineered Streptomyces bacteria in media, wherein the S. eitanensis bacteria is engineered to express a plasmid encoding PmaD and PmaG, and wherein the culture media is supplemented with a complex protein, such as soybean meal.

[0129] In some embodiments, premarineosin is extracted from a culture comprising an engineered Streptomyces bacteria using extraction and isolation protocols known in the art, wherein Streptomyces biomass is separated from media via vacuum filtration and cell pellets are broken with a lysing mixture. Disclosed herein are various embodiments wherein premarineosin is extracted from a culture comprising an engineered Streptomyces bacteria using methanol (MeOH), optionally wherein a lysing solution comprises a mixture of MeOH in dichloromethane (DCM). In some embodiments, premarineosin is extracted from a culture comprising an engineered Streptomyces bacteria using a lysing solution comprising acetone.EXAMPLESExample 1

[0130] Disclosed herein is a previously unexplored (-)-premarineosin A (pma) biosynthetic gene cluster (BGC) in Streptomyces eitanensis, with pathway engineering employed to improve production. Access to a suitable source of (-)-premarineosin A enabled development of high-yield strategies for the late-stage derivatization of (-)-premarineosin A via unique semi-synthetic and biocatalytic approaches. The new (-)-premari neosin derivatives were investigated for potency and toxicity in vitro, which identified a novel brominated analog with improved activity against Plasmodium falciparum.

[0131] Genome and metabolome analysis identified a (-)-premarineosin A biosynthetic gene cluster in S. eitanensis. Production of 23-hydroxy undecyl prodigi ni ne was recently reported in Streptomyces eitanensis strain engineered for improved production of concanamycin A.21Close inspection of the strain genome revealed a biosynthetic gene cluster (BGC) with 90% similarity to the canonical marineosin BGC (Ml BiG:BGC0000091).11’22However, it lacked the putative acyltransferase (MarE) and reductase (MarA) needed to reduce the central pyrrole (B-ring) in the final step of marineosin biosynthesis (FIG. 1, A-C).11UntargetedDocket No. 30275 / 70841metabolomic analysis revealed the production of a compound with the same molecular weight and MS fragmentation pattern as premarineosin A, while marineosins production was not observed. Thus, it was reasoned that the identified pma BGC likely encoded premarineosin assembly, suggesting that S. eitanensis could serve as a uniquely capable chassis strain for optimizing its large-scale production.

[0132] Overexpression of pma cluster-situated regulator (pmaD) and Rieske oxygenase (pmaG) improved (-)-premari neosin A production in S. eitanensis. Culturing wildtype S. eitanensis resulted in the identification of trace amounts of (-)-premari neosin A (FIG. 1 D). Annotation of pmaBGC identified a predicted regulatory protein PmaD, which is a homolog (40% protein identity, 52% similarity) of the RedD transcriptional activator from the undecylprodigiosin (UDP) pathway in Streptomyces coelicolor A3(2).23Overexpression of redD in both its native host and Streptomyces lividans enabled overproduction of UDP23-25, suggesting that PmaD may similarly regulate (-)-premarineosin A production in S. eitanensis. Inspired by previous work21, S. eitanensis was engineered for enhanced (-)-premarineosin A production using a regulatory gene expression strategy. The pmaD gene was amplified from S. eitanensis genome and used to assemble a construct in pSET152k under the strong constitutive promoter KasOp*, yielding pSET152k-pmaD. This plasmid was integrated at the $031 actinophage integrase attB site in the S. eitanensis genome, generating the pmaD overexpressing strain Se_D. To ensure that the producing phenotype is directly attributed to the overexpression of the pma genes, empty vector was integrated into the S. eitanensis genome, generating the strain Se_V. Under non-optimized culture conditions, using corn gluten meal, the Se_D strain produced significantly more (-)-premarineosin A (14.72 ± 1.44 mg / L) than the wild-type strain (below the limit of detection) (FIG. 1 D). Production of 2 was also observed in the vector control Se_V strain (1.05 ± 0.40 mg / L), albeit at a 14-fold lower production compared to the engineered Se_D strain.

[0133] The last step of (-)-premarineosin A biosynthesis involved the bicyclization of 23-HUP into (-)-premarineosin A by the putative Rieske oxygenase PmaG.711To improve the conversion of 23-HUP to (-)-premarineosin A, the pSET152k-pmaDG plasmid was designed for constitutive overexpression of pmaG and pmaD. This plasmid was integrated into S. eitanensis wildtype, yielding the Se_DG strain. Constitutive overexpression of pmaD and pmaG significantly enhanced (-)-premarineosin A production (23.26 ± 0.45 mg / L) in the Se_DG strain, a 1.58-fold increase compared to pmaD alone (FIG. 1 D). The metabolic shift towards production of 2 is also reflected by the distinct color difference between the Se_V / wildtype strains (green) and the Se_D / Se_DG strains (red) (data not shown).

[0134] Secondary metabolite production in Streptomyces sp. can vary appreciably depending on media composition and environmental factors.2627Crude protein sources have been shown to modulate undecylprodigiosin production in Streptomyces sp. JS520.28While the corn-gluten-meal-based GICYE medium resulted in optimal CMA production in S. eitanensis2', soybean meal was identified as the ideal complex protein source for enhanced (-)-premarineosin A production in S. eitanensis (FIG. 2). Large-scale (1L) cultivation of the Se_DG strain in soybean meal medium (GISYE) significantly improved (-)-premarineosin A production by 1.39-fold in comparison to GICYE cultivation (FIG. 1 D). Production of (-)-premarineosin A and 23-HUP was assessedDocket No. 30275 / 70841throughout cultivation, and while (-)-premari neosin A titers peaked around day 6, the titers of 23-HUP were lowest on day 7 (FIG. 1 E). Both acetone and methanol were investigated as extraction solvents, with acetone generating, on average, higher (-)-premarineosin A and 23-HUP titers (FIG. 1 D).

[0135] Under optimal conditions (soybean meal media at 22 °C for 7 days followed by extraction with acetone) the engineered Se_D and Se_DG strains produced comparable amounts of (-)-premarineosin A (34.75 + 1.58 mg / L and 33.73 + 5.13 mg / L, respectively), over 200-fold more than S. eitanensis wild-type (0.16 + 0.08 mg / L) (FIG. 1 D). The Se_DG strain, however, produces nearly twice (p < 0.05) as much (-)-premarineosin A per dry cell weight (5.05 mg / g DOW for Se_DG vs. 2.60 mg / g DOW for Se_D) (FIG. 1 E). As with the corn gluten media, the Se_V strain produced nearly 20-fold more (-)-premarineosin (3.01 +2.14 mg / L) compared to wildtype.Curiously, the only observed significantly (p < 0.005) increased production was of the red (-)-premarineosin A precursor (23-HUP) in the Se_DG strain cultured (FIG. 1 D). (-)-Premarineosin A was purified and structurally characterized using NMR (data not shown), X-ray crystallography (FIG. 1 C), and polarimetry. While the relative stereochemistry of (-)-premarineosin A matches what was previously described,7’13’14the X-ray / crystal data showed that absolute stereochemistry of (-)-premarineosin A isolated from S. eitanensis is not in accordance to what was observed by total synthesis;1920the optical rotation value for the (-)-premarineosin A isolated from S. eitanensis was [O]D24(C 0.4758 in MeOH) = -104.0 +0.1°.

[0136] Isolated compounds 23-Hydroxyundecylprodiginine and (-)-Premarineosin A were tested against the malarial parasites drug-sensitive 3D7 and drug-resistant Dd2 Plasmodium falciparum in an in vitro assay (Table B). The compounds were concomitantly evaluated for cytotoxicity against the HEK293 embryonic kidney and MOLT4 T-cell leukemia cell lines. (-)-Premarineosin A isolated from S. eitanensis exhibited nanomolar potency against 3D7 (IC50 = 4 ± 0.059 nM) and Dd2 (IC50 = 1 ± 0.7 nM) P. faciparum with minimal toxicity to mammalian cells (Table B). In comparison, (-)-premarineosin A was 144- and 352-fold more potent than 23-HUP, and over 10- (IC50 = 41 ± 3 nM) and 300-fold (IC50 = 382 ± 22 nM) more potent than chloroquine, respectively (Table B).Table B: Bioactivity of (-)-premarineosin A and derivativesDocket No. 30275 / 70841Compounds were tested against P. falciparum 3D7 (drug-sensitive) and Dd2 (drug-resistant) for antimalarial activity and the HEK293 embryonic kidney and MOLT4 T-cell leukemia cell lines for cytotoxicity to mammalian cells. Selectivity Index (SI) = Mammalian Cell Line IC50 / P. falciparum IC50.

[0137] Acid-catalyzed electrophilic aromatic substitution enabled late-stage derivatization of (-)-premarineosin A. With access to substantial quantities of (-)-premarineosin A, a semi-synthetic approach was pursued to functionalize its scaffold for a structure-activity relationship analysis. Natural product analogs can and have been strategically designed to improve potency, reduce toxicity, optimize metabolism, or increase oral bioavailability in comparison to their initial scaffolds.30-34Ultimately, the benefit of diversifying the core premarineosin A scaffold is two-fold, offering swift access to potentially promising lead compounds, and developing molecular probes that could be used to identify drug target(s) and elucidate the (-)-premarineosin A mechanism of action.

[0138] Previous synthetic studies established that 2,5-dimethylpyrroles are susceptible to acid-catalyzed condensation with ketones, such as acetone, to form dimerized substrates at the 3-position in an electrophilic aromatic substitution-like reaction.35To date, this synthetic approach has not been previously applied to any natural product scaffold. Thus, it was reasoned that exploration of the breadth and flexibility of this chemistry has the advantage of enabling access to previously untapped chemical diversity. Applying similar methodologies, it was found that (-)-premarineosin A readily dimerized under acidic conditions with both ketones and aldehydes at the C12 position of its C-ring, forming premarineosin A dimers with a gem-dimethyl-bridge (Compound A1- LC-MS: calculated for C53H70N6O4 [M+H+] 855.5537 m / z; found 855.5551 m / z), a methylene-bridge (Compound A2- LC-MS: calculated for51H66N6O4 [M+H+] 827.5224 m / z; found 827.5216 m / z.), and trifluoromethyl-bridge (Compound A3- LC-MS: calculated for C53H67F3N6O4 [M+H+] 909.5254 m / z; found 909.5257 m / z). A crystal structure of gem-dimethyl-bridged premarineosin A was obtained to confirm its structure (FIG. 3 B).Docket No. 30275 / 70841

[0139] The dimerization reaction of gem-dimethyl-bridged premarineosin A (Compound A1) with trifluoroacetone yielded a monomeric derivative (12-trifluoroacetyl premarineosin A, Compound A4- LC-MS: calculated for C28H36F3N3O3 [M+H+] 450.2757 m / z; found 450.2798 m / z.), likely due to early reaction termination before complete substrate consumption. Early termination with acetone or formaldehyde as the electrophile did not lead to monomeric intermediates, suggesting that the isolation of these modified premarineosin A monomers is only possible with bulky groups.35To synthesize other monomeric derivatives of (-)-premarineosin A, acid chlorides were employed as the electrophile, mimicking a Friedel-Crafts acetylation to form 12-formyl premarineosin A (Compound A5- LC-MS: calculated for C26H33N3O3 [M+H+] 436.2600 m / z; found 436.2636 m / z.) and 12-acetyl premarineosin A (Compound A6- LC-MS: calculated for C27H35N3O3 [M+H+] 450.2757 m / z; found 450.2798 m / z). Further reduction of 12-formyl premarineosin A with sodium borohydride provided the desired primary alcohol, forming 12-hydroxymethyl-premarineosin A (Compound A7-LC-MS: calculated for C26H35N3O3 [M+H+] 438.2757 m / z; found 438.2754 m / z .

[0140] The antiparasitic activity and cytotoxicity of these synthetic derivatives were evaluated in comparison to (-)-premarineosin A (Table B). The monomeric derivatives 12-trifluoroacetyl premarineosin A (Compound A4), 12-formyl premarineosin A (Compound A5) and 12-acetyl premarineosin A (Compound A6) exhibited submicromolar antiplasmodial activity against the drug-sensitive (3D7) and multidrug-resistant (Dd2) P. falciparum strains. Despite the reduced activity compared to (-)-premarineosin A, derivatives 12-trifluoroacetyl premarineosin A, 12-formyl premarineosin A, and 12-acetyl premarineosin A (Compounds A4, A5, and A6 respectively) perform better against Dd2 than chloroquine (IC50 = 382 ± 22 nM). 12-trifluoroacetyl premarineosin A is the most promising semi-synthetic derivative in terms of selectivity, as it is quite potent against Dd2 (IC50 = 87 ± 15 nM) with limited apparent toxicity to HEK293 cells (IC50 = 3730 nM). Methylene-bridged premarineosin A (Compound A2) (IC50 = 548 ± 76 nM) and 12-hydroxymethyl premarineosin A (Compound A7) (IC50 = 704 ± 304 nM) also exhibited sub-micromolar potency against the multidrug-resistant strain yet had limited potency against the drug-sensitive strain (Table B). While synthetic modifications at the C-ring reduced antimalarial potency of the premarineosin A scaffold, all the analogs show decreased cytotoxicity.

[0141] The results disclosed herein affirm and expand on the promise of premarineosin A as an effective scaffold for inhibiting malaria parasites in vitro. While the nanomolar-level potency of premarineosin A was initially reported over ten years ago, further investigation of its pharmacologic potential has been stymied by limited access to the molecule.7’11’19Reported herein is the identification of a (-)-premarineosin A BGC in S. eitanensis. By pairing genome engineering and media optimization strategies, (-)-premari neosin A titers were increased over 200-fold (from 0.16 + 0.08 mg / L to 34.75 + 1.58 mg / L) in comparison to S. eitanensis wildtype. This is the highest titer reported to date, as previous heterologous expression efforts resulted in less than 5 mg / L (-)-premarineosin A and production was not observed in fermentation cultures larger than 100mL.11While the difference in (-)-premari neosin A titer between the engineered Se_D and Se_DG strains under optimized conditions was not statistically significant, it was observed that Se_DG produces nearly twice as much (-)-premarineosin A per gram of dry cell weight. As the accumulation of (-)-premarineosin A and 23-HUP in theDocket No. 30275 / 70841engineered Se_DG strain has the potential to inhibit cell growth, it was reasoned that further culture optimization or adaptive laboratory evolution to increase cell viability in this engineered strain would enhance production titers. Interestingly, the strain integrating the empty vector produced nearly 20x more (-)-premarineosin A than the wildtype. Prodiginine production in the engineered Se_V strain could be a result of plasmid integration at the cpC31 integrase (attB) site, which has been shown to both alter the availability of malonyl-CoA and acetyl-CoA40and induce a stress response (stimulating secondary metabolite production).41

[0142] The stereocenters of the (-)-premarineosin A isolated herein have the same relative configuration as previously reported structures of marineosin Afrom both isolative711'14and total synthetic1920studies. However, the crystallographic data for both (-)-premari neosin A and the gem-dimethyl-bridged dimer (Compound A1) supports an absolute stereochemistry of 8S, 9R, 21 R, 23R, which is the enantiomer of the previously reported premarineosin A structure.19

[0143] Overall, this engineering approach yielded a sustainable source of (-)-premarineosin A that enabled semi-synthetic derivatization of the (-)-premarineosin A scaffold, culminating in the synthesis of seven new analogs. The strategy employed herein focused on diversifying the C12 position of the (-)-premarineosin A C ring. This approach led to the surprising discovery of pyrrole dimerization chemistry involving (-)-premarineosin A. While electrophilic substitution has been utilized previously to functionalize the p-position of pyrroles,4243this chemistry has yet to be explored with any natural product scaffold. Thus, the current work revealed the robust nature of acid-catalyzed electrophilic aromatic substitution of (-)-premarineosin A and a potential road map for the derivatization of prodiginines and other pyrrole-containing natural products. Of the seven semi-synthetic premarineosin A derivatives (Compounds A1-A7), five show sub-micromolar potency against the chloroquine-resistant P. falciparum Dd2, with three having a lower IC50 than chloroquine (Table B). However, they all have reduced bioactivity compared to (-)-premarineosin A, suggesting that the C12 position is crucial for the potent antimalarial activity of the premarineosin A scaffold (FIG. 5). Remarkably, all seven synthetic premarineosin A analogs are less toxic to both HEK293 and MOLT4 cells than (-)-premarineosin A, which is consistent with the bioactivity of other C-ring functionalized prodiginines (Table B).44-46Hence, functionalizing the 012 position of the C-ring is an encouraging avenue to increase the selectivity index (SI) of premarineosin A and improve its therapeutic potential.Example 2

[0144] Biocatalysts enabled late-stage C-H functionalization of (-)-premarineosin A. The biocatalytic bromination of (-)-premarineosin A represents a sustainable and innovative approach to producing novel derivatives with enhanced biological activity. Bromine, as a halogen, holds significant value in medicinal chemistry due to its capacity to improve key pharmacokinetic properties, such as membrane permeability, target binding affinity, and metabolic stability— factors crucial for combating Plasmodium species effectively.3637To harness the potential of selective C-H bromination of the (-)-premarineosin A scaffold, two bacterial flavindependent halogenases (FDHs) were computationally screened. Molecular docking of (-)-premarineosin A wasDocket No. 30275 / 70841performed with D3, an orphan FDH from Saccharophagus degradans38, and RebH, a FDH from the rebeccamycin biosynthetic pathway.39While RebH showed low substrate binding and minimal contact with the active site (data not shown), D3 revealed strong substrate binding (-8.6 kcal / mol), suggesting that the enzyme may be capable of regioselective bromination at the A and B pyrrole rings (FIG.4 A-B). Reacting D3 with (-)-premarineosin A in vitro resulted in a brominated derivative, 1 -bromo premarineosin A (Compound A9-LC-MS: calculated for C25H32BrN3O2 [M+H+] 486.1756 m / z; found 486.1771 m / z.)(FIG.4 C).

[0145] The antiparasitic activity and cytotoxicity of brominated premarineosin A derivatives were assessed.12-Bromo premarineosin A (Compound A11) demonstrated potent activity against 3D7 (IC50 = 2 ± 0 nM) and Dd2 (IC50 = 22 ± 11 nM) and low cytotoxicity (Table B). Selectivity against HEK293 cells was 785 (HEK293 / 3D7) and 71.4 (HEK293 / Dd2), while lower SI values were obtained relative to MOLT4 (328 for 3D7, and 29.8 for Dd2) (FIG. 5). Furthermore, the compound's relatively high clogP (6.21) suggests favorable lipophilicity, which may contribute to its potent antiparasitic activity and selectivity. 1-Bromo premarineosin A exhibited moderate antiparasitic activity, with IC50 values of 53 ± 11 nM (3D7) and 61 ± 33 nM (Dd2). Despite lower activity compared to 12-bromo premarineosin A, 1-bromo premarineosin A (Compound A9) showed limited cytotoxicity against both cell lines. These findings highlight 12-bromo premarineosin A (Compound A11) as a lead compound, combining exceptional antimalarial potency and lower cytotoxicity with remarkable selectivity.

[0146] To modify (-)-premarineosin A beyond the C12 position (at the A- and B-rings), late stage biocatalytic C-H bromination utilizing FDHs was explored, for the purpose of modifying complex pyrrole-containing scaffolds. While previous studies have demonstrated the utility of FDHs like PrnC for the selective early stage halogenation of simple pyrroles, such efforts have been limited to significantly smaller precursor molecules, including the chemoenzymatic generation of site-selective derivatives like a chlorinated analog of Fludioxonil.47Notably, no prior work has explored the enzymatic bromination of complex pyrrole scaffolds. Use of the orphan FDH D3 from S. degradans to selectively brominate (-)-premari neosin A on both the spiroaminal (B-ring) and minimally substituted (A-ring) pyrroles with approximately 50% substrate conversion efficiency sets significant promise for expanding the scope of enzymatic halogenation (FIG. 4 C). The regioselective bromination observed with D3 highlights the enzyme's capacity to target distinct pyrrole rings within the (-)-premarineosin A scaffold. The observed selectivity supported computational docking studies with the AlphaFold-generated structure of D3 exhibiting strong substrate binding and a preference for bromination at the A- and B-rings (FIG. 4 A-B). This specificity likely arises from the structural features of D3, particularly its large and flexible active site pocket, which can accommodate the bulky (-)-premarineosin A framework (FIG. 4 A-B).

[0147] The 12-bromo (Compound A11) and 1-bromo (Compound A9) derivatives were substantially more potent (over 17-fold and 6-fold, respectively) than chloroquine against the multidrug-resistant P. falciparum Dd2, underscoring their potential as lead compounds for antimalarial drug development. While neither outperformed the outstanding potency and selectivity of (-)-premarineosin A against Dd2, 12-bromo premarineosin A was twice as potent as 2 against the drug-sensitive P. falciparum 3D7 with improved selectivity relative to both HEK293 and MOLT4 mammalian cells (FIG. 5, Table B). As this potency also exceeds the efficacy of both artemisinin andDocket No. 30275 / 70841chloroquine in 3D7, these results suggest that 12-bromo premarineosin A (Compound A8) is a promising therapeutic candidate for the treatment of this parasite. 1 -Bromo premarineosin A also exhibited comparable potency to chloroquine against the drug-sensitive P. falciparum 3D7 and was less cytotoxic than (-)-premarineosin A against both cell lines, but had decreased selectivity overall (FIG. 5). The results disclosed herein suggest that modifications at the B-ring position have the potential to decrease cytotoxicity while maintaining the single-digit nanomolar potency of (-)-premarineosin A against P. falciparum. Interestingly, modification of B-ring in linear prodiginines also improved selectivity against malarial parasites.9

[0148] The instant study supports halogenation as a powerful tool in medicinal chemistry, offering opportunities to enhance drug efficacy, stability, and bioavailability.48Bromine's unique physicochemical properties, including its ability to modulate target binding affinity and metabolic stability, make it particularly valuable in antimalarial drug discovery.49The ability to regioselectively brominate (-)-premarineosin A opens the door to systematic C-H diversification of its structure, providing a platform for the development of derivatives with tailored bioactivity against Plasmodium species. The remarkable selectivity of 12-bromo-premarineosin A (Compound A11) against P. falciparum (FIG. 5) warrants further investigation into its mechanism of action and in vivo efficacy studies. By integrating strain engineering, biocatalysis, computational modeling, and semi-synthesis, disclosed herein is the foundation for the rational design of next-generation antimalarial agents, harnessing the full potential of the premarineosin A scaffold to address global health challenges.Example 3

[0149] Synthesis of Compounds A10-A13. Additional halogenated (-)-premarineosin A derivatives discussed herein were synthesized, structure-confirmed, and tested in both mammalian cell cytotoxicity and antiplasmodial assays.12-Chloro premarineosin (Compound A10)

[0150] To a solution of (-)-premarineosin A in chloroform at room temperature was added 2-Chloro-1 ,3-bis(methoxycarbonyl) guanidine (CBMG or "Palau'chlor”) (1.2 eq) drop wise. The mixture was stirred at room temperature. After 12 hours the reaction was quenched with sodium bicarbonate. Following a liquid-liquid extraction with DCM, the organic layer was extracted and dried. The material then was purified using flash column chromatography using basic conditions: 20-50% of a (3% ammonia (7N in methanol) in DCM solution) in hexane gradient. The resulting material was further purified by preparative HPLC with a phenyl hexyl column (5 pm, 100 A, 250 x 10 mm) using a gradient of 10-55% of acetonitrile and water, both modified with 0.1% formicDocket No. 30275 / 70841acid, over 50 minutes at a flow rate of 5 mL / min. LC-MS: calculated for C25H32CIN3O2 [M+H+] 442.2256 m / z; found 442.2242 m / z.

[0151] 12-Bromo premarineosin (Compound A11)

[0152] To a solution of (-)-premarineosin A (13.8mg, 5mmol) in dichloromethane at 4 °C was added N-Bromo succinimide (10 mg, Wmmol, DCM) drop wise. The mixture was stirred at 4 °C. After 2 hours the reaction was quenched with sodium bicarbonate. Following a liquid-liquid extraction with DCM, the organic layer was extracted and dried. The resulting material was further purified by preparative HPLC with a phenyl hexyl column (5 m, 100 A, 250 x 10 mm) using a gradient of 10-55% of acetonitrile and water, both modified with 0.1% formic acid, over 50 minutes at a flow rate of 5 mL / min. LC-MS: calculated for C25H32BrN3O2 [M+H+] 486.1756 m / z; found 486.1811 m / z.

[0153] 12-lodo premarineosin (Compound A12)

[0154] To a solution of 10mM (-)-premarineosin A in DMSO at room temperature was added excess ethyl acetate with 1% acetic acid and 2M Nal (to final concentrations of 0.02 mM (-)-premarineosin A, 4 mM Nal). The reaction was vortexed and incubated for 1 hours at room temperature, then dried. Following liquid-liquid extraction with ethyl acetate and water, the organic layer was further purified by preparative HPLC with a phenyl hexyl column (5 pm, 100 A, 250 x 10 mm) using a gradient of 10-55% of acetonitrile and water, both modified with 0.1% formic acid, over 50 minutes at a flow rate of 5 mL / min. LC-MS: calculated for C25H32IN3O2 [M+H+] 534.1612 m / z; found 534.1607 m / z.1 ,12-Dibromo premarineosin (Compound A13)Docket No. 30275 / 70841

[0155] To a solution of 10mM 1-bromo premarineosin A (Compound A11) in DMSO at room temperature was added excess ethyl acetate with 1% acetic acid and 2M NaBr (to final concentrations of 0.02 mM (-)-premarineosin A, 4 mM NaBr). The reaction was vortexed and incubated at room temperature, then dried.Following liquid-liquid extraction with ethyl acetate and water, the organic layer was further purified by preparative HPLC with a phenyl hexyl column (5 pm, 100 A, 250 x 10 mm) using a gradient of 10-55% of acetonitrile and water, both modified with 0.1% formic acid, over 50 minutes at a flow rate of 5 mL / min. LC-MS: calculated for C25H31Br2N3O2 [M+H+] 564.0856 m / z; found 564.0851 m / z.Example 4

[0156] Synthesis of Compound A15. An oct-7-ynoyl premarineosin derivative was also synthesized.

[0157] To a solution of premarineosin A in dichloromethane was added oct-7-ynoyl chloride and BFa'OEt?, and the solution was stirred at room temperature for 2.5 hours to yield oct-7-ynoyl premarineosin A, Compound A15.Example 5

[0158] Strains and culture conditions. All strains that were designed and / or cultured in this work are listed in Table C. Plasmid assembly, replication, and preservation were performed using high efficiency Escherichia coli DH5o (NEB). E. coli S17-1 was used as the mobilization host for conjugation with Streptomyces eitanensis. All E. coli strains were cultivated in LB medium (1 Og / L tryptone, 10 g / L NaCI, and 5 g / L yeast extract in ultrapure water) at 37 °C. The LB media was supplemented with kanamycin for plasmid maintenance and selection. The premarineosin-producing Streptomyces eitanensis strain was a gift from Fermentek. All Streptromyces strains were cultivated in 2xYT (16 g / L tryptone, 10 g / L yeast extract, and 5 g / L NaCI in ultrapure water) for three days at 28 °C for seed culture. Strains were sporulated in OPAH (1 g / L oatmeal, 1 g / L pharmamedia, 1 g / L arabinose, 0.5 g / L humic acid, 0.5 mM KH2PO4, 0.5 mM CaCh, 0.5 mM MgSO4, 1.9 mg / L Na2-EDTA'2H20, 1.4 mg / L FeSO4'7H2O, 0.2 mg / L H3BO3, 0.05 mg / L MnSO4'H2O, 0.01 mg / L ZnSO4'7H2O, 0.01 mg / L Na2MoO4'2H2O, 0.01 mg / L CUSO4, and 0.01 mg / L C0CI2 in ultrapure water). Media was supplemented with 50 pig / mL kanamycin (Goldbio, K-120-25) and / or 25 pig / mL nalidixic acid (Cayman, 19807) as needed.Docket No. 30275 / 70841Table C: Strains and Plasmids<

[0159] Plasmid design and genome editing. All strains and plasmids generated in this study are described in Tables C and D. The pmaD and pmaG genes were amplified from wildtype S. eitanensis gDNA, then assembled via Gibson Assembly in the plasmid pSET152k- asO*p between the BamHI and EcoRI restriction sites (New England Biolabs, R0136S and R0101S), as previously described in our concanamycin engineering work.22Synthetic ribosomal binding sites45were used as overlapping regions for multi-gene vector design. Primers were designed with guidance from pyDNA46(Table E). An integrative system, mediated by the attP site of the Streptomyces phage $C3143, was used for the transfer of DNA from E. coli S17144to S. eitanensis via interspecies conjugation. Following incubation (12 h, 28 °C), each plate was overlaid with 1 mL sterilized water containing 1.25 mg kanamycin and 0.5 mg nalidixic acid. After 3-5 days, recombinants were transferred to OPAH plates with 25 pg / mL nalidixic acid and 50 pg / mL kanamycin. The resulting antibiotic resistant strains were whole-genome sequenced. Cluster comparison and visualization were performed with clinker47and DNAviewer48.Table D: PlasmidsTable E: PrimersDocket No. 30275 / 70841

[0160] Genome extraction, sequencing, and assembly. High-quality genomic DNA was prepared using reagents from the MasterPure Complete DNA and RNA Purification Kit (Lucigen, MC85200). The wildtype and engineered Streptomyces strains were cultivated in 2xYT for three days at 28 °C. Cells were pelleted via centrifugation, resuspended in 480 piL EDTA (50 mM, pH 8, sterile filtered) and 120 piL of lysozyme (10 mg / mL, DotScientific DSL38100-10), and incubated for 35 min at 37 °C. Post-incubation, the cells were centrifuged (1 min, 17,000 x g, RT) and resuspended in 200 piL MasterPure Tissue and Cell Lysis Solution treated with 1 piL Proteinase K (Qiagen RP107B-5). Cells were incubated for 15 min at 65 °C, incubated for 2 min at 95 °C, then cooled to RT. 30 piL of RNase A (10 mg / mL, Sigma Aldrich R5503-1G) was added prior to incubation for 1.5 hr at 37 °C. The genomic DNA was precipitated following the MasterPure Kit Precipitation of Total Nucleic Acids protocol. Genomic DNA quality was assessed using gel electrophoresis. S. eitanensis bacterial genome sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation.

[0161] Premarineosin production conditions. Spores from fresh OPAH plates were inoculated into 50 mL of peanut meal & starch media (10 g / L glucose, 30 g / L starch, 5 g / L bacto peptone, 10 g / L peanut meal, 5 g / L yeast extract, 2 g / L CaCOs in ultrapure water, adjusted to pH=7.0) and incubated for three days at 28 °C and 200 rpm.10 mL of this pre-inoculum culture was used to inoculate 1 L of soybean-meal-based producing media (GISYE: 10 g / L glucose, 30 g / L inulin, 5 g / L bacto peptone, 10 g / L soybean meal, 5 g / L yeast extract, 2 g / L CaCOs in ultrapure water, adjusted to pH=7.0) in 2.8 L Fernbach flasks. Cultures were incubated for 7 days at 22 °C and 170 rpm.

[0162] Sample preparation and quantification of premarineosin. Quantification of premarineosin production in 1L cultures was performed using representative 1 mL biomass samples from three independent replicates. To estimate growth and production over time, biomass was collected and extracted every day for 7 days. Compound quantification refers to extracts collected on Day 7 unless stated otherwise. On each collection day, 1mL was collected from each 1L culture and the biomass was pelleted via centrifugation (4 min, 20800 rpm, RT). The cell pellet was resuspended in 1 mL of solvent (methanol or acetone) and 100 pL of glass beads. Supernatant was combined 50:50 with solvent and stored at -20 °C until quantification. Tubes were vortexed (2 hours, 4 °C), followed by centrifugation (4 min, 20800 rpm, RT). The supernatant was retained as the extract and was stored at -20 °C until quantification. Quantification of premarineosin production was performed using analytical HPLC (Shimadzu) equipped with a PDA detector analyzed with a Phenyl-hexyl column (Luna 5 uM Phenyl-Hexyl 100 A, LC Column 250 x 4.6mm, heated to 40 °C). Water + 0.1% Formic Acid (A) I Acetonitrile + 0.1% Formic Acid (B) (10% to 100% B) was used as mobile-phase at 2 ml / min. Calibration curves were preparedDocket No. 30275 / 70841with known concentrations of pure premarineosin and 23-HUP. Production titers were determined by comparing the calibration curve and sample peak areas (AUG) at 346 nm (premarineosin) and 520 nm (red compounds). Premarineosin and 23-HUP were detected in culture supernatants at trace levels for all tested strains. Therefore, quantifications reported in this document correspond to the intracellular concentration of premarineosin.Statistical analysis and visualization were performed with GraphPad Prism v.10.2.2.

[0163] Biomass quantification. Throughout cultivation, daily 1mL samples were collected from each of the three independent replicates and vacuum filtered through a pre-dried membrane filter. The retained cells were dried in a microwave oven for 1.5 min.50As S. eitanensis is known to form small, spherical pellets in liquid culture, sampling was performed using sterile wide-bore pipette tips and serological pipettes.

[0164] Isolation and purification of premarineosin. The total biomass (from 1 L culture) was separated via vacuum filtration. Cell pellets were broken by coating the cells with 750 mL of 100% acetone and then shaking overnight. The acetone was obtained by further vacuum filtration then concentrated. The dried extract was concentrated with silica to be dry loaded for normal phase purification on a Biotage Isolera flash column system. Initial rounds of purification utilized a 40 g silica column with an ethyl acetate / hexane gradient (15-100%) with 1% acetic acid; premarineosin eluted at 34-50%. Premarineosin peak(s) were further purified on a 40 g Basic Alumina column with a 3% Ammonia (7N) Methanol in DCM I hexane gradient (15-100%); premarineosin eluted at 24%. Purity assessment of isolated compounds was performed using HPLC, NMR, and LC-MS / MS. All samples for bioactivity testing were >95% pure.

[0165] NMR data of premarineosin.1H NMR (599 MHz, Acetone) 5 13.65 (s, 1H), 12.05 (s, 1H), 9.80 (s, 1H), 7.65 (ddd, J= 3.3, 2.3, 1.3 Hz, 1H), 7.45 (s, 1H), 6.50 (dt, J = 4.0, 2.1 Hz, 1H), 6.26 (d, J= 1.7 Hz, 1H), 5.72 (t, J = 2.9 Hz, 1H), 5.52 (t, J= 2.9 Hz, 1H), 4.46 -4.38 (m, 1H), 4.14 (s, 3H), 3.03 (d, J= 12.5 Hz, 1H), 2.69 (dp, J = 13.4, 4.9 Hz, 1H), 2.39 (dt, J= 14.8, 4.4 Hz, 1H), 2.28 (ddd, J = 14.8, 11.5, 3.3 Hz, 1H), 1.95 (dt, J= 13.8, 4.2 Hz, 1H), 1.92 - 1.83 (m, 2H), 1.70 (ddd, J= 14.6, 10.0, 5.2 Hz, 1H), 1.45 (d, J = 6.8 Hz, 3H), 1.36 (dtdd, J = 25.8, 18.0, 13.5, 7.1 Hz, 4H), 1.25- 1.17 (m, 1H), 1.11 - 1.01 (m, 2H), 0.89 -0.77 (m, 2H), 0.54 (p, J = 7.0 Hz, 1H).13C NMR (151 MHz, Acetone) 5 181.77 (d, J = 5.5 Hz), 165.87 (d, J = 22.6 Hz), 135.05, 134.18, 127.55, 125.09, 121.64, 114.62, 110.64, 105.99, 97.24, 93.84, 71.23 (d, J = 4.3 Hz), 60.92, 46.32, 38.27, 33.56, 28.70, 28.19, 27.26, 25.87, 25.23 (2 carbons), 24.82, 20.92 (d, J = 4.3 Hz). LC-MS: calculated for C25H33N3O2 [M+H+] 408.265 m / z; found 408.265 m / z.Example 6

[0166] Premarineosin derivatization general procedure. Premarineosin was dissolved in acetonitrile (MeCN) before trifluoroacetic acid (TFA) and a carbonyl-containing substrate was added at RT. Reaction was run until TLC (50% ethyl acetate in hexanes with 1% acetic acid) showed consumption of starting material (15 min - 6 h). After completion, the reaction was diluted with ethyl acetate and washed with 1M NaOH to quench the TFA. The aqueous layer was further extracted with ethyl acetate (x3) and organics were combined, dried over sodium sulfate, filtered, and concentrated. The material then was purified through a combination of acidic and / or basicDocket No. 30275 / 70841conditions. Acidic conditions: 50% ethyl acetate (with 1% acetic acid) and hexanes (with 1% acetic acid). Basic conditions: 20-50% of a (3% ammonia (7N in methanol) in DCM solution) in hexanes gradient.

[0167] D3 halogenase expression and purification. For large scale halogenase production, 10 mL culture tubes containing 5 mL LB with kanamycin were inoculated with BL-21(ADE3) E. coll cells harboring pET28b containing the D3 halogenase gene. Construction of the D3 halogenase expression plasmids was performed by the Jared Lewis group, Indiana University. The cultures were incubated overnight at 37 °C, 250 rpm. The next day, 750 mL TB with kanamycin was inoculated with the entire overnight culture. The inoculated expression cultures were incubated at 37 °C, 225 rpm until CD600 was between 0.6 and 0.8. The incubator was allowed to cool to 30 °C, and gene expression was induced with 100 piM IPTG, and the expression culture was incubated for 20 hours. Cells were harvested by centrifugation at 6000xg at 4°C for 15 min and the cell pellet was stored at -80 °C until purification of the protein. Cells were lysed via sonication with a total processing time of 5 minutes with 1 minute on / off cycles. Cell lysate was centrifugated (60,000 xg for 25 min), and the resulting clarified lysate was transferred to a fresh 50 mL centrifuge tube and added to pre-equilibrated Ni-NTA (equilibration buffer: 20 mM phosphate, 300 mM NaCI, 10 mM imidazole pH 7.4). Clarified lysate was incubated with resin for approximately 30 minutes at which point it was transferred to uncapped spin columns and the lysate was allowed to flow through. The resin was washed with at least 5 CV wash buffer (20 mM phosphate, 300 mM NaCI, 25 mM imidazole pH 7.4), at which point the spin column was transferred to a new centrifuge tube and the resin was washed with elution buffer (20 mM phosphate, 300 mM NaCI, 250 mM imidazole, pH 7.4). Eluted protein was concentrated via diafiltration using Amicon spin filters Ultra 30K MWCO spin filters and the buffer was exchanged for storage buffer (25 mM HEPES and 10% glycerol, pH 7.4). For long term storage, proteins were immediately frozen in liquid nitrogen and stored at - 80 °C until use.

[0168] HpaC Flavin Reductase Expression and Purification. For large scale HpaC production, 10 mL culture tubes containing 5 mL LB with ampicillin were inoculated with BL-21(ADE3) pRare E. coll cells harboring pET21 a containing the HpaC halogenase gene. The HpaC flavin reductase (phaC plasmid) was obtained from Prof. David Ballou (University of Michigan). The cultures were incubated overnight at 37 °C, 250 rpm. The next day, 750 mL TB with ampicillin was inoculated with the entire overnight culture. The inoculated expression cultures were incubated at 37 °C, 225 rpm until CD600 was between 0.6 and 0.8. The incubator was allowed to cool to 20 °C, and gene expression was induced with 100 piM IPTG, and the expression culture was incubated for 20 hours. Cells were harvested by centrifugation at 6000xg at 4°C for 15 min and the cell pellet was stored at -80 °C until purification of the protein. Cells were lysed via sonication with a total processing time of 5 minutes with 1 minute on / off cycles. Cell lysate was centrifugated (60,000 xg for 25 min), and the resulting clarified lysate was transferred to a fresh 50 mL centrifuge tube and added to pre-equilibrated Ni-NTA (equilibration buffer: 20 mM HEPES, 300 mM NaCI, 50 pM FAD, 10% glycerol pH 7.4). Clarified lysate was incubated with resin for approximately 30 minutes at which point it was transferred to uncapped spin columns and the lysate was allowed to flow through. The resin was washed with at least 5 CV wash buffer (20 mM HEPES, 300 mM NaCI, 50 mM imidazole, 10% glycerol pH 7.4), at which point the spin column was transferred to a new centrifuge tube and theDocket No. 30275 / 70841resin was washed with elution buffer (20 mM HEPES, 300 mM NaCI, 500 mM imidazole, 10% glycerol pH 7.4). Eluted protein was concentrated via diafiltration using Amicon spin filters Ultra 30K MWCO spin filters and the buffer was exchanged for storage buffer (20 mM HEPES, 100 mM NaCI, 10% glycerol pH 7.4). For long term storage, proteins were immediately frozen in liquid nitrogen and stored at - 80 °C until use.

[0169] Analytical scale D3 halogenase reaction conditions. Analytical reactions were performed using 20 pM D3, 45 pM HPAC flavin reductase, 100 pM FAD, 50 mM sodium bromide, and 400 pM of premarineosin dissolved in DMSO was added then diluted to a final volume of 250 pL with reaction buffer (10 mM HEPES, pH 7.4 containing 10% glycerol) and initiated by adding NADH (1 mM). Two control reactions were performed which included all contents except D3. Reactions were incubated at 30 °C for 4 h agitating at 600 rpm in a thermoshaker (Multithermoshaker, Benchmark) and quenched via addition 750 pL of methanol, followed by vortexing at full speed S3 for 30 s. Quenched reactions were centrifuged at 17,000 x gravity to remove insoluble material and supernatant was analyzed with chromatographic conditions identical to analytical reactions and were performed using an Agilent G6230B time-of-flight (TOF) mass spectrometer system operating in positive mode, monitoring a mass range of 200 to 1200 amu with ESI-MS, and UV (195-400 nm) detection. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 11 L / min and the first 1 min of flow was diverted to waste.

[0170] Preparatory scale D3 halogenase reaction conditions. D3 was expressed as previously reported, and conversion of premarineosin by D3 was performed under identical conditions as those used for analytical scale reactions with 500 pM of premarineosin and a final volume of 10 mL. The reaction was quenched via addition of 20 mL of HPLC-grade methanol, and vortexed on the highest setting for 1 minute. This mixture was then passed through a pad of Celite© and the filter cake was washed with an additional 20 mL of methanol. To purify the biocatalytic byproduct, the aqueous solution was first extracted with ethyl acetate (x3). The organics were combined, washed with brine, dried over sodium sulfate, filtered and concentrated. The material was then passed through a silica plug to remove residual flavin using 50% ethyl acetate (with 1% acetic acid) in hexanes. The material was then further purified by pTLC using a 80% (3% ammonia (7N in methanol) in DCM) in hexanes. The major top band was pure, ring A derivative (2 mg), while the major lower band was a mixture of premarineosin and ring-B brominated material. The impure ring B derivative was further purified by HPLC with a phenyl hexyl column (5 pm, 100 A, 250 x 10 mm). A gradient of 10-55% of acetonitrile and water, both modified with 0.1% formic acid, over 50 minutes at a flow rate of 5 mL / min.Table F: Sequence TableDocket No. 30275 / 70841Docket No. 30275 / 70841References:(1) Hu, D. X. et al. Chemical reviews 2016, 116 (14), 7818.Docket No. 30275 / 70841(2) Anwar, M. M.et al. Cancer Cell International 2022, 22 (1), 419.(3) Williamson, N. R. et al. Future Microbiol 2007, 2 (6), 605-618.(4) Klein, A. S. et al. ChemBioChem 2018, 19 (14), 154^1552.(5) Alihosseini, F. et al. Biotechnology Progress 2008, 24 (3), 742-747.(6) You, Z. et al. Appl Microbiol Biotechnol 2019, 103 (7), 2873-2887.(7) Kancharla, P. et al. J. Org. Chem. 2014, 79 (23), 11674-11689.(8) Kancharla, P. et al. Journal of medicinal chemistry 2015, 58 (18), 7286.(9) Kumar, A.; et al. J. Med. Chem. 2024, 67, 21, 19755-19776.(10) Papireddy, K. et al. J. Med. Chem. 2011, 54 (15), 5296-5306.(11) Salem, S. M. et al. J. Am. Chem. Soc. 2014, 136 (12), 4565-4574.(12) de Rond, T. et al. Nat Chem Biol 2017, 13 (11), 1155-1157.(13) Jones, B. T. et al. J. Nat. Prod. 2013, 76, 10, 1937-1945.(14) Boonlarppradab, C. et al. Org Lett 2008, 10 (24), 5505-5508.(15) Dereje, N. et al. Nat Med 2025, 1-2. https: / / doi.org / 10.1038 / s41591-024-03439-z(16) CDC. Malaria's Impact Worldwide. Malaria, https: / / www.cdc.gov / malaria / php / impact / index.html (accessed 2024-10-10).(17) Kancharla, P. et al. J. Med. Chem. 2024, 67 (10), 8323-8345.(18) Kancharla, P. et al. J. Med. Chem. 2021, 64 (12), 8739-8754.(19) Feng, Z. et al. J. Am. Chem. Soc. 2019, 141 (6), 2274-2278.(20) Xu, B. et al. Org. Lett. 2016, 18 (9), 2028-2031.(21) Pereira, F. et al. Metabolic Engineering 2025, 88, 63-76.(22) Zdouc, M. M. et al. Nucleic Acids Research 2025, 53 (D1), D678-D690.(23) Takano, E. et al. Mol Microbiol 1992, 6 (19), 2797-2804.(24) Malpartida, F. et al. Gene 1990, 93 (1), 91-99.(25) Malpartida, F. et al. Nature 1984, 309 (5967), 462-464.(26) Ferraiuolo, S. B. et al. Applied Microbiology and Biotechnology 2021, 105 (2), 551.(27) Evangelista-Martinez, Z. et al. Revista especializada en ciencias quimico-bioldgicas 2022, 25. (28) Stankovic, N. et al. Appl Microbiol Biotechnol 2012, 96 (5), 1217-1231.(29) Gilchrist, C. L. M. et al. Bioinformatics 2021, 37 (16), 2473-2475.(30) Barnes, E. C. et al. Nat. Prod. Rep. 2016, 33 (3), 372-381.(31) Majhi, S. et al. Tetrahedron 2021, 78, 131801.(32) Camp, D. et al. Future Medicinal Chemistry 2012, 4 (9), 1067-1084.(33) Camp, D. et al. J Nat Prod 2015, 78 (6), 1370-1382.(34) Pascolutti, M. et al. Drug Discovery Today 2014, 19 (3), 215-221.(35) White, J. D. Chem. Commun. (London) 1966, No. 20, 711-712.(36) Sleebs, B. E. et al. J. Med. Chem. 2014, 57 (18), 7644-7662.(37) Jiang, X. et al. Cell 2020, 183 (1), 258-268.e12.(38) Lewis, J. C.. Acc. Chem. Res. 2024, 57 (15), 2067-2079.(39) Yeh, E. et al. Proceedings of the National Academy of Sciences 2005, 102 (11), 3960-3965. (40) Tala, A. et al. Metabolic Engineering 2018, 48, 254-268.(41) Fayed, B. et al. BMC Biotechnology 2014, 14 (1), 51.(42) Hunjan, M. K. et al. The Chemical Record 2021, 21 (4), 715-780.(43) Tsuchimoto, T. Chemistry - A European Journal 2011, 17 (15), 4064-4075.(44) Jenkins, S. et al. CrystEngComm 2009, 11 (2), 242-245.(45) Regourd, J. et al. J. Med. Chem. 2007, 50 (7), 1528-1536.(46) Rastogi, S. et al. Org Biomol Chem 2013, 11 (23), 3834-3845.(47) Peh, G. et al. Commun Chem 2024, 7 (1), 1—9.(48) Mendez, L. et al. Molecules 2017, 22 (9), 1397.(49) Wilcken, R. et al. J. Med. Chem. 2013, 56 (4), 1363-1388.(50) Borodina, I. et al. Journal of Biological Chemistry 2008, 283 (37), 25186-25199.

Claims

Docket No. 30275 / 70841What is claimed is:

1. A compound, or pharmaceutically acceptable salt thereof, having a structure of formula I:whereinX1is N or N+-O-;each RNis independently H, OH, Ci-ealkyl, C^alkenyl, or C(O)-Ci-3alkyl;R1is halo, H, OH, OHO, C(O)-Ci-3alkyl, C(O)-Ci-3haloalkyl, C1-6 alkyl optionally substituted with 1-3 R4, C2-6alkenyl, or NO2;R2and R3are independently H, OH, halo, C^alkenyl, or NO2; andeach R4is independently OH or Ci-shaloalkyl,with the proviso that at least one of R1, R2, and R3is not H.

2. The compound or salt of claim 1, wherein R1is H, OH, OHO, C(O)-Ci-3alkyl, C(O)-Ci-3haloalkyl, C1-6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2.

3. The compound or salt of claim 1 or o2, wherein R1is H, OH, OHO, C(O)-Ci-3alkyl, C1-6 alkyl optionally substituted with 1-3 R4, C^alkenyl, or NO2.

4. The compound or salt of claim 1 , wherein R1is halo.

5. The compound or salt of claim 4, wherein R1is F.

6. The compound or salt of claim 4, wherein R1is Cl, Br, or I.

7. The compound or salt of claim 6, wherein R1is Cl.

8. The compound or salt of claim 6, wherein R1is Br.

9. The compound or salt of claim 6, wherein R1is I.

10. The compound or salt of any one of claims 1 to 3, wherein R1is H.

11. The compound or salt of claim 1 or 2, wherein R1is C(O)CF3.

12. The compound or salt of any one of claims 1 to 3, wherein R1is OHO or C(O)CH3.

13. The compound or salt of any one of claims 1 to 3, wherein R1is Ci-ealkyl optionally substituted with 1 to 3 R4.

14. The compound or salt of claim 13, wherein R1is unsubstituted Ci-ealkyl.

15. The compound or salt of claim 13, wherein R1is Ci-ealkyl substituted with 1-3 R4.

16. The compound or salt of claim 15, wherein each R4is independently OH or CF3.Docket No. 30275 / 7084118. A compound, or pharmaceutically acceptable salt thereof, having a structure of formula II:whereineach of X1and X2is independently N or N+-O;each RNis independently H, OH, Ci-ealkyl, C2-6 alkenyl, or C(O)-Ci-3alkyl;L is a linker; andeach of R2, R2', R3, and R3' is independently H or halo.

19. The compound or salt of any one of claims 1 to 18, wherein X1is N.

20. The compound or salt of any one of claims 1 to 19, wherein X2is N.

21. The compound or salt of any one of claims 1 to 20, wherein at least one RNis H.

22. The compound or salt of any one of claims 1 to 21 , wherein each RNis H.

23. The compound or salt of any one of claims 1 to 22, wherein R2is H.

24. The compound or salt of any one of claims 1 to 22, wherein R2is halo.

25. The compound or salt of claim 24, wherein R2is Br.

26. The compound or salt of any one of claims 1 to 25, wherein R3is H.

27. The compound or salt of any one of claims 1 to 25, wherein R3is halo.

28. The compound or salt of claim 27, wherein R3is Br.

29. The compound or salt of any one of claims 18 to 28, wherein R2' is H.

30. The compound or salt of any one of claims 18 to 28, wherein R2' is halo.

31. The compound or salt of claim 30, wherein R2' is Br.

32. The compound or salt of any one of claims 18 to 31, wherein R3' is H.

33. The compound or salt of any one of claims 18 to 31, wherein R3' is halo.

34. The compound or salt of claim 33, wherein R3' is Br.

35. The compound or salt of any one of claims 18 to 34, wherein L is optionally substituted Ci. ealkylene.

36. The compound or salt of claim 35, wherein L is Chalky lene or Ci-shaloalky lene.VCF337. The compound or salt of claim 35 or 36, wherein L is, ' / ,or.

38. A compound or salt having a structure as shown in Table A.

39. A pharmaceutical composition comprising the compound or salt of any one of claims 1 to 38 and a pharmaceutically-acceptable excipient.Docket No. 30275 / 7084140. A method for producing a semi-synthetic derivative of premarineosin or a pharmaceutically acceptable salt thereof, comprising contacting premarineosin or a derivative thereof with an acid catalyst, a halogenase, or both, under conditions suitable for carrying out an electrophilic aromatic substitution reaction on one or more pyrrole rings of the premarineosin or derivative thereof.

41. The method of claim 40, wherein the electrophilic aromatic substitution reaction comprises an acylation reaction, a condensation reaction, a halogenation reaction, or a combination thereof.

42. The method of claim 40 or 41 , comprising contacting premarineosin or a derivative thereof with an acid catalyst.

43. The method of any one of claims 40 to 42, wherein the acid catalyst comprises trifluoroacetic acid.

44. The method of any one of claims 41 to 43, wherein the acylation or condensation reaction further comprises contacting the premarineosin or derivative thereof with a ketone or aldehyde reagent.

45. The method of claim 44, wherein the ketone or aldehyde reagent is acetone, trifluoroacetone, or formaldehyde.

46. The method of claim 44 or 45, wherein the reaction is an acylation reaction.

47. The method of claim 46, wherein the acylation reaction is stopped before complete consumption of the premarineosin or derivative thereof.

48. The method of claim 46 or 47, further comprising a reduction step.

49. The method of claim 48, wherein the reduction step comprises reducing a carbonyl group of the semi-synthetic derivative of premarineosin or salt thereof.

50. The method of claim 44 or 45, wherein the reaction is a condensation reaction.

51. The method of claim 40 or 41 , wherein the reaction is a halogenation reaction.

52. The method of claim 51 , comprising contacting the premarineosin or derivative thereof with a halogenase.

53. The method of claim 51 or 52, comprising contacting the premarineosin or derivative thereof with an indole halogenase.

54. The method of claim 53, wherein the indole halogenase is a flavin-dependent halogenase (“FDH”).

55. The method of claim 54, wherein the flavin-dependent halogenase ("FDH”) is FDH 4V or FDH D3.

56. The method of any one of claims 40 to 55, wherein the semi-synthetic derivative of premarineosin or pharmaceutically acceptable salt thereof is the compound or salt of any one of claims 1 to 38.

57. A method of inhibiting a Plasmodium falciparum parasite, comprising contacting the parasite with the compound or salt of any one of claims 1 to 38 or the pharmaceutical composition of claim 39.

58. The method of claim 57, wherein the parasite is a drug-resistant or drug-sensitive Plasmodium falciparum parasite.Docket No. 30275 / 7084159. The method of claim 57 or 58, wherein the parasite is a Dd2 or 3D7 Plasmodium falciparum parasite.

60. A method of treating or preventing malaria in a patient, comprising administering to the patient a therapeutically effective amount of the compound or salt of any one of claims 1 to 38 or the pharmaceutical composition of claim 39.

61. The method of claim 60, wherein malaria is caused by a drug-resistant or a drug-sensitive Plasmodium.

62. The method of claim 60 or 61 , wherein malaria is caused by a Dd2 or 3D7 Plasmodium falciparum parasite.

63. An engineered Streptomyces bacteria having increased expression of a PmaD protein, wherein the engineered Streptomyces bacteria produces high levels of premarineosin.

64. An engineered Streptomyces bacteria having increased expression of a PmaD protein and a PmaG protein, wherein the engineered Streptomyces bacteria produces high levels of premarineosin.

65. The engineered Streptomyces bacteria of claim 63 or 64, wherein the PmaD protein comprises:I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1;ii) the amino acid sequence of SEQ ID NO: 1;ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3; oriv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 3.

66. The engineered Streptomyces bacteria of claim 63 or 64, wherein the bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the nucleotide sequence of SEQ ID NO: 3, operably linked to a constitutive promoter.

67. The engineered Streptomyces bacteria of any one of claims 64 to 66, wherein the PmaG protein comprises:I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2;ii) the amino acid sequence of SEQ ID NO: 2;ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4; oriv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 4.

68. The engineered Streptomyces bacteria of claim 64, wherein the bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4, operably linked to a constitutive promoter.Docket No. 30275 / 7084169. The engineered Streptomyces bacteria of any one of claims 64 to 68, wherein the PmaD protein comprises the amino acid sequence of SEQ ID NO: 1, and the PmaG protein comprises the amino acid sequence of SEQ ID NO: 2.

70. The engineered Streptomyces bacteria of any one of claims 64 to 69, wherein the PmaD protein is encoded by the nucleotide sequence of SEQ ID NO: 3, and the PmaG protein is encoded by the nucleotide sequence of SEQ ID NO: 4.

71. A culture comprising the engineered Streptomyces bacteria of any one of claims 63 to 70.

72. The engineered Streptomyces bacteria of any one of claims 63 to 71, wherein the bacteria is Streptomyces eitanensis, Streptomyces sp. CNQ617, Streptomyces smyrnaeus, Streptomyces sp. RK75, Streptomyces spirodelae, Streptomyces diacarni, Streptomyces tubbatahanensis, Streptomyces sp. JV178, Streptomyces NPDC008159, Streptomyces scabiei, Streptomyces sp. 11x1, Streptomyces sp. MNU76, or Streptomyces roseoverticillatus.

73. A plasmid comprising a nucleotide sequence encoding a PmaD protein operably linked to a constitutive promoter.

74. A plasmid comprising I) a nucleotide sequence encoding a PmaD protein and ii) a nucleotide sequence encoding a PmaG protein, wherein the nucleotide sequences are operably linked to a constitutive promoter.

75. The plasmid of claim 73 or 74, wherein the nucleotide sequence encoding the PmaD protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the sequence of SEQ ID NO: 3.

76. The plasmid of claim 73 or 74, wherein the PmaD protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1.

77. The plasmid of claim 74, wherein the nucleotide sequence encoding the PmaG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4.

78. The plasmid of claim 74, wherein the PmaG protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2.

79. The plasmid of any one of claims 73 to 78, wherein the constitutive promoter is KasO*p.

80. The plasmid of claim 79, wherein the constitutive promoter is SEQ ID NO: 5.

81. A method of engineering a bacteria comprising contacting a Streptomyces bacteria with the plasmid of any one of claims 73 to 80.

82. A method of producing premarineosin comprising culturing the engineered Streptomyces bacteria of any one of claims 63 to 70.

83. The method of claim 72, wherein the culture comprises soybean meal as the complex protein source in media.Docket No. 30275 / 7084174. The method of claim 73, wherein the method further comprises extraction and isolation of premarineosin using 25% methanol in dichloromethane.

85. The method of claim 83, wherein the method further comprises extraction and isolation of premarineosin using acetone.

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