Systems and methods for detecting and / or screening protein aggregation and / or disaggregation

Abstract

The present disclosure provides systems and methods for screening and measuring aggregation and / or disaggregation of an aggregable biomolecule. In one embodiment, a protein aggregation platform disclosed herein uses intact and split TEM-1 β-lactamase proteins as reporters of protein aggregation. The combination of the intact and split lactamase constructs allows the distinction of aggregation modulators targeting oligomerization from those targeting overall aggregation. The low-cost bacterial cell-based and biochemical assays disclosed herein are suitable for high-throughput screening of aggregation inhibitors targeting oligomers of various amyloid proteins.

Description

P-620504-PC SYSTEMS AND METHODS FOR DETECTING AND / OR SCREENING PROTEIN AGGREGATION AND / OR DISAGGREGATION CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 424,046, filed November 9, 2022, which is incorporated herein by reference in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant Number AG050687 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD

[0003] The present disclosure relates to systems and methods for detecting and screening biomolecules. BACKGROUND

[0004] The deposition of aggregated proteins such as amyloid fibrils is a primary pathological feature in a wide variety of over 50 mammalian pathologies, including, e.g., Alzheimer’s disease and type II diabetes. In addition to forming aqueous-insoluble fibrils, there are two other species of amyloid protein: monomers, and aqueous-soluble oligomers. The oligomers are believed to be more toxic and more pathogenic than insoluble fibrils.

[0005] Lately there has been growing research interest in soluble amyloid oligomers as a cause of disease. In practice, however, it is challenging to screen specifically for presence and / or activity of soluble amyloids, separate from the insoluble fibrils. An effective screening platform must be capable of distinguishing and quantifying monomers, fibrils, and oligomers. Further, a screening platform should be able to measure and distinguish changes in a particular amyloid protein species. The most pressing unmet need is for a screening platform that can specifically monitor the changes in soluble amyloid-β (Aβ) oligomers, for example in in vitro cell-based screening assays.

[0006] Faced with this unmet need, the present disclosure provides new and surprising methods and systems for screening and monitoring aggregation and disaggregation of proteins.P-620504-PC SUMMARY

[0007] The present disclosure relates to systems and methods for detecting and / or screening aggregation and disaggregation of an aggregable biomolecule of interest.

[0008] In an aspect, a system is provided for monitoring oligomerization or fibrillization of oligomers of an aggregable biomolecule, the system comprising: a. a first fusion biomolecule comprising the biomolecule of interest bound to an N- terminus portion of an enzyme, said first fusion biomolecule having no enzyme activity; and b. a second fusion biomolecule comprising the biomolecule of interest bound to a C- terminus portion of an enzyme, said second fusion biomolecule having no enzyme activity; wherein an oligomerization of the biomolecule of interest correlates with a first measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule.

[0009] In an embodiment, oligomerization of the biomolecule correlates with an increase in enzymatic activity, and fibrillization of oligomers correlates with a reduction in enzymatic activity. In an embodiment, an inhibitor of oligomerization inhibits the increase in enzymatic activity. In an embodiment, an accelerator of fibrillization of oligomers decreases enzymatic activity. In an embodiment, an agent that disaggregates oligomers and fibrils to monomers decreases activity. In an embodiment, an agent that aggregates monomers to oligomers increases activity. In an embodiment, an agent that disaggregates fibrils to oligomers increases activity.

[0010] In an aspect, the forgoing system further comprises a third fusion biomolecule comprising the biomolecule of interest bound to an enzyme comprising a full-length enzyme or homolog thereof, wherein an oligomerization of the biomolecule of interest correlates with first measurable change in enzymatic activity of third fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the third fusion biomolecule.P-620504-PC

[0011] In an embodiment, the third fusion biomolecule comprises full-length enzyme or homolog thereof.

[0012] In an embodiment, oligomerization of the biomolecule correlates with no change in enzymatic activity of the third fusion biomolecule, and fibrillization of oligomers correlates with a reduction in enzymatic activity of the third fusion biomolecule. In an embodiment, an inhibitor of oligomerization does not change the enzymatic activity of the third fusion biomolecule. In an embodiment, an accelerator of fibrillization of oligomers decreases activity of the third fusion biomolecule.

[0013] In an embodiment, an inhibitor of oligomerization that does not accelerate fibrillization inhibits the increase in enzymatic activity of the first and second fusion biomolecules and does not decrease enzymatic activity of the third fusion biomolecule. In an embodiment, an agent that disaggregates oligomer to monomers decreases activity of the first and second fusion biomolecules and does not change the enzymatic activity of the third fusion biomolecule. In an embodiment, an agent that disaggregates fibrils to monomers does not change the activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule. In an embodiment, an agent that aggregates monomers to oligomers increases activity of the first and second fusion biomolecules and does not change activity of the third fusion biomolecule. In an embodiment, an agent that disaggregate fibrils to oligomers increases activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.

[0014] In an embodiment or any of the foregoing, the aggregable biomolecule is an aggregable protein. In an embodiment, the aggregable protein is an amyloid protein. In an embodiment, aggregation of the amyloid protein forms aqueous-soluble oligomers or aqueous-insoluble fibrils. In an embodiment, the amyloid protein is wild-type Aβ42 or F19D mutant Aβ42.

[0015] In an embodiment, the enzyme is a β-lactamase.

[0016] In an embodiment, the enzymatic activity is measurable via a growth rate of a non- antibiotic-resistant Gram-negative bacteria grown in antibiotic-containing growth media. In an embodiment, the antibiotic is ampicillin.

[0017] In an embodiment, the enzymatic activity is measurable via β-lactamase activity.

[0018] In an embodiment, the β-lactamase is a TEM-1 β-lactamase.P-620504-PC

[0019] In an embodiment, the first fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:39 and SEQ ID NO: 41; and the second fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO:8, SEQ ID NO:13, and SEQ ID NO:43.

[0020] In an embodiment, the first fusion biomolecule is a polypeptide consisting of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:39 or SEQ ID NO: 41; and the second fusion biomolecule is a polypeptide consisting of: SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:43.

[0021] In an embodiment, the third fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; and SEQ ID NO:37.

[0022] In an embodiment, the third fusion biomolecule is a polypeptide consisting of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; or SEQ ID NO:37.

[0023] In an aspect, a method is provided for screening oligomerization or fibrillization of oligomers of an aggregable biomolecule, the method comprising the steps of: providing a cell culture in a culture medium, the cells producing: a. a first fusion biomolecule comprising the biomolecule of interest bound to an N-terminus portion of an enzyme, said first fusion biomolecule having no enzyme activity; and b. a second fusion biomolecule comprising the biomolecule of interest bound to a C-terminus portion of an enzyme, said second fusion biomolecule having no enzyme activity; measuring the activity of the enzyme, wherein an oligomerization of the biomolecule of interest correlates with a first measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule.P-620504-PC

[0024] In an embodiment, oligomerization of the biomolecule correlates with an increase in enzymatic activity, and fibrillization of oligomers correlates with a reduction in enzymatic activity. In an embodiment, an inhibitor of oligomerization inhibits the increase in enzymatic activity. In an embodiment, an accelerator of fibrillization of oligomers decreases enzymatic activity. In an embodiment, an agent that disaggregates oligomers and fibrils to monomers decreases activity. In an embodiment, an agent that aggregates monomers to oligomers increases activity. In an embodiment, an agent that disaggregates fibrils to oligomers increases activity.

[0025] In an embodiment, the above method further comprises the steps of: a. providing a cell culture in a culture medium, the cells producing a third fusion biomolecule comprising the biomolecule of interest bound to an enzyme comprising a full-length enzyme or homolog thereof, and b. measuring the activity of third fusion biomolecule, wherein an oligomerization of the biomolecule of interest correlates with first measurable change in enzymatic activity of third fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the third fusion biomolecule.

[0026] In an embodiment, the third fusion biomolecule comprises full-length enzyme or homolog thereof.

[0027] In an embodiment, oligomerization of the biomolecule correlates with no change in enzymatic activity of the third fusion biomolecule, and fibrillization of oligomers correlates with a reduction in enzymatic activity of the third fusion biomolecule. In an embodiment, an inhibitor of oligomerization does not change the enzymatic activity of the third fusion biomolecule. In an embodiment, an accelerator of fibrillization of oligomers decreases activity of the third fusion biomolecule. In an embodiment, an inhibitor of oligomerization that does not accelerate fibrillization inhibits the increase in enzymatic activity of the first and second fusion biomolecules and does not decrease activity of the third fusion biomolecule. In an embodiment, an agent that disaggregates oligomer to monomers decreases activity of the first and second fusion biomolecules and does not change the enzymatic activity of the third fusion biomolecule. In an embodiment, an agent that disaggregates fibrils to monomers does not change the activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule. In an embodiment, an agent that aggregates monomers to oligomersP-620504-PC increases activity of the first and second fusion biomolecules and does not change activity of the third fusion biomolecule. In an embodiment, an agent that disaggregate fibrils to oligomers increases activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.

[0028] In an embodiment, the aggregable biomolecule is an aggregable protein. In an embodiment, the aggregable protein is an amyloid protein. In an embodiment, aggregation of the amyloid protein forms aqueous-soluble oligomers or aqueous-insoluble fibrils. In an embodiment, the amyloid protein is wild-type Aβ42 or F19D mutant Aβ42.

[0029] In an embodiment, the enzyme is a β-lactamase.

[0030] In an embodiment, the cultured cells are non-antibiotic resistant Gram-negative bacteria, and the growth media contains a β-lactam-containing antibiotic. In an embodiment, the antibiotic is ampicillin.

[0031] In an embodiment, the enzymatic activity is measurable via a growth rate of the cells.

[0032] In an embodiment, the enzymatic activity is measurable via β-lactamase activity of whole-cell lysate.

[0033] In an embodiment, the β-lactamase is a TEM-1 β-lactamase.

[0034] In an embodiment, the first fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:39 and SEQ ID NO:41; and the second fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO:8, SEQ ID NO:13, and SEQ ID NO:43.

[0035] In an embodiment, the first fusion biomolecule is a polypeptide consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:39 or SEQ ID NO:41; and the second fusion biomolecule is a polypeptide consisting of: SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:43.

[0036] In an embodiment, the third fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; and SEQ ID NO:37.

[0037] In an embodiment, the third fusion biomolecule is a polypeptide consisting of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; or SEQ ID NO:37.P-620504-PC

[0038] In an aspect, a polypeptide is provided comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:39 or SEQ ID NO:41.

[0039] In an aspect, a polypeptide is provided consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:11; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:39 or SEQ ID NO:41.

[0040] In an aspect, a polypeptide is provided comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:43.

[0041] In an aspect, a polypeptide is provided consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:43.

[0042] In an aspect, a polypeptide composition is provided comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; or SEQ ID NO:37.

[0043] In an aspect, a polypeptide composition is provided consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO:5; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:15; or SEQ ID NO:37.

[0044] In an aspect, an expression vector or plasmid is provided encoding any of the foregoing.

[0045] In an aspect, an expression vector or plasmid is provided comprising a nucleic acid sequence of any one of SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO: 40 or SEQ ID NO:42.

[0046] In an aspect, an expression vector or plasmid is provided comprising a nucleic acid sequence of any one of SEQ ID NO:26, SEQ ID NO:30 or SEQ ID NO:44.

[0047] In an aspect, an expression vector or plasmid is provided comprising a nucleic acid sequence of any one of SEQ ID NO:25; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32 or SEQ ID NO:38.P-620504-PC BRIEF DESCRIPTION OF THE FIGURES

[0048] Some embodiments of the disclosure are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the disclosure. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the disclosure may be practiced.

[0049] Figs. 1A–1B depict the design of intact and split β-lactamase constructs to study Aβ oligomer and fibril formation. Fig. 1A: Fusion protein construct of Aβ and intact β-lactamase (BLA). For the intact β-lactamase construct, lactamase activity exists in both monomers and oligomers. Fig. 1B: Fusion protein constructs of Aβ and split β-lactamase. For the split construct, lactamase activity exists only in oligomers, not monomers or fibrils..

[0050] Figs. 2A–2C show growth curve studies of the intact β-lactamase constructs. Fig.2A: Fusion with Aβ42 wild-type. Fig. 2B: Fusion with the Aβ42 F19D mutant. E. coli cells were grown at 37°C in the presence of 0.2 mM IPTG unless indicated otherwise. Fig. 2C: Plot of growth recovery time extracted from growth curves as a function of the ampicillin concentration.

[0051] Figs.3A–3B show growth curve studies of the intact β-lactamase construct containing the M180T mutation. Fig.3A: Aβ42 wild-type. Fig.3B: Aβ42 with the F19D mutation. E. coli cells were grown at 37°C in the presence of 0.2 mM IPTG and various concentrations of ampicillin as indicated.

[0052] Figs. 4A–4D show growth curve studies of the split β-lactamase constructs. Four different β-lactamase constructs, wild-type (Fig. 4A), insertion of tripeptide NGR (Fig. 4B), M180T (Fig.4C), and the combination of M18T and NGR (Fig.4D), were used to express the fusion proteins with either wild-type Aβ42 or the F19D mutant. E. coli cells were grown at 37°C in the presence of 0.2 mM IPTG and various concentrations of ampicillin as indicated.

[0053] Figs.5A–5D show only soluble fractions of the bacterial cell lysate contain β-lactamase activity. Figs 5A-5B: Measurement of β-lactamase activity in E. coli cells expressing the intact (Fig. 5A) or split (Fig. 5B) lactamase-Aβ42. Whole cell lysate was centrifuged to separate supernatant from pellet. The supernatant was then filtered through a 0.2 µm ultrafiltration filter to obtain the 0.2 µm filtrate. Nitrocefin was used as the substrate and the activity was measured by monitoring absorbance at 492 nm. The β-lactamase activity was represented as theP-620504-PC hydrolysis rate of nitrocefin. Figs 5C-5D: For both the intact (Fig. 5C) and split (Fig. 5D) lactamase constructs, there are no significant differences between whole cell lysate, supernatant, and 0.2 µm filtrate, suggesting that the lactamase activity is present only in the soluble fraction of the cell lysate. Mean and standard deviation calculated from three technical repeats are shown.

[0054] Figs. 6A-6C show distribution of β-lactamase activity in bacterial cell lysate fractionated using size exclusion chromatography. Cell lysate of the split lactamase-Aβ42 (Fig. 6A), intact lactamase-Aβ42 (Fig. 6B), and split lactamase without Aβ (Fig. 6C) was run through an ENrich SEC 650 column, which has a separation size range of 5-650 kD. Fractions were collected between elution volumes of 7 and 20 mL. Lactamase activities were determined for each of these fractions in triplicate. Mean and standard deviations are plotted as a function of elution volume. Arrowheads and vertical lines indicate the peak positions of gel filtration standards. Notably, the split lactamase-Aβ construct shows a significant activity peak corresponding Aβ oligomers of 28 subunits (Fig. 6A), while the oligomer peak contributes minimally to the total lactamase activity in the intact lactamase-Aβ lysate (Fig.6B). The split lactamase without Aβ does not show the oligomer peak (Fig.6C), suggesting that the formation of lactamase-Aβ oligomers is not driven by the aggregation of the split lactamase

[0055] Fig. 7 shows the DNA (SEQ ID NO:38) and protein (SEQ ID NO: 37) sequences of BLA-Aβ42. The DNA sequence shown is located on the pET28b plasmid between NcoI and XhoI sites. Residue M180 is indicated with an arrow. BLA protein is 286-residue long, but residues 2-23 of the BLA protein are not included in the construct.

[0056] Fig. 8 shows the DNA (SEQ ID NO: 40) and protein (SEQ ID NO: 39) sequences of NBLA-Aβ42. The DNA sequence shown here is located on the pET28b plasmid between NcoI and XhoI sites. NBLA contains residues 24-195 of the BLA protein.

[0057] Fig. 9 shows the DNA (SEQ ID NO: 42) and protein (SEQ ID NO: 41) sequences of NBLA-Aβ42 containing the NGR tripeptide insertion. The DNA sequence shown here is located on the pET28b plasmid between NcoI and XhoI sites. NBLA contains residues 24-195 of the BLA protein. NGR tripeptide is inserted after residue 196 of the BLA protein.

[0058] Fig.10 shows the DNA (SEQ ID NO: 44) and protein (SEQ ID NO: 43) sequences of CBLA-Aβ42. The DNA sequence shown here is located on the pCDFDuet-1 plasmid between NcoI and HindIII sites. CBLA contains residues 196-286 of the BLA protein.P-620504-PC

[0059] Fig. 11 shows the DNA (SEQ ID NO: 46) and protein (SEQ ID NO: 45) sequences of NBLA-M180T. The DNA sequence shown here is located on the pET28b plasmid.

[0060] Fig. 12 shows the DNA (SEQ ID NO: 48) and protein (SEQ ID NO: 47) sequences of CBLA. The DNA sequence shown here is located on the pCDFDuet-1 plasmid.

[0061] Figs.13A-13B show the calibration of the ENrich SEC 650 size exclusion column. Fig. 13A: Gel filtration standard containing thyroglobulin (670 kD), γ-globulin (158 kD), ovalbumin (44 kD), myoglobin (17 kD), and vitamin B12 (1.35 kD) was run at 1 mL / min in PBS buffer (50 mM phosphate, 140 mM NaCl, pH 7.4). Fig.13B: Standard curve using a linear fit to the log molecular mass of the gel filtration standard (excluding vitamin B12) versus elution volume. The molecular mass of the two activity peaks for the split lactamase-Aβ42 cell lysate was estimated to be 600 and 22 kD. DETAILED DESCRIPTION

[0062] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure: Singleton, et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger, et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0063] Amyloid protein Aβ is also referred to herein as Aβ42, or Aβ1-42, or Aβ.

[0064] The deposition of aggregated biomolecules in the form of amyloid fibrils is a principal pathological feature in a wide range of mammalian disorders, including Alzheimer's disease and Type II diabetes. However, some aggregable biomolecules that form insoluble aggregate fibrils may also form smaller soluble oligomers.

[0065] In certain research and clinical contexts, it may be of great importance to detect and measure the difference and changes in aggregated form and disaggregated form of such aggregable biomolecules. For example, presently, it is challenging to screen for inhibitors of the formation of oligomeric form of β-amyloid. The most pressing knowledge gap is the unavailability of a screening tool that can specifically monitor changes in amyloid-β aggregation, between the monomeric form, oligomeric form and fibril form.P-620504-PC

[0066] In the aggregation process of Aβ and other amyloid proteins, there are three main aggregable / aggregated species: monomers, soluble aggregates (mostly oligomers), and insoluble aggregates (mostly fibrils). Oligomers are believed to be more toxic and more pathogenic than insoluble fibrils. The toxicity of amyloid oligomers can be reduced by either disaggregation to monomers, which are non-toxic and can be cleared by protein degradation machineries in the cell, or by further aggregation to form fibrils. The fibrils themselves are generally believed to be non-toxic, but they catalyze the formation of toxic oligomers by acting as seeds of aggregation. Because monomers, oligomers, and fibrils are in equilibrium, fibrils are also reservoirs of amyloid monomers and oligomers. A halo of oligomers is often found surrounding the amyloid plaques of Alzheimer’s disease in vivo. Therefore, disaggregation of both oligomers and fibrils to monomers is the preferred avenue of therapeutic intervention. An effective screening platform needs to distinguish among these three aggregable / aggregated species and distinguish the changes in a particular species. A pressing knowledge gap is a screening tool that can specifically monitor the changes in Aβ oligomers, such as for identifying agents that can interfere with Aβ oligomerization in vivo, inducing disaggregation of oligomers, or disaggregation of fibrils to monomers, or any combination thereof, wherein such agents are potentially useful to treat and / or prevent any of the Aβ-related diseases known or described herein. For the purposes disclosed herein, such species are referred to as monomers, oligomers and fibrils, the latter two formed by oligomerization and fibrillization, respectively. Such nomenclature is equally applicable to other monomers of potential pathobiological importance wherein oligomerization and / or fibrillization thereof is responsible for a pathological process.

[0067] Faced with this unmet need, the present disclosure relates to systems and methods for detecting and / or screening aggregation and disaggregation of an aggregable biomolecule of interest. This is achieved through the use of a split enzyme construct, based on the principle of a protein fragment complementation assay (PCA). The system may further include an intact enzyme construct.

[0068] To untangle oligomerization from fibrillization for Aβ, disclosed herein is a protein aggregation reporter system utilizing Aβ42 fused to each of the two halves of split TEM-1 β- lactamase. Only the formation of Aβ42 oligomers can lead to the reconstitution of β-lactamase activity, while both Aβ42 monomers and insoluble aggregates (e.g., fibrils) result in inactive β-lactamase (see Figure 1B). An inhibitor of oligomerization would lead to reduction in β- lactamase activity. However, an aggregation accelerator that accelerates oligomer-fibrilP-620504-PC conversion would also cause reduction in β-lactamase activity. To distinguish an oligomer inhibitor from an aggregation accelerator, an additional assay is used utilizing Aβ42 fused with the intact β-lactamase (see Figure 1A). While aggregation accelerators would lead to reduction in β-lactamase activity for the intact β-lactamase construct, an oligomer inhibitor would have a muted effect on β-lactamase activity because β-lactamase is active in both Aβ oligomers and monomers. A combination of the intact and split lactamase systems allows the selective identification of inhibitors of amyloid oligomerization. Such principles may be applied to other systems in which distinguishing between monomers, oligomers and aggregates can be achieved, for the purpose, for example, of identifying agents that inhibit oligomerization.

[0069] The ensuing descriptions of assay designs, assay components, constructs, and examples are based on the non-limiting example of oligomerization and fibrillization of the amyloid protein monomer Aβ; however, the disclosure is not so limited and is applicable to other biomolecule monomers that undergo oligomerization and fibrillization in vivo, and for which the former (and / or latter) is pathophysiologically responsible for disease and for which methods of identifying agents that modulate one or the other process are needed. The methods disclosed herein are useful for detecting modulation (e.g., increase, decrease or no change) in either or both of the steps from oligomerization of monomers to fibrillization of oligomers, whether or not oligomerization is an intermediate step therein. Such systems and assays are useful wherein the pathological species is a monomer, an oligomer or a fibril, as the disclosure herein provides methods for identifying agents that modulate any part of the pathway. The skilled artisan will readily adapt the teachings herein to other monomers.

[0070] As noted herein, a fusion biomolecule having no enzymatic activity, or similar terms as used herein, refer to a biomolecule that is inactive with respect to the full length enzyme from which the fusion biomolecule was derived, such as full length β-lactamase or active variants thereof. The N-terminus portion or the C-terminus portion, as described herein in respective fusion biomolecules, are inactive with respect to β-lactamase activity, or have no β-lactamase activity, or are substantially inactive with respect to β-lactamase activity, or essentially have no β-lactamase activity. The skilled artisan will readily understand from the teachings herein the objective of the β-lactamase fragment complementation disclosed herein the meaning of an inactive fusion biomolecule. Split Enzyme Based Assay

[0071] For example, in an embodiment, the system of the present disclosure may comprise a split TEM-1 β-lactamase construct fused to β-amyloid (1–42) (human) protein (Aβ42). By wayP-620504-PC of non-limiting example, the system comprising Aβ42 is fused to the N-terminus portion of TEM-1 β-lactamase (“NBLA”), providing an Aβ42–NBLA fusion construct; and Aβ42 is fused to the C-terminus half of TEM-1 β-lactamase (“CBLA”), providing an Aβ42–CBLA fusion construct. Such NBLA and CBLA are provided, for example, in vitro for a cell-free system (in which β-lactamase activity is measured).

[0072] In an embodiment, NBLA and CBLA are grown in a cell culture system, for example, non-antibiotic-resistant cells in an β-lactam-antibiotic-containing growth medium (e.g., an ampicillin-containing growth medium). The Aβ42–NBLA and Aβ42–CBLA fusion constructs may each freely move through the intracellular space, and in the absence of an inhibitor of oligomerization, the NBLA and CBLA halves may come together, forming oligomers with reconstituted β-lactamase activity and cleave the ampicillin β-lactam, thus conferring antibiotic resistance on the cells and permitting them to grow more readily. Detection of cell growth in the presence of the antibiotic (e.g., resistance) indicates no inhibition of Aβ oligomerization; addition of a candidate Aβ oligomerization inhibitor will prevent reconstitution of β-lactamase activity and the cells will be susceptible to the antibiotic and growth affected.

[0073] Alternatively, in the absence of bacterial or other cells expressing the NBLA and CBLA, β-lactamase activity can be measured in vitro using an assay. Similar to the above, in the absence of an inhibitor, NBLA and CBLA will oligomerize, reconstituting β-lactamase activity detectable by enzymatic activity. In the presence of an Aβ oligomerization inhibitor, NBLA and CBLA are unable to oligomerize and thus not reconstitute β-lactamase, such reduced enzymatic activity detectable in an enzymatic assay.

[0074] While the foregoing assay distinguishes oligomerization of Aβ or lack thereof in the presence of an oligomerization inhibitor, a detected reduction of β-lactamase activity indicating a suspected inhibitor may also be a result of aggregation of NBLA-CBLA oligomers to form fibrils, which also exhibit reduced β-lactamase activity. In order to distinguish reduced β- lactamase activity resulting only from reduced oligomerization and not aggregation of oligomers into fibrils, a separate assay is utilized using a related construct: an intact enzyme construct, and related methods. As with the split enzyme assay, the intact enzyme assay may also be used for other aggregable monomer assay systems. Such intact enzyme constructs are described further below. Split Enzyme ConstructsP-620504-PC

[0075] In one non-limiting example, the monomer used to assess oligomerization or fibrillization is Aβ. In one embodiment, the split β-lactamase constructs, the N-terminal and C- terminal halves of β-lactamase, which may be referred to as “NBLA” and “CBLA” respectively, were split between residues 195 and 196 based on the studies of Galarneau et al. (2002) and Wehrman et al. (2002). Structural studies have shown that the C-terminal region of Aβ is generally structured in the amyloid fibrils, while the N-terminal region is more disordered. Therefore, in one embodiment, the Aβ sequence is placed at the C-terminal end of β-lactamase so that the C-terminal regions of Aβ can freely interact with each other during aggregation (e.g., oligomerization and / or fibrillization). A 20-residue (GGGGS)4 (SEQ ID NO:9) linker is inserted between β-lactamase and Aβ sequences. Alternatives to the forgoing construction of the split β-lactamase including other linkers are fully embraced by the disclosure herein. A His tag may be included in the constructs described herein, and may be used for purification. Such His tag may be inserted in the protein sequence with a SS sequence before and after (e.g. SSHHHHHHSS; SEQ ID NO:20).

[0076] The N-terminal β-lactamase in the constructs described herein did not include the N- terminal secretory sequence, amino acids 1-23, though this is not limiting, and the methods described here may be carried out with amino acids 1-23 present.

[0077] As described in the examples herein, growth curve studies on E. coli C41 cells expressing NBLA–Aβ42 and CBLA–Aβ42 show that the cell growth did not recover within the timeframe of 18 hours at ampicillin concentrations of 50 μg / mL or higher (Figure 4A), showing that the split β-lactamase construct has much lower stability than the intact β- lactamase. To mitigate the reduced β-lactamase enzyme activity in the split β-lactamase system, in one embodiment, the M180T mutant (SEQ ID NO:2) and the addition of a tripeptide sequence, NGR, at the end of the β-lactamase fragment in NBLA, were evaluated in addition to an NBLA construct containing both M180T and NGR (SEQ ID NO:4). As shown in the examples herein, the growth curve studies show that the presence of NGR peptide did not change the growth curve; the presence of the M180T mutation (SEQ ID NO:19) led to growth recovery for both 200 μg / mL and 400 μg / mL ampicillin concentrations. When both M180T and NGR are introduced to the NBLA construct (SEQ ID NO:4), the cell growth recovered, but the recovery was slower than for the M180T construct. Thus, in an embodiment, the NBLA construct comprises M180T and NGR (SEQ ID NO:4).

[0078] The assays disclosed herein and the particular constructs of the N-terminal split β- lactamase can be designed by one of skill in the art following the teaching herein, withoutP-620504-PC limitation to the particular modifications if any to the β-lactamase sequence. In some embodiments, more aggregation-prone proteins may utilize a construct that is more stable, such as the one with M180T mutation; in some embodiments, less aggregation-prone proteins may utilize a less stable construct, such as the wild-type β-lactamase. Use of an assay system that employs the split N-terminal β-lactamase fragment construct without the M180T mutation or NGR is also embodied herein, as well as other modifications of the β-lactamase fragment to enable detectability of recovered β-lactamase activity in any other β-lactamase activity detection system is embraced herein.

[0079] Because the Aβ42 F19D mutant aggregates more slowly than wild-type Aβ42, it is reasoned that the Aβ42 F19D mutant may accumulate a different amount of oligomers than wild-type Aβ42. The growth curve assay would be able to show the difference in oligomerization between wild-type Aβ42 and the F19D mutant Aβ42. As shown in the examples, the growth recovery of cells expressing the Aβ42 F19D mutant also requires the M180T mutation in β-lactamase constructs, as both the NBLA wild-type and NBLA-NGR did not lead to cell recovery in the presence of 50 μg / mL and 100 μg / mL ampicillin. With the NBLA-M180T construct, the Aβ42 F19D mutant had a growth recovery time of 2.6 hours and 3.8 hours with 200 μg / mL and 400 μg / mL ampicillin, respectively. In comparison, the growth recovery time for Aβ42 wild-type in the same NBLA-M180T background is 6.1 hours and 9.0 hours with 200 μg / mL and 400 μg / mL ampicillin, respectively. The NBLA-M180T-NGR construct also showed that Aβ42 F19D mutant resulted in faster growth recovery compared to wild-type Aβ42. Comparing NBLA-M180T-NGR and NBLA-M180T for Aβ42 F19D, the presence of NGR peptide also slowed down cell recovery. Intact Enzyme Based Assay

[0080] The split-enzyme fusion constructs based assay may be further complemented with an assay using an intact-enzyme fusion construct, wherein Aβ is fused to the intact β-lactamase. As noted above, while the split enzyme system can distinguish monomers from oligomers, such as Aβ monomers (e.g., not oligomerized) from Aβ oligomers, it cannot distinguish between monomers and aggregated oligomers, because neither has detectable β-lactamase activity. To complement the split enzyme assay to confirm that the lack of detectable β-lactamase activity is due to inhibition of oligomerization or inhibition of oligomer aggregation (fibrillization), the intact enzyme system is used. In an embodiment, using Aβ as the example, Aβ fused to intact β-lactamase and evaluated in a bacterial system or in vitro, provides the distinction between oligomerization and fibrillization: Aβ fused to intact β-lactamase has β-lactamase activity as aP-620504-PC monomer and when oligomerized, but not when fibrillized. Thus, in the presence of a candidate oligomerization inhibitor in either the bacterial or in vitro system, the lack of change in β- lactamase activity of the intact construct indicates monomers or formation of oligomers, but a reduced activity indicates fibrillization. A candidate compound that only inhibits oligomerization but does not inhibit fibrillization of oligomers would show no change in the intact enzyme assay but a reduction in enzyme activity in the split enzyme system. Intact β-Lactamase Constructs

[0081] In one non-limiting example wherein the monomer to be evaluated for oligomerization or fibrillization is Aβ, in an embodiment, the intact β-lactamase construct consists of residues 24–286 with human amyloid-β (1–42) (Aβ42) sequence (SEQ ID NO:5) fused to the C- terminal end via a (GGGGS)4 (SEQ ID NO:9) linker. The first 23 residues of β-lactamase form a signal for secretion to periplasm and were not included in the fusion construct; this allows the fusion protein to be trapped in the cytoplasm. As shown in the Examples, a growth curve study of E. coli C41 cells expressing the fusion protein of β-lactamase and Aβ42 was performed, after growth and being supplemented with isopropyl β-D-1-thiogalactopyranoide (IPTG) and ampicillin. As shown herein, cell density decreased initially with increasing concentrations of ampicillin, and recovered after some time. A “growth recovery time” was established as the time from the beginning of the decline in cell density to the time the cell density recovers to the same level. A plot of growth recovery time as a function of ampicillin concentration is shown in Figure 2C. The growth recovery time is the time needed for the active lactamase to reduce the ampicillin in the culture medium to a growth-permissive concentration threshold. In some embodiments, a M180T mutation or other mutation may be included in the β-lactamase used in the intact fusions embodied herein. In some embodiments, the M180T mutant may be needed in the assay system for highly aggregation-prone proteins fused to the intact β-lactamase Such modification to provide an assay system as disclosed herein will be readily apparent to one of skill in the art, and any such intact fusion constructs are embraced herein.

[0082] As will be shown in the Examples, to establish that changes in growth curve can be used as a proxy for changes in Aβ aggregation, an F19D mutation was introduced to the Aβ42 sequence (SEQ ID NO:6). Previous studies showed that the F19D mutation markedly decreased the formation of insoluble Aβ42 fibrils both in vivo and in vitro. When the Aβ42 F19D mutation is introduced to the β-lactamase fusion construct, the cell density decreased to a lesser extent than Aβ42 wild-type and it recovered much faster. The growth recovery time wasP-620504-PC shortened by approximately 1.5 hours at all studied ampicillin concentrations (50–800 μg / ml) by the F19D mutation. The difference in growth recovery time reflects a higher level of lactamase activity in the F19D construct. These results confirm that, with the intact β-lactamase construct, one can use bacterial cell growth as a proxy for Aβ aggregation. Other Constructs

[0083] As described herein, an M180T mutation to the TEM-1 β-lactamase sequence is SEQ ID NO:2. (The M180T mutation is sometimes also referred to as M182T due to the use of a numbering system that is based on the consensus sequence of class A β-lactamases. Several previous studies show that the M180T mutation suppresses defects of protein folding and improves lactamase stability. One intent was to evaluate if the M180T mutation would improve the ability of the β-lactamase-Aβ construct to respond to changes in Aβ aggregation. Surprisingly, it was found that the M180T mutation in β-lactamase dramatically increased the ampicillin resistance of the fusion protein constructs with both wild-type Aβ42 and the Aβ42 F19D mutant. Neither 500 μg / ml nor 1000 μg / ml ampicillin caused any decrease in cell density as determined by OD600nm. These results suggest that the M180T mutation greatly stabilized the β-lactamase protein, making it active even when sequestered within the aggregated Aβ42. Therefore, the wild-type β-lactamase is more suitable than the M180T mutant as a reporter of Aβ aggregation when used as the intact protein. In other embodiments, other mutations in the β-lactamase protein can be provided to optimize the assay as disclosed herein.

[0084] Aggregation of at least 36 proteins have been identified in a wide range of human disorders. In addition to Aβ, many other amyloid proteins, such as but not limited to α- synuclein and tau have also been found to form soluble oligomers that play critical roles in pathogenesis. In some embodiments, similar methods as disclosed herein can be used for identifying small molecule inhibitors or biologics that specifically target such other oligomers, by way of non-limiting examples, and represent an important step in developing effective therapeutic interventions. The aggregation reporting platform reported in the present disclosure can be used to study any amyloid proteins or other proteins that form intracellular aggregates in E. coli or other cellular systems. Using E. coli cells, both cell growth and lactamase activity in cell lysate can be used to screen for small molecule inhibitors or biologics of oligomerization. While mammalian cells are not subject to the selection pressure exerted by ampicillin, the cell lysate of mammalian culture can still be used to screen for inhibitors using the lactamase activity assay.

[0085] In some embodiments, the sequences disclosed herein are as follows.P-620504-PC SEQ ID NO SEQUENCE SGK SPV t HVT l ME TG TTT CT t CAG l GA CCG GTT AAG ACT AC GAA TG TCC AC AG GGT TAT AGC SGK SPV t HVT l ME TG TTT CT t CAG l GA CCG GTT AAG ACT AC GAA TGG CCC CTT GCC TAA GG CAT SGK SPV HVTP-620504-PC (GGGGS)4 Aβ42 RLDRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGENGRGSGGGGSGGGG (wt) SGGGGSGGGGSDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV GCG AA CGG ( CA ( GGC AG AGA AC AC TGA CA AGG GC CA TCA SGK SPV ( HVT t GGS ( VVI ( GCG AA ( CGG t CA ( GGC ( AG AGA AC AC TGA CAA GGC CG AA AT SGK SPV HVT ( ME TG RH GCG AA CGG ( CA GGC AGP-620504-PC TACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGA ATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTAC AC TGA CA CGG TCT CGG AGC AT TTG GGT TGA CA RGII NK AT CTG GT AT TC AGG CC ATT AC ( s SGK SPV ( HVT t GGS ( VVI GCG AA ( CGG t CA ( GGC AG AGA AC AC TGA CAAP-620504-PC CAACGTTGCGCAAACTATTAACTGGCGAAAACGGTCGTGGATCAGGAGGC GGAGGATCAGGAGGAGGTGGTTCTGGTGGAGGTGGTAGTGGGGGCGGCG AA ATT SGK SPV ( HVT t GGG GCG AA ( CGG t CA GGC AG AGA AC AC TGA CA AT GA GG TCA SGK SPV HVT ( ME TG RH GCG AA CGG ( CA GGC AG AGA AC AC TGA CA CGG TCT CGG AGC AT TTG GGT TGA CAP-620504-PC SEQ ID NO:13 MGGLLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGII β-lactamase (196- AALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW NK AT CTG GT AT TC AGG CC ATT ACT SGK SPV HVT ME TG RH GC TGA GC GAG CG GTT AA AA TGC CT GCA AGC GA TCT AG GCA GA TGG GAC TGT TC SGK SPV HVT ME TG RH GCG AA CGG CAP-620504-PC Aβ42(wt) CTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGC AAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAG AGA AC AC TGA CAA GGC CTG GGT GCC TG TGG GTA GAA AA SGK SPV HVT ( GG A GCG AA CGG ( CA GGC AG AGA AC AC TGA CA AT GA GG TC SGK SPV HVT ( GG IA GCG AA CGG ( CA GGC AG AGA AC AC TGA CAP-620504-PC ACAACGTTGCGCAAACTATTAACTGGCGAAGGATCAGGAGGCGGAGGAT CAGGAGGAGGTGGTTCTGGTGGAGGTGGTAGTGGGGGCGGCGGATCCGA GG CTC SGK SPV HVT ( GG GVV GCG AA CGG ( CA GGC AG AGA AC AC TGA CA AGG GC CA CAT SGK SPV ( HVT t GGG GCG AA ( CGG t CA GGC AG AGA AC AC TGA CA AT GA GG TC SGK SPV HVT MEP-620504-PC Aβ42(wt) ADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTG and XhoI site SQATMDERNRQIAEIGASLIKHWGSGGGGSGGGGSGGGGSGGGGSDAEFRH GCG AA CGG CA GGC s AG AGA AC AC TGA CA CGG TCT CGG AGC AT TTG GT TGA CA GA SGK SPV ( HVT t GGG AS s GCG AA ( CGG t CA GGC s AG AGA AC AC TGA CA AT GA GG TC CT SGK SPV ( HVT t GGS ( VVI (P-620504-PC SEQ ID NO:42 ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCG β-lactamase 24-195 CGGCAGCCATATGCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA ( CGG t CA ( GGC ( AG AGA AC AC TGA CAA GGC CG AA AT GC RGII NK s AT CTG GT AT s TC AGG CC ATT AC SGK SPV ( HVT GGG GCG AA ( CGG ( CA t GGC AG AGA AC AC TGA CA AT GA RGIIP-620504-PC and BamH1 site SEQ ID NO:48 ATGGGCGGACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT CTG GT a AT TC AGG CC AATEXAMPLE 1 Materials And Methods Generation of Intact And Split β-Lactamase Constructs

[0086] The TEM-1 β-lactamase (BLA) gene encoding residues 24-286 was amplified from the pET19b plasmid using PCR. Restriction enzyme recognition sites for NdeI and BamHI were introduced to the 5′- and 3′-end of the β-lactamase sequence via the overhang of the PCR primers. DNA sequence encoding a (GGGGS)4linker (SEQ ID NO:9) and the Aβ42 protein (e.g., SEQ ID NO:6) was chemically synthesized by Integrated DNA Technologies. BclI and XhoI sites were introduced at the 5′- and 3′-end of the (GGGGS)4-Aβ42 sequence during DNA synthesis. The TEM-1 β-lactamase was inserted between the NdeI and BamHI sites of the pET28b plasmid to generate pET28b-BLA. Then the DNA fragment consisting of the (GGGGS)4-Aβ42 sequence was digested with BclI and XhoI, and inserted into the BamHI and XhoI sites of pET28b-BLA to generate pET28b-BLA-(GGGGS)4-Aβ42, which is abbreviated as BLA-Aβ42. The BLA M180T mutation was introduced to the BLA-Aβ42 construct using QuikChange site-directed mutagenesis. Sanger DNA sequencing was performed to confirm each step of molecular cloning process. The final protein and DNA sequences, respectively, for the BLA-Aβ42 constructs are shown in Figure 7 (with XhoI site, SEQ ID NOs:37 and 38) and in SEQ ID NOs:5 and 25; SEQ ID NOs:12 and 29 (with F19D Aβ42); SEQ ID NOs:15 and 32 (with M180T); and SEQ ID NOs:14 and 31 (with both M180T and F19D Aβ42).

[0087] To generate the split β-lactamase constructs, deletion mutagenesis was performed using the QuikChange mutagenesis kit (Agilent). For the N-terminal half of the BLA construct (NBLA), residues 196-286 were deleted from the BLA-Aβ42 construct. The final DNA and protein sequences, respectively, of NBLA-Aβ42 construct are shown in Figure 8 (with XhoI site, SEQ ID NOs:40 and 39). The NBLA-M180T-Aβ42 was generated by performing the same deletion of residues 196-286 from the BLA-M180T-Aβ42 plasmid. The NGR tripeptide insertion mutant of NBLA-Aβ42 was created by including the NGR coding sequence in theP-620504-PC PCR primers. The final DNA and protein sequences, respectively, of NBLA-NGR-Aβ42 are shown in Figure 9 (with XhoI site, SEQ ID NOs:42 and 41). For the C-terminal half of the BLA construct, residues 24-195 of the β-lactamase together with the His-tag sequence between NcoI and NdeI sites were deleted. Then the CBLA-Aβ42 sequence was cut out of pET28b with NcoI and HindIII and inserted to the pCDFDuet-1 plasmid (Millipore Sigma) digested with the same restriction enzymes. The final DNA and protein sequences, respectively, of CBLA-Aβ42 are shown in Figure 10 (with HindIII site, SEQ ID NOs:44 and 43). DNA sequences of all constructs were confirmed using Sanger sequencing.

[0088] The F19D mutation was introduced to the Aβ42 sequence using site-directed mutagenesis and the sequence was confirmed with Sanger sequencing.

[0089] The NBLA-M180T construct was generated by performing a deletion mutagenesis to remove Aβ42 sequence from the BLA-M180T-Aβ42 plasmid. The final DNA and protein sequences, respectively, of NBLA-M180T are shown in Figure 11 (with BamH1 site, SEQ ID NOs:46 and 45). To create the CBLA construct, the CBLA fragment was excised from CBLA- Aβ42 using NcoI / BamHI and inserted to the pCDFDuet-1 plasmid digested with the same restriction enzymes. The final DNA and protein sequences, respectively, of CBLA are shown in Figure 12 (with BamH1 site, SEQ ID NOs:48 and 47). Growth Curve Studies of the Intact β-Lactamase Constructs

[0090] The pET28b plasmid containing BLA-Aβ42 constructs were transformed to E. coli C41 competent cells (Lucigen). There are two variants of BLA: wild-type (WT) and the M180T mutant. There are also two variants of the Aβ42 protein: WT and the F19D mutant.

[0091] For the growth curve studies of BLA-WT fused with either Aβ42 WT or F19D, a single colony was picked to inoculate 20 mL of lysogeny broth (LB) containing 30 µg / mL of kanamycin. The culture was incubated at 37°C with shaking at 220 rpm. The next morning, the cell density of the overnight cultures was determined using optical density at 600 nm (OD600nm). Then the overnight cultures were diluted with LB-kanamycin broth to an OD600nm of 0.35 for both BLA-Aβ42 and BLA-Aβ42-F19D. In a 15-mL tube, 8 µL of isopropyl-β-D-1- thiogalactopyranoside (IPTG, 0.2 M in deionized water) was mixed with 8 mL of diluted cells (OD600nm of 0.35) to achieve the final IPTG concentration of 0.2 mM. Then 990 µL of these cells was pipetted to each of the six 1.5-mL microcentrifuge tubes, which were labeled 0, 50, 100, 200, 400, and 800 µg / mL ampicillin. Ten µL of deionized water was added to the 0 µg / mL ampicillin tube, and 10 µL of ampicillin stock solutions at 5, 10, 20, 40, and 80 mg / mL wereP-620504-PC added to the tubes labeled 50, 100, 200, 400, and 800 µg / mL ampicillin to achieve the final ampicillin concentration. Different stock ampicillin solutions were made by serial dilutions from the 80 mg / mL solution (in deionized water). Then 50 µL of the cells was transferred to a 384-well white microplate with clear bottom (Greiner Bio-One, product number 781095). Each experimental condition consists of four technical repeats. The microplate was sealed with a Breathe-Easy cell culture membrane (Electron Microscopy Sciences, product number 7053610) and incubated in a SpectraMax iD3 microplate reader (Molecular Devices) at 37°C with 1 min of shaking following every 4 min of quiescent incubation. Absorbance at 600 nm was measured every 5 min.

[0092] For the growth curve studies of BLA-M180T fused with either Aβ42 WT or F19D, the same procedure for BLA-WT was followed except the final ampicillin concentrations were adjusted to 0, 500, and 1000 µg / mL. Growth Curve Studies of the Split β-Lactamase Constructs

[0093] For the split β-lactamase constructs, the NBLA-Aβ42 construct on pET28b and the CBLA-Aβ42 construct on pCDFDuet-1 were co-transformed to E. coli C41 cells (Lucigen) and selected using kanamycin and streptomycin double resistance. There are four variants of NBLA: NBLA-WT, NBLA-M180T, NBLA-NGR, and NBLA-M180T-NGR. The CBLA fragment of lactamase was not modified. When studying the Aβ42 F19D mutant, the F19D mutation is present in both the NBLA-Aβ42 and CBLA-Aβ42 constructs.

[0094] For the growth curve studies of the split β-lactamase constructs, a single colony was picked from the transformation plate to inoculate 20 mL of LB broth containing 30 µg / mL of kanamycin and 50 µg / mL of streptomycin. The culture was incubated at 37°C with shaking at 220 rpm. The next morning, the cells were diluted to an OD600nm of 0.60 using LB- kanamycin-streptomycin. The rest of the procedure is similarly performed as for the intact β- lactamase constructs with the exception of different ampicillin concentrations as indicated in the results. Cell Lysate Preparation

[0095] A single colony for the intact β-lactamase construct (BLA-Aβ42), the split β-lactamase- Aβ construct (NBLA-M180T-Aβ42 + CBLA-Aβ42), and the split β-lactamase without Aβ, was used to inoculate 20 mL of LB broth containing the appropriate antibiotics and incubated at 37°C with shaking at 220 rpm. The next morning, 200 µL of the overnight culture was used to inoculate 20 mL of LB broth. IPTG was added to a final concentration of 0.2 mM when theP-620504-PC cells grew to an OD600nmof 0.4. The cells were grown for another hour and then pelleted down with centrifugation at 4500 rpm for 20 min. Fresh LB broth was added to the cell pellet and the cells were sonicated on ice using a Branson 450 Sonifier (standard tip, 90% amplitude, pulse mode with 5 s on and 10 s off, 3 min of total on time). An aliquot of the sonicated cell lysate was saved as the total cell lysate. The rest of the cell debris was pelleted by centrifugation (20,000 g, 30 min, 4°C). Ultrafiltration filters with 0.2 µm cutoff were pre-washed by adding 400 µL of water and centrifuged at 14,000 g for 5 min. After removing all the water from the filters, 500 µL of the cell lysate supernatant was added and obtained the 0.2 µm filtrate after centrifugation at 14,000 g at 4°C. Centrifugation was stopped when the volume of the retentate corresponded to the dead volume of the filters. Lactamase activity assays were then performed without delay. Size Exclusion Chromatography

[0096] A Bio-Rad ENrich SEC 650 10×300 column was used for size exclusion chromatography (SEC) studies of E. coli cell lysates. The column was equilibrated with PBS buffer (50 mM phosphate, 140 mM NaCl, pH 7.4), and calibration was performed using the gel filtration standard (Bio-Rad product number 1511901) containing thyroglobulin (670 kD), γ-globulin (158 kD), ovalbumin (44 kD), myoglobin (17 kD), and vitamin B12 (1.35 kD). The sample volume for the gel filtration standard and the cell lysate is 100 and 250 µL, respectively. The flow rate for all chromatography runs was at 1 mL / min. For cell lysate, 0.5 mL fractions were collected for β-lactamase activity assays. TEM-1 β-Lactamase Activity Assay

[0097] Nitrocefin (TOKU-E product number N005, >95% purity) was dissolved in DMSO to obtain a 5 mM stock concentration. Then a 200 µM working solution was prepared in PBS buffer. The TEM-1 β-lactamase activity was determined by mixing 25 µL of the cell lysate with 25 µL of the nitrocefin working solution in a microplate (Greiner Bio-One product number 781095) and measuring the absorbance at 492 nm every min for 10 min using a SpectraMax iD3 microplate reader (Molecular Devices). The lactamase activity assay was performed on: (1) total cell lysate, (2) supernatant after centrifugation to remove cell debris, (3) supernatant filtered through a 0.2 µm ultrafiltration filter, and (4) various SEC fractions. Three technical repeats for each lysate sample were performed. The sample was diluted as needed to obtain the linear range of absorbance change for accurate activity measurements. The β-lactamase activity is represented by the slope of the kinetic measurements, which were obtained using the LINEST function of Microsoft Excel.P-620504-PC EXAMPLE 2 Design of a Protein Aggregation Platform Based on Intact And Split β-Lactamase Constructs

[0098] In the aggregation process of Aβ and other amyloid proteins, there are three main aggregated species: monomers, soluble aggregates (mostly oligomers), and insoluble aggregates (mostly fibrils). An effective screening platform distinguishes these three aggregated species, and more importantly, it needs to be able to distinguish the changes in a particular species. The most pressing need is a screening tool that can specifically monitor the changes in Aβ oligomers. To achieve this goal, a combination of two cell-based and biochemical assays was utilized. The first system is a fusion protein of Aβ and intact TEM-1 β-lactamase (Figure 1A). The second is a fusion protein between Aβ and split β-lactamase (Figure 1B). TEM-1 β-lactamase is encoded by the ampicillin resistance gene in E. coli and other Gram-negative bacteria. The fusion constructs with β-lactamase allow for the use of E. coli cell growth or activity assays with cell lysate to screen for aggregation inhibitors or modulators.

[0099] The rationale of using the full-length β-lactamase is that formation of insoluble Aβ fibrils will sequester lactamase proteins in the Aβ aggregates and may also cause misfolding of lactamase proteins, and thus reduce the lactamase activity in E. coli cells (Figure 1A). As a result, increased Aβ aggregation would lead to reduced lactamase activity and poor cell growth. Previously, Aβ has been fused to reporter proteins such as green fluorescent protein and dihydrofolate reductase, and it has been shown that these reporters can be used to monitor Aβ aggregation. Aggregation inhibitors can be screened using the Aβ-lactamase fusion system for changes in the E. coli growth curve or lactamase activity. The shortcoming of the intact β- lactamase construct is that Aβ monomers and oligomers are not distinguished because lactamase proteins fused to either are expected to retain activity.

[0100] The split lactamase system has one copy of Aβ fused to the N-terminal half of TEM-1 β-lactamase (NBLA), and another copy of Aβ fused to C-terminal half of β-lactamase (CBLA) (Figure 1B). The split β-lactamase system is based on the principle of protein fragment complementation assay, which has been widely used to study protein-protein interactions. The activity of β-lactamase is restored only when Aβ proteins interact with each other in soluble oligomers. In fibrils, even though the two halves of β-lactamase can come together, formation of insoluble aggregates would render the enzyme inactive or sequester the enzyme from its substrates.P-620504-PC

[0101] The combination of the split and intact lactamase constructs can distinguish Aβ oligomer formation from fibrillization. When used for drug screening, inhibitors of Aβ oligomerization would lead to reduced lactamase activity and cell growth for the split β- lactamase constructs. However, increased fibril formation would have the same effect. For the intact β-lactamase construct, only molecules that promote fibrillization would reduce lactamase activity, while oligomer inhibitors would have a muted effect. Therefore, a compound that reduces lactamase activity and cell growth in the split lactamase system, but does not do so in the intact lactamase system, would be a potential drug candidate that specifically inhibits oligomerization. EXAMPLE 3 Intact β-Lactamase Constructs

[0102] The intact β-lactamase construct consists of residues 24-286 with Aβ42 sequence fused to the C-terminal end via a 20-residue (GGGGS)4 linker (Figure 1A). The first 23 residues of β-lactamase form a signal for secretion to periplasm and are not included in the fusion construct. This allows the fusion protein to be trapped in the cytoplasm. Structural studies have shown that the C-terminal region of Aβ is structured in the amyloid fibrils, while the N-terminal region is more disordered. Therefore, the Aβ sequence is placed at the C-terminal end of β- lactamase so that the C-terminal region of Aβ can freely interact with each other during aggregation.

[0103] A growth curve study of E. coli cells expressing the intact lactamase-Aβ42 was performed. The E. coli cells were supplemented with IPTG to induce protein expression, and the cell growth was monitored using absorbance measurements at 600 nm in a microplate reader. The cells with 0.2 mM IPTG (Figure 2A, red traces) had similar growth curves as the cells without IPTG (Figure 2A, black traces), suggesting that overexpression of Aβ fusion protein does not have significant effect on cell growth. In the presence of ampicillin, cell density decreased initially and recovered after a lag time (Figure 2A). Eventually, the cell density in the presence of ampicillin reached similar levels as the cells without ampicillin. With increasing concentrations of ampicillin, the cells grew slower and slower, suggesting that the amount of lactamase was in a range sensitive to changes in ampicillin concentrations.

[0104] To establish that one can use the changes in growth curve as a proxy for changes in Aβ aggregation, a F19D mutation was introduced to the Aβ42 sequence. Previous studies have shown that the F19D mutation markedly decreased the formation of insoluble Aβ42 fibrils bothP-620504-PC in vivo and in vitro. With the F19D mutation, the cell density decreased to a lesser extent than Aβ42 wild-type and the cell growth also recovered faster (Figure 2B). The difference in growth recovery reflects a higher level of lactamase activity in the F19D construct. These results confirm that, with the intact lactamase-Aβ construct, one can use bacterial cell growth as a proxy for Aβ aggregation.

[0105] To quantify the effect of ampicillin on cell growth, the “growth recovery time” is defined as the time from the beginning of the decline in cell density to the time when cell density recovers to the same level (Figure 2C, inset). A plot of growth recovery time as a function of ampicillin concentration is shown in Figure 2C. It is rationalized that the growth recovery time represents the time needed for the active lactamase to reduce the ampicillin in the culture medium below a growth-permissive concentration threshold. There appears to be a noticeable trend that the difference in growth recovery time between Aβ42 wild-type and F19D mutant increases with increasing concentrations of ampicillin. As a result, using higher concentrations of ampicillin may help detect smaller changes in protein aggregation when performing growth curve studies. Because the cell growth in the presence of ampicillin depends on the total lactamase activity, and because lactamase proteins in both oligomers and monomers are active, the cell growth curve alone does not distinguish contributions from Aβ oligomers or monomers. The relationship between different Aβ aggregates and lactamase activity are further investigated below using size exclusion chromatography of the cell lysates.

[0106] To improve the construct, an M180T mutation was introduced to the β-lactamase sequence. The M180T mutation is also referred to as M182T in a numbering system that is based on the consensus sequence of class A β-lactamases. Several previous studies show that the M180T mutation suppresses defects of protein folding and improves lactamase stability. Whether the M180T mutation would better detect changes in Aβ aggregation was examined. It was found that the M180T mutation in β-lactamase dramatically increased the ampicillin resistance of the fusion protein constructs with both wild-type Aβ42 (Figure 3A) and the Aβ42 F19D mutant (Figure 3B). Neither 0.5 or 1 mg / mL ampicillin caused any decrease in cell density. These results suggest that, when an enzyme is used as a reporter of protein aggregation, too much enzyme activity makes it insensitive to changes in protein aggregation. For the intact lactamase construct, the wild-type β-lactamase is more suitable than the M180T mutant to monitor Aβ aggregation. EXAMPLE 4 Split β-Lactamase ConstructsP-620504-PC

[0107] For the split β-lactamase constructs, the N- and C-terminal halves of β-lactamase, NBLA and CBLA (Figure 1B), were split between residues 195 and 196 based on the studies of Galarneau et al. and Wehrman et al. The relative positions of protein of interest and the split lactamase constructs may affect the complementation of enzyme activity. Using GCN4 leucine zippers (ZIP), Galarneau et al. tested various combinations of having the protein of interest at either N- or C-terminal side of NBLA and CBLA. They found that the order of complementation efficiency is NBLA-ZIP / ZIP-CBLA > ZIP-NBLA / ZIP-CBLA > NBLA- ZIP / CBLA-ZIP > ZIP-NBLA / CBLA-ZIP. Wehrman et al. put interacting protein partners at the C-terminal side of NBLA, but N-terminal side of CBLA. In designing the split lactamase- Aβ constructs, a main concern is how the attachment of a fusion protein affects Aβ aggregation. Because the C-terminal end of Aβ is more ordered in Aβ oligomers and fibrils, Aβ was placed at the C-terminal side of both NBLA and CBLA constructs so that movement of Aβ C-terminal region is not restricted by the fusion protein.

[0108] Growth curve studies of E. coli cells expressing NBLA-Aβ42 and CBLA-Aβ42 were performed. The results showed that cell growth did not recover within the timeframe of 18 h at ampicillin concentrations of 50 µg / mL or higher (Figure 4A). The cells grew poorly in the presence of 25 µg / mL ampicillin. The poor growth of cells expressing wild-type split lactamase constructs may be due to lower protein expression levels because the cells need to express both NBLA-Aβ42 and CBLA-Aβ42, in comparison with just one protein for the intact lactamase- Aβ42 construct. It is also expected that the split lactamase is less stable than the intact lactamase due to the entropy cost of complementation.

[0109] To overcome the low lactamase activity in the split β-lactamase system, two previously reported constructs of β-lactamase were used. One is the M180T mutant discussed above. The other construct is the insertion of a tripeptide sequence, NGR, at the C-terminus of NBLA, which was found to increase the activity of the complemented lactamase. In addition, an NBLA construct containing both M180T and NGR was made. Growth curve studies show that the presence of NGR tripeptide did not significantly change the growth curves for the split lactamase-Aβ42 constructs (Figure 4B). The presence of the M180T mutation, however, led to growth recovery at both 200 and 400 µg / mL ampicillin concentrations (Figure 4C). When both M180T and NGR are introduced to the NBLA construct, the cell growth also recovered at 200 and 400 µg / mL ampicillin (Figure 4D), but with slower recovery than the M180T construct (Figure 4C). The findings that M180T mutation is critical for good cell growth areP-620504-PC in line with previous studies on the split lactamase system showing that the M180T mutation increased the reconstituted lactamase activity 10- to 250-fold over background levels.

[0110] Next whether the split β-lactamase constructs can detect the aggregation difference between Aβ42 WT and the F19D mutant was investigated. It was found that the growth recovery of cells expressing the Aβ42 F19D mutant also requires the M180T mutation in β- lactamase, as both the NBLA-WT (Figure 4A) and NBLA-NGR (Figure 4B) did not lead to cell recovery in the presence of 50 and 100 µg / mL ampicillin. With the NBLA-M180T construct, the Aβ42 F19D mutant had a growth recovery time of 2.6 and 3.8 h with 200 and 400 µg / mL ampicillin, respectively (Figure 4C). In comparison, the growth recovery time for Aβ42 wild-type in the same NBLA-M180T background was 6.1 and 9.0 h with 200 and 400 µg / mL ampicillin, respectively (Figure 4C). The NBLA-M180T-NGR construct also showed that F19D mutation resulted in faster growth recovery compared to wild-type Aβ42 (Figure 4D). Comparing NBLA-M180T-NGR and NBLA-M180T for Aβ42 F19D, the presence of NGR peptide also slowed down cell recovery (Figures 4C, 4D). However, the NGR construct amplified changes in Aβ aggregation as a result of the F19D mutation. At 200 µg / mL ampicillin, the difference in growth recovery time between Aβ42 WT and F19D is 3.5 h for the M180T construct (Figure 4C), and 5.1h for the M180T-NGR construct (Figure 4D). And this is consistent with previous studies that show NGR improved signal-to-noise ratio in using the split lactamase to study protein-protein interactions.

[0111] The results presented herein suggest that, when an enzyme is used to report protein aggregation, the enzyme activity needs to be tuned to match the aggregation profile of the amyloid protein. For the intact lactamase construct, wild-type is better at detecting protein aggregation than the M180T mutant because the M180T mutant of β-lactamase is too active (Figures 2 and 3). For the split lactamase construct, the M180T mutant is critical for detecting Aβ aggregation because the wild-type β-lactamase does not provide enough activity (Figure 4). The collection of different β-lactamase constructs provides a toolbox to accommodate the aggregation properties of different amyloid proteins. EXAMPLE 5 Split β-Lactamase Constructs Are Sensitive to the Formation of Soluble Aβ Oligomers

[0112] To investigate the contribution to β-lactamase activity from Aβ aggregates of different sizes, the cell lysate activity expressing intact and split β-lactamase constructs was studied. Total cell lysate was obtained by sonicating the bacterial cells. The total cell lysate wasP-620504-PC separated to supernatant and pellet using centrifugation. The supernatant was then filtered through a 0.2 µm ultrafiltration filter to obtain 0.2 µm filtrate. The β-lactamase activity was determined using nitrocefin as a substrate. For both the intact and split β-lactamase constructs (Figure 5), it was found that there is no significant difference between total cell lysate, supernatant, and 0.2 µm filtrate, suggesting that the β-lactamase activity exists only in the soluble fraction of the cell lysate. In other words, formation of insoluble Aβ aggregates leads to loss of β-lactamase activity.

[0113] To further investigate whether Aβ forms oligomers in the cell lysate and how oligomer formation contributes to total β-lactamase activity, the cell lysate was fractionated using a Bio- Rad ENrich SEC 650 size exclusion column. Molecular weight calibration was performed using a gel filtration standard containing thyroglobulin (670 kD), γ-globulin (158 kD), ovalbumin (44 kD), myoglobin (17 kD), and vitamin B12 (1.35 kD). Excellent linearity (R2= 0.9992) was obtained with the gel filtration standard (Figure 13). β-lactamase activity assays were performed on all protein-containing fractions, ranging from elution volumes of 7-20 mL.

[0114] As shown in Figure 6A, the split lactamase-Aβ42 construct showed two main activity peaks, centered at elution volumes of 10.25 and 15 mL. Based on the calibration standard, these two activity peaks have an apparent molecular mass of 600 and 22 kD (Figure 13). The molecular mass of the NBLA-Aβ42 is 27 kD and CBLA-Aβ42 is 16 kD. Therefore, the 600- kD activity peak may contain 14 heterodimers of NBLA-Aβ42 and CBLA-Aβ42, assuming an equal molar mixture in the oligomer. And this corresponds to 28 molecules of Aβ in the 600- kD activity peak. This is consistent with a previous study that shows Aβ42 oligomers consisting of 24-36 subunits. A previous study of Aβ oligomers from Alzheimer’s disease brain tissues showed an SEC peak near 158-kD, corresponding to ~35 Aβ molecules. The 22-kD activity peak likely represents a mixture of NBLA-Aβ42 and CBLA-Aβ42, which give an average molecular mass of 21.5 kD. The high level of lactamase activity in the 22-kD activity peak may arise from a high background complementation of the split lactamase, or from the formation of Aβ42 dimers. It is also possible that self-complementation of the split lactamase constructs can drive Aβ dimerization. Aβ dimers have been found in both in vivo and in vitro preparations. It has been shown that Aβ forms dimers even in the presence of 8 M urea. The lactamase activity from the 600-kD activity peak accounts for ~20% of total activity in the cell lysate. Therefore, it is a viable option to screen for oligomer-targeting compounds by monitoring lactamase activity in the cell lysate or using cell growth assays.P-620504-PC

[0115] The intact lactamase-Aβ42 construct also showed two main activity peaks at similar elution volumes (Figure 6B). In contrast to the split lactamase construct, the 22-kD activity peak accounts for 97% of total lysate activity. The much higher activity of the 22-kD activity peak is because the intact lactamase does not require fragment complementation. As a result, changes in the oligomers (600-kD activity peak) would have negligible effect on the total lactamase activity for the intact lactamase construct.

[0116] To further characterize the split lactamase construct, NBLA-M180T (SEQ ID NO: 45) and CBLA (SEQ ID NO: 47) were prepared without Aβ fusion and the cell lysate was fractionated using the size exclusion column. As shown in Figure 6C, the split lactamase without Aβ shows only one activity peak near 22 kD. This confirms that NBLA-M180T and CBLA are capable of forming an active lactamase in the absence of Aβ oligomers, representing the background activity in protein fragment complementation assays. The absence of additional activity peaks at higher molecular masses suggests that the 600-kD activity peaks observed in the lactamase-Aβ fusion construct (Figure 6A) are a result of Aβ aggregation, not driven by lactamase aggregation.

[0117] The SEC studies confirmed the design of the split lactamase construct (Figure 1), which is sensitive to the formation of amyloid oligomers. At the same time, the high activity peak at 22-kD suggests high background activity due to spontaneous, in contrast to oligomerization-assisted, complementation of the split lactamase (Figure 6A). This is partly caused by the high level of protein expression in the bacterial expression system. Mutations can be introduced near the binding interface of the split lactamase construct to lower binding affinity, and thus reduce spontaneous complementation. The SEC activity profile described in Figure 6 can be used to evaluate the effects of these mutations in future improvement of the split lactamase construct.

[0118] Amyloid oligomers are key drug targets in Alzheimer’s disease and other amyloid- related disorders. Due to the difficulty in untangling the processes of oligomerization and fibrillization, few aggregation systems are designed specifically for the screening of oligomer inhibitors. For example, Hecht and co-workers designed a fusion protein construct of Aβ and green fluorescent protein. Aβ aggregation led to reduced fluorescence when overexpressed in E. coli, allowing high-throughput screening of aggregation modulators with fluorescence as a readout. Liebman and co-workers developed a fusion protein of Aβ and the C-domain of yeast prion Sup35p, a translation termination factor. Aβ aggregation causes ade1-14 gene read- through and allows the yeast cells to grow on medium lacking adenine. As a result, the growthP-620504-PC of yeast cells can be used to screen for modulators of Aβ aggregation. Stains and co-workers developed a fusion protein construct of Aβ and NanoLuc. Aggregation of Aβ led to reduced bioluminescence, which can be used to screen for aggregation modulators. The Ventura group created a fusion construct using human dihydrofolate reductase and used yeast cell survival as a reporter of protein aggregation. Radford, Brockwell, and co-workers used the refolding of TEM-1 β-lactamase in the periplasm of E. coli to screen for aggregation inhibitors. This lactamase system puts the amyloid protein in between the two split lactamase fragments and is fundamentally different from the design of the present work. All these systems are based on overall Aβ aggregation, without explicit distinction of fibrillization and oligomerization.

[0119] Previously, Hyman and co-workers used a split Gaussia luciferase complementation assay to study Aβ oligomer formation in human cell lines. Using luciferase activity assay, they found that the split luciferase-Aβ fusion formed oligomers corresponding to 24-36 Aβ molecules. This is consistent with the present split lactamase-Aβ fusion oligomers that gave an activity peak corresponding to 28 Aβ molecules (Figure 6). Although the luciferase system is a very sensitive assay, Gaussia luciferase displays “flash” kinetics represented by an initial burst of activity and then a rapid decay. The flash properties of Gaussia luciferase has been shown to be caused by covalent inactivation of the enzyme. Because luciferase is not related to cell survival, detecting changes in Aβ aggregation can only be achieved through bioluminescence measurements, limiting the use of the split Gaussia luciferase system in high- throughput screening.

[0120] Overall, the present work presents a novel protein aggregation platform that combines the use of split and intact β-lactamase to distinguish processes of oligomerization from fibrillization. Changes in the aggregation of amyloid proteins can be monitored through survival of bacterial cells in the presence of ampicillin or lactamase activity assays. For mammalian and other eukaryotic cells, intracellular lactamase activity can be monitored using a membrane-permeable fluorescent substrate. The low-cost assays using bacterial cells are especially suitable for initial high-throughput screening targeting oligomerization of Aβ and other amyloid proteins.

[0121] While certain features of the antibiotic-recombinant bacteriophage conjugates and methods of use thereof have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the disclosure.

Claims

P-620504-PC What is claimed is:

1. A system for monitoring oligomerization or fibrillization of oligomers of an aggregable biomolecule, the system comprising: a. a first fusion biomolecule comprising the biomolecule of interest bound to an N-terminus portion of an enzyme, said first fusion biomolecule having no enzyme activity; and b. a second fusion biomolecule comprising the biomolecule of interest bound to a C-terminus portion of an enzyme, said second fusion biomolecule having no enzyme activity; wherein an oligomerization of the biomolecule of interest correlates with a first measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule.

2. The system of claim 1 wherein oligomerization of the biomolecule correlates with an increase in enzymatic activity, and fibrillization of oligomers correlates with a reduction in enzymatic activity.

3. The system of claim 1 wherein an inhibitor of oligomerization inhibits the increase in enzymatic activity.

4. The system of claim 1 wherein an accelerator of fibrillization of oligomers decreases enzymatic activity.

5. The system of claim 1 wherein an agent that disaggregates oligomers and fibrils to monomers decreases activity.

6. The system of claim 1 wherein an agent that aggregates monomers to oligomers increases activity.

7. The system of claim 1 wherein an agent that disaggregates fibrils to oligomers increases activity.P-620504-PC 8. The system of any one of claims 1-7 further comprising a third fusion biomolecule comprising the biomolecule of interest bound to an enzyme comprising a full-length enzyme or homolog thereof, wherein an oligomerization of the biomolecule of interest correlates with first measurable change in enzymatic activity of third fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the third fusion biomolecule.

9. The system of claim 8 wherein the third fusion biomolecule comprises full-length enzyme or homolog thereof.

10. The system of claim 8 wherein oligomerization of the biomolecule correlates with no change in enzymatic activity of the third fusion biomolecule, and fibrillization of oligomers correlates with a reduction in enzymatic activity of the third fusion biomolecule.

11. The system of claim 8 wherein an inhibitor of oligomerization does not change the enzymatic activity of the third fusion biomolecule.

12. The system of claim 8 wherein an accelerator of fibrillization of oligomers decreases activity of the third fusion biomolecule.

13. The system of any one of claims 8-12 wherein an inhibitor of oligomerization that does not accelerate fibrillization inhibits the increase in enzymatic activity of the first and second fusion biomolecules and does not decrease enzymatic activity of the third fusion biomolecule.

14. The system of any one of claims 8-12 wherein an agent that disaggregates oligomer to monomers decreases activity of the first and second fusion biomolecules and does not change the enzymatic activity of the third fusion biomolecule.

15. The system of any one of claims 8-12 wherein an agent that disaggregates fibrils to monomers does not change the activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.P-620504-PC 16. The system of any one of claims 8-12 wherein an agent that aggregates monomers to oligomers increases activity of the first and second fusion biomolecules and does not change activity of the third fusion biomolecule.

17. The system of any one of claims 8-12 wherein an agent that disaggregate fibrils to oligomers increases activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.

18. The system of any one of claims 1-18 wherein the aggregable biomolecule is an aggregable protein.

19. The system of claim 18 wherein the aggregable protein is an amyloid protein.

20. The system of claim 19 wherein aggregation of the amyloid protein forms aqueous- soluble oligomers or aqueous-insoluble fibrils.

21. The system of claim 19 wherein the amyloid protein is wild-type Aβ42 or F19D mutant Aβ42.

22. The system of any of claims 1–21 wherein the enzyme is a β-lactamase.

23. The system of claim 22 wherein the enzymatic activity is measurable via a growth rate of a non-antibiotic-resistant Gram-negative bacteria grown in antibiotic-containing growth media.

24. The system of claim 22 wherein the enzymatic activity is measurable via β-lactamase activity.

25. The system of claim 24 wherein the β-lactamase is a TEM-1 β-lactamase.

26. The system of claim 23 wherein the antibiotic is ampicillin.

27. The system of any one of claims 1-4 wherein a. the first fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:39 and SEQ ID NO:41; andP-620504-PC b. the second fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 08, SEQ ID NO: 13, and SEQ ID NO:

43.

28. The system of any of claims 8–26 wherein the third fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 05; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 15 and SEQ ID NO:

37.

29. A method for screening oligomerization or fibrillization of oligomers of an aggregable biomolecule, the method comprising the steps of: providing a cell culture in a culture medium, the cells producing a. a first fusion biomolecule comprising the biomolecule of interest bound to an N-terminus portion of an enzyme, said first fusion biomolecule having no enzyme activity; and b. a second fusion biomolecule comprising the biomolecule of interest bound to a C-terminus portion of an enzyme, said second fusion biomolecule having no enzyme activity; measuring the activity of the enzyme, wherein an oligomerization of the biomolecule of interest correlates with a first measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the first fusion biomolecule and second fusion biomolecule.

30. The method of claim 29 wherein oligomerization of the biomolecule correlates with an increase in enzymatic activity, and fibrillization of oligomers correlates with a reduction in enzymatic activity.

31. The method of claim 29 wherein an inhibitor of oligomerization inhibits the increase in enzymatic activity.

32. The method of claim 29 wherein an accelerator of fibrillization of oligomers decreases enzymatic activity.

33. The method of claim 29 wherein an agent that disaggregates oligomers and fibrils to monomers decreases activity.P-620504-PC 34. The method of claim 29 wherein an agent that aggregates monomers to oligomers increases activity.

35. The method of claim 29 wherein an agent that disaggregates fibrils to oligomers increases activity.

36. The method of any one of claims 29-35 further providing the steps of: a. providing a cell culture in a culture medium, the cells producing a third fusion biomolecule comprising the biomolecule of interest bound to an enzyme comprising a full-length enzyme or homolog thereof, and b. measuring the activity of third fusion biomolecule, wherein an oligomerization of the biomolecule of interest correlates with first measurable change in enzymatic activity of third fusion biomolecule, and wherein fibrillization of oligomers of the biomolecule of interest correlates with a second measurable change in enzymatic activity of the third fusion biomolecule.

37. The method of claim 36 wherein the third fusion biomolecule comprises full-length enzyme or homolog thereof.

38. The method of claim 36 wherein oligomerization of the biomolecule correlates with no change in enzymatic activity of the third fusion biomolecule, and fibrillization of oligomers correlates with a reduction in enzymatic activity of the third fusion biomolecule.

39. The method of claim 36 wherein an inhibitor of oligomerization does not change the enzymatic activity of the third fusion biomolecule.

40. The method of claim 36 wherein an accelerator of fibrillization of oligomers decreases activity of the third fusion biomolecule.

41. The method of any one of claims 36-40 wherein an inhibitor of oligomerization that does not accelerate fibrillization inhibits the increase in enzymatic activity of the first and second fusion biomolecules and does not decrease activity of the third fusion biomolecule.P-620504-PC 42. The method of any one of claims 36-40 wherein an agent that disaggregates oligomer to monomers decreases activity of the first and second fusion biomolecules and does not change the enzymatic activity of the third fusion biomolecule.

43. The method of any one of claims 36-40 wherein an agent that disaggregates fibrils to monomers does not change the activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.

44. The method of any one of claims 36-40 wherein an agent that aggregates monomers to oligomers increases activity of the first and second fusion biomolecules and does not change activity of the third fusion biomolecule.

45. The method of any one of claims 36-40 wherein an agent that disaggregate fibrils to oligomers increases activity of the first and second fusion biomolecules and increases activity of the third fusion biomolecule.

46. The method of any one of claims 29-45 wherein the aggregable biomolecule is an aggregable protein.

47. The method of claim 46 wherein the aggregable protein is an amyloid protein.

48. The method of claim 47 wherein aggregation of the amyloid protein forms aqueous- soluble oligomers or aqueous-insoluble fibrils.

49. The method of claim 47 wherein the amyloid protein is wild-type Aβ42 or F19D mutant Aβ42.

50. The method of any of claims 29–49 wherein the enzyme is a β-lactamase.

51. The method of any one of claims 29-50 wherein the cultured cells are non-antibiotic resistant Gram-negative bacteria, and the growth media contains a β-lactam-containing antibiotic.

52. The method of any one of claims 29-51 wherein the enzymatic activity is measurable via a growth rate of the cells.

53. The method of any one of claims 29-50 wherein the enzymatic activity is measurable via β-lactamase activity of whole-cell lysate.P-620504-PC 54. The method of any one of claims 29-53 wherein the β-lactamase is a TEM-1 β- lactamase.

55. The method of claim 51 wherein the antibiotic is ampicillin.

56. The method of any one of claims 29-55 wherein the first fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, and SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 39 and SEQ ID NO:41; and the second fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 08, SEQ ID NO: 13 and SEQ ID NO:

43.

57. The method of any of claims 36-56 wherein the third fusion biomolecule is a polypeptide comprising a sequence selected from the list consisting of: SEQ ID NO: 05; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 15; and SEQ ID NO:

37.

58. A polypeptide composition comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:39 or SEQ ID NO:

41.

59. A polypeptide composition consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 10, SEQ ID NO: 11; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:39 or SEQ ID NO:

41.

60. A polypeptide composition comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 08, SEQ ID NO: 13, or SEQ ID NO:

43.

61. A polypeptide composition consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 08, SEQ ID NO: 13, or SEQ ID NO:

43.

62. A polypeptide composition comprising an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 05; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 15; or SEQ ID NO:37.P-620504-PC 63. A polypeptide composition consisting of an amino acid sequence, or homolog thereof, defined by any of: SEQ ID NO: 05; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 15; or SEQ ID NO:

37.

64. An expression vector or plasmid encoding any of the polypeptides of claims 58-60.

65. An expression vector or plasmid comprising a nucleic acid sequence of any one of SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 35; SEQ ID NO: 36; SEQ ID NO:40 or SEQ ID NO:

42.

66. An expression vector or plasmid comprising a nucleic acid sequence of any one of: SEQ ID NO: 26, SEQ ID NO: 30 or SEQ ID NO:

44.

67. An expression vector or plasmid comprising a nucleic acid sequence of any one of SEQ ID NO: 25; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; or SEQ ID NO:38.

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