High-throughput drug screening method

Abstract

Provided herein are methods suitable for high-throughput multiplexing that use, in part, modified enzyme complementation assays that can be used to screen libraries of test compounds and identify compounds that inhibit the degradation of a target polypeptide of interest.

Description

[Technical Field]

[0001]

[0003] Embodiments of the present invention relate to a novel high-throughput screening method for detecting and identifying test compounds that interact with or bind to a target polypeptide of interest. The method described herein is relatively rapid and suitable for automation and multiplexing. [Overview of the project]

[0002]

[0004] The method provided herein is based in part on a modified enzyme complementation assay that requires the assembly of a nuclease acceptor and a nuclease donor, which are two components that can be assembled into a functional nuclease complex capable of cleaving a labeled nucleic acid substrate. The nuclease acceptor is prepared as a fusion protein comprising a nuclease acceptor (e.g., an S-tag acceptor peptide) and a target polypeptide of interest (e.g., a viral coat protein). In certain embodiments, the assay is carried out in the presence of a denaturing factor (e.g., heat) that denatures the test compound and the fusion protein and prevents the assembly of the active nuclease complex. When the test compound interacts with and / or binds to the target polypeptide and inhibits the denaturation of the fusion protein, the active nuclease complex is formed, and the cleavage of the labeled substrate can be detected, thereby identifying a potential drug candidate (i.e., the test compound) that interacts with the target polypeptide (e.g., a viral coat protein).

[0003]

[0005] In some embodiments, a method for determining whether a test compound can interact with a target polypeptide is described herein, comprising the steps of (a) contacting a fusion protein with (i) a test compound, (ii) a denaturing factor, (iii) a nuclease donor, and (iv) a nucleic acid substrate; and (b) detecting the amount of cleavage products produced from the nucleic acid substrate or the amount of uncleaved substrate remaining from the cleavage reaction.

[0004]

[0006] In certain embodiments, the contacting in (a) comprises contacting a cell or a cell-derived lysate with (i) a test compound, (ii) a denaturing factor, (iii) a nuclease donor, and / or (iv) a nucleic acid substrate, wherein the cell or cell lysate comprises a fusion protein. In some embodiments, the nucleic acid substrate comprises a pair of FRET labels. In some embodiments, determining the amount of the cleavage product comprises detecting the amount of a fluorescence signal emitted from the cleavage product and obtaining a data point, wherein the fluorescence signal enables identification of a target saturation dose, an apparent equilibrium dissociation constant (K D ), and a half-maximal effective concentration (EC50) for target engagement between the target polypeptide and the test compound.

[0005]

[0007] In some aspects, provided herein is a high-throughput assay comprising performing any of the methods set forth herein in a plurality of containers, wherein each container comprises a fusion protein. In some embodiments, each container comprises a different fusion protein. In some embodiments, the plurality of containers comprises at least 96 containers, at least 384 containers, or at least 1536 containers.

[0006]

[0008] In some aspects, provided herein is a kit comprising a plurality of containers, wherein a first container comprises a fusion protein, a second container comprises a nuclease donor, and a third container comprises a nucleic acid substrate.

[0007]

[0009] Certain aspects of the present technology are further illustrated in the following description, examples, claims, and drawings.

[0008]

[0010] The drawings are provided to illustrate embodiments of the present technology and are not intended to limit it. For clarity and ease of description, the drawings are not drawn to scale, and in some instances various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] [Figure 1]This is a schematic diagram illustrating one embodiment of the S-tag complementary strategy. [Figure 2] This figure shows the chemical structure of one embodiment of the optimized fluorescence-generating substrate 6-FAM-dArUdAdA-6-TAMRA, where 6-FAM refers to 6-carboxyfluorescein and 6-TAMRA refers to 6-carboxytetramethylrhodamine. A "cleavable bond" that is cleaved by active RNase S during S-tag complementation has been identified. [Figure 3] This schematic diagram illustrates fluorescence emission resulting from the cleavage of a fluorescence-generating substrate by active RNase S, which is formed during the complementation of an S protein and an S-tagged acceptor peptide. The diagram shows a pair of FRET-labeled acceptor fluorescent dyes (A) and donor fluorescent dyes (D). [Figure 4] This figure shows an immunoblot illustrating exemplary expression in HEK-293 cells, where the fusion protein comprises an S-tag acceptor peptide (S-tag) and a target polypeptide (MTH1) with or without an optional 3-amino acid linker (3aa) or 10-amino acid linker (10aa). [Figure 5] This figure shows a comparison of fluorescence signals (y-axis) from fusion proteins without linkers (MTH1-S tag) or fusion proteins with linkers of different sizes (MTH1-3aa-S tag or MTH1-10aa-S tag) as a function of temperature (x-axis). In this assay, the S tag alone (i.e., the S tag not incorporated into the fusion protein) does not generate any signal. While not theoretically bound, S-tagged peptides can be thermodynamically unstable and therefore lack a thermal fusion profile under thermal load when not incorporated into the fusion protein. This characteristic minimizes the contribution of the S tag to the fusion target protein and makes the S tag ideal for target engagement studies. [Figure 6] This figure shows the relationship between the amount of nuclease donor (S protein, x axis) and the fluorescence signal (relative light units, RLU) (y axis) for each different MTH1-S tag fusion protein construct. [Figure 7]This figure shows the results of dilution tests with different cell counts (legend), where the fluorescence signal (Y-axis) is shown as a function of time (x-axis). The cells in this assay overexpressed the MTH1-3aa-S tagged fusion protein. [Figure 8] This figure shows the results of a gradual increase in temperature (x axis) versus fluorescence signal (y axis) to identify the aggregation temperature (Tagg) of MTH1-3aa-S tags within cells. [Figure 9] This figure shows the results of gradually increasing inhibitor (test compound) concentration (x axis) versus fluorescence signal (y axis) after heating cells in Tag in the presence of the inhibitor (test compound). [Figure 10] This figure shows the results of increasing inhibitor concentration (x axis) versus protein stabilization (y axis) at 50°C. [Figure 11] This figure shows the effect of incubation time (legend) as a function of temperature (x axis) on signal separation when 50 μg of cell lysate prepared from MTH1-3aa-S tagged cells is heated. [Figure 12] This figure shows the effect of incubation time (legend) on the fluorescence signal (y axis) as a function of crizotinib (test compound and MTH1 inhibitor) concentration (x axis) when incubated with 50 μg of cell lysate prepared from MTH1-3aa-S tagged cells. [Figure 13] This figure shows the effect of incubation time (legend) on the fluorescence signal (y axis) as a function of crizotinib (test compound and MTH1 inhibitor) concentration (x axis) when incubated with 10 μg of cell lysate prepared from MTH1-3aa-S tagged cells. [Figure 14] This figure shows the effect of the amount of lysate (legend) (prepared from cells expressing the MTH1-3aa-S tag) on ​​the fluorescence signal (y axis) as a function of the crizotinib (test compound and MTH1 inhibitor) concentration (x axis) after incubation for 0.5 minutes. [Figure 15]This figure shows the effect of the amount of lysate (legend) (prepared from cells expressing the MTH1-3aa-S tag) on ​​the fluorescence signal (y axis) as a function of the crizotinib (test compound and MTH1 inhibitor) concentration (x axis) after incubation for 5 minutes. [Figure 16] This figure shows the effect of the amount of lysate (legend) (prepared from cells expressing the MTH1-3aa-S tag) on ​​the fluorescence signal (y axis) as a function of the crizotinib (test compound and MTH1 inhibitor) concentration (x axis) after incubation for 10 minutes. [Figure 17] This figure shows an example of how this method can be used to generate a distributed array and display all SARS-CoV-2 virus proteins on a single plate. [Figure 18] This figure shows an example of how this method can be used to generate a columnar array and display all SARS-CoV-2 virus proteins on a single plate. [Figure 19] This figure illustrates an exemplary strategy for screening an array of pathogen proteins, for example, derived from coronaviruses. A microfluidic chip is another embodiment of the array designed for faster signal generation and high-content drug screening. [Figure 20] This figure illustrates an exemplary strategy for screening an array of peptides derived from various pathogens, showing how multiple drugs (Panel B) can be tested using a pathogen array (Panel A) (e.g., Figures 17-19) to measure the target engagement response (Panels C and D) of each protein (i.e., target polypeptide) of the arrayed pathogens. [Figure 21]This figure illustrates an exemplary strategy for screening an array of small molecule compounds (test compounds) for their ability to interact with pathogen-derived peptides, with the aim of identifying potential drug candidates for drug discovery. As shown, using an array (see, for example, Figure 20) allows for ranking viral proteins (i.e., targets) based on their overall response to denaturing factors and test compounds. Using the array, multiple drugs (test compounds) can be ranked by their engagement potency (effective dose and / or EC50, etc.). Therefore, this system makes it possible to select the most effective drug against the most responsive target of the pathogen. Existing therapeutics can also be screened using pathogen arrays, so that existing therapeutics can be repurposed, for example, to treat COVID-19. [Figure 22] This figure illustrates real-time monitoring of fluorescence signal generation over time in an assay. It shows a kinetic trace of S-tag (microtag) fluorescence signal generation over a 20-minute period. The signal kinetics were the result of the enzymatic cleavage reaction of the FRET-labeled substrate after enzymatic complementation of the nuclease donor (S protein) with the target polypeptide (MTH1) fused to the nuclease acceptor (S tag). [Figure 23A] Figures 23A–23C show exemplary thermal profiles of several microtagged (S-tagged) fusion protein constructs revealing the unique thermal signatures of such tagged proteins. Figure 23A shows that the maximum signal temperature (Tmax) of EGFR microtagged proteins under thermal load was identified as 59°C. [Figure 23B] Figures 23A–23C show exemplary thermal profiles of several microtagged (S-tagged) fusion protein constructs revealing the unique thermal signatures of such tagged proteins. Figure 23B shows that the aggregation temperature (Tagg) of the MTH1 microtagged protein under thermal load was identified as 55°C. [Figure 23C]Figures 23A–23C show exemplary thermal profiles of several microtagged (S-tagged) fusion protein constructs revealing the unique thermal signatures of such tagged proteins. Figure 23C shows that the minimum signal temperature (Tmin) for the BCL6 microtagged protein under thermal load was identified as 46.5°C. [Figure 24] This figure shows the time-dependent exemplary fluorescence signals (relative light units (RLU) per minute) of MTH1 microtagged (S-tag) fusion proteins heated at the Tagg temperature and those not heated. In the unheated samples, the fluorescence signal rapidly developed, peaking within the first three minutes, followed by a decrease in fluorescence signal development due to depletion (cleavage) of the FRET-labeled substrate. In the heated samples, there were fewer microtagged proteins (MTH1-3aa-S-tag fusion proteins) and substrate depletion (cleavage) was slower. [Figure 25A] Figures 25A-25B show exemplary tests of BCL6-microtags (BCL6-S tag fusion proteins) using the BI-3812 inhibitor and the BI-5273 inhibitor. Microtagged BCL6 was expressed in HEK293 cells, and lysates from these cells were treated with the inhibitor (Figure 25A) BI-3812 and the inactive analog (Figure 25B) BI-5273. The samples were heated in Tmin, and after a 4-minute incubation with S protein (nuclease donor) and FRET-labeled substrate, the fluorescence signal (relative light units) was detected. [Figure 25B] Figures 25A-25B show exemplary tests of BCL6-microtags (BCL6-S tag fusion proteins) using the BI-3812 inhibitor and the BI-5273 inhibitor. Microtagged BCL6 was expressed in HEK293 cells, and lysates from these cells were treated with the inhibitor (Figure 25A) BI-3812 and the inactive analog (Figure 25B) BI-5273. The samples were heated in Tmin, and after a 4-minute incubation with S protein (nuclease donor) and FRET-labeled substrate, the fluorescence signal (relative light units) was detected. [Figure 26A]Figures 26A-26C show that the target saturation dose was identified after 15 minutes by the fluorescence peak. Figure 26A shows the 15-minute examination of the reaction shown in Figure 25A to identify the target saturation dose. [Figure 26B] Figures 26A-26C show that the target saturation dose was identified at 15 minutes by the fluorescence peak. Figure 26B shows that by excluding data points above the saturation dose, it was possible to identify the EC50 of the same target engagement as identified at the initial point (Figures 25A-25C) by fitting the sigmoid dose-response curve. [Figure 26C] Figures 26A-26C show that the target saturation dose was identified after 15 minutes by the fluorescence peak. Figure 26C shows that the apparent equilibrium dissociation constant (apparent KD) of drug binding to the protein target could also be identified by fitting the observable fluorescence signal data to a saturation binding equation (total of 1 site) using GraphPad Prism. [Figure 27A] Figures 27A–27E illustrate exemplary identification of the target saturation dose, Emax (maximum effect (maximum fluorescence signal)), EC50 of target engagement, and apparent KD. Figure 27A shows a kinetic trace of fluorescence over time at each concentration of the tested inhibitor. [Figure 27B] Figures 27A–27E show exemplary identification of the target saturation dose, Emax, EC50 of target engagement, and apparent KD. Figure 27B shows that at higher drug concentrations, the signal at the later time point (5 minutes) was lower than the signal at the earlier time point (0 minutes, the first detection at the start of the dynamics trace). This decrease in signal at higher drug concentrations resulted in a bell-shaped curve. [Figure 27C] Figures 27A–27E illustrate the exemplary identification of the target saturation dose, Emax, EC50 of target engagement, and apparent KD. Figure 27C shows a bell curve in which the saturation concentration (target saturation dose) of the drug that produced the maximum effect (Emax) was identified. [Figure 27D]Figures 27A–27E show exemplary identifications of the target saturation dose, Emax, EC50 of target engagement, and apparent KD. Figure 27D shows that the EC50 of target engagement could be determined by fitting the sigmoid dose-response curve to the initial time point data. [Figure 27E] Figures 27A–27E illustrate exemplary identification of the target saturated dose, Emax, EC50 of target engagement, and apparent KD. Figure 27E shows that a saturated binding curve can be generated by identifying the target saturated dose, and that the apparent equilibrium dissociation constant KD could be identified by nonlinear regression analysis of the curve fitting. It was shown that the EC50 of target engagement and the apparent KD were identical, and that the fluorescence readings were directly proportional to drug binding and could be used to determine the apparent affinity binding constant. [Figure 28A] Figures 28A–28C illustrate exemplary identification of the target saturation dose, Emax, EC50 of target engagement, and apparent KD of (S)-crizotinib binding to the MTH1 microtagged protein. Figure 28A shows the EC50 of target engagement as detected by fluorescence after 2 minutes of enzyme complementation reaction following heating of the MTH1 microtagged protein (MTH1-3aa-S tag fusion protein) at 55°C in the presence of gradually increasing concentrations of the inhibitor (S)-crizotinib. [Figure 28B] Figures 28A–28C illustrate the exemplary identification of the target saturation dose, Emax, EC50 of target engagement, and apparent KD of (S)-crizotinib binding to the MTH1 microtagged protein. Figure 28B shows fluorescence detection of the response after 10 minutes and bell curve fitting to identify the target saturation dose and Emax. [Figure 28C]Figures 28A–28C illustrate exemplary identification of the target saturation dose, Emax, EC50 of target engagement, and apparent KD of (S)-crizotinib binding to the MTH1 microtagged protein. Figure 28C shows saturation binding curve fitting (total 1 site) using GraphPad Prism to determine apparent KD. [Figure 29A] Figures 29A-29B illustrate exemplary denaturation and UV-inducible loss of protein drug stabilization induced by UV-B exposure. In Figure 29A, cell lysates were exposed to UV-B for the indicated time, and the aggregated insoluble fraction was removed by centrifugation. Immunoblots of the proteins GSK3 beta and actin are shown. Both proteins denatured and became insoluble with increasing exposure to UV-B. [Figure 29B] Figures 29A-29B illustrate drug stabilization of proteins in the case of exemplary denaturation and UV-induced loss of activity induced by UV-B exposure. In Figure 29B, target engagement in the presence of the GSK3 beta inhibitor (CHIR99021) is represented by the stabilization of the GSK3 beta protein after lysate exposure to UV-B for 4 minutes. Nonspecific control GAPDH was not stabilized from UV-B-induced denaturation by the GSK3 beta-specific inhibitor. [Figure 30] This figure illustrates how exemplary microwave irradiation (MWI) induces protein denaturation. Cell lysates were exposed to microwave irradiation for the indicated time, and denatured and aggregated insoluble proteins were removed by centrifugation. The soluble fraction was used for immunoblotting analysis of the protein GSK3 beta. [Modes for carrying out the invention]

[0010]

[0041] In some embodiments, this specification provides a target-independent platform for monitoring drug-target engagement within cells. In some embodiments, the platform uses a modified cell thermal shift assay (CTSA) based on the premise that heating causes proteins to begin unfolding, denaturing, and exposing buried hydrophobic regions, as well as forming insoluble aggregates. The mean temperature, arithmetic mean temperature, or absolute temperature at which protein melting and aggregation occur is T agg (or T m It is often described as follows: Thermally inducible aggregation is altered by small molecules that can indirectly interact with or directly bind to polypeptides. agg This can cause a detectable shift (called a thermal shift). In some CTSA assays, insoluble aggregated proteins are removed by centrifugation, leaving soluble proteins stabilized by small molecule interactions or binding in the soluble fraction. The amount of residual soluble protein can be determined by various protein detection methods, such as Western blotting. Such methods are often time-consuming, expensive, and labor-intensive, and require special training to perform. Therefore, they are not suitable for high-throughput drug screening. High-throughput drug discovery requires a rapid and inexpensive method that can be used to screen a large number of chemical compounds in a relatively short time to identify new drug candidates.

[0011]

[0042] This specification provides a modified enzyme complementation assay that can be used for high-throughput screening of compounds that stabilize selected target polypeptides upon exposure to heat (e.g., CTSA) or another denaturing factor (e.g., ultraviolet light, microwaves, radiation, or chemical denaturants). The modified enzyme complementation assay used herein is partially based on the assembly of (i) a nuclease donor and (ii) a nuclease acceptor, which, when assembled, form a functional nuclease complex capable of cleaving small nucleic acid substrates that have been flanked by a pair of FRET labels (e.g., FRET-labeled nucleic acid substrates). In some embodiments, the modified enzyme complementation assay used herein is partially based on the assembly of (i) a ribonuclease (RNase) donor and (ii) an S-tag acceptor peptide, which, when assembled, form a functional nuclease complex capable of cleaving a nucleic acid substrate flanked by a pair of FRET labels (e.g., FRET-labeled DNA / RNA substrates). This assay is monitored by detecting the presence or amount of the cleaved nucleic acid FRET-labeled substrate (Figure 1). This substrate yields a detectable signal when cleaved. The S-tag acceptor peptide is prepared as a fusion protein containing the S-tag acceptor peptide and the target polypeptide of interest. If the target polypeptide is denatured and aggregated in the presence of a denaturing factor, the S-tag acceptor peptide portion of the aggregated fusion protein cannot be associated with the RNase acceptor. Therefore, if the target polypeptide is denatured, the active RNase complex is not formed, and no cleavage product is detected. When a test compound interacts with a target polypeptide, and this interaction prevents the target polypeptide from denaturing, a functional RNase enzyme complex is formed, and the cleavage of the FRET-labeled DNA / RNA substrate is detected. This method can be used to identify test compounds that interact with target polypeptides of interest, such as pathogen proteins, and to identify, for example, drug candidates.The assay methods described herein can be used as a high-throughput platform for rapidly and efficiently screening libraries of test compounds.

[0012]

[0043] In some embodiments, the assay fusion protein can be expressed in cells using a preferred method. The cells containing the fusion protein can then be brought into contact with the test compound in the presence of a denaturing factor (e.g., heat) to determine whether the test compound can prevent the denaturation of the target polypeptide. The assay method described herein offers numerous advantages over conventional screening methods. For example, (i) the assay method described herein is very rapid and can detect cleaved FRET-labeled DNA / RNA substrates in seconds to minutes; (ii) the S-tag acceptor peptide is relatively small and does not interfere with the denaturation of the larger target polypeptide portion of the fusion protein, as determined herein; (iii) this assay does not require Western blot analysis; (iv) this assay is relatively inexpensive; (v) this assay can be implemented as a multiplex assay screening hundreds or even thousands of test compounds; (vi) this assay can be monitored in real time; (vii) the assay design and real-time monitoring can lead to quantitative measurement of drug binding to the target polypeptide; and (viii) this assay is suitable for automation.

[0013] Fusion protein

[0044] In some embodiments, the method includes contacting a fusion protein with one or more of the following: a test compound, a denaturing factor, a nuclease donor, and a nucleic acid substrate. In some embodiments, the method includes contacting a fusion protein with one or more of the following: a test compound, a denaturing factor, an RNase donor, and a nucleic acid substrate flanked by a pair of FRET labels (e.g., a FRET-labeled RNA substrate). In certain embodiments, the fusion protein comprises a target polypeptide and a nuclease acceptor. In some embodiments, the nuclease acceptor is an S-tag acceptor domain. In some embodiments, the fusion protein comprises a target polypeptide and an S-tag acceptor peptide.

[0014]

[0045] The fusion protein may contain one or more or two or more nuclease acceptors. In some embodiments, the fusion protein may contain one or more or two or more S tags. Any suitable target polypeptide of interest can be used in the methods herein. The target polypeptide and nuclease acceptors (e.g., S-tag acceptor peptides) can be attached by suitable covalent bonds or linkers. Non-limiting examples of linkers include one or more amino acids, peptide linkers, alkanes, PEGs, optionally substituted C1-C50 alkyls, optionally substituted C2-C50 alkenyls, alkynyls, acyls, acyloxys, alkoxys, aryloxys, cycloalkyls, cycloalkenyls, cycloalkoxys, aryls, aminocarbonyls, azides, carboxys, silanes, thiols, sulfoxides, sulfones, sulfonic acid esters, cyanos, amides, aminos, esters, phosphonic acids, other suitable polymers, derivatives thereof, and combinations thereof. In some embodiments, the linker contains a peptide comprising two or more amino acids, 2 to 100 amino acids, 5 to 100 amino acids, 2 to 50 amino acids, 5 to 50 amino acids, 2 to 25 amino acids, 5 to 25 amino acids, 2 to 20 amino acids, 5 to 20 amino acids, 2 to 10 amino acids, or 5 to 10 amino acids. In some embodiments, the linker contains a peptide comprising 1 to 20, 1 to 10, or 1 to 5 amino acids. In some embodiments, the fusion protein is assembled by attaching the target polypeptide to a nuclease acceptor (e.g., an S-tag acceptor peptide) using, for example, a suitable linking chemistry. In some embodiments, the fusion protein is expressed using a suitable expression system as a single continuous polypeptide comprising a nuclease acceptor (e.g., an S-tag acceptor peptide) and the target polypeptide. In some embodiments, the target polypeptide is attached to the C-terminus of the nuclease acceptor (e.g., an S-tag acceptor peptide).In some embodiments, the target polypeptide is attached to the N-terminus of a nuclease acceptor (e.g., an S-tagged acceptor peptide).

[0015]

[0046] In some embodiments, the fusion protein comprises a target polypeptide and a nuclease acceptor. In some embodiments, the fusion protein comprises a target polypeptide and a nuclease. In some embodiments, the fusion protein comprises a target polypeptide, the N-terminal domain of a nuclease, and a first domain that enables dimerization of the N-terminal domain with the C-terminal domain of the same nuclease, wherein the C-terminal domain is fused to a second domain complementary to the domain that enables dimerization. In some embodiments, the nuclease acceptor is an S-tagged donor peptide, and the nuclease is an S protein. In some embodiments, a split Cas9 system can be designed in which the nuclease domain and helix domain are independently cloned and expressed, and then complemented in a controlled reaction. This allows for greater control of the nuclease activity of the Cas9 enzyme. These domains or any of their subdomains can be fused with a target polypeptide and then complemented in a cell target engagement system (Wright AV et al., "Rational design of a split-Cas9 enzyme complex." Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 10 (2015): pp. 2984-2989. doi:10.1073 / pnas.1501698112). In some embodiments, the nuclease is selected from the group consisting of Cas9, Micrococcal nuclease, RNase H, unnatural nuclease hybrids such as Cas9-Fok1, and Cpf1 / Cas12a. In some embodiments, the nuclease is Cas9, with the first domain enabling dimerization being Coh2 and the second domain being DocS. Coh2 and DocS are two C. thermocellum proteins that interact with each other with high affinity.A Coh2-DocS complementary system can be designed in which one of these proteins, or one of their domains or subdomains, is fused to a target polypeptide and complements the rest of the Coh2-DocS complex in a cell target engagement system (Yu Y et al. Engineering a far-red light-activated split-Cas9 system for remote-controlled genome editing of internal organs and tumors. Sci Adv. 2020; vol. 6 (no. 28): eabb1777. Published July 10, 2020. doi:10.1126 / sciadv.abb1777).

[0016]

[0047] In some embodiments, the signal regulator is any optional compound or stimulant that can control the enzymatic activity of the complementary active enzyme, the initiation of the target engagement reaction, the rate of this reaction, and / or the duration / maturity of this reaction. In some embodiments, the signal regulator is an antibody, a chemical, a peptide, temperature, UV, microwave, or light. In some embodiments, the signal regulator is far-red light. In some embodiments, binding of the Coh2 domain and the DocS domain is enabled by the signal regulator, in which case the signal regulator is far-red light.

[0017]

[0048] The fusion protein may contain one or more linkers. In some embodiments, the fusion protein contains one or more linkers between the target polypeptide and the nuclease acceptor domain. In some embodiments, the fusion protein contains one or more linkers between the target polypeptide and the nuclease. In some embodiments, the fusion protein contains one or more linkers between the target polypeptide, the N-terminal domain of the nuclease, and a first domain that enables dimerization of the N-terminal domain with the C-terminal domain of the same nuclease, wherein the C-terminal domain is fused to a second domain complementary to the domain that enables dimerization.

[0018] Nuclease

[0049] The method described herein partially relies on the assembly of a functional nuclease enzyme complex, wherein the N-terminal domain of a first nuclease enzyme and the C-terminal domain of a second nuclease enzyme assemble to form an active enzyme complex capable of cleaving nucleic acid substrates. The method described herein partially relies on the assembly of a functional RNase enzyme complex, wherein the N-terminal domain of a first RNase enzyme and the C-terminal domain of a second RNase enzyme assemble to form an active enzyme complex capable of cleaving FRET-labeled RNA or DNA / RNA substrates. In some embodiments, a preferred RNase may (i) have an N-terminal portion of the protein separated from the C-terminal portion of the protein, (ii) have isolated N-terminal and C-terminal portions that lack enzymatic activity, and (iii) have a domain structure such that the isolated N-terminal and C-terminal portions of the RNase can self-assemble non-covalently to form a functional RNase enzyme. Non-limiting examples of suitable RNase proteins that can be used in the methods described herein include bovine RNase A (acceptance number AAB35594; UniprotKB P61823)); human RNase A (NCBI acceptance number NP_002924.1); chimpanzee RNase A (NCBI acceptance number XP_520673.1); canine RNase A (NCBI acceptance number XP_532618.2); mouse RNase A (NCBI acceptance number NP_035401.2); rat RNase A (NCBI acceptance number XP_223969.2); their homologs, etc., and their derivatives having RNase activity. In some embodiments, the RNase is bovine pancreatic RNase A (e.g., UniProtKB P61823) or a derivative thereof having the following mature protein sequence: KETAAAKFERQHMDSSTSA ASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV (Sequence ID 21), the underlined portion represents the S-tag peptide sequence of the protein.

[0019]

[0050] In some embodiments, the nuclease is a ribonuclease or a deoxyribonuclease. In some embodiments, the nuclease is a ribonuclease. In some embodiments, the nuclease is a deoxyribonuclease.

[0020]

[0051] In some embodiments, the nuclease is a sequence-specific nuclease. In some embodiments, the nuclease is a clustered, regularly interspaced short palindromic repeat (CRISPR) related protein (i.e., a Cas protein). In some embodiments, the Cas protein is Cas9 (Csn1), Cas12a (Cpf1), Cas12b (C2c1), Cas13a (C2c2), Cas13b (C2c6), or Cas13c (C2c7). In some embodiments, the Cas protein is Cas9.

[0021]

[0052] In some embodiments, the nuclease is a non-natural nuclease hybrid. In some embodiments, the non-natural nuclease hybrid is Cas9-Fok1.

[0022]

[0053] In some embodiments, the nuclease is an RNase. In some embodiments, the RNase is RNase A, RNase H, or RNase S.

[0023]

[0054] In some embodiments, the nuclease is a micrococcal nuclease.

[0024] Nuclease donor

[0055] In some embodiments, the nuclease donor is a nuclease. In some embodiments, the nuclease donor comprises or consists of the C-terminal portion of a preferred nuclease. In some embodiments, the nuclease donor is an RNase donor. In some embodiments, the RNase donor comprises or consists of the C-terminal portion of a preferred RNase. In some embodiments, the RNase donor is an RNase S protein. In some embodiments, the RNase donor is the S protein of an RNase S complex. In some embodiments, RNase S is a complex comprising two proteolytic fragments of RNase A. The RNase donor (e.g., the RNase S protein) can be prepared using preferred methods. In one non-limiting example, the RNase donor is prepared by treating RNase A with subtilisin, which, under appropriate conditions, cleaves a single peptide bond of the RNase, thereby yielding an N-terminal portion (i.e., the S peptide, e.g., about 15-25 amino acids) and a C-terminal portion (i.e., the S protein, e.g., about 90-120 amino acids). In another non-limiting example, RNase donors are prepared using recombinant techniques so that the C-terminal (S protein) portion of the RNase is expressed using a suitable expression system. The isolated RNase donor is substantially devoid of enzymatic activity (e.g., RNase activity) until it comes into contact with a suitable S-tagged acceptor peptide.

[0025]

[0056] In some embodiments, the RNase donor is an amino acid sequence MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV (Sequence ID 19) or MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFD(Sequence ID 20) The amino acid sequence has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or 100% identity. In some embodiments, the RNase donor comprises a polypeptide containing at least 85, at least 90, at least 95, or at least 100 consecutive amino acids of the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20.

[0026]

[0057] Derivatives of nuclease donors may contain conservative amino acid substitutions or amino acid analogs. In some embodiments, the nuclease donor contains a suitable amino acid tag (e.g., a histidine tag or a flag tag). In some embodiments, derivatives of RNase donors may contain conservative amino acid substitutions or amino acid analogs. In some embodiments, the RNase donor contains a suitable amino acid tag (e.g., a histidine tag or a flag tag).

[0027] Nuclease acceptor

[0058] In some embodiments, a nuclease acceptor or nuclease acceptor domain is a domain that enables the assembly of a functional nuclease enzyme complex, where the N-terminal domain of a first nuclease and a second nuclease or the C-terminal domain of the same first nuclease assemble to form an active enzyme complex capable of cleaving nucleic acid substrates. In some embodiments, the nuclease acceptor is an acceptor peptide containing a suitable S tag. Nuclease acceptor peptides often contain or consist of a relatively small N-terminal portion of a nuclease protein. S-tagged acceptor peptides often contain or consist of a relatively small N-terminal portion of an RNase protein. When an S-tagged peptide is non-covalently associated with an RNase donor (e.g., an S protein), it confers RNase enzyme activity. Any suitable combination of S-tagged acceptor peptide and RNase donor can be used in the method herein. Various combinations of S-tagged acceptor peptide and RNase donor can be readily tested for use in the method herein without requiring excessive experimental work. S-tagged acceptor peptides derived from one species often form functional enzyme complexes in association with RNase donors derived from another species. In some embodiments, derivatives of S-tagged peptides and / or derivatives of RNase donors can be used in the methods described herein.

[0028]

[0059] In some embodiments, the length of the nuclease acceptor peptide is in the range of 10–60 amino acids, 10–40 amino acids, 15–30 amino acids, 15–25 amino acids, 10–25 amino acids, or 8–25 amino acids. In some embodiments, the nuclease acceptor peptide contains, consists of, or is essentially composed of about 15–25 amino acids. In certain embodiments, the nuclease acceptor peptide does not have a detectable secondary structure. In certain embodiments, the nuclease acceptor peptide is highly soluble. In certain embodiments, the nuclease acceptor peptide has no net charge at neutral pH.

[0029]

[0060] In some embodiments, the length of the S-tag acceptor peptide is in the range of 10–60 amino acids, 10–40 amino acids, 15–30 amino acids, 15–25 amino acids, 10–25 amino acids, or 8–25 amino acids. In some embodiments, the S-tag acceptor peptide contains, consists of, or is essentially composed of about 15–25 amino acids. In certain embodiments, the S-tag acceptor peptide does not have a detectable secondary structure. In certain embodiments, the S-tag acceptor peptide is highly soluble. In certain embodiments, the S-tag acceptor peptide has no net charge at neutral pH.

[0030]

[0061] In some embodiments, the S-tag acceptor peptide is KETAAAKFERQHMDSSTSAA (SEQ ID NO: 1), KETNWAWFWDQHMDSSTSA (SEQ ID NO: 2), KETGWALFVQQHMDSSTSA (SEQ ID NO: 3), KETVMANFQMQHMDSSTSA (SEQ ID NO: 4), KETGDAVFARQHMDSSTSA (SEQ ID NO: 5), KETGWAAFVKQHMDSSTSA (SEQ ID NO: 6), KETGWATFVEQHMDSSTSA (SEQ ID NO: 7), KETKLAFFLKQHMDSSTSA (SEQ ID NO: 8), KETWWAWFFGQHMDSSTSA (SEQ ID NO: 9), KETTWAEFTWQHMDSSTSA (SEQ ID NO: 10), KETPWASFNKQHMDSSTSA ( Peptides and derivatives thereof having amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or 100% identical to amino acid sequences selected from SEQ ID NO: 11), KETAMAMFVTQHMDSSTSA (SEQ ID NO: 12), KETLWAWFMWQHMDSSTSA (SEQ ID NO: 13), KETAAAKFERQHMDS (SEQ ID NO: 14), KETAAAKFERQHMNS (SEQ ID NO: 15), NRAWSEFLWQHLAPV (SEQ ID NO: 16), NRGWSEFLWQHHAPV (SEQ ID NO: 17), and NRAWSVFQWQHIAPA (SEQ ID NO: 18). In some embodiments, the S-tag acceptor peptide contains at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 consecutive amino acids from an amino acid sequence and its derivatives selected from SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, SEQ ID NOs: 12, SEQ ID NOs: 13, SEQ ID NOs: 14, SEQ ID NOs: 15, SEQ ID NOs: 16, SEQ ID NOs: 17, and SEQ ID NOs: 18.In some embodiments, the S-tag acceptor peptide is the S-peptide disclosed in Backer et al. (2002) Protein Expression and Purification Vol. 26: pp. 455-461; Dwyer et al. (2001) Biochemistry Vol. 40 (No. 45): pp. 13491-500; Kim and Raines (1993) Protein Science Vol. 2: pp. 348-356; and Beintema, JJ (1987) Life Chem. Rep. Vol. 4: pp. 333-389. These documents are incorporated herein by reference. Derivatives of the S-tag acceptor peptide may include conservative amino acid substitutions or amino acid analogs. In certain embodiments, the S-tag acceptor peptide can be prepared and / or selected using the methods described in Yu and Smith (1996) Methods in Enzymology Vol. 267, pp. 3–27; and Goldberg et al. (1999) PNAS Vol. 96: pp. 2019–2024.

[0031] Targeted polypeptide

[0062] The fusion protein may contain a preferred target polypeptide. In some embodiments, the target polypeptide has a length in the range of 10 to 1000, 10 to 500, 10 to 250, 10 to 125, or 10 to 50 amino acids. In certain embodiments, the target polypeptide comprises a polypeptide or portion thereof derived from a preferred pathogen, non-limiting examples of which include viruses, bacteria, fungi, and parasites. Non-specific examples of viruses include the families Adenoviridae, Papovaviridae, Parvoviridae, Herpesviridae, Poxviridae, Anelloviridae, Pleolipoviridae, Reoviridae, Picornaviridae, Caliciviridae, Togaviridae, Arenaviridae, and Flaviviridae. Examples include the families Orthomyxoviridae (e.g., influenza virus), Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae, Coronaviridae (e.g., SARS, SARS-CoV-2, MERS, HKU1), Astroviridae, Bornaviridae, Arteriviridae, Rotavirus, and Hepeviridae. In certain embodiments, the virus is the SARS-CoV-2 coronavirus. In certain embodiments, the virus is the influenza virus. In certain embodiments, the virus is the hepatitis A, B, or C virus. In certain embodiments, the virus is the herpesvirus. In some embodiments, the pathogen is a bacterium.In certain embodiments, the bacteria are Helicobacter pylori, Mycobacterium tuberculosis, or Mycobacterium.

[0032]

[0063] In some embodiments, the target polypeptide is modified. Non-limiting examples of polypeptide modification include the substitution, deletion, or addition of one or more amino acids. For example, in some embodiments, the method herein is carried out as a multiplex assay or high-throughput assay using multiple containers (e.g., microtiter wells), where each well contains a different fusion protein, and each fusion protein contains a different target polypeptide. The different target polypeptides may be different proteins and / or modifications of the target polypeptide. In one non-limiting example, the target polypeptide is a viral capsid protein (e.g., the spike protein of SARS-CoV-2, or the hemagglutinin protein of the influenza virus), and each container or microtiter well contains a different modification of the viral capsid protein (e.g., a random mutation or a computer-generated mutation).

[0033]

[0064] In some embodiments, the target polypeptide is a native polypeptide or a portion thereof. In some embodiments, the target polypeptide is synthetic. In some embodiments, the target polypeptide is naturally occurring or recombinantly produced. In some embodiments, the target polypeptide comprises a protein or a portion of a protein that has been isolated, purified, and / or recombinantly expressed as a soluble protein (e.g., isolated, purified, or expressed as a soluble fusion protein). In some embodiments, the target polypeptide or fusion protein is expressed intracellularly or on the surface of a cell. In some embodiments, the target polypeptide or fusion protein is expressed on the surface of a cell. In certain embodiments, the cell is a preferred eukaryotic cell (e.g., a mammalian cell). In certain embodiments, the cell is a prokaryotic cell (e.g., a bacterium).

[0034]

[0065] In some embodiments, the target polypeptide is mutT homolog 1 (MTH1). In some embodiments, the nuclease acceptor is an S-tagged peptide. In some embodiments, there is no linker between MTH1 and the S-tagged peptide. In some embodiments, the amino acid sequence of the MTH1-S-tagged construct is shown in SEQ ID NO: 25.

[0035] Test compound

[0066] The method described herein can be used to screen any suitable test compound or library of test compounds.

[0036]

[0067] As used herein, the term “test compound” refers to any suitable compound that can be screened for its ability to interact with or bind to a target polypeptide of interest using the methods described herein. Non-limiting examples of test compounds include small compounds (e.g., small organic or inorganic molecules), large compounds (e.g., larger than 5000 Da), polysaccharides, carbohydrates, sugars, fatty acids, lipids, biological macromolecules (e.g., peptides, polypeptides, proteins, peptide analogs and derivatives, peptide mimetic compounds, nucleic acids, nucleotides, nucleotide analogs), natural or synthetic compounds, binders (e.g., antibodies), or non-natural and synthetic binders (e.g., TandAb, nanobodies, aptamers, BiTE, SMIP, DARPin, DNL, ​​aphibodies, Duocalin, adnectin, fynomers, Knitz domains). Examples include Albu-dab, DART, DVD-IG, Covx-body, peptidebody, scFv-Ig, SVD-Ig, dAb-Ig, Knob-in-Hole, and triomAb, etc., their binding fragments, their derivatives, their polymers, their salts, their isomers, their polymorphs, and combinations thereof. In some embodiments, the test compound is contained in an extract made from a biological material, such as an extract of bacteria, plants, fungi, animal cells, or animal tissue. In some embodiments, the test compound is contained in a biological liquid. Therefore, in some embodiments, the test compound comprises an extract or a biological liquid. Examples of small compounds include molecules with a molecular weight greater than about 40 Daltons (Da) but less than 5000 Da, less than 3000 Da, or less than 1000 Da. The miniature compounds may contain any suitable chemical moieties or groups, non-limiting examples of which include alkanes, alkenes, alkynes, alcohols, halogens, ketones, aldehydes, carboxylic acids, ethers, esters, amines, amides, saturated, partially saturated, or unsaturated ring structures, nucleotides, nucleosides, polyatomic nonmetals (e.g., P, S, Se), transition metals, post-transition metals, metalloids, salts thereof, and combinations thereof.

[0037]

[0068] In certain embodiments, test compounds may include synthetic or natural compounds of a suitable library. Numerous small molecule libraries are known in the art, some of which are commercially available. Commercially available compound libraries can be found, for example, from ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, Biomol International, and Oxford. Methods for developing small molecule, polymer, and genome-based libraries are described, for example, in Ding et al., J Am. Chem. Soc. Vol. 124: pp. 1594-1596 (2002) and Lynn et al., J. Am. Chem. Soc. Vol. 123: pp. 8155-8156 (2001). Compound libraries from the NIH Roadmap and the Molecular Library Screening Center Network (MLSCN) can also be used, for example. Small compound libraries can be prepared using any suitable method. Compound libraries can be screened using the suitable methods described herein.

[0038]

[0069] In certain embodiments, the test compounds include molecular weights of 40-500,000 Da, 40-200,000 Da, 40-100,000 Da, 40-50,000 Da, 40-25,000 Da, 40-10,000 Da, 40-5000 Da, or 40-1000 Da. In certain embodiments, the test compounds include molecular weights of 5000-500,000 Da, 10,000-500,000 Da, 25,000-500,000 Da, or 5000-100,000 Da.

[0039]

[0070] The test compound can be tested at any preferred concentration. In some embodiments, the test compound is tested at concentrations of at least 1 pM, at least 10 pM, at least 100 pM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1 μM, at least 10 μM, at least 100 μM, or at least 1 mM. In some embodiments, the test compound is tested at concentrations in the range of 1 pM to 100 mM, 1 pM to 10 mM, 1 pM to 1 mM, 10 pM to 100 mM, 10 pM to 10 mM, 10 pM to 1 mM, 100 pM to 100 mM, 100 pM to 10 mM, or 100 pM to 1 mM. In some embodiments, the test compound is tested at concentrations less than 100 mM, less than 10 mM, less than 1 mM, or less than 100 nM. In some embodiments, the test compound is tested or assayed at one or more different concentrations.

[0040] substrate

[0071] In some embodiments, a fusion protein and / or nuclease donor, or a mixture or cell containing a fusion protein and a nuclease donor, is contacted with a suitable nucleic acid substrate. In some embodiments, a fusion protein and / or RNase donor, or a mixture or cell containing a fusion protein and an RNase donor, is contacted with a suitable nucleic acid substrate. In some embodiments, the nucleic acid substrate is a nucleic acid that can be cleaved by a nuclease disclosed herein or by an assembled nuclease enzyme complex described herein. In some embodiments, the nucleic acid substrate is a nucleic acid that can be cleaved by an RNase disclosed herein or by an assembled RNase enzyme complex described herein (e.g., including an S-tag acceptor peptide and an RNase donor). In some embodiments, the nucleic acid substrate includes RNA and / or DNA. In some embodiments, the nucleic acid substrate includes ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The nucleic acid substrate may be single-stranded or double-stranded. In certain embodiments, the nucleic acid substrate includes two or more, three or more, five or more, or ten or more nucleotides. In certain embodiments, the nucleic acid substrate comprises at least one pyrimidine nucleotide. In certain embodiments, the nucleic acid substrate comprises at least two, at least three, or at least four adjacent pyrimidine nucleotides. In some embodiments, the fusion protein comprises a nuclease acceptor domain in contact with a nuclease or nuclease donor, the nuclease or nuclease donor being a ribonuclease such as Cas9 or Cpf1. In some embodiments, the ribonuclease, e.g., Cas9 or Cpf1, is brought into contact with a guide RNA. In some embodiments, the guide RNA (gRNA) directs the ribonuclease to the target nucleic acid substrate.

[0041]

[0072] In certain embodiments, the nucleic acid substrate includes a suitable detectable label. In certain embodiments, the detectable label of the substrate results in a detectable signal, a change in the detectable signal (e.g., an increase or decrease in the signal or a wavelength shift), or loss of the detectable signal upon cleavage of the labeled substrate by a nuclease (e.g., RNase). In some embodiments, the detectable signal emitted from the nucleic acid substrate label is undetectable until after substrate cleavage. In some embodiments, the detectable signal emitted from the nucleic acid substrate label is enhanced after substrate cleavage. In some embodiments, the detectable signal emitted from the nucleic acid substrate label is reduced after substrate cleavage.

[0042]

[0073] Non-limiting examples of detectable labels include metallic labels, fluorescent labels, fluorescent proteins (e.g., green fluorescent protein (GFP)), pH-sensitive proteins or pH-sensitive GFP (e.g., PHLourin), any suitable fluorophore (e.g., mCherry), chromophores, chemiluminescent labels, electrochemiluminescent labels (e.g., Origen®), phosphorescent labels, quenchers (e.g., fluorophore quenchers), fluorescence resonance energy transfer (FRET) pairs (e.g., donor and acceptor), proteins (e.g., enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, and alkaline phosphatase)), antigens or parts thereof, linkers, members of binding pairs), enzyme substrates, small molecules (e.g., biotin, avidin), mass tags, quantum dots, nanoparticles, or combinations thereof. Any suitable fluorophore or luminescent substance can be used as a detectable label. Non-limiting examples of fluorescent labels include fluorescein, rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2,7-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM), or cyanine dyes such as N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), Cy3, Cy5, Alexa542, Bodipy630 / 650, fluorescent particles, fluorescent semiconductor nanocrystals, and combinations thereof. Detectable labels can be detected and / or quantified by a variety of suitable techniques, such as digital photography, flow cytometry, gel electrophoresis, chip analysis (e.g., any chip methodology), microarrays, mass spectrometry, cell fluorescence analysis, fluorescence microscopy, confocal laser scanning microscopy, laser scanning cytometry, suitable plate readers, and combinations thereof.

[0043]

[0074] In some embodiments, the nucleic acid substrate includes a preferred fluorescent energy transfer (FRET) label. One advantage of FRET-labeled nucleic acids is that they can readily enter intact cells, allowing for detection of substrate cleavage in complete cells. In certain embodiments, the nucleic acid substrate includes a pair of FRET labels comprising a preferred fluorescent donor / acceptor pair separated by a polynucleotide containing an RNase-cleavable sequence, such that the donor's fluorescence emission is quenched until the substrate is cleaved by an assembled RNase complex. In certain embodiments, the pair of FRET labels comprises 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (6-TAMRA) (Figure 2) separated by two or more consecutive nucleotides. In some embodiments, the nucleic acid substrate comprises (6-FAM)-X-(6-TAMRA), where X comprises a polynucleotide containing 2 to 10 nucleotides. In some embodiments, the nucleic acid substrate comprises 6-FAM-dArUdAdA-6-TAMRA (Figure 2), where rU is uridine and dA is deoxyadenine.

[0044]

[0075] In certain embodiments of the methods described herein, the amount of substrate cleavage is determined using a preferred method. In certain embodiments, the presence or absence of cleavage products of the nucleic acid substrate is determined using a preferred method. In some embodiments, the presence, absence, or amount of cleavage of a nucleic acid substrate labeled with a pair of FRET labels is determined using a preferred method. The presence, absence, or amount of cleavage of the labeled nucleic acid substrate can be determined at a preferred time after contacting cells or a mixture with the nucleic acid substrate or nuclease donor. The presence, absence, or amount of cleavage of the labeled nucleic acid substrate can be determined at a preferred time after contacting cells or a mixture with the nucleic acid substrate or RNase donor. In some embodiments, the presence, absence, or amount of cleavage of the labeled nucleic acid substrate is determined dynamically over a period of time, for example, to determine the cleavage rate. A predetermined amount of cleavage products can be determined using a preferred positive control. For example, the positive control may be a fusion protein containing a known protein, and a test compound known to interact with the known protein and stabilize it when exposed to a denaturing factor, thereby enabling complementation of the S-tag acceptor peptide by the RNase donor protein to form an active nuclease complex. The presence or amount of nucleic acid substrate cleaved by the positive control active nuclease complex can be used as a baseline for detecting other test compounds that interact with the target polypeptide.

[0045]

[0076] The cleavage and detection of cleavage of nucleic acid substrates labeled with a pair of FRET labels are extremely rapid (often requiring less than 30 seconds) (see, for example, Figures 11-13). Therefore, in certain embodiments, FRET-labeled substrates are used in the high-throughput methods described herein, enabling automation of the methods described herein.

[0046] Degenerative factors

[0077] In some embodiments, the fusion protein is brought into contact with a denaturing agent. Non-limiting examples of denaturing agents include (i) heat, (ii) ultraviolet light, (iii) microwaves, (iv) radiation, and (iv) chemical denaturing agents.

[0047]

[0078] In certain embodiments, contacting a fusion protein with a denaturing factor includes contacting the fusion protein, or cells or mixtures containing the fusion protein, with heat. In some embodiments, the fusion protein is contacted with an amount of heat sufficient to denaturate and / or aggregate the fusion protein. In certain embodiments, contacting a fusion protein with heat includes heating the fusion protein, or cells or mixtures containing the fusion protein, to temperatures in the range of 40°C to 90°C, 40°C to 80°C, 40°C to 75°C, 45°C to 75°C, 50°C to 75°C, or 55°C to 70°C. In certain embodiments, contacting a fusion protein with heat includes heating the fusion protein, or cells or mixtures containing the fusion protein, to temperatures of at least 40°C, at least 50°C, at least 60°C, at least 60°C, at least 65°C, or at least 70°C. In certain embodiments, contacting a fusion protein with heat includes heating the fusion protein, or cells or mixtures containing the fusion protein, to temperatures from about 30°C to 40°C to about 50°C to 70°C. In certain embodiments, contacting the fusion protein with heat includes exposing the fusion protein or cells or mixtures containing the fusion protein to a temperature gradient in the range of 30°C to 90°C, 30°C to 80°C, 30°C to 75°C, 37°C to 75°C, or 37°C to 70°C. In some embodiments, contacting the fusion protein with a heat gradient includes increasing the temperature of the fusion protein or cells or mixtures containing the fusion protein at a rate of at least about 1°C to 10°C per minute or about 1 to 5°C per minute. In some embodiments, contacting the fusion protein with heat includes exposing the fusion protein or cells or mixtures containing the fusion protein to heat or a gradually increasing temperature gradient for a period of at least 30 seconds, at least 1 minute, or at least 30 seconds, at least 3 minutes, or at least 5 minutes.

[0048]

[0079] In certain embodiments, the fusion protein is brought into contact with or exposed to a denaturing factor for a period of time. In some embodiments, the fusion protein is brought into contact with or exposed to a denaturing factor for a period of at least 20 seconds, at least 30 seconds, at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, or at least 30 minutes.

[0049]

[0080] In certain embodiments, contacting the fusion protein with a denaturing factor includes subjecting the fusion protein or cells or mixtures containing the fusion protein to one or more freeze-thaw cycles.

[0050]

[0081] In certain embodiments, contacting a fusion protein with a denaturing factor involves exposing the fusion protein or cells or mixtures containing the fusion protein to a suitable amount of electromagnetic radiation sufficient to denature the protein, non-limiting examples of which electromagnetic radiation includes ultraviolet light (e.g., Figures 29A and 29B), microwaves (e.g., Figure 30), or radiation (e.g., beta rays or gamma rays). In some embodiments, protein denaturation occurs for 5 minutes or 0.1 joules / cm². 2 This can be carried out using 250nm UV light.

[0051]

[0082] In certain embodiments, contacting a fusion protein with a denaturing factor includes contacting the fusion protein or cells or mixtures containing the fusion protein with a denaturing agent, not limited to oxidizing agents, but including oxidizing agents, toxins, acids, bases, carcinogens, and chemotherapeutic agents. Simple routine tests can be performed to quickly determine the amount of denaturing agent required to denaturate the fusion protein. In some embodiments, the fusion protein or cells or mixtures containing the fusion protein are contacted with a denaturing agent in the presence of a test compound, and after substantially removing the denaturing agent (e.g., by incorporating a washing step), the fusion protein is contacted with a nuclease donor (e.g., an RNase donor protein). In certain embodiments, contacting a fusion protein with a denaturing agent includes contacting the fusion protein or cells or mixtures containing the fusion protein with a denaturing agent at concentrations in the range of 1 fM to 500 mM, 1 pM to 100 mM, 1 nM to 10 mM, 1 nM to 1 mM, or 1 nM to 100 μM.

[0052] Exemplary Method

[0083] In some embodiments, the fusion protein is brought into contact with the test compound and the denaturing factor. In some embodiments, an isolated fusion protein is brought into contact with the test compound and / or the denaturing factor. In some embodiments, a fusion protein present in or on a cell or in a mixture (e.g., a cell lysate) is brought into contact with the test compound and / or the denaturing factor. In some embodiments, the fusion protein is brought into contact with the test compound before or after contacting the denaturing factor. In some embodiments, the fusion protein is brought into contact with the test compound and the denaturing factor at the same time or substantially simultaneously. In some embodiments, the fusion protein is brought into contact with the test compound and the denaturing factor at the same time. In some embodiments, the fusion protein is brought into contact with the test compound and the denaturing factor simultaneously. In some embodiments, the fusion protein is brought into contact with the test compound and the denaturing factor substantially simultaneously (e.g., the fusion protein is brought into contact with the test compound and the denaturing factor consecutively within minutes of a previous contact). In some embodiments, the fusion protein is expressed in or on a cell (e.g., on the cell surface) and the cell is brought into contact with the test compound and / or the denaturing factor.

[0053]

[0084] In certain embodiments, the method includes the step of contacting cells with a nucleic acid that encodes or induces the expression of a fusion protein. For example, in some embodiments, the method includes transfecting or transforming cells with a nucleic acid (e.g., a vector) that encodes or induces the expression of a fusion protein, and subsequently contacting the cells or cell lysates with a test compound, a denaturing factor, a nuclease donor (e.g., an RNase donor), and / or a nucleic acid substrate. In certain embodiments, the method includes the step of introducing the nucleic acid that encodes or induces the expression of a fusion protein into cells using a preferred method. For example, the nucleic acid may be introduced into eukaryotic cells using a viral vector, or into bacterial cells using a phage.

[0054]

[0085] In some embodiments, the fusion protein is brought into contact with a ribonuclease (RNase) donor and / or a nucleic acid substrate. In certain embodiments, a mixture containing the fusion protein, a test compound, and / or a denaturing factor is brought into contact with the RNase donor and / or a nucleic acid substrate. In some embodiments, the fusion protein, which is present in or on a cell or in a mixture (e.g., a cell lysate, e.g., a mixture containing the fusion protein, a test compound, and / or a denaturing factor), is brought into contact with the RNase donor and / or a nucleic acid substrate. In some embodiments, the fusion protein is brought into contact with the RNase donor before or after contacting the fusion protein with the nucleic acid substrate. In some embodiments, the fusion protein is brought into contact with the RNase donor and the nucleic acid substrate at the same time or substantially simultaneously.

[0055]

[0086] In certain embodiments, cells containing a fusion protein are brought into contact with a denaturing factor and a test compound, the denaturing factor is optionally removed or eliminated, the cells are exposed to a nuclease donor and a nucleic acid substrate, and the cleavage of the nucleic acid substrate is detected or quantified. In certain embodiments, cells containing a fusion protein are brought into contact with a denaturing factor and a test compound, the denaturing factor is optionally removed or eliminated, the cells are exposed to an RNase donor and a nucleic acid substrate, and the cleavage of the nucleic acid substrate is detected or quantified. In some embodiments, a mixture or cells containing a fusion protein are brought into contact substantially simultaneously with a test compound, an RNase donor, a nucleic acid substrate, and a denaturing factor to detect and / or quantify the cleavage of the substrate. For example, cells can be recombinantly produced to express a fusion protein, an RNase donor, and / or a nucleic acid substrate, their expression being controlled in some embodiments by one or more inducible promoters. The cells or their lysate are then brought into contact with the test compound and the denaturing factor, for example by adding the test compound and applying heat, while monitoring the cleavage of the nucleic acid substrate containing a pair of FRET labels in real time. Variations of such methods are also intended herein.

[0056]

[0087] In some embodiments, during or after contacting the fusion protein with heat, the absolute aggregation temperature, average aggregation temperature, or arithmetic mean aggregation temperature (T agg ), the arithmetic mean temperature of maximum signal (T max ), or the arithmetic mean temperature of minimum signal (T min ) is determined. In some embodiments, T agg , T max , or T min is determined in the absence of a test compound. In some embodiments, T agg , T max , or T min is determined in the presence of a solvent control or a control compound (e.g., a compound known to have no effect on the T agg , T max , or T min of the fusion protein, or a compound known to increase or decrease the T agg , T max , or T min of the fusion protein). Such a control can be used to determine a threshold T agg , T max , or T min (e.g., a predetermined threshold), and in some embodiments, such a threshold is used to identify a compound that interacts with or binds to a target polypeptide. For example, in certain embodiments, a test compound that changes or shifts the T agg , T max , or T min of the fusion protein to an amount above a predetermined threshold is often identified as a test compound that interacts with or binds to the target polypeptide.

[0057]

[0088] When electromagnetic radiation, freeze-thaw, or chemical substances are used as denaturing factors, a similar method can be used to identify test compounds that interact with or bind to the target polypeptide. For example, the critical exposure time required for denaturation of the fusion protein, the concentration of the denaturing factor, the thaw temperature, wavelength, or energy can be determined in the absence of the test compound and / or in the presence of a control compound to determine a predetermined threshold. In some embodiments, any test compound that causes a shift or change in the threshold is determined to be a compound that interacts with or binds to the target polypeptide of the fusion protein.

[0058]

[0089] In certain embodiments, the method or assay described herein is carried out as a multiplex assay and / or high-throughput assay, comprising the step of carrying out the method in at least 96, at least 100, at least 384, at least 500, at least 1000, at least 1536, at least 5000, or at least 10,000 separate containers. In certain embodiments, the method or assay described herein is carried out as a multiplex assay and / or high-throughput assay, and some or all of the steps of the method are carried out substantially simultaneously or concurrently in multiple containers. In some embodiments, some or all of the multiple separate containers used in the multiplex assay and / or high-throughput assay are in contact with or contain different fusion proteins, different denaturing factors, different test compounds, different nuclease donors, and / or different nucleic acid substrates. In some embodiments, some or all of the multiple separate containers used in the multiplex assay and / or high-throughput assay are in contact with or contain different fusion proteins, different denaturing factors, different test compounds, different RNase donors, and / or different nucleic acid substrates. In some embodiments, some or all of the separate containers used in multiplex assays and / or high-throughput assays are in contact with or contain the same fusion protein, the same denaturing factor, the same test compound, the same nuclease donor, and / or the same nucleic acid substrate. “Container” as used herein refers to any suitable container, tube, or well. A container may be a well, for example, a well in a microtiter plate.

[0059]

[0090] In certain embodiments, the methods or assays described herein are carried out as multiplex and / or high-throughput assays using an array of surface-bound fusion proteins, which are often located at addressable positions on a suitable substrate (e.g., a chip). In some embodiments, the array comprises at least 20, at least 96, at least 100, at least 384, at least 500, at least 1000, at least 1536, at least 5000, or at least 10,000 different fusion proteins bound to the surface of a suitable substrate.

[0060]

[0091] The methods and assays described herein result in increased responsiveness and sensitivity compared to other types of complementary assays. The methods provided herein can detect the presence of active test compounds at levels below nanomolar (nM).

[0061]

[0092] In some embodiments, the nucleic acid substrate in the method herein comprises one or more detectable labels. In some embodiments, the nucleic acid substrate comprises one or more FRET labels. In some embodiments, the nucleic acid substrate comprises a pair of FRET labels, and the amount of cleavage products comprises detecting the amount of fluorescence signal emitted from the cleavage products and obtaining a data point, wherein the fluorescence signal is the target saturation dose between the target polypeptide and the test compound, the apparent equilibrium dissociation constant (K). D ), enabling the identification of the maximum half-dose effective concentration (EC50) for target engagement.

[0062]

[0093] In some embodiments, the target saturation dose of the test compound is determined by the peak fluorescence value (Emax) after cleavage / depletion of the nucleic acid FRET-labeled substrate in the enzymatic reaction. In some embodiments, the apparent equilibrium dissociation constant (K) between the target polypeptide and the test compound is determined. D The EC50 of the target engagement is determined by plotting the saturated binding curve, and data points exceeding Emax are excluded from the plot. In some embodiments, the EC50 of the target engagement is determined from the initial data points of the reaction where there is an excess of nucleic acid FRET-labeled substrate.

[0063] kit

[0094] In some embodiments, a kit is provided. In some embodiments, the kit comprises a plurality of containers, the first container comprising a fusion protein, the second container comprising a nuclease donor, and the third container comprising a nucleic acid FRET-labeled substrate.

[0064]

[0095] In some embodiments, the fusion protein comprises a target polypeptide and a nuclease acceptor. In some embodiments, the nuclease acceptor is an S-tagged donor peptide, and the nuclease is RNase S. [Examples]

[0065] Example 1 Materials: Reagents, cell lines, and constructs

[0096] The following antibodies were obtained from Cell Signaling Technology: rabbit mAb S-Tag(D2K2V)XP (catalog number 12774) and anti-MTH1(D6V4O) rabbit mAb (catalog number 43918). HEK-293 cells were obtained from ATCC (catalog number CRL-1573) and cultured in DMEM (Millipore SIGMA catalog number D5796) supplemented with 10% fetal bovine serum (FBS) (Millipore SIGMA catalog number F2442) and 1× penicillin / streptomycin (Millipore SIGMA catalog number P4333). Trypsin-EDTA solution 1× (0.05% trypsin, 0.02% EDTA) was obtained from Millipore SIGMA (catalog number 59417C), and 20× TBS solution was obtained from Teknova (catalog number T1680). Triton-X-100 was obtained from Millipore SIGMA (catalog number X100). MTH1 inhibitor (S)-crizotinib (crizotinib) was obtained from Millipore SIGMA (catalog number PZ0240). DMSO was obtained from Millipore SIGMA (catalog number 673439). 96-well plates were obtained from CELLTREAT Scientific (catalog number 229196), white PCR plates from BIORAD (catalog number MLL9651), Microseal "B" film for sealing PCR plates from BIORAD (catalog number MSB1001), and black 96-well plates from COSTAR (catalog number 3915). For cell transfection, lipofectamine 2000 transfection reagent (Thermo Fisher Scientific Cat#11668027) was used according to the manufacturer's recommended protocol. I purchased an optimized fluorescence-generating substrate (5'-6FAM / ArUAA / 3'TAMRA_(NHS ester)(v3)) from IDT.The S protein was cloned by Synbio Technologies Inc. using the KPN1 GGTACC and BamH1 GGATCC cloning sites in the bacterial expression vector pET-30a(+), with a 6×His tag (SEQ ID NO: 34) attached to the carboxyl terminus. The S protein His tag fusion protein was expressed and purified from bacteria by Synbio Technologies Inc.

[0066]

[0097] The sequence of the clonal construct encoding the RNase donor is as follows:

[0067]

[0098] [ka] The sequence encoding the 6×His tag (sequence number 34) is in bold, and the TAG termination codon is underlined.

[0068]

[0099] The corresponding translated amino acid sequence for the His-tagged RNase donor protein (S protein) is as follows: MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASVHHHHHH(Sequence ID 23)

[0069]

[0100] The cloning of the S-tag acceptor peptide containing the mutT homolog (MTH1) protein (i.e., the target polypeptide) was performed by Synbio Technologies Inc. Cloning of the S-tag acceptor peptide with the carboxyl terminus of MTH1 was performed at the vector pcDNA3.1(+): cloning sites KPN1 GGTACC and BamH1 GGATCC (bold), as shown below.

[0070]

[0101] The inventors have generated a coding construct that does not have a linker between MTH1 and the S-tagged peptide (underlined), as shown below.

[0071]

[0102] [ka]

[0103] The translated amino acid sequence of the resulting fusion protein containing MTH1 and a carboxy-terminal S-tag acceptor peptide is shown below. The 20-amino acid sequence of the S-tag acceptor peptide is underlined. It was confirmed that the S-tag can be shortened to the first 15 amino acids and still function as an S-tag acceptor peptide.

[0072]

[0104] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTV KETAAAKFERQHMDSSTSAA (Sequence ID 25).

[0073]

[0105] Furthermore, the inventors generated a coding construct of a fusion protein containing a three-amino acid linker (the part enclosed in a square) between the MTH1 target polypeptide and the S-tag acceptor peptide (underlined) as shown below.

[0074]

[0106] [ka]

[0075]

[0107] The translated amino acid sequence of the resulting fusion protein containing an MTH1-targeted polypeptide with a three-amino acid spacer and a carboxy-terminal S-tagged acceptor peptide is shown below. The S-tag is underlined, and the three-amino acid spacer is enclosed in a square.

[0076]

[0108] [ka]

[0077]

[0109] Furthermore, an coding construct containing a 10-amino acid linker (enclosed in a square) between the MTH1 target polypeptide and the S-tag acceptor peptide (underlined) was constructed as shown below.

[0078]

[0110] [ka]

[0079]

[0111] The translated amino acid sequence of the MTH1-target polypeptide having a 10-amino acid spacer and a carboxy-terminal S-tagged acceptor peptide is shown below. The S-tag is underlined, and the 10-amino acid spacer is enclosed in a square.

[0080]

[0112] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTVAAAAAAAAAAKETAAAKFERQHMDSSTSAA (Sequence ID 29).

[0081] Methods: A protocol for evaluating drug target engagement using S-tags (microtags). A. Number of test cells in dynamics readings

[0113] HEK-293 cells were transfected with an MTH1-S tag fusion construct using Lipofectamine 2000. This protocol was modified to use reverse transfection, in which the cells were floated and re-seeded with the transfection reagent along with the DNA. 24 hours after transfection, the cells were floated with trypsin, washed with 1× TBS, and counted. Dilution series of cell numbers were tested.

[0082]

[0114] Cells are measured in 1×TBS (Chilled TBS) for 1×10 6 The solution was diluted to cells / ml (i.e., 1000 cells / μl). Cell counts ranging from 50,000 to 1000 cells were tested in 96-well plates with a final volume of 50 μl in chilled TBS. To each well containing 50 μl of cells, 1% Triton-X-100 was added in 50 μl of room temperature TBS to obtain a final concentration of 0.5% Triton-X-100. The wells were mixed by pipetting up and down and incubated at room temperature for 10 minutes to allow for gentle lysis of the cell membranes. 80 μl of each well was transferred to a black Costar plate, avoiding air bubbles, and 10 μl of 10×S protein (RNase donor) was added to a final concentration of 1 ng / μl, followed by 10 μl of 10× substrate (nucleic acid FRET-labeled substrate) to obtain a final substrate concentration of 50 nM. 10×S protein (10 ng / μl) was prepared immediately before use at room temperature using 0.5% triton-X in TBS. 10× substrate (500 nM) was prepared immediately before use at room temperature using dH2O.

[0083]

[0115] Plates were immediately read using a PolarStar Omega96-well plate reader in dynamic mode (excitation = 485 nm, emission = 520 nm). All plates were read every minute for 10 minutes (11 time points were generated). An appropriate cell dilution was selected that yielded the highest signal within 6 minutes without signal saturation.

[0084] B. Experimental T by applying a temperature gradient agg Identification

[0116] Cells are measured in 1×TBS (Chilled TBS) for 1×10 6 The solution was diluted to cells / ml (i.e., 1000 cells / µl).

[0085]

[0117] Accurate dilutions of cells were prepared in a white PCR plate using chilled TBS to a final volume of 50 μl. Using a thermal cycler (MJ Research PTC-200 Peltier thermal cycler), the plate was exposed to a temperature gradient of 40°C to 64°C for 15 minutes, allowed to stand at room temperature for 1 minute, and then 50 μl of 1% Triton-X-100 in room temperature TBS was added to obtain a final 0.5% Triton-X-100. The wells were mixed by pipetting up and down, and then incubated at room temperature for 10 minutes.

[0086]

[0118] 80 μl of lysate from each well was transferred to a black Costar plate, avoiding air bubbles. Subsequently, 10 μl of 10×S protein (RNase donor) was added to obtain a final concentration of 1 ng / μl, followed by the addition of 10 μl of 10× substrate (nucleic acid substrate) to obtain a final substrate concentration of 50 nM. The plate was immediately read using a PolarStar Omega96-well plate reader in dynamic mode (excitation = 485 nm, emission = 520 nm). The plate was read every minute for 10 minutes. T of MTH1-S tagged fusion protein agg It was determined that the temperature is approximately 50°C.

[0087] C. Given cell number and T agg Inhibitor dosage in the study

[0119] The inhibitor (i.e., test compound) dose was determined based on the optimal cell number and T level determined in the above experiment. agg The tests were conducted using 1×TBS (Chilled TBS) with cells measured in 1×10⁶ cells. 6 The cells were diluted to cells / ml (i.e., 1000 cells / µl). Accurate cell dilutions were prepared to a final volume of 50 µl in a white PCR plate using chilled TBS. 0.5 µl of 100 × crizotinib (a multi-target small molecule tyrosine kinase inhibitor) (or any other inhibitor to be tested) was added in DMSO. For crizotinib, EC50 was tested in the range of approximately 50 nM. In the control well, Tagg The sample included DMSO tested at 50°C (0% stability control) and DMSO tested at 40°C (100% stability control).

[0088]

[0120] The cells were incubated on ice with the inhibitor for 40 minutes, then T agg The cells were placed in a thermal cycler for 15 minutes (control wells were heated separately at 40°C for 15 minutes), followed by 1 minute at room temperature. Then, 50 μl of 1% Triton-X-100 was added to room temperature TBS to obtain a final concentration of 0.5% Triton-X-100. The cells were mixed by pipetting up and down, followed by incubation at room temperature for 10 minutes. 80 μl was transferred to a black Costar plate, avoiding air bubbles, followed by the addition of 10 μl of 10×S protein to obtain a final concentration of 1 ng / μl, and then 10 μl of 10× substrate to obtain a final concentration of 50 nM. The plate was immediately read every minute for 10 minutes using a PolarStar Omega96-well plate reader in dynamic mode (excitation = 485 nm, emission = 520 nm). The fluorescence units were plotted against the corresponding inhibitor (test compound) dose to determine the EC50 of protein stabilization.

[0089] result

[0121] Three MTH-1-S tagged peptide fusion protein constructs were transfected into HEK293 mammalian cells to determine whether the spacer length between the target polypeptide and the S tagged acceptor peptide affects the detection of functional RNase S complementation. Figure 4 shows that the expression of the constructs in HEK293 cells was equal, as determined by Western blotting.

[0090]

[0122] When a 50 μg dose of transfected cell lysate was tested and spacers of various lengths were compared, it was shown that spacer length did not have a significant effect on the fluorescence signal (Figure 5). Spacer length does not affect the signal level of the target protein or the aggregation temperature (T aggIt was also considered that it did not play a significant role in this regard. The structure, inactivity, and thermal insensitivity of the S tag did not contribute to the biophysical stability of the target protein. This significantly improved the downstream applications and use of this S tag technology with a wide range of target proteins. Furthermore, the data showed that in the absence of S tag fusion expression, there was no detectable signal indicating the specificity of S tag S protein complementation.

[0091]

[0123] To determine whether S protein concentration affects the fluorescence signal, gradually increasing concentrations of S protein were tested with various S-tagged fusions. Figure 6 shows that the signal began to saturate at S protein concentrations of approximately 1–2 ng / µl. This data demonstrates the sensitivity of this system and shows that only minimal levels of S protein are required to drive enzyme complementation.

[0092]

[0124] Similarly, when FRET nucleic acid substrates were tested at progressively increasing concentrations, it was observed that the ideal concentration was in the range of 50 nM or higher (Table 1). [Table 1]

[0093]

[0125] The kinetics of cleavage of the fluorescence-generating substrate were rapid, and detection was highly sensitive. The change in fluorescence signal with respect to cell number is shown in Figure 7. The optimal cell number was determined, and then the T of the fusion protein was determined by performing an improved cell thermal shift assay (CTSA). agg This was identified. In Figure 8, in the case of the MTH1-S tag fusion, T agg It was shown that the temperature was approximately 50°C. aggDose-response curves were constructed using [a specific method] to identify the binding of small molecule crizotinib to the fusion protein. Cells transfected with the MTH1-S tag fusion protein were incubated with escalating concentrations of the MTH1 inhibitor (S) crizotinib (Figure 9). Crizotinib stabilized the MTH1 fusion protein, as indicated by an increase in fluorescence signal (Figure 9). The EC50 was determined using the protein stabilization formula shown below, and it was found that the EC50 of the stabilized protein was approximately 25 nM (Figure 10). Formula for protein stabilization: (1-((RLU 40℃ -RLU 50℃ )-(RLU x -RLU 50℃ )) / (RLU 40℃ -RLU 50℃ )))*100 In the formula, RLU is the relative light unit.

[0094] [Table 2]

[0095] [Table 3]

[0096]

[0126] A short incubation time between the lysate, S protein, and substrate was sufficient for complete signal generation (Figure 11). The shortest incubation time (0.5 minutes) was sufficient for complete signal generation. Signal quality was not compromised with longer incubation times. Such rapid signal generation is important for downstream applications of this technology, particularly array and chip applications.

[0097]

[0127] The combination of low lysate / cell input and short incubation time yielded a highly sensitive signal, demonstrating the thawing profile of the target protein (MTH1-3aa-S tag) when bound to its ligand (crizotinib) (Figures 12 and 13). For crizotinib-MTH1 engagement, the signal pattern was identical for both high and low lysate inputs. These figures show that the signal pattern persists with longer incubation times despite signal saturation. This flexibility in the combination of lysate / cell input and incubation time allows for the detection of engagement EC50 for ligands with varying target engagement potencies.

[0098]

[0128] Figures 14-16 show the effects of combinations of lysate / cell input volume and incubation time on signal pattern and persistence, and the thawing profile of the target protein (MTH1-3aa-S tag) when bound to its ligand (crizotinib). Note that in the case of crizotinib-MTH1 engagement, the signal pattern was the same between short and long incubation times (Figures 14-16). Note that the signal pattern persisted with longer incubation times despite signal saturation. Also note that signal separation was broad at short incubation times of 0.5 minutes and narrower at longer incubation times. Note the interrelationship between lysate input volume and incubation time. This flexibility in the combination of lysate / cell input volume and incubation time allows for the detection of ligand-bound EC50s with various target engagement efficiencies, as well as a variety of applications where different amounts of lysate input or incubation time are preferred.

[0099] Example 2 - Target engagement in plate array format

[0129] In one embodiment of the method or assay described herein, the target polypeptide is a protein encoded by a pathogen (non-limiting examples include coronaviruses, influenza viruses, hepatitis viruses, and bacteriophages) or a modified protein thereof. The fusion protein comprises a target pathogen protein (e.g., an S-tagged acceptor peptide) expressed with an N-terminal S-tag and / or a C-terminal S-tag. The expression construct of the pathogen protein would include codon-optimized cDNA that enables maximal expression of the fusion protein. In this example, an array is used to provide the DNA encoding the fusion protein to cells in a multi-well format (96-well, 384-well, or 1536-well format) for expression in the cell line of interest. The DNA can be transfected into cells placed in each well of the multi-well plate to enable protein expression. Using this plate array, one or more agonists (drugs, i.e., test compounds) can be tested against all of the fusion proteins in a single run. In addition, this array can be tested with multiple doses of one or more test compounds. This array can be used in high-throughput methods to test compound libraries against the entire proteome of pathogens. Using this array, test compound binding can be screened under various physiological conditions (e.g., different buffers, presence of serum components, growth factors, or other irritants). The plate can then be subjected to a gradient heating denaturation step, followed by the addition of an RNase donor for enzyme complementation, and detection of FRET-labeled substrate cleavage can be performed. Fluorescence detection using a plate reader will be performed in dynamic mode to monitor any potential increases in fluorescence over time.

[0100] Example 3 - Incorporation of potential viral mutations into a target engagement array

[0130] In this example, the array contains various computer-generated combinations of highly mutagenic / mutagenic regions of the SARS-CoV-2 coronavirus protein or another pathogen. For example, various potential mutations of the spike protein are generated using in silico tools, and the target polypeptide variants fused to an S-tagged acceptor peptide are presented on the array for drug engagement. The array will consist of plasmid DNA encoding the tagged protein. This is followed by transfection of the DNA into selected cells and drug screening with one or more test compounds. Random or concentrated libraries of drug compounds can be screened for engagement using a thermal gradient denaturation step, followed by enzyme complementation, substrate cleavage, and fluorescence detection.

[0101]

[0131] Such arrays can identify test compounds that bind to one or more of various viral protein targets. These arrays can address the viral mutation response to therapeutic agents. They can also explore therapeutic options to circumvent viral mutations, potentially preventing future pandemics caused by the same or related coronaviruses.

[0102] Example 4 - Array of either complete or partial influenza viruses

[0132] Each protein of the influenza virus is fused to an S-tagged peptide acceptor. The repertoire of selected influenza virus proteins may be limited to highly mutant proteins of the influenza virus family. This array can be made to present highly mutant targets from past influenza pandemics and seasonal epidemics, such as hemagglutinin (HA), neuraminidase (NA), M1, nucleoprotein, or other influenza targets. Similarly, this array may also contain computer-generated versions of highly mutant influenza virus proteins. This array is used to screen a panel of drug compounds for direct engagement with viral proteins.

[0103]

[0133] Such arrays can identify drugs that bind to various potential targets of influenza viruses, both existing and predicted. This array assay can address the response of potential viral mutations to potential therapeutic test compounds. It can also address the effect of specific mutations on the toxicity of future variants of the pathogen. Both drugs and viral proteins can be ranked in terms of engagement potency. This array can also identify therapeutic options to circumvent viral mutations, potentially preventing future pandemics caused by the same influenza viridae. The most effective and broadly specific drugs can also be identified as potential treatments for future pandemics.

[0104] Example 5 - Combination array of pathogen families

[0134] This array enables the combined presentation of hepatitis A, B, and C viruses in a single multiplex assay. Therapeutic targets of the three viruses are presented and expressed in host hepatocytes. A panel of drugs is tested for target engagement using single-dose or multi-dose patterns. This array can also be extended to other pathogen families, such as herpesviruses and rotaviruses. Target engagement will be determined using S-tagged complementary assays for each target.

[0105]

[0135] Combining pathogen testing can accelerate the discovery of broad-spectrum therapeutics. Drugs are ranked based on their target engagement efficacy for each pathogen subclass. In this particular case, this approach enables the discovery of multispecific / broad-spectrum drugs against hepatitis viridae. This is especially useful for testing, discovering, ranking, and validating existing and novel therapies for clinical and research purposes.

[0106] Example 6 - Array of bacteriophage proteins

[0136] The selected bacteriophages may infect specific pathogenic bacteria, such as Helicobacter pylori, multidrug-resistant Mycobacterium tuberculosis, or a broad class of pathogens including Mycobacterium. In any case, individual proteins of the bacteriophage are cloned as S-tagged fusion proteins and presented on an array as shown above. This array may contain variants of a single or multiple class of bacteriophages. Similarly, this array may contain proteins that are bacterial surface receptors. Using S-tagged complementation assays for each target, a panel of drugs can be tested for engagement with individual bacteriophage proteins or bacteriophage-bacterial protein pairs.

[0107]

[0137] Such arrays are used to discover proteomic stabilizers that enhance bacteriophage activity against host bacteria. They are also used to identify agonists that can enhance the infectivity of bacterial hosts by enhancing bacteriophage-receptor interactions. Using this method, it is possible to discover drugs and therapeutic agents that can enhance bacteriophage activity against clinically important pathogens such as multidrug-resistant Mycobacterium tuberculosis. Such drugs could be used clinically to eliminate drug-resistant pathogens from human hosts.

[0108] Example 7 - Array of genetically randomized pathogen targets

[0138] Selected protein targets of the SARS-CoV-2 coronavirus, such as the spike protein, Nuc protein, or Memb protein, are genetically randomized using genome editing tools such as CRISPR-Cas9 and fused to S-tag acceptor peptides. A library of such proteins is presented in an array, each representing an individual randomized edit of a viral target. A panel of drugs can then be screened for engagement with these viral proteins.

[0109]

[0139] This array will identify drugs that bind to various potential pathogen target types, both existing and predicted. It can address the response of potential viral mutations to therapeutic agents. Furthermore, this array assay can address the effect of specific mutations on the toxicity of future variants of the pathogen. Both drugs and viral proteins can be ranked in terms of engagement potency. This array will also identify therapeutic options to circumvent viral mutations, potentially preventing future pandemics caused by the same coronavirus family. It can identify the most effective drugs and identify broadly specific drugs as potential treatments for future pandemics caused by the same pathogen family.

[0110] Example 8 - Array of SARS-CoV-2 proteome for screening drug target engagement

[0140] In one embodiment of the array, the format may include the proteome of the SARS-CoV-2 proteome. This virus expresses 28 predictive proteins from its viral RNA genome. The array may include individual expression constructs for each of the viral proteins tagged with an S-tag acceptor peptide (see, for example, Figure 17). The expression constructs can then be placed in a multi-well plate format for expression in the cell line of interest. The viral target engagement array plate can then be screened for direct binding with one or more test agonists using S-tag complementation assays for each target. The viral array may include several rows of plates having the same tagged viral proteins for expression, allowing testing of agonists in each row of the plate under different physiological conditions (see, for example, Figure 18).

[0111] Example 9 - An array for discovering drug formulations against selected individual pathogen targets, such as the spike protein of the SARS-CoV-2 coronavirus.

[0112]

[0141] This type of array represents a limited number of pathogen targets. A panel of drug compounds can be tested with various formulations representing realistic pharmaceutical protocols. Such formulations may include enhancers, volume expanders, co-active ingredients, and combinations thereof. The engagement of such drug formulations can be tested against viral targets in host cell models using S-tag complementation assays.

[0113]

[0142] Predicted Result 6: Such screening will identify formulations that enhance the activity of active drug compounds that bind to pathogen targets. Such screening is drug development and can accelerate the transition from therapeutic research to clinical trials. Such screening will be particularly used for repurposing existing drugs.

[0114] Example 10 - Multiviral Array for Drug Target Engagement

[0143] In some embodiments of the array, several viral pathogen proteins derived from distinct viruses can be tagged with an S-tag acceptor peptide and combined in a single array. The array would consist of a multi-well format with expression constructs of proteins encoded from different viruses, with each fusion protein expressed in an individual well of the plate (see, for example, Figure 19). This array can then be used to screen for single or multiple test agents across proteins encoded by different viral pathogens (e.g., coronaviruses: SARS, MERS, HKU1, or proteins derived from hepatitis A, B, and C).

[0115] Example 11 - Combination array of pathogen families

[0144] This array enables the combined presentation of hepatitis A, B, and C viruses in a single configuration. It presents therapeutically important targets of the three viruses and expresses them in host hepatocytes. A panel of drugs is tested for target engagement using single-dose or multi-dose patterns. This array can also be extended to other pathogen families such as herpesviruses or rotaviruses. Target engagement will be read using an S-tag complementation assay.

[0116]

[0145] Combining pathogen testing can accelerate the discovery of broad-spectrum therapeutics. Drugs are ranked based on their target engagement efficacy within each pathogen subclass. In this particular case, this approach enables the discovery of multispecific / broad-spectrum drugs against hepatitis viridae. This is especially useful for testing, discovering, ranking, and validating existing and novel therapies for clinical and research purposes.

[0117] Example 12 - Array of host proteins required for viral entry

[0146] In this embodiment, the array may include the expression of human or mammalian proteins necessary for pathogen binding and / or infection, and may include a mixture of both human and viral proteins known to be necessary for infection. As an example, the array may include human angiotensin-converting enzyme 2 (ACE2) and human transmembrane serine protease 2 (TMPRSS2) as target polypeptides. These proteins are expressed on the surface of endothelial cells and play a crucial role in SARS-CoV-2 virus entry and infection. The assay then tests one or more agonists at various doses to identify direct target engagement with one or both targets.

[0118]

[0147] This array allows for the multiplexing of protein targets tested with a single or multiple test agents. The viral target engagement array can be screened using multiple test agents at varying doses. Then, by reading the fluorescence of each individual viral protein, it is possible to identify which proteins a test agent binds to and stabilizes the fusion protein under a thermal gradient (see, e.g., Figures 20A–20C). Furthermore, this array allows for the identification and ranking of drugs based on their ability to bind to and stabilize targets under a thermal gradient, by focusing on specific wells where a particular viral protein is tested with multiple agents at multiple doses (see, e.g., Figure 20D).

[0119] Example 13 - Array of virus-host interaction proteome

[0148] Representative proteins derived from both the SARS-CoV-2 coronavirus and the human host are presented as fusion proteins containing S-tag acceptor peptides. For example, viral membrane proteins (Nuc, Memb, Spike) are presented together with the receptor ACE2, other members of the ACE receptor family, the AT1 receptor, the AT2 receptor, and other potential cell surface receptors. Various host receptors can be included to represent various host tissues. Viral proteins may be presented individually or paired with host proteins. A panel of drugs can be tested for engagement with single viral or host proteins or pairs thereof.

[0120]

[0149] This presentation allows for the discovery of therapeutic options that would prevent viral docking and integration into various host cell tissues. This array can also explore the potential infectivity of the virus to other less explored host tissues, such as the kidneys, heart, spleen, brain, or liver.

[0121] Example 14 - Whole Live Virus Array

[0150] In this embodiment, the array may include the expression of chimeric live viral particles that enable assembly with one or more constituent proteins fused with an S-tag acceptor peptide. This array may be useful for evaluating drug engagement with the target protein in the context of live viral particles.

[0122] Example 15 - Array of Chimeric Live Virus Particles

[0151] This embodiment of the array represents S-tagged fusion viral proteins assembled within live viral particles. Lysates from cells expressing these individual viral proteins are systematically added to cells that replicate virulent SARS-CoV-2 coronavirus. The S-tagged viral proteins are incorporated by the host cells into the live assembled coronavirus particles. This generates an array of live chimeric coronaviruses. This array is expandable. This array allows for direct testing of targets against individual viral targets in the context of live viruses.

[0123]

[0152] Such arrays are used to test drug engagement with individual viral proteins in the context of live viruses. The advantage of this system is that S-tagged proteins can be incorporated into live viruses, allowing drugs to be tested against these live viral particles within the host. Using S-tagged complementarity, it is possible to 1) measure viral replication as a result of drug interactions, 2) correlate target engagement with viral replication, 3) identify the viral proteins that have the greatest impact on viral replication, and 4) discover the most clinically relevant drugs that have the greatest impact on viral replication.

[0124] Example 16 - Various thermal profile signatures of fusion proteins with S-tags (microtags) Materials and methods:

[0153] HEK-293 cells were obtained from ATCC (catalog number CRL-1573) and cultured in DMEM (Millipore SIGMA catalog number D5796) supplemented with 10% fetal bovine serum (FBS) (Millipore SIGMA catalog number F2442) and 1× penicillin / streptomycin (Millipore SIGMA catalog number P4333). A 1× trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA) was obtained from Millipore SIGMA (catalog number 59417C), and a 20× TBS solution was obtained from Teknova (catalog number T1680). Triton-X-100 was obtained from Millipore SIGMA (catalog number X100). The surfactant n-dodecyl beta-D-maltoside (DDM) was obtained from Millipore SIGMA (catalog number D4641). The MTH1 inhibitor (S)-crizotinib was obtained from Millipore SIGMA (catalog number PZ0240). The BCL6 inhibitors BI-3812 and BI-5273 were obtained from Boehringer Ingelheim. DMSO was obtained from Millipore SIGMA (catalog number 673439). 96-well plates were obtained from CELLTREAT Scientific (catalog number 229196), white PCR plates from BIORAD (catalog number MLL9651), Microseal "B" film for sealing PCR plates from BIORAD (catalog number MSB1001), and black 96-well plates from COSTAR (catalog number 3915). Cell transfection was performed using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific catalog number 11668027) according to the manufacturer's recommended protocol. I purchased an optimized fluorescence-generating substrate (5'-6FAM / ArUAA / 3'TAMRA_(NHS ester)(v3)) from IDT.The S protein was cloned by Synbio Technologies Inc. using the KPN1 GGTACC and BamH1 GGATCC cloning sites into the bacterial expression vector pET-30a(+), with a 6×His tag (SEQ ID NO: 34) attached to the carboxyl terminus. The S protein His tag fusion protein was expressed and purified from bacteria by Synbio Technologies Inc.

[0125]

[0154] The sequence of the clonal construct encoding the RNase donor is as follows: [ka]

[0126]

[0155] The sequence encoding the 6×His tag (sequence number 34) is in bold, and the TAG termination codon is underlined.

[0127]

[0156] The corresponding translated amino acid sequence for the His-tagged RNase donor protein (S protein) is as follows:

[0128]

[0157] MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASVHHHHHH(Sequence ID 23)

[0129]

[0158] The cloning of the S-tag acceptor peptide containing the mutT homolog (MTH1) protein (i.e., the target polypeptide) was performed by Synbio Technologies Inc. Cloning of the S-tag acceptor peptide to the carboxyl terminus of MTH1 was performed at the vector pcDNA3.1(+): cloning sites KPN1 GGTACC and BamH1 GGATCC (bold), as shown below.

[0130]

[0159] The inventors have generated a coding construct that does not have a linker between MTH1 and the S-tagged peptide (underlined), as shown below.

[0131]

[0160] [ka]

[0132]

[0161] The translated amino acid sequence of the resulting fusion protein containing MTH1 and a carboxy-terminal S-tagged acceptor peptide is shown below. The 20-amino acid sequence of the S-tagged acceptor peptide is underlined. It was confirmed that the S-tag can be shortened to the first 15 amino acids and still function as an S-tagged acceptor peptide.

[0133]

[0162] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTV KETAAAKFERQHMDSSTSAA (Sequence ID 25).

[0134]

[0163] Furthermore, the inventors constructed an coding construct of a fusion protein containing a three-amino acid linker (enclosed in a square) between the MTH1 target polypeptide and the S-tag acceptor peptide (underlined), as shown below.

[0135]

[0164] [ka]

[0136]

[0165] The synthesis of the BCL6 (B-cell lymphoma 6) S-peptide microtag construct was performed at Twist Biosciences, and the corresponding nucleotide sequence was as follows:

[0137]

[0166]

[0138]

[0167] Lowercase letters indicate the carboxyl-terminal S peptide nucleotide residue. The obtained protein amino acid sequence of the expressed protein is shown below. The S peptide sequence is underlined.

[0139]

[0168] MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCE SRAFAPSLYSGLSTPPASYSMYSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNA CILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGPEQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSE EASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC KETAAAKFERQHMDSSTSAA -(Sequence ID 31)

[0140]

[0169] The synthesis of the EGFR S peptide tag construct was performed at Twist Biosciences, and the corresponding nucleotide sequence was as follows:

[0141]

[0170] [ka] JPEG0007927708000013.jpg81149

[0142]

[0171] The carboxyl-terminal S peptide nucleotide sequence is underlined. The resulting amino acid sequence of the expressed protein is as follows:

[0143]

[0172] KETAAAKFERQHMDSSTSAA -(Sequence ID 33)

[0144]

[0173] The S peptide nucleotide sequence at the carboxyl terminus is underlined.

[0145] A. Cell transfection and lysate preparation

[0174] HEK293 cells were transfected with lipofectamine 2000 according to the manufacturer's protocol. Two days after transfection, the medium was removed and the cells were floated by pipetting up and down with 1× TBS. The cells were centrifuged at 400g for 4 minutes. The TBS wash was removed and the cells were lysed with either 1% Triton X 100 in TBS for MTH1 and BCL6 cells, or 0.5% DDM in TBS for EGFR-expressing cells. The cells were lysed in a rotator at 4°C for 1 hour, and cell residue was removed by gently centrifuging at 6000 rpm for 1 minute. The lysates were transferred to new tubes, and aliquots not used immediately in the assay were frozen at -80°C for future use.

[0146] B. Thermal Profile

[0175] Lysates prepared from cells overexpressing the construct were diluted 1 / 10 with 1×TBS, 40 μl were ali-coated into PCR tubes or plates, and heated in a thermal cycler with a heat gradient for 15 minutes. Immediately after heating, the samples were assayed for enzyme complementarity. A mixture of 1.25 μg / ml of S protein and 250 nM of FRET-labeled nucleic acid substrate in 1×TBS containing 0.5% DMSO was combined with the heated samples, and fluorescence was detected using a microplate reader. Plates were read using a PolarStar Omega96-well plate reader in dynamic mode (excitation = 485 nm, emission = 520 nm). All plates were read every minute for 20 minutes (21 time points were generated).

[0147]

[0176] Determine the thermal profile, T agg , T max , or T minTo determine this, the biofluorescence or fluorescence signal generated every minute was plotted against temperature. The sigmoid plot identifies the temperature at which aggregation occurs, or the maximum or minimum signal.

[0148] C. Small molecule target engagement screening

[0177] Small molecule compounds were dissolved in DMSO to a 10 mM concentration, and 100× concentration DMSO series dilutions were prepared from this stock. Lysates of cells expressing microtagged (S-peptide-tagged) proteins were diluted 1 / 10 with cooled 1× TBS, 39.6 μl was aliquoted onto a PCR plate, 0.4 μl of 100× small molecule compounds were added, the plate was sealed, vortexed, and centrifuged for 2 minutes. The plate was then heated at a suitable temperature for 15 minutes, followed by cooling at 25°C for 1 minute. A reaction buffer (1.25 μg / ml of S protein binding partner and 250 nM of FRET-labeled substrate in 1× TBS with 0.5% DMSO) was prepared, and 120 μl was transferred to a black 96-well plate. The heated sample (30 μl) was added, and fluorescence was detected using a microplate reader. The plate was read using a PolarStar Omega 96-well plate reader in dynamic mode (excitation = 485 nm, emission = 520 nm).

[0149]

[0178] For analysis, GraphPad Prism was used to plot the biofluorescence signal or fluorescence change every minute against the inhibitor dose on a semi-logarithmic scale. Nonlinear regression analysis was used for curve fitting to determine the EC50 or apparent K of target engagement. D They identified it.

[0150] result

[0179] The application of microtags (S-tags) to cell target engagement relies on the principle of protein thermal fusion. Cells expressing microtag constructs or cell lysates were subjected to a thermal gradient to identify the aggregation temperature. This is the temperature at which the protein portion denatures and aggregates, and therefore becomes insoluble. In thermal shift assays, the binding of ligands that affect the protein's three-dimensional structure can rescue the protein from this thermal denaturation, making the protein more stable under denaturing conditions such as thermal loading.

[0151] A. Thermal profile of a microtagged target

[0180] To screen ligands that would bind to the microtagged constructs, we subjected cells expressing the constructs, or undenatured lysates prepared from such cells, to a thermal gradient to determine the protein aggregation temperature. However, an interesting feature of this microtagging system is that the thermal load on the tagged protein results in several different thermal signatures: aggregation temperature (T agg ), maximum signal temperature (T max ), and minimum signal temperature (T min The key finding was that the [specified entity] was identified (Figures 23A-23C).

[0152]

[0181] Cells or cell lysates overexpressing MTH1 microtagged (S-peptide tagged) protein at 55°C (T aggUsing heating in the assay, it is possible to screen for molecules that will bind to and stabilize MTH1 microtagged proteins, resulting in a higher fluorescence signal. In the microtagged (S peptide) assay system, an increase in fluorescence directly proportional to the amount of microtagged protein during the reaction was detected. The ability to monitor fluorescence signal generation in real time, combined with the rapid enzymatic kinetics of the active whole RNase enzyme (S peptide complemented by the S protein), provided a unique feature not possible with other similar target engagement strategies: monitoring of FRET-labeled substrate depletion. As shown in Figure 24, this system could be used to investigate the time-dependent fluorescence generation (relative light units (RLU) change per minute) directly resulting from the cleavage of the FRET-labeled substrate. The increase in fluorescence signal within the first 3 minutes of the reaction, the fluorescence peak at approximately 3-4 minutes, and the decrease in fluorescence after 4 minutes were all directly proportional to the amount of microtagged protein in the assay. This demonstrated the flexibility of this assay in that it allows for various methods to directly quantify the level of microtagged protein.

[0153] B. Screening of molecules that bind to microtagged proteins

[0182] The maximum signal temperature (T) of the target protein max ), agglomeration temperature (T agg ), or minimum signal temperature (T minAfter identifying the target engagement EC50, we were able to evaluate ligand binding at these temperatures. When a ligand binds to a microtagged protein, a conformational change occurs due to a binding event that stabilizes the microtagged protein under heat or other denaturing loads. Here, we used a BCL6 microtagged protein with the well-characterized Boehringer Ingelheim inhibitor BI-3812, which has an in vitro IC50 of approximately 3 nM. A related structural analog of BI-3812 is the inactive compound BI-5273, which has an in vitro IC50 of approximately 10 μM. By incubating these inhibitors with cells or lysates derived from HEK293 cells overexpressing the BCL6 microtag, we were able to identify the target engagement EC50 (Figures 25A-25B).

[0154]

[0183] After a 4-minute incubation, the detected fluorescence could be plotted on a semi-logarithmic scale against the dose of the tested inhibitor. The EC50 of target engagement was identified by using nonlinear regression analysis and fitting a sigmoid dose-response curve (variable slope) using GraphPad Prism. The BCL6-specific inhibitor BI-3812 bound to the BCL6 microtagged protein at an EC50 of 0.63 nM target engagement (Figures 25A-25B). Inactive analogs did not bind to the target. Depletion of the FRET-labeled substrate was induced, as indicated by the decrease in the minute-by-minute fluorescence increase (RLU / min) at BI-3812 concentrations that stabilized the target, allowing for a longer duration (10-15 minutes) of the S protein reaction. The dose of the inhibitor that saturates the microtagged protein target was identified by the peak fluorescence signal. At inhibitor concentrations above this saturation dose, rapid cleavage of the FRET-labeled substrate occurred, resulting in a time-dependent loss of the fluorescence signal (relative light units per minute (RLU / min)) and a decrease in detectable fluorescence. Below this saturation dose, the microtag construct denatured under thermal load, resulting in fewer microtags available for enzyme complementation and a lower fluorescence signal (Figure 26A). Removing data points above the target saturation dose allowed for (1) determination of the EC50 of target engagement and (2) determination of the apparent equilibrium dissociation constant (apparent K) of the drug binding to the protein target. D This made it possible to determine (Figures 26B and 26C). The EC50 of the target engagement determined by this method is the apparent K D These values ​​were very close to 0.9 nM and 1.6 nM, respectively.

[0155] C. Quantitative coupling dynamics data in physiological situations

[0184] This method can be used to identify the drug dose at which the target protein is saturated and elicits maximum fluorescence. Using this method, the target saturation dose was identified, as shown over time in Figures 27A-27C. With longer incubation times, an inflection point was identified by the decrease in fluorescence signal over time, occurring at 5 minutes (Figure 27B). The dose at which the signal peaked after a longer incubation was the target saturation dose (Figure 27C). The inflection point was where longer incubations began to result in a decrease in the fluorescence signal over time. Peak fluorescence refers to the Emax value (maximum effect). Knowing the drug concentration that saturates a given target is an important parameter for drug discovery because it can be used to define target occupancy. The EC50 of target engagement was determined from a sigmoid dose-response curve of fluorescence signal versus drug concentration on a semi-logarithmic scale, using the initial time point (0 minutes) before any signal decrease occurred at higher drug concentrations (Figure 27D). The EC50 of target engagement was identified by nonlinear regression analysis fitted to sigmoid dose-response curves using GraphPad Prism software (Figures 27D and 26B). Observable fluorescence signal data could also be fitted to a saturated binding equation (total of 1 site) using GraphPad Prism (Figures 27E and 26C). This allowed for the identification of the apparent equilibrium dissociation constant (apparent K) of the drug binding to the protein target. D A specific EC50 was identified, which was similar to the observed EC50 of target engagement (Figures 27E and 26C). Since the relationship between target binding and fluorescence response was expected to be directly proportional up to the drug's saturation concentration, it was possible to fit the observable fluorescence data to the saturation binding equation.

[0156]

[0185] Target saturation dose, Emax, and apparent K DAs another example of identifying the target engagement, we tested the MTH1 microtagged protein and the inhibitor (S)-crizotinib. Incubating the inhibitor with the protein under thermal load, followed by enzyme complementation and fluorescence detection, led to the identification of the EC50 of the target engagement after 2 minutes (Figure 28A). With longer incubation times (10 minutes), fluorescence decreased over time at higher drug doses. The dose at which the signal peaked after longer incubation was the target saturation dose (Figure 28B). Peak fluorescence indicates the Emax value (Figure 28B). Nonlinear regression analysis, fitted to the sigmoid dose-response curve using GraphPad Prism software, identified the EC50 of the target engagement as approximately 43 nM (Figure 28A). Observable fluorescence signal data could also be fitted to a saturation binding equation (total 1 site) using GraphPad Prism (Figure 28C). This resulted in an apparent equilibrium dissociation constant of 32 nM (apparent K D A similar EC50 (43 nM) was identified, which was similar to the EC50 of the target engagement identified at 2 minutes using sigmoid dose-response curve fitting.

[0157] Consideration

[0186] The method using S peptide tags (microtags) offered several advantages compared to other enzyme complementation strategies. Firstly, these short tags (15–20 amino acids) were small enough not to interfere with the folding, localization, protein-protein interactions, and function of the tagged protein target. Secondly, the use of enzyme complementation with S proteins and FRET-labeled nucleic acid substrates for fluorescence signal generation offered the advantage of a rapid reaction that could be monitored in real time (Figure 22). The addition of fluorescence detection provided several unique features to this technique. For some targets, thermal profiles that identified the maximum and minimum temperatures of protein fusion were advantageous (Figures 23A–23C). max and T minWe were able to screen proteins possessing this characteristic for ligand binding at temperatures significantly lower than the typical aggregation temperature.

[0158]

[0187] A key feature of this technique was the reaction rate (Figure 24). Rapid enzymatic cleavage of the FRET-labeled substrate allowed for the determination of the EC50 of target engagement within the first 5 minutes of enzyme complementation. This could then be tracked over time to identify the dose at which the small molecule saturated the protein target. The saturation dose was determined using the peak fluorescence value (Emax) after depletion of the FRET-labeled substrate. The rate of this RNase cleavage of the FRET-labeled nucleic acid substrate led to depletion of the FRET-labeled substrate within 10–20 minutes. This was detected in real time as a decrease in the fluorescence signal over time. This peak fluorescence (target saturation dose) occurred when the increase in signal due to stabilization of the target protein balanced with the decrease in signal from the excessively stabilized protein target (e.g., rapid FRET depletion). The dose at which the small molecule saturated its target was useful for quantifying target occupancy, a feature highly relevant to drug discovery.

[0159]

[0188] The ability to perform quantitative kinetic analysis of small molecules binding to protein targets in a physiological setting was provided as fluorescence readings, which were a direct result of ligand-bound, stabilized, microtagged proteins. Since the target saturation dose was identified in this reaction, the apparent equilibrium dissociation constant (K) was determined. D We were able to determine the apparent K of the small molecule that binds to the target by removing doses exceeding the saturation dose. D We were able to obtain a saturated binding curve that yielded the desired result. The EC50 of target engagement, determined from the initial point before the depletion of the FRET-labeled substrate, was the apparent K determined from the saturated binding curve. D It correlated with the measured value.

[0160]

[0134] Every patent, patent application, publication, or any other reference or document cited herein is incorporated by reference in its entirety. In case of any conflict, this specification, including its definitions, shall prevail.

[0161]

[0135] No citation of any patent, patent application, publication, or any other document shall constitute an endorsement of any of the foregoing as relevant prior art, nor shall it constitute any endorsement of the content or date of such publication or document.

[0162]

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0163]

[0137] All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly stated, the disclosed features (e.g., antibodies) are examples of the types of equivalent or similar features.

[0164]

[0138] As used herein, all numbers or numerical ranges include integers and values ​​or fractional parts of integers within such ranges unless the context clearly indicates otherwise. Furthermore, if a list of values ​​is provided herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), that list includes all of its intermediate and fractional values ​​(e.g., 54%, 85.4%). Thus, for example, a reference to “80% or more” includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., and 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0165]

[0139] References to integers that are "greater than" or "less than" include any number greater than or less than the reference number, respectively. For example, a reference to "less than 100" includes numbers from 99, 98, 97, etc., down to the number 1, and a reference to "less than 10" includes numbers from 9, 8, 7, etc., down to the number 1.

[0166]

[0140] As used herein, unless the context clearly indicates otherwise, all numbers or ranges include the values ​​and fractional parts of integers within such ranges, and the fractional parts of integers within such ranges. For example, references to numerical ranges such as 1 to 10 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Similarly, references to the range 1 to 50 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., up to 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., and 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0167]

[0141] A reference to a range includes a range that combines the boundary values ​​of different ranges within that range. For example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,50 References to ranges such as 0-3,000, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include ranges such as 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

[0168]

[0142] Modifications can be made to the foregoing without departing from the basic aspects of the present technology. Although the present technology is described in considerable detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to embodiments specifically disclosed in this application, and that such modifications and improvements will still fall within the scope and spirit of the present technology.

[0169]

[0143] The present invention is generally disclosed herein using assertive language to describe a number of embodiments and aspects. The present invention also particularly includes embodiments in which certain subjects, such as substances or materials, method steps and conditions, protocols, or procedures, are completely or partially excluded. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Accordingly, the present invention also discloses aspects that are not expressly excluded in the present invention, even if they are not generally expressed herein in relation to things that are not included in the present invention.

[0170]

[0144] The techniques described herein by reference can be suitably carried out in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each example herein, the terms “comprising,” “consisting essentially of,” and “consisting of” can each be replaced with any of the other two terms. The terms and expressions used are used as descriptive terms, not restrictive terms, and the use of such terms and expressions does not preclude any equivalents of the illustrated and described features or segments thereof, and various modifications are possible within the scope of the claimed techniques. The terms “a” or “an” can refer to one or more of the elements it modifies unless it is evident from the context that one or more of the elements it modifies are described (for example, “reagent” can mean one or more reagents). As used herein, the term “about” refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and the use of the term “about” at the beginning of a string of values ​​modifies each of the values ​​(i.e., “about 1, 2, and 3” refers to about 1, about 2, and about 3). For example, a weight of “about 100 grams” can include weights from 90 grams to 110 grams. The term “substantially” as used herein refers to a value modifier meaning “at least 95%”, “at least 96%”, “at least 97%”, “at least 98%”, or “at least 99%”, and can include 100%. For example, a composition substantially free of X may contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of X, and / or X may not be present in the composition or may be undetectable. The phrase “substantially at the same time” means occurring at that point in time or within a time frame of a few seconds (e.g., within a 0-10 second frame).

[0171]

[0145] Accordingly, although the present technology is disclosed in detail by representative embodiments and optional features, those skilled in the art will understand that modifications and variations of the concepts disclosed herein may be used, and such modifications and variations will be considered to be within the scope of the present technology.

[0172] [Simplified Sequence List]

[0173]

[0189] KETAAAKFERQHMDSSTSAA(Sequence ID 1)

[0174]

[0190] KETNWAWFWDQHMDSSTSA(Sequence ID 2)

[0175]

[0191] KETGWALFVQQHMDSSTSA(Sequence ID 3)

[0176]

[0192] KETVMANFQMQHMDSSTSA(Sequence ID 4)

[0177]

[0193] KETGDAVFARQHMDSSTSA(Sequence ID 5)

[0178]

[0194] KETGWAAFVKQHMDSSTSA(Sequence ID 6)

[0179]

[0195] KETGWATFVEQHMDSSTSA(Sequence ID 7)

[0180]

[0196] KETKLAFFLKQHMDSSTSA (Sequence ID 8)

[0181]

[0197] KETWWAWFFGQHMDSSTSA (Sequence ID 9)

[0182]

[0198] KETTWAEFTWQHMDSSTSA(Sequence ID 10)

[0183]

[0199] KETPWASFNKQHMDSSTSA(Sequence ID 11)

[0184]

[0200] KETAMAMFVTQHMDSSTSA (Sequence ID 12)

[0185]

[0201] KETLWAWFMWQHMDSSTSA (Sequence ID 13)

[0186]

[0202] KETAAAKFERQHMDS (Sequence ID 14)

[0187]

[0203] KETAAAKFERQHMNS (Sequence ID 15)

[0188]

[0204] NRAWSEFLWQHLAPV (Sequence ID 16)

[0189]

[0205] NRGWSEFLWQHHAPV (Sequence ID 17)

[0190]

[0206] NRAWSVFQWQHIAPA (Sequence ID 18)

[0191]

[0207] MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV (Sequence ID 19)

[0192]

[0208] MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFD(Sequence ID 20)

[0193]

[0209] KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV (Sequence ID 21)

[0194]

[0210] GGTACCATGAGCAGCTCCAACTACTGTAACCAGATGATGAAGAGCCGGAACCTGACCAAAGATCGATGCAAGCCAGTGAACACCTTTGTGCACGAGTCCCTGGCTGATGTCCAGGCCGTGTGCTCCCAGAAAAATGTTGCCTGCAAGAATGGGCAGACCAATTGCTACCAGAGCTACTCCACCATGAGCATCACCGACTGCCGTGAGACCGGCAGCTCCAAGTACCCCAACTGTGCCTACAAGACCACCCAGGCGAATAAACACATCATTGTGGCTTGTGAGGGAAACCCGTACGTGCCAGTCCACTTTGATGCTTCAGTGCATCACCATCACCATCACTAGGGATCC(SEQ ID NO: 22)

[0195]

[0211] MSSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASVHHHHHH(SEQ ID NO: 23)

[0196]

[0212] GGTACCATGGGCGCCTCCAGGCTCTATACCCTGGTGCTGGTCCTGCAGCCTCAGCGAGTTCTCCTGGGCATGAAAAAGCGAGGCTTCGGGGCCGGCCGGTGGAATGGCTTTGGGGGCAAAGTGCAAGAAGGAGAGACCATCGAGGATGGGGCTAGGAGGGAGCTGCAGGAGGAGAGCGGTCTGACAGTGGACGCCCTGCACAAGGTGGGCCAGATCGTGTTTGAGTTCGTGGGCGAGCCTGAGCTCATGGACGTGCATGTCTTCTGCACAGACAGCATCCAGGGGACCCCCGTGGAGAGCGACGAAATGCGCCCATGCTGGTTCCAGCTGGATCAGATCCCCTTCAAGGACATGTGGCCCGACGACAGCTACTGGTTTCCACTCCTGCTTCAGAAGAAGAAATTCCACGGGTACTTCAAGTTCCAGGGTCAGGACACCATCCTGGACTACACACTCCGCGAGGTGGACACGGTCAAGGAAACTGCAGCAGCCAAGTTTGAGCGGCAGCACATGGACTCCAGCACTTCCGCTGCCTAGGCTGCCTAGGGATCC (SEQ ID NO: 24)

[0197]

[0213] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTVKETAAAKFERQHMDSSTSAA (SEQ ID NO: 25)

[0198]

[0214] GGTACCATGGGCGCCTCCAGGCTCTATACCCTGGTGCTGGTCCTGCAGCCTCAGCGAGTTCTCCTGGGCATGAAAAAGCGAGGCTTCGGGGCCGGCCGGTGGAATGGCTTTGGGGGCAAAGTGCAAGAAGGAGAGACCATCGAGGATGGGGCTAGGAGGGAGCTGCAGGAGGAGAGCGGTCTGACAGTGGACGCCCTGCACAAGGTGGGCCAGATCGTGTTTGAGTTCGTGGGCGAGCCTGAGCTCATGGACGTGCATGTCTTCTGCACAGACAGCATCCAGGGGACCCCCGTGGAGAGCGACGAAATGCGCCCATGCTGGTTCCAGCTGGATCAGATCCCCTTCAAGGACATGTGGCCCGACGACAGCTACTGGTTTCCACTCCTGCTTCAGAAGAAGAAATTCCACGGGTACTTCAAGTTCCAGGGTCAGGACACCATCCTGGACTACACACTCCGCGAGGTGGACACGGTCGCCGCCGCCAAGGAAACTGCAGCAGCCAAGTTTGAGCGGCAGCACATGGACTCCAGCACTTCCGCTGCCTAGGCTGCCTAGGGATCC(SEQ ID NO: 26)

[0199]

[0215] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTVAAAKETAAAKFERQHMDSSTSAA(SEQ ID NO: 27)

[0200]

[0216] GGTACCATGGGCGCCTCCAGGCTCTATACCCTGGTGCTGGTCCTGCAGCCTCAGCGAGTTCTCCTGGGCATGAAAAAGCGAGGCTTCGGGGCCGGCCGGTGGAATGGCTTTGGGGGCAAAGTGCAAGAAGGAGAGACCATCGAGGATGGGGCTAGGAGGGAGCTGCAGGAGGAGAGCGGTCTGACAGTGGACGCCCTGCACAAGGTGGGCCAGATCGTGTTTGAGTTCGTGGGCGAGCCTGAGCTCATGGACGTGCATGTCTTCTGCACAGACAGCATCCAGGGGACCCCCGTGGAGAGCGACGAAATGCGCCCATGCTGGTTCCAGCTGGATCAGATCCCCTTCAAGGACATGTGGCCCGACGACAGCTACTGGTTTCCACTCCTGCTTCAGAAGAAGAAATTCCACGGGTACTTCAAGTTCCAGGGTCAGGACACCATCCTGGACTACACACTCCGCGAGGTGGACACGGTCGCCGCCGCCGCCGCCGCCGCCGCCGCCAAGGAAACTGCAGCAGCCAAGTTTGAGCGGCAGCACATGGACTCCAGCACTTCCGCTGCCTAGGCTGCCTAGGGATCC(SEQ ID NO: 28)

[0201]

[0217] MGASRLYTLVLVLQPQRVLLGMKKRGFGAGRWNGFGGKVQEGETIEDGARRELQEESGLTVDALHKVGQIVFEFVGEPELMDVHVFCTDSIQGTPVESDEMRPCWFQLDQIPFKDMWPDDSYWFPLLLQKKKFHGYFKFQGQDTILDYTLREVDTVAAAAAAAAAAKETAAAKFERQHMDSSTSAA(SEQ ID NO: 29)

[0202]

[0218]

[0203]

[0219] MASPADSCIQFTRHASDVLLNLNRLRSRDILTDVVIVVSREQFRAHKTVLMACSGLFYSIFTDQLKCNLSVINLDPEINPEGFCILLDFMYTSRLNLREGNIMAVMATAMYLQMEHVVDTCRKFIKASEAEMVSAIKPPREEFLNSRMLMPQDIMAYRGREVVENNLPLRSAPGCESRAFAPSLYSGLSTPPASYSMYSHLPVSSLLFSDEEFRDVRMPVANPFPKERALPCDSARPVPGEYSRPTLEVSPNVCHSNIYSPKETIPEEARSDMHYSVAEGLKPAAPSARNAPYFPCDKASKEEERPSSEDEIALHFEPPNAPLNRKGLVSPQSPQKSDCQPNSPTESCSSKNACILQASGSPPAKSPTDPKACNWKKYKFIVLNSLNQNAKPEGPEQAELGRLSPRAYTAPPACQPPMEPENLDLQSPTKLSASGEDSTIPQASRLNNIVNRSMTGSPRSSSESHSPLYMHPPKCTSCGSQSPQHAEMCLHTAGPTFPEEMGETQSEYSDSSCENGAFFCNECDCRFSEEASLKRHTLQTHSDKPYKCDRCQASFRYKGNLASHKTVHTGEKPYRCNICGAQFNRPANLKTHTRIHSGEKPYKCETCGARFVQVAHLRAHVLIHTGEKPYPCEICGTRFRHLQTLKSHLRIHTGEKPYHCEKCNLHFRHKSQLRLHLRQKHGAITNTKVQYRVSATDLPPELPKAC KETAAAKFERQHMDSSTSAA -(SEQ ID NO: 31)

[0204]

[0220]

Chemical

[0205]

[0221] KETAAAKFERQHMDSSTSAA -(Sequence ID 33)

[0206]

[0222] HHHHHH (Sequence ID 34)

[0207] [Related patent applications]

[0001] This patent application claims the interests of U.S. Provisional Patent Application No. 63 / 114,927, filed on 17 November 2020, the contents of which, including the text, tables and drawings, are incorporated herein by reference in their entirety.

[0208] [Sequence List]

[0002] The sequence listing is submitted with this specification as an ASCII-compliant text file named "062554-0562965_SL_ST25.txt" with a size of 43,786 bytes, created on November 17, 2021, in accordance with U.S. Patent Rules §1.821(c). The contents of the above file are incorporated herein by reference in their entirety.

Claims

1. A method for determining whether a test compound can interact with a target polypeptide, (a) A fusion protein comprising the target polypeptide and the S-tag acceptor peptide (i) Test compounds which are small compounds having a molecular weight of 40 Da to 5000 Da, (ii) Heat as a denaturing factor, (iii) Nuclease donors containing the S protein of the RNase S complex, and (iv) Nucleic acid substrate The step of bringing it into contact, (b) Step of detecting the amount of cleavage products produced from the nucleic acid substrate. A method that includes this.

2. The method according to claim 1, comprising the step of contacting the fusion protein with (v) a signal control factor selected from the group consisting of an antibody, a chemical substance, a peptide, temperature, UV, microwave, or light.

3. The method according to claim 2, wherein the signal control factor is far-red light.

4. The method according to claim 1, wherein the contact in (a) comprises contacting a cell or a lysate derived from the cell with (i) the test compound, (ii) the denaturing factor, (iii) the nuclease donor, and / or (iv) the nucleic acid substrate, wherein the cell or cell lysate comprises the fusion protein.

5. The method according to claim 4, wherein the fusion protein is expressed by the cells.

6. The method according to claim 4, wherein the contact in (a) includes contacting the cells or lysates derived from the cells with the denaturing factor or the nucleic acid substrate.

7. The method according to claim 1, wherein the fusion protein is brought into contact with the denaturing factor, or simultaneously with the fusion protein being brought into contact with the nuclease donor and / or the nucleic acid substrate.

8. The method according to claim 4, wherein the cells contain the nuclease donor, or the nuclease donor is expressed by the cells.

9. The method according to claim 1, wherein the contact in (a) comprises heating the fusion protein to a temperature of at least 40°C, at least 50°C, or at least 60°C.

10. The method according to claim 9, wherein the temperature is sufficient to denature or aggregate the fusion protein or the target polypeptide in the absence of the test compound.

11. The method according to claim 9, wherein the heating is performed for a period of at least 20 seconds, at least 30 seconds, at least 1 minute, at least 3 minutes, at least 5 minutes, at least 10 minutes, or at least 15 minutes.

12. The method according to claim 9, wherein the heating includes exposing the cells to a temperature gradient that increases the temperature at a rate of at least 5°C per minute.

13. The method according to claim 1, wherein the nucleic acid substrate comprises a ribonucleotide and / or a deoxyribonucleotide.

14. The method according to claim 1, wherein the nucleic acid substrate includes a detectable label.

15. The method according to claim 14, wherein the detectable marker is a fluorescent marker.

16. The method according to claim 14, wherein the nucleic acid substrate comprises a pair of FRET labels.

17. The method according to claim 16, wherein the nucleic acid substrate comprises 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (6-TAMRA).

18. The method according to claim 16, wherein the nucleic acid substrate comprises (6-FAM)-X-(6-TAMRA), where X is a polynucleotide having the sequence Y-dA-R, Y is one or more pyrimidines, dA is deoxyadenosine, and R is one or more purines.

19. The method according to claim 16, wherein the nucleic acid substrate comprises (6-FAM)-dA-rU-dA-dA-(6-TAMRA), where rU is uridine.

20. The method according to claim 1, wherein the S-tag acceptor peptide comprises 8 to 20 amino acids.

21. The method according to claim 20, wherein the S-tag acceptor peptide comprises the S peptide of RNase S.

22. The method according to claim 21, wherein the RNase S is pancreatic RNase.

23. The S-tag acceptor peptide is a peptide having an amino acid sequence that is at least 90% identical to the amino acid sequence KETAAAKFERQHMDSSTSAA (SEQ ID NO: 1), or a peptide containing at least 14 consecutive amino acid sequences of the amino acid sequence KETAAAKFERQHMDSSTSAA (SEQ ID NO: 1), or KETNAWFWDQHMDSSTSA (SEQ ID NO: 2), KETGWALFVQQHMDSSTSA (SEQ ID NO: 3), KETVMANFQMQHMDSSTSA (SEQ ID NO: 4), KETGDAVFARQHMDSSTSA (SEQ ID NO: 5), KETGWAAFVKQHMDSSTSA (SEQ ID NO: 6), KETGWATFVEQHMDSSTSA (SEQ ID NO: 7), KETKLAFFLKQHMDSSTS The method according to claim 20, comprising a peptide having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from A (SEQ ID NO: 8), KETWWAWFFGQHMDSSTSA (SEQ ID NO: 9), KETTWAEFTWQHMDSSTSA (SEQ ID NO: 10), KETPWASFNKQHMDSSTSA (SEQ ID NO: 11), KETAMAMFVTQHMDSSTSA (SEQ ID NO: 12), KETLWAWFMWQHMDSSTSA (SEQ ID NO: 13), KETAAAKFERQHMDS (SEQ ID NO: 14), KETAAAKFERQHMNS (SEQ ID NO: 15), NRAWSEFLWQHLAPV (SEQ ID NO: 16), NRGWSEFLWQHHAPV (SEQ ID NO: 17), and NRAWSVFQWQHIAPA (SEQ ID NO: 18).

24. The method according to claim 1, wherein the S-tag acceptor peptide is covalently bound to the N-terminus of the target polypeptide.

25. The method according to claim 1, wherein the S-tag acceptor peptide is covalently bonded to the C-terminus of the target polypeptide.

26. The method according to claim 1, wherein the target polypeptide includes a linker between the target polypeptide and the S-tag acceptor peptide.

27. The method according to claim 26, wherein the linker is a peptide containing 1 to 10 amino acids.

28. The method according to claim 1, wherein the test compound is determined to interact with the target polypeptide in proportion to the amount of the cleavage product detected in (b).

29. The method according to claim 28, wherein the detection of the amount of the cleavage product in (b) includes detecting the presence or amount of a fluorescent signal emitted from the cleavage product or a fluorescent signal emitted from the uncleaved nucleic acid substrate.

30. The method according to claim 29, wherein the amount of the cleavage product detected in (b) is greater than a predetermined threshold.

31. The method according to claim 30, wherein the amount of the cleavage product detected in (b) is at least 20%, at least 30%, or at least 50% greater than a predetermined threshold.

32. The method according to claim 30, wherein the predetermined threshold is determined by (i) carrying out steps (a) and (b) in the absence of the test compound, or by (ii) carrying out steps (a) and (b) in the presence of a compound that neither interacts with nor binds to the target polypeptide.

33. The method according to claim 4, further comprising the step of contacting a cell with a nucleic acid encoding and / or inducing the expression of the fusion protein.

34. The method according to claim 33, wherein the cell is a bacterium, and the method comprises the step of contacting the bacterium with a bacteriophage comprising the nucleic acid encoding and / or inducing the expression of the fusion protein.

35. The method according to claim 1, wherein the target polypeptide is a protein derived from or expressed by a pathogen, a modified protein, or a portion thereof.

36. The method according to claim 35, wherein the pathogen is a virus or a bacterium.

37. A high-throughput assay comprising the step of performing the method according to claim 1 in a plurality of containers, each container containing the fusion protein.

38. The assay according to claim 37, wherein each container contains a different fusion protein.

39. The assay according to claim 37, wherein each of the containers contains cells expressing different fusion proteins.

40. The assay according to claim 37, wherein part or all of the container is brought into contact with a different test compound.

41. The assay according to claim 37, wherein the plurality of containers comprises at least 96 containers, at least 384 containers, or at least 1536 containers.

42. The assay according to claim 37, wherein the container is a well of a microtiter plate.

43. The assay according to claim 37, wherein contact between the fusion protein in each of the containers and the test compound, the denaturing factor, the nuclease donor, and / or the nucleic acid substrate occurs substantially simultaneously or concurrently.

44. The assay according to claim 37, wherein the detection step of (b) includes detecting the amounts of the nucleic acid substrate cleavage products in each of the containers substantially simultaneously or concurrently.

45. The assay according to claim 37, comprising the step of bringing cells in each of the containers into contact with a nucleic acid that expresses, encodes, or induces the expression of the fusion protein.

46. The nucleic acid substrate comprises a pair of FRET labels, and the amount of the cleavage product is used to detect the amount of fluorescence signal emitted from the cleavage product and to obtain a data point, wherein the fluorescence signal is used to determine the target saturation dose, apparent equilibrium dissociation constant (K) between the target polypeptide and the test compound. Ｄ The method according to claim 1, which enables the identification of the maximum half-size effective concentration (EC50) of target engagement.

47. The method according to claim 46, wherein the target saturation dose of the test compound is determined by the peak fluorescence value (Emax) after the depletion of the nucleic acid substrate.

48. The apparent equilibrium dissociation constant (K) between the target polypeptide and the test compound Ｄ The method according to claim 46, wherein the saturation coupling curve is identified by plotting the Emax, and data points exceeding Emax are excluded from the plot.

49. The method according to claim 46, wherein the EC50 of the target engagement is determined from an initial data point before the depletion of the nucleic acid substrate.

50. A kit for use in the method according to any one of claims 1 to 49, comprising a plurality of containers, the first container comprising a fusion protein, the second container comprising a nuclease donor, and the third container comprising a nucleic acid substrate.

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