WO2020014518A1

WO2020014518A1 - Methods for identifying compounds that inhibit repair of frame-shift mutations by mismatched repair system

Info

Publication number: WO2020014518A1
Application number: PCT/US2019/041458
Authority: WO
Inventors: Richard A. Fishel; Brooke M. BRITTON; James A. London; Juana V. MARTIN-LOPEZ
Original assignee: Ohio State Innovation Foundation
Priority date: 2018-07-11
Filing date: 2019-07-11
Publication date: 2020-01-16

Abstract

Disclosed herein is a method of treating a cancer in a subject that involves treating the subject with an agent that inhibits mismatch repair (MMR) in the cancer followed by treatment with a PD-1 or PDL-1 inhibitor to make the cancer hypersensitive to immune-surveillance. Also disclosed is a method for screening candidate agents to identify an MMR inhibitor that specifically inhibits the recognition/repair of frame-shift mutations by the MMR system.

Description

METHODS FOR IDENTIFYING COMPOUNDS THAT INHIBIT REPAIR OF FRAME- SHIFT MUTATIONS BY MISMATCHED REPAIR SYSTEM

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 62/696,662, filed July 11 , 2018, which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government Support under Grant No. CA67007 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

Tumors with defective mismatch repair (MMR) display an enormous increase in genomic mutations (Vogelstein, B., et al. (2013) Science, 339, 1546-155). Those mutations lead to altered proteins that are often presented on the surface of the tumor cell (Kelderman, S., et al. (2015) Cancer Cell, 28, 11-13). These tumors evade the natural immune response by a number of mechanisms. However, treatment of these tumors with the PD-1 inhibitor Pembrolizumab reactivates the immune response and makes them hypersensitive to immune-surveillance (Le, D.T., et al. (2015) N Engl J Med, 372, 2509-25). This is nearly curative in MMR-defective tumors (Le, D.T., et al. (2015) N Engl J Med, 372, 2509-2520; Germano, G., et al. (2017) Nature, 552, 116-120; Le, D.T., et al. (2017) Science, 357, 409- 413). However, there remains a need for therapies effective in treating cancers with active

MMR genes.

SUM MARY

Disclosed herein is a method of treating a cancer in a subject that involves treating the subject with an agent that selectively inhibits mismatch repair (MMR) in the cancer followed by treatment with a PD-1 or PDL-1 inhibitor to make the cancer hypersensitive to immune-surveillance. PD-1 and PDL-1 inhibitors are known in the art, and include, for example, cemiplimab, nivolumab, pembrolizumab, pidilizumab, AMP-224 AMP-514, and PDR001.

Also disclosed is a method for screening candidate agents to identify an MMR inhibitor. In some embodiments, the MMR inhibitor of the disclosed methods inhibits MMR component interactions and/or binding to one another. In some embodiments, the MMR inhibitor of the disclosed methods inhibits the normal interaction and/or binding of MMR components to normal DNA, DNA containing a mismatch, or DNA containing nucleotide oxidative or methylation lesion in cancer cells.

As disclosed herein, the altered proteins that are most effective for PD-1/PDL-1 immune-surveillance are the result of frame-shift mutations that result in altered peptides or additional nucleotides within non-coding RNA sequences. These are relatively rare and are recognized differently from the majority of nucleotide errors that result in mutations (single nucleotide changes), which are the largest drivers of cancer in MMR-deficient cells.

Therefore, in some embodiments, the disclosed MMR inhibitor specifically inhibits the recognition/repair of frame-shift mutations by the mismatch repair system.

Therefore, in some embodiments, the method is a rapid screening system to identify candidate agents that inhibit the recognition/repair of frame-shift mutations by the mismatch repair system.

MMR proteins binding to a mismatch can be shown several ways. FRET analysis using labelled proteins and a labelled DNA close to the mismatch can show interaction between the protein and DNA. This analysis can be done in a bulk study as well as looking at individual interactions using single molecule total internal reflection microscopy (smTIRF). Biacore analysis of bulk interactions between a mismatch DNA bound to a surface and MMR proteins will also show recognition of mismatches.

Conformational transitions have been shown with MMR protein TaqMuts using a FRET pair on either subunit, but the domains may not be useable for the human proteins. Structural analysis using X-ray chrystallography or possibly electron microscopy could be used to look at the detailed structure of MMR proteins bound to a mismatch.

A FRET assay could be used to confirm interaction between MutS and MutL homologs. When ATP is present, this assay becomes a sliding clamp system. The method can monitor conformational transitions in MMR proteins by monitoring FRET. DNA binding would be a high FRET. The conformational transition would result in a lower FRET.

Interaction between MutS and MutL can also be monitored with FRET based assay with donor fluorophore on the MutS homolog and acceptor fluorophore on the MutL homolog. Excitation of donor that results in acceptor signal indicates interaction of MutS and MutL homologues.

In some embodiments, the method involves labeling a panel of MMR proteins with fluorophores and using fluorescence resonance energy transfer (FRET) to evaluate the effect of candidate agents on, for example, mismatch binding, conformational transitions associated with mismatch recognition by MutS homologs, interaction between MutS homologs and MutL homologs, or the formation of a sliding clamp by MutL homologs. FRET pair fluorophores can be placed in strategic locations that position them within 10 nm of each other. Candidate FRET pairs might include but are not limited to Alexa488 with Cy3, Cy3 with Alexa647, or Cy3 with Cy5.

Conformational transitions by MutS and MutL homologs are driven by ATP binding. ATP-binding conformational changes may be altered by competitive inhibitors, but are not specific to distinguish single nucleotide versus frame-shift DNA lesions. Nor are they specific enough to inhibit explicit progressions in the mismatch repair process. The method can involve designating FRET pairs and locations that can specifically inhibit well-defined steps in the mismatch repair progression to distinguish functional steps. Inhibitors of these singular steps then target specific MMR functions.

Therefore, disclosed herein is a method for identifying an agent that selectively inhibits mismatch repair (MMR) in a cell, the method comprising contacting MMR proteins with a candidate agent, and assaying the MMR proteins for binding to a DNA

oligonucleotide, interaction between MutS homologs and MutL homologs, ATP binding to the MMR proteins, or the formation of a sliding clamp by MutS and/or MutL homologs. In some embodiments, the MMR proteins and/or DNA oligonucleotide are labeled with fluorescence resonance energy transfer (FRET) fluorophore pairs.

In some embodiments, assaying the MMR proteins first involves providing a sliding clamp system, contacting the sliding clamp system with a candidate agent, and measuring fluorescence resonance energy transfer (FRET) resonance of the sliding clamp system. For example, a decrease or increase in FRET resonance compared to a control can be an indication that the candidate agent inhibited MMR.

In some embodiments, the sliding claim system involves an MMR heterodimer conjugated to a donor fluorophore, ATP, and an oligonucleotide comprising a mismatched nucleotide linked at one end to a surface, blocked at the other end with a macromolecule, and comprising a acceptor fluorophore conjugated to the mismatched oligonucleotide a first MMR heterodimer comprising MutS homologs, and a second MMR heterodimer comprising MutL homologs. In some embodiments, the oligonucleotide has at least one mismatched nucleotide, wherein the oligonucleotide is linked at one end to a surface and blocked at the other end with a macromolecule. In some embodiments, an acceptor fluorophore conjugated to the first or second MMR heterodimer. In some embodiments, a donor fluorophore is conjugated to the first MMR heterodimer, the second MMR heterodimer, or the mismatched nucleotide.

In some embodiments, the acceptor fluorophore is conjugated to the first MMR heterodimer and the donor fluorophore is conjugated to the mismatched nucleotide. In some embodiments, the acceptor fluorophore is conjugated to a first protein of the first MMR heterodimer and the donor fluorophore is conjugated to a second protein of the first MMR heterodimer. In some embodiments, the acceptor fluorophore is conjugated to the first MMR heterodimer and the donor fluorophore is conjugated to the second MMR heterodimer. In some embodiments, the acceptor fluorophore is conjugated to a first protein of the second MMR heterodimer and the donor fluorophore is conjugated to a second protein of the second MMR heterodimer.

In some embodiments, the first MMR heterodimer comprises MutSa, MutSy, or Muΐqb heterodimers. In some embodiments, the first MMR heterodimer comprises i) MSH2 and MSH3 or ii) MSH 2 and MSH6. In some embodiments, the second MMR heterodimer comprise MutLa, Mutl-b, or MutLy heterodimers. In some embodiments, the second MMR heterodimer comprises i) MLH1 and PMS1 or ii) MLH1 and PMS2.

In some embodiments, the DNA oligonucleotide comprises a single nucleotide mismatch. Possible permutations of single nucleotide mismatches include G/T or A, C/T or A, A/G or C, T/G or C.

In some embodiments, the DNA oligonucleotide comprises an insertion and deletion loop (IDL). The IDL can range in size from 1 to 12 nucleotides. Larger than 12 nt, MSH2- MSH3 will bind but NOT form a sliding clamp and may be relevant to TNR diseases.

Types of end modifications that can be used as blocking macromolecules include 5’ or 3’ single or dual biotin conjugated with streptavidin, neutravidin, or avidin. Virtually anything that one can stably be placed on the end of a DNA can be used as a block.

Additional examples include Holliday junction and Lacl. To retain the MLH sliding clamp one needs something larger than a nucleosome. In some embodiments, the blocking

macromolecule is a 5’ or 3’ digoxigenin conjugated with anti-digoxigenin. In some embodiments, the blocking macromolecule is reversible DNA-End Blocking of a transcription factor binding to its cognate promotor. An example includes Lacl to the Lac Operon. Another example includes circular mismatch DNA substrates. In some embodiments, the blocking macromolecule comprises antibody-bound 5’-digoxygenin.

In some embodiments, the oligonucleotide comprises 20 to 200 bp, including 20 to 100 bp, 100 to 200 bp, and 50 to 150bp. Therefore, the oligonucleotide can have 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 bp.

In some embodiments, the sliding clamp system has a ratio of first or second MMR heterodimer to DNA oligonucleotide greater than 0.1 , such as between 0.25 and 0.5.

In some embodiments, the sliding clamp system is disposed in a solution comprising less than 0.5% DMSO. In some embodiments, the cell a prokaryotic cell or an animal cell. For example, the animal cell can be a human cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a pathogen. For example, the pathogen can be one of a Enterococcus, Staphylococcus, Klebsiellai,

Acinetobacter, Pseudomonas, or Enterobacter genus. In some embodiments, the pathogen is Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, or Enterobacter cloacae species.

Also disclosed herein is a method of treating a cancer in a subject that involves treating the subject with an agent that selectively inhibits mismatch repair (MMR) in the subject followed by or in combination with treatment with a checkpoint inhibitor to make the cancer hypersensitive to immune-surveillance. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof. In some embodiments, the cancer has intact or functional DNA mismatch repair. In some embodiments, the subject has been diagnosed with Lynch syndrome, Huntington diease, myotonic dystrophy, or fragile-X syndrome.

Also disclosed is a method to specifically label proteins with a chemical fluorophore that involves the use of thiolester-fluorophore and ligation (TFAL) to an N-terminal Cys residue created by TEV protease cleavage. TFAL combined with Formylglycine Generating Enzyme (FGE)/Hydrazino-Pictet-Spengler (HIPS) flourophore addition (Liu et al., Sci Rep 5:16883, 2015) and Sortase A C-terminal Gly-fluorophore exchange (Dorr et al., PNAS

111 :13343, 2014) are capable of placing a fluorophore in virtually any non-destructive location on any mismatch repair component.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration of FRET-Based Assay for Distinguishing HsMSH2-HsMSH3 and HsMSH2-HsMSH6 Activities. The assay requires labeling the DNA with an FRET- Acceptor (Cy5 or similar), and the MSH heterodimer with a FRET-Donor (Cy3 or similar. Mismatch recognition by MSH proteins results in High FRET and the formation of an ATP- bound MSH sliding clamp results in a time-averaged Intermediate FRET.

FIGs. 2A to 2H show FRET analysis of mismatch recognition by HsMSH2-HsMSH3 and HsMsh2-HsMSH6. FIG. 2A shows FRET calculations using 20 nM HsMSH2-HsMSH3 with 80 nM DNA substrates. FIG. 2B are representative examples of corrected S1c/R1c intensity profiles used for FRET calculations in FIG. 2A. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. FIG. 2C shows FRET calculations using 20 nM HsMSH2-HsMSH6 with 80 nM DNA substrates. FIG. 2D shows representative examples of corrected S1c/R1c intensity profiles used for FRET calculations in FIG. 2C. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. FIG. 2E shows FRET calculations using 20 nM HsMSH2-HsMSH3 with 40 nM DNA substrates. FIG. 2F shows representative examples of corrected S1c/R1c intensity profiles used for FRET calculations in FIG. 2E. Note that genuine FRET entails anti correlated intensity changes of acceptor and donor peaks. FIG. 2G shows FRET calculations using 20 nM HsMSH2-HsMSH6 with 40 nM DNA substrates. FIG. 2H shows representative examples of corrected S1c/R1c intensity profiles used for FRET calculations in FIG. 2G. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. Error bars from at least two separate experiments are shown.

FIG. 3 shows corrected S1c/R1c intensity profiles used in determination of a correction Factor for the contribution of Cy5-DNA to 670 nm emission.

FIGs. 4A and 4B are bar graphs showing comparison of uncorrected and corrected FRET at various protein-to-DNA ratios. FIG. 4A shows uncorrected FRET (E_App) in assays utilizing 20 nM HsMSH2-HsMSH6 with 40 nM (1 :2), 80 nM (1 :4), 400 nM (1 :10) and 800 nM (1 :20) G/T mismatch substrate DNA without and with ATR FIG. 4B shows corrected FRET (E_App) in assays utilizing 20 nM HsMSH2-HsMSH6 with 40 nM (1 :2), 80 nM (1 :4), 400 nM (1 :10) and 800 nM (1 :20) G/T mismatch substrate DNA without and with ATP.

FIGs. 5A to 5D are graphs showing the Effect of DMSO on fluorescence excitation and emission. FIG. 5A shows DMSO alone has an intrinsic effect on 510 nm excitation over the wavelengths that are used to calculate FRET. This intrinsic emission may become significant depending on the MSH:DNA ratio and should be determined prior to any screen utilizing FRET analysis. FIG. 5B shows DMSO affects -560 nm and -670 nm emission of assays that include HsMSH2-HsMSH6 and G/T DNA. FIG. 5C shows DMSO reduces HsMSH2-HsMSH3 emission at -560 nm, but has no effect on -670 emission. FIG. 5D shows DMSO reduces HsMSH2-HsMSH6 emission at -560 nm, but has no effect on -670 emission.

FIGs. 6A to 6B are graphs showing determination of a correction factor for the contribution of Cy5-DNA to 670 nm emission in the presence of DMSO. FIG. 6A shows the effect of DMSO on 40 nM Cy5-G/C DNA. FIG. 6B shows the effect of DMSO on 80 nM Cy5- G/C DNA. FIGs. 7A and 7B are bar graphs showing the Effect of DMSO on FRET recognition by

HsMSH2-HsMSH6.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the

publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be

independently confirmed.

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

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise.

The term“subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term“patient” refers to a subject under the treatment of a clinician, e.g., physician.

The term“treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the

improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

The term“agent” or“compound” as used herein refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition. The chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, or any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi, such as siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies, aptamers, polypeptides, nucleic acid analogues or variants thereof.

The term“inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

Treatment

Disclosed herein is a method of treating a cancer in a subject that involves treating the subject with an agent that selectively inhibits mismatch repair (MMR) in the cancer followed by treatment with a checkpoint inhibitor to make the cancer hypersensitive to immune-surveillance.

The two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of cosignaling molecules that play important roles throughout all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern. When the ligands bind to PD-1 , an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation. Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A,

MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016). Human monoclonal antibodies to programmed death 1 (PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other

immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies.

Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

In some embodiments, the PDL1 inhibitor comprises an antibody that specifically binds PDL1 , such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds PD1 , such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca). Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies. Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552,154, which is incorporated by reference for these antibodies. Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is incorporated by reference for these antibodies.

Screening Assay

Disclosed herein is a method for screening candidate agents to identify an MMR inhibitor. In some embodiments, the MMR inhibitor of the disclosed methods inhibits MMR component interactions and/or binding to one another. In some embodiments, the MMR inhibitor of the disclosed methods inhibits the normal interaction and/or binding of MMR components to normal DNA, DNA containing a mismatch, or DNA containing nucleotide oxidative or methylation lesion in cancer cells.

Therefore, in some embodiments, the MMR inhibitor specifically inhibits the

recognition/repair of frame-shift mutations by the mismatch repair system.

In humans, seven DNA mismatch repair (MMR) proteins (MLH1 , MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2) work coordinately in sequential steps to initiate repair of DNA mismatches. A heterodimer between MSH2 and MSH6 (MutSa) or MSH2 and MSH3 (Mίΐΐqb) first recognizes the mismatch. The formation of the MutSa or Miiΐqb heterodimer accommodates a second heterodimer of MLH1 and PMS2 (MutLa), MLH1 and PMS1 (Mutl-b), or MLH1 and MLH3 (MutLy). This protein complex formed between the 2 sets of heterodimers enables initiation of repair of the mismatch defect. Other gene products involved in mismatch repair (subsequent to initiation by MMR genes) in humans include DNA polymerase delta, PCNA, RPA, HMGB1 , RFC and DNA ligase I, plus histone and chromatin modifying factors.

Therefore, in some embodiments, the disclosed method identifies agents that inhibits an interaction selected from the group comprising MSH2-MSH6, MSH2-MSH3, MLH1- PMS1 , MLH1-PMS2, and MLH1-MLH3.

In some embodiments, the method involves assaying for the ability of a candidate agent to affect the ability of MutS and MutL homologs to recognize a mismatch in an oligonucleotide. For example, the method can involve linking one end of a mismatched oligonucleotide to as surface, such as the surface of a 396-well plate. The other end can then be blocked by a 5’-digoxygenin linked to the DNA and bound by an anti-digoxigenin antibody. A larger end-block can have the anti-digoxigenin antibody linked to a

paramagnetic bead (e.g. 2 pm bead). In this embodiment, the larger end-block is required to retain a MutL homolog sliding clamp, while the smaller end block is sufficient to retain a MutS homolog sliding clamp. Time averaged FRET fluorescence between a donor fluorophore (Cy3) on a MutS homolog sliding clamp and an acceptor fluorophore (Alexa647 or Cy5) on the mismatched nucleotide can then be an indicator that the MutS homolog has successfully recognized a mismatch and formed a sliding clamp.

Different types of mismatches (single base pair mismatch versus insertion/ deletion loop, IDL, type mismatch) can be compared and a compound identified that alters the efficiency of one type of mismatch compared to the other. Similarly, in human cells there are two major MutS homolog heterodimers, hMSH2-hMSH6 and hMSH2-hMSH3. The hMSH2- hMSH3 heterodimer is far more active at recognizing IDL mismatched nucleotides.

Identifying a compound that specifically inhibits hMSH2-hMSH3 can increase the number of IDLs that remain in the DNA compared to single basepair mismatches.

In some embodiments, the method involves a FRET assay that identifies the precise interaction between hMSH2-hMSH6 or hMSH2-hMSH3 and one of the human MutL homologs (hMLH1-hPMS1 , hMLH1-hPMS2, or hMLH1-hMLH3). This assay can be used to identify compounds that are specific for downstream mismatch repair processes after recognition of the mismatch. One embodiment would be to place a donor fluorophore (Cy3) on hMSH6 or hMSH3 and record FRET with an acceptor (Alexa647 or Cy5) located on hPMS1 , hPMS2 or hMLH3.

In general, candidate agents can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) used.

Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods or by standard synthetic methods in combination with solid phase organic synthesis, micro-wave synthesis and other rapid throughput methods known in the art to be amenable to making large numbers of compounds for screening purposes. Furthermore, if desired, any library or compound, including sample format and dissolution is readily modified and adjusted using standard chemical, physical, or biochemical methods.

When a crude extract is found to have a desired activity, further fractionation of the positive lead extract can be used to isolate chemical constituents responsible for the observed effect. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value may be subsequently analyzed using in vitro cell based models and animal models for diseases or conditions, such as those disclosed herein.

Candidate agents encompass numerous chemical classes, but are most often organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Candidate agents can include functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, for example, at least two of the functional chemical groups. The candidate agents often contain cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1 : Method for Distinguishing MSH2-MSH3 and MSH2-MSH6 DNA Recognition and Mismatch Repair Activities

Background

Mismatch repair (MMR) is a highly conserved excision-resynthesis system that maintains genome integrity by principally correcting polymerase misincorporation errors. MMR excision commonly begins at a single-stranded DNA (ssDNA) break that marks the error-containing strand and may be several kb distant from the mismatch. Resynthesis of the resulting ssDNA gap is independent of the excision process and generally completed in a“do- over” event by the replicative polymerase machinery. MMR defects result in a significant increase in spontaneous mutations that result from the unrepaired replication errors (termed: Mutator or Mut). This phenotype was used to identify the MMR components in bacteria, including MutS and MutL. The vast majority of terrestrial organisms utilize conserved MutS homolog (MSH) and MutL homolog (MLH/PMS) proteins to coordinate MMR. In Archea and prokaryotes the MSH and MLH/PMS proteins function as homodimers. In eukaryotes, there appears to have been a separation of function between dimer halves resulting in heterodimers that arose from gene duplication and separate evolution. The MSH proteins are fundamentally responsible for mismatch recognition and then providing a platform for attracting the MLH/PMS proteins. The increasing complexity of the eukaryotic genome has resulted in two MSH mismatch-recognition heterodimers that function in MMR. The MSH2-MSH6 heterodimer primarily recognizes single nucleotide mismatches and small nucleotide insertion/deletion loop-type (IDL) mismatches, while the MSH2-MSH3 heterodimer primarily recognizes small to large (12 nt) IDL mismatches.

Mutation of the human (Hs) MMR genes HsMSH2, HsMSH6, HsMLHI and HsPMS2 are the major cause of Lynch syndrome also known as hereditary nonpolyposis colorectal cancer (LS/HNPSS). Mutation of any one of these MMR genes results in significantly elevated mutation rates from which the numerous genetic alterations that cause cancer may evolve. In addition to the common hereditary cancer LS/HNPCC, numerous sporadic colorectal, endometrial, ovarian and upper urinary tract tumors are causes by inactivation of MMR genes, with the principal cause being methylation-inactivation of the HsMLHI promoter.

One of the tumor phenotypes that originally implicated MMR defects in LS/HNPCC was the common observation of microsatellite instability (MSI) in tumors. Studies in several organisms including bacteria, yeast and human cells demonstrated that MSI occurred when the polymerase machinery stalled or dissociated during replication through short repeated DNA sequences (microsatellite sequences). Subsequent replication restart often leaves insertion or deletion mismatches in the DNA as a result of strand slippage. Under normal circumstances, these mismatched nucleotides would be recognized and repaired by the MMR machinery. However, in MMR-defective cells these IDL mismatches remain in the DNA and following a subsequent round of replication result in insertion or deletion of nucleotides from the microsatellite sequence and the observation of MSI.

Huntington’s Disease, Fragile X syndrome, X-linked Spinal and Bulbar Muscular Atrophy (Kennedy’s syndrome), and Myotonic Dystrophy Type 1 are examples of trinucleotide repeat (TNR) diseases. These are caused by dramatic expansion of trinucleotide repeats that are often but not exclusively within the coding sequence of a gene. The expansion generally occurs during germ cell development. However, significant pathology associated with these diseases occurs after birth in somatic tissues where TNR expansion appears to continue to occur leading to disease-associated phenotypes. Several genetically engineered mouse models (Mm) have indicated that mutation of MmMsh2, MmMsh3 and MmMIhl dramatically reduce or eliminate germline TNR expansions. Genetic screens in human cell model systems have correspondingly indicated that HsMSH2, HsMSH3, HsMLHI , and apparently the crosslink-repair associated gene FAN1 reduce/eliminate TNR expansion. The observation that at subset of MMR genes reduce TNR expansion seems counterintuitive to the observation of MSI in MMR-defective tumors. However, the mechanical role of MMR genes in TNR expansion is likely to be different than the mechanical role of these genes in MMR. The fact that mutation/inactivation of HsMSH2, HsMSH3, and HsMLHI reduces/eliminates TNR expansion makes them potential drug targets for obviating the somatic pathologies of TNR diseases.

The mechanics of MMR over the last nearly 25 yrs has been described (J. Mol. Biol. 430:4456, 2018). The bottom line is that the mechanism for E.coli and human MMR over the years is very different from what is found in most textbooks and on the 2015 Nobel website. However, the mechanism (termed: the MolecularSwitch/Sliding Clamp model) is the reason that an embodiment of the disclosed drug screen assay works.

PD1 and PDL1 inhibitors have been at the vanguard of immunotherapy since they appear to reactivate a native immune response against tumors. While their effectiveness as a therapy for multiple tumor types has been quite variable, they appear to be nearly curative for MMR-deficient tumors. It is believed by most in the field that this superb response is a result of the elevated mutational burden, which may be up to 10-fold greater in MMR-deficient tumors compared to most other solid tumors. Elevated genomic mutations presumably increase the frequency of novel antigens (neoantigens) that may be presented to the immune system resulting in an enhanced tumor immune response. These observations seem to make HsMSH2, HsMSH6, HsMLHI and HsPMS2 potential tumor-specific drug targets that might elevate neoantigens and enhance an immunotherapy response.

Rationale for Developing Drugs that Specifically Target HsMSH2-HsMSH3

While defects in HsMSH2, HsMSH6, HsMLHI and HsPMS2 have been clearly linked to hereditary and sporadic cancer development there is no convincing evidence that defects in HsMSH3, HsPMSI or HsMLH3 similarly cause cancer. There may be several reasons for these differences. In the case of HsMLH3, it is likely that it plays either no role or an extremely minor role in MMR, but rather functions during meiosis with the meiosis-specific MSH homologs MSH4-MSH5 in linking homologous chromosomes prior to meiosis I metaphase/anaphase. Similarly, HsPMSI either has no function in MMR and functions specifically in the HsMSH2-HsMSH3 pathway that is responsible for the recognition and repair of IDL mismatches.

IDL mismatches that are specifically recognized by the HsMSH2-HsMSH3 heterodimer occur with significantly less frequency than single basepair mismatches. Moreover, the cellular concentration of the HsMSH2-HsMSH3 protein is at least 5-fold less than HsMSH2-HsMSH6. These two reasons could potentially account for the lack of any convincing link between HsMSH3 defects and cancer. However, a more intriguing possibility is that HsMSH3 defects would specifically lead to a significant increase in frame-shift mutations resulting in entirely new peptides presented on a cell surface containing multiple and/or potent neoantigens. These multiple/potent neoantigenes could then elicit an innate immune response eliminating a preneoplasia before it really got started.

One of the major problems in selecting MMR as a drug target is that by eliminating MMR one runs the risk of accelerating spontaneous mutations resulting from unrepaired replication misincorporation errors with a corresponding induction of tumorigenesis. This would almost surely be the case if HsMSH2, HsMSH6, HsMLHI or HsPMS2 were targeted by drug therapy. However, this would likely not likely be the case if HsMSH3 were targeted. Ultimately specifically targeting HsMSH3 means specifically targeting the HsMSH2-HsMSH3 heterodimer since there is no evidence that any of these MMR proteins exist alone in the cell. Moreover, to make the drug specific it must target HsMSH2-HsMSH3 alone and not HsMSH2- HsMSH6, insuring that the common core component HsMSH2 remains fully functional. This is because aging evidence has shown that HsMSH2 regulates the ATP binding cycle that leads to the formation of a sliding clamp by HsMSH2-HsMSH6 (J. Biol. Chem. 286:40287, 201 1). It is likely that HsMSH2 plays a similar functional role within the HsMSH2-HsMSH3 heterodimer, although this hypothesis remains untested.

Assay Development

The MSH binding and sliding clamps formation comparison assay is based on a FRET comparison of MSH2-MSH6 heterodimer activity on a blocked-end G/T mismatch-containing oligonucleotide compared to MSH2-MSH3 heterodimer activity on a double blocked-end oligonucleotide containing a (CA)₄ I DL mismatch (Fig. 1). The assay may be used to distinguish mismatch recognition (high FRET) from sliding clamp formation (intermediate FRET). A G/T and G/+(CA)₄ may not be the only distinguishing mismatches for HsMSH2- HsMSH3 and HsMSH2-HsMSH6.

DNA Flourophore Labeling and Preparation - DNA oligonucleotides may be easily labeled with near 100% efficiency with fluorescent (Cy/AF) dyes. To increase homogeneity between multiple substrates, the G-strand is always labeled with the acceptor fluorophore and then annealed with a complementary strand containing no mismatch (G/C), a single nucleotide mismatch (G/T) or an IDL mismatch [G/+(CA)₄]

1. Use ~10 nmol of DNA in 100 pl_ aqueous solution.

2. Add 10 mI_ of 3 M sodium acetate pH 5.2 buffer, and 250 mI_ of 95% EtOH, mix well, keep the mixture in -80 °C for 2 hours (Ethanol precipitation).

3. Centrifuge at 14000 rpm for 1 hour. Usually a white pellet of DNA is formed at the bottom of the tube.

4. Wash the precipitate with 70% EtOH, centrifuged for 20 min.

5. Repeat step 4.

6. Air dry the pellet.

7. Resuspend DNA pellet in 70-100 uL of 0.1 M sodium tetraborate pH8.5 and add Cy- Dye previously dissolved in dimethylformamide. The Cy-dye should be 10-30x molar excess.

8. Mix very well.

9. Keep in the thermo mixture overnight at 23°C and 500 rpm. 10. Repeat steps 2-6, before the HPLC purification. If the pellet is not fluorescent, repeat the labeling process again to get a high labeling efficiency.

11. The flourophore-labeled single-stranded DNA oligonucleotide is separated from unlabeled oligonucleotide and unincorporated labeled by C18 reverse-phase HPLC chromatography.

Note: For ~20 nmoles of DNA, the resuspended solution tends to be cloudy during the labeling. As a solution total reaction volume can be increased to 100 pL to get efficient labeling. The step# 7 is then modified as; Dissolve the pellet in 87 uL of 0.1 M sodium tetraborate, pH 8.5 labeling buffer, and to that add -200 nmol of Cy dye dissolved in 13 pL of DMSO.

The flourophore-labeled oligonucleotide is annealed with it complementary unlabeled DNA in a 1 : 1 ratio by heating to 95°C and slow step cooling. The duplex DNA product is then purified from any remaining single stranded DNA substrates by ion exchange HPLC chromatography.

Fluorescent Labeling of MSH Proteins - MSH proteins may be labeled on the N- or C- terminus by introducing a Sortase recognition sequence (LPXTG) and utilizing the Sortase transpeptidase enzyme. An Example of N-terminal HsMSH2-HsMSH3 flourophore labeling is:

Preparation of the Cv3-CLPETGG Peptide

10mg of lyophilized CLPETGG peptide (SEQ ID NO: 1 , GenScript) is dissolved in 200ul of degassed reaction buffer (50 mM Tris 7.0 and 5 mM TCEP) and incubated at room temperature for 30mins. The reaction was then added to 3 mg of lyophilized Sulfo-Cy3 maleimide (Lumiprobe) and incubated overnight at 4°C. The labeled peptide was then purified with reverse phase chromatography using a C18 column. The reaction mixture was loaded on the column in Buffer A (water and 0.1 % TFA) and eluted over a gradient with Buffer B (acetonitrile and 0.1 % TFA). Peak fractions were lyophilized, and stored at -80 °C until needed. For labeling the peptide is dissolved in Sortase reaction buffer (25 mM HEPES pH 7.8, 150 mM NaCI and 10% glycerol) and optimized with small scale Sortase labeling reactions.

MSH Sortase Tag Addition and Expression

MSH proteins may be modified on the N-terminus to contain a Sortase recognition sequence with two serine spacers, a hexa-histidine metal affinity chromatography tag, and a linker to MSH3 (HHHHHH-SS-LPETG-GGGS-MSH protein, SEQ ID NO:2 for underlined portion). Similarly, a C-terminal modification may include a linker to the protein, the Sortase recognition sequence with two serine spacers and a hexa-histidine metal affinity chromatography tag (MSH protein-GG-LPETG-SS-HHHHHH. SEQ I D NO:3 for underlined portion). MSH proteins were cloned into pFastBad (Invitrogen). The proteins were expressed in insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Cells were pelleted, washed with washing buffer (25 mM HEPES pH 7.8, 150 ml, 10% glycerol, 0.5 mM EDTA, 0.5 mM PMSF, 2.3 mM leupeptin and 1.2 mM pepstatin) and stored in freezing buffer (25mM HEPES pH 7.8, 300mM NaCI, 10% glycerol, 20 mM Imidazole, 0.5 mM PMSF, 2.3 pM leupeptin and 1.2 pM pepstatin) at -80°C.

Example of HsMSH2-HsMSH3 Purification and Labeling

The frozen pellets were thawed and lysed by passing through a 25G needle three times and clarified by centrifuging at 120,000 x g for 1 hr at 4°C. The supernatant was loaded onto a 3ml Ni-NTA column pre-equilibrated in Ni-A1 buffer (25 mM HEPES pH 7.8, 200 mM NaCI, 10% glycerol, 20 mM Imidazole, 0.5 mM PMSF, 2.3 pM leupeptin and 1.2 pM pepstatin). The column was washed with 15 ml of Ni-A2 buffer (25 mM HEPES pH 7.8, 1 M NaCI, 10% glycerol, 20mM Imidazole, 0.5 mM PMSF, 2.3 pM leupeptin and 1.2 pM pepstatin) and then washed with 10 ml of Ni-A1 buffer. The protein was then eluted with 10 ml of Ni-B buffer (25 mM HEPES pH 7.8, 200 mM NaCI, 10% glycerol, 200 mM Imidazole, 0.5 mM PMSF, 2.3 uM leupeptin and 1.2 pM pepstatin). Peak fractions were collected and loaded onto a 1 ml Heparin-Sepharose column pre-equilibrated in Heparin-A buffer (25 mM HEPES pH 7.8, 150 mM NaCI, 10% glycerol). The column was washed with 15 ml of heparin-A buffer and then eluted with 10 ml of heparin-B buffer (25 mM HEPES pH 7.8, 1 M NaCI, 10% glycerol). The peak fractions were pooled and 4-times molar ratio of Sortase (purified as described in Chen et al., 201 1) and 50 times molar ratio of Cy3-CLPETGG probe were combined with the 2.9 nmoles of the purified HsMSH2-HsMSH3 supplemented with 10 mM CaCh and allowed to react for 30mins at 4°C. The reaction was quenched with 20 mM EDTA and loaded onto to a 2 ml 40 kDa MWCO Zeba spin desalting column pre-equilibrated with 25 mM HEPES pH 7.8, 200 mM NaCI, 10% glycerol. The eluent was then loaded back onto the Heparin-Sepharose and eluted as described above. Peak fractions were combined and dialyzed in storage buffer (25 mM HEPES pH 7.8, 150 mM NaCI, 20% glycerol and 1 mM DTT). Labeling efficiencies vary from 50-70%.

Validation of the FRET Mismatch Recognition Assay

FRET detection was performed utilizing a FlouroMax-4 (Horiba) according to manufacturers recommendations [Melhuish, M.H., Absolute Spectroflourometry. J. Res. Nat. Bureau. Stand. - Sec. C (Engineering and Instrumentation). A76:547-560, 1972] The Flouromax-4 is a corrected photon counting system where the Intensity (counts per sec / mA) is: corrected signal detector (S1c) / corrected reference detector (R1c) or S1c/R1 c. Because FRET calculations are ratios of acceptor and donor intensities, the S1c/R1c read-out from the FluoroMax (or any other corrected photon counting reader) may be used directly as measures of fluorescence intensity (I). While peak intensity at specific acceptor (-560) and donor (-670) wavelengths may be used as an initial screen for FRET efficiency calculations, increased accuracy may be obtained by fitting the scanned peak intensities to a Gaussian and taking the area under the curve. Apparent FRET efficiency (EA_pp) is calculated by: EA = (U - U-DNA omy) / [(IA - IA-DNA only) + (ID - ID-DNA omy)]; where l_A is the MSH+DNA acceptor intensity, U-DNA omy is the acceptor intensity of the DNA alone, I D is the MSH+DNA donor intensity, and ID-DNA om_y is the donor intensity of the DNA alone (see: Fig. 3). Thus, EA corrects for background contribution of 510 nm excitation that results in the acceptor Cy5-DNA Gaussian emissions with peaks at -560 nm and at -670 nm in the absence of donor Cy3-MSH protein. The donor Cy3-MSH protein alone does not contribute to background Gaussian emission with a peak at -670 nm, and therefore does not require a correction factor (see: Fig. 5C and 5D).

Figure 2. FRET Analysis of Mismatch Recognition by HsMSH2-HsMSH3 and HsMsh2- HsMSH6. The assay is designed to detect and compare FRET on different DNA substrates as described in the text. In the absence of ATP FRET is a measure of steady-state substrate recognition, while of the presence of ATP FRET is a measure of stable sliding clamp formation. A) FRET calculations using 20 nM HsMSH2-HsMSH3 with 80 nM DNA substrates. B) Representative examples of corrected S1c/R1 c intensity profiles used for FRET calculations in panel A. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. C) FRET calculations using 20 nM HsMSH2-HsMSH6 with 80 nM DNA substrates. D) Representative examples of corrected S1 c/R1c intensity profiles used for FRET calculations in panel C. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. E) FRET calculations using 20 nM HsMSH2-HsMSH3 with 40 nM DNA substrates. F) Representative examples of corrected S1c/R1 c intensity profiles used for FRET calculations in panel E. Note that genuine FRET entails anti-correlated intensity changes of acceptor and donor peaks. G) FRET calculations using 20 nM HsMSH2- HsMSH6 with 40 nM DNA substrates. H) Representative examples of corrected S1 c/R1 c intensity profiles used for FRET calculations in panel G. Note that genuine FRET entails anti correlated intensity changes of acceptor and donor peaks. Error bars from at least two separate experiments are shown.

It should be noted that while there appears to be background recognition by HsMSH2- HsMSH6 of G/+(CA)₄ +/-ATP, there is essentially zero recognition of a G/T mismatch by HsMSH2-HsMSH3. This difference and a comparison of potential drug effects on these differences is the basis of the high-throughput drug screen. Moreover and comparison analysis of the effect of a potential drug +/-ATP informs the differential effect on mismatch binding versus sliding clamp formation.

Figure 3. Determining a Correction Factor for the Contribution of Cy5-DNA to 670 nm Emission. Excitation of the Cy5 acceptor located on the DNA substrate by 510 nm (Cy3 excitation wavelength) results in a concentration-dependent increase in -670 nm emission. Unless corrected, this “false FRET” will contribute to FRET calculations and erroneously increase the background. In the FRET system described, the complementary strand (G- strand) is always the one labeled with the acceptor fluorophore and always at the same labeling efficiency. Thus, correcting for the DNA concentration effect on 670 nm emission from 510 nm excitation may be done with any one of the DNA substrates utilized in the FRET comparison assay.

Figure 4. Comparison of Uncorrected and Corrected FRET at Various Protein-to-DNA Ratios. A) Uncorrected FRET (EA_pp) in assays utilizing 20 nM HsMSH2-HsMSH6 with 40 nM (1 :2), 80 nM (1 :4), 400 nM (1 : 10) and 800 nM (1 :20) G/T mismatch substrate DNA without (black) and with (grey) ATP. B) Corrected FRET (E_App) in assays utilizing 20 nM HsMSH2- HsMSH6 with 40 nM (1 :2), 80 nM (1 :4), 400 nM (1 : 10) and 800 nM (1 :20) G/T mismatch substrate DNA without (black) and with (grey) ATP. Note that at lower ratios of MSH:DNA (1 :2 and 1 :4) the FRET difference between +/-ATP remain clear, while at elevated ratios the contribution of the DNA alone contributes to significant error.

Figure 5. The Effect of DMSO on Fluorescence Excitation and Emission. A) DMSO alone has an intrinsic effect on 510 nm emission over the wavelengths that are used to calculate FRET. This intrinsic emission may become significant depending on the MSH:DNA ratio and should be determined prior to any screen utilizing FRET analysis. B) DMSO affects -560 nm and -670 nm emission of assays that include HsMSH2-HsMSH6 and G/T DNA. C) DMSO reduces HsMSH2-HsMSH3 emission at -560 nm, but has no effect on -670 emission. D) DMSO reduces HsMSH2-HsMSH6 emission at -560 nm, but has no effect on -670 emission.

Figure 6. Determining a Correction Factor for the Contribution of Cy5-DNA to 670 nm Emission in the Presence of DMSO. DMSO affects fluorescence emission of Cy5-DNA at both 560 nm and 670 nm. A suitable concentration-dependent correction factor may be obtained by scanning across wavelength in the presence of DMSO. A) The effect of DMSO on 40 nM Cy5-G/C DNA. B) The effect of DMSO on 80 nM Cy5-G/C DNA.

Figure 7. The Effect of DMSO on FRET Recognition by HsMSH2-HsMSH6. DMSO at indicated concentrations was added to the HsMSH2-HsMSH6 FRET analysis with Cy5- labeled duplex (G/C) and mismatched (G/T) DNA substrate. A) 20 nM HsMSH2-HsMSH6 with 40 nM DNA. B) 20 nM HsMSH2-HsMSH6 with 80 nM DNA. We observe a consist high FRET with HsMSH2-HsMSH6 that resolves to a clearly discernable intermediate FRET state that is well above the duplex DNA binding background without DMSO and in the presence of 0.1% DMSO. This well-defined difference begins to disappear by 0.5% DMSO. We conclude that the FRET assay is relatively insensitive to DMSO below 0.5%. Since most compound libraries are dissolved in DMSO, these data demonstrate the limits of compound inclusion that is dissolved in DMSO.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying an agent that selectively inhibits mismatch repair (MMR) in a cell, the method comprising contacting MMR proteins with a candidate agent, and assaying the MMR proteins for binding to a DNA oligonucleotide, interaction between MutS homologs and MutL homologs, ATP binding to the MMR proteins, or the formation of a sliding clamp by MutS and/or MutL homologs.

2. The method of claim 1 , wherein the MMR proteins and/or DNA oligonucleotide are labeled with fluorescence resonance energy transfer (FRET) fluorophore pairs.

3. The method of claim 1 or claim 2, wherein assaying the MMR proteins comprises:

(a) providing a sliding clamp system comprising

(1) a first MMR heterodimer comprising MutS homologs;

(2) a second MMR heterodimer comprising MutL homologs;

(3) the oligonucleotide comprising at least one mismatched nucleotide, wherein the oligonucleotide is linked at one end to a surface and blocked at the other end with a macromolecule;

(4) an acceptor fluorophore conjugated to the first or second MMR heterodimer; and

(5) a donor fluorophore conjugated to the first MMR heterodimer, the second MMR heterodimer, or the mismatched nucleotide;

(b) contacting the sliding clamp system with a candidate agent; and

(c) measuring fluorescence resonance energy transfer (FRET) resonance of the sliding clamp system.

4. The method of claim 3, wherein the acceptor fluorophore is conjugated to the first MMR heterodimer and the donor fluorophore is conjugated to the mismatched nucleotide.

5. The method of claim 3, wherein the acceptor fluorophore is conjugated to a first protein of the first MMR heterodimer and the donor fluorophore is conjugated to a second protein of the first MMR heterodimer.

6. The method of claim 3, wherein the acceptor fluorophore is conjugated to the first MMR heterodimer and the donor fluorophore is conjugated to the second MMR heterodimer.

7. The method of claim 3, wherein the acceptor fluorophore is conjugated to a first protein of the second MMR heterodimer and the donor fluorophore is conjugated to a second protein of the second MMR heterodimer.

8. The method of claim 3, wherein a decrease or increase in FRET resonance compared to a control is an indication that the candidate agent inhibited MMR.

9. The method of any one of claims 1 to 8, wherein the sliding clamp system further comprises the presence of ATR

10. The method of any one or claims 1 to 9, wherein the first MMR heterodimer comprises MutSa, MutSy, or Muΐqb heterodimers.

11. The method of claim 10, wherein the first MMR heterodimer comprises i) MSH2 and MSH3 or ii) MSH 2 and MSH6.

12. The method of any one of claims 1 to 11 , wherein the second MMR heterodimer comprise MutLa, Mutl-b, or MutLy heterodimers.

13. The method of claim 12, wherein the second MMR heterodimer comprises i) MLH1 and PMS1 or ii) MLH1 and PMS2.

14. The method of claim any one of claims 1 to 13, wherein the DNA oligonucleotide comprises a single nucleotide mismatch.

15. The method of claim any one of claims 1 to 14, wherein the DNA oligonucleotide comprises an insertion and deletion loop (IDL).

16. The method of claim any one of claims 1 to 15, wherein the blocking macromolecule comprises biotin-streptavidin.

17. The method of claim any one of claims 1 to 16, wherein the blocking macromolecule comprises antibody-bound 5’-digoxygenin.

18. The method of claim any one of claims 1 to 17, wherein the oligonucleotide comprises 20 to 200 bp.

19. The method of claim any one of claims 3 to 18, wherein the sliding clamp system has a ratio of first or second MMR heterodimer to DNA oligonucleotide greater than 0.1.

20. The method of claim 19, wherein the sliding clamp system has a ratio of first or second MMR heterodimer to DNA oligonucleotide between 0.25 and 0.5.

21. The method o of claim any one of claims 2 to 20 wherein the sliding clamp system is disposed in a solution comprising less than 0.5% DMSO.

22. The method of any one of claims 1 to 21 , wherein the cell a prokaryotic cell or an animal cell.

23. The method of claim 22, wherein the animal cell is a human cell.

24. The method of claim 22, wherein the animal cell is a cancer cell.

25. The method of claim 22, wherein the prokaryotic cell is pathogenic.

26. The method of claim 25, wherein the pathogenic cell is one of a Enterococcus,

Staphylococcus, Klebsiellai, Acinetobacter, Pseudomonas, or Enterobacter genus.

27. The method of claim 26, wherein the pathogenic prokaryotic cell is Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,

Pseudomonas aeruginosa, or Enterobacter cloacae species.

28. A method of treating a cancer in a subject, comprising treating the subject with an agent that selectively inhibits mismatch repair (MMR) in the subject followed by or in combination with treatment with a checkpoint inhibitor to make the cancer hypersensitive to immune-surveillance.

29. The method of claim X, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or a combination thereof.

30. The method of claim X, wherein cancer has intact or functional DNA mismatch repair. The method of claim X, wherein the subject has been diagnosed with Lynch syndrome.