Protein interfaces

Abstract

The present disclosure provides methods to treat conditions, including cancer, using compounds that can target resistant cancer cells. The compounds can be used to sensitize resistant cancer cells or decrease the proliferation of cells. The compounds can target proteins in the DNA damage repair pathway leading to a decrease in DNA damage repair in target cells.

What is claimed is: 
     
         1 . A method for causing cancer cell death, the method comprising:
 contacting a cancer cell with a non-naturally occurring polypeptide that inhibits to RAD51, thereby causing cell death.   
     
     
         2 . The method of  claim 1 , wherein the polypeptide comprises an amino acid sequence according to the following formula or an invert thereof:
   X 1 -X 2 -X 3 -X 4 -X 5 -X 6      wherein:   X 1  and X 2  are present or absent, and are independently selected from T/K/R/Q;   X 3  is T/K/R/Q;   X 4  is L/I/V/F/M/W/Y;   X 5  is R/G/S; and   X 6  is L/I/V/F/M/W/Y (SEQ ID NO.:70),   wherein any one or more of the amino acids are optionally (D) amino acids.   
     
     
         3 . The method of  claim 1 , wherein the polypeptide comprises an amino acid sequence according to the following formula or an invert thereof:
   X 1 -L-G-X 4 , wherein   X 1  is R/K and   X 4  is M/V (SEQ ID NO.:71),   wherein any one or more of the amino acids are optionally (D)-amino acids.   
     
     
         4 . The method of  claim 1 , wherein the polypeptide comprises an amino acid sequence according the following formula or an invert thereof:
   R-L-G-V-X 5 -X 6 , wherein   X 5  is M/V, and   X 6  is selected from L/I/V/F/M/A/W/Y (SEQ ID NO.: 72),   wherein any one or more of the amino acids are optionally (D)-amino acids.   
     
     
         5 . The method of  claim 1 , wherein the polypeptide comprises an amino acid sequence according to the following formula or an invert thereof:
   X 1 -X 2 -R-L-G-V-X 7 -X 8 , wherein   X 1  is present or absent, and is selected from T/K/R/Q,   X 2  is selected from T/K/R/Q,   X 7  is selected from M/V, and   X 8  is selected from L/I/V/F/M/A/W/Y (SEQ ID NO: 73);   wherein any one or more of the amino acids are optionally (D)-amino acids.   
     
     
         6 . The method of  claim 1 , wherein the polypeptide has a binding constant to RAD51 with a K d  value of 10 −4  M or less. 
     
     
         7 . The method of  claim 1 , wherein the polypeptide further comprises a cell-penetrating peptide sequence. 
     
     
         8 . The method of  claim 1 , wherein the contacting the cell results in an increase in free calcium concentration within the cell, thereby causing cell death. 
     
     
         9 . The method of  claim 1 , wherein the polypeptide comprises at least one (D)-amino acid. 
     
     
         10 . The method of  claim 1 , wherein the polypeptide is a non-naturally occurring polypeptide. 
     
     
         11 . The method of  claim 1 , wherein contacting the cell with the polypeptide results in altered assembly of RAD51 monomers on DNA. 
     
     
         12 . The method of  claim 1 , wherein contacting the cell with the polypeptide results in inhibition of cellular homologous recombination within the cell. 
     
     
         13 . The method of  claim 1 , wherein the peptide does not inhibit RAD51 ATPase activity. 
     
     
         14 . The method of  claim 1 , wherein the polypeptide specifically binds to RAD51-AP1's binding site on RAD51. 
     
     
         15 . The method of  claim 1 , further comprising administration of a chemotherapeutic agent, wherein contacting the cell with the polypeptide sensitizes the cell to the chemotherapeutic agent. 
     
     
         16 . A method for screening for polypeptide inhibitors of a protein interaction between a first test protein and a second test protein, the method comprising:
 expressing, in a plurality of host cells, a first fusion protein comprising a first test protein and a DNA-binding moiety; and   expressing, in the plurality of host cells, a second fusion protein comprising the second test protein and a gene-activating moiety;   expressing, in each of the plurality of host cells, a randomized polypeptide;   wherein each of the plurality of host cells comprises a polynucleotide that encodes (1) a cytotoxic agent and (2) a promoter for the cytotoxic agent, wherein the DNA-binding moiety is configured to bind to the promoter;   
       wherein binding of the first fusion protein to the second fusion protein activates expression of the death agent, and wherein disruption of binding of the first fusion protein to the second fusion protein permits identification, based on cell viability, of polypeptide inhibitors of the interaction between the first test protein and the second test protein. 
     
     
         17 . The method of  claim 16 , wherein the polynucleotide encodes a plurality of cytotoxic agents whose expression is activated by binding of the first fusion protein to the second fusion protein. 
     
     
         18 . The method of  claim 16 , wherein the cytotoxic reporter is a ribosomally-encoded xenobiotic agent, a ribosomally-encoded poison, a ribosomally-encoded endogenous or exogenous gene that results in severe growth defects upon mild overexpression, a ribosomally-encoded recombinase that excises an essential gene for viability, a limiting factor involved in the synthesis of a toxic secondary metabolite, or any combination thereof 
     
     
         19 . The method of  claim 18 , wherein the cytotoxic reporter is Cholera toxin, SpvB toxin, CARDS toxin, SpyA Toxin, HopU1, Chelt toxin, Certhrax toxin, EFV toxin, ExoT, CdtB, Diphtheria toxin, ExoU/VipB, HopPtoE, HopPtoF, HopPtoG, VopF, YopJ, AvrPtoB, SdbA, SidG, VpdA, Lpg0969, Lpg1978, YopE, SptP, SopE2, SopB/SigD, SipA, YpkA, YopM, Amatoxin, Phallacidin, Killer toxin KP1, Killer toxin KP6, Killer Toxin K1, Killer Toxin K28 (KHR), Killer Toxin K28 (KHS), Anthrax lethal factor endopeptidase, Shiga Toxin, Ricin Toxin, or any combination thereof. 
     
     
         20 . The method of  claim 16 , wherein the plurality of host cells are prokaryotic or eukaryotic. 
     
     
         21 . The method of  claim 16 , wherein the plurality of host cells are fungal cells. 
     
     
         22 . A method for inducing expression in a host cell, comprising:
 (a) expressing in the host cell a first fusion protein comprising the first test protein and a DNA-binding moiety;   (b) expressing in the host cell a second fusion protein comprising the second test protein and a gene activating moiety; and   (c) expressing in the host cell a plurality of cytotoxic genes, wherein the plurality of cytotoxic genes are activated by promoter DNA sequences specific for the DNA binding moiety,   wherein interaction between the first protein and the second protein activates expression of the plurality of cytotoxic genes.   
     
     
         23 . The method of  claim 22 , further comprising expressing a test gene in the host cell, wherein the test gene comprises a DNA sequence that encodes a randomized polypeptide library. 
     
     
         24 . The method of  claim 23 , wherein the randomized polypeptide library comprises polypeptides 60 or fewer amino acids in length. 
     
     
         25 . The method of  claim 23 , wherein the test gene comprises a 3′ UTR of a short protein. 
     
     
         26 . The method of  claim 22 , wherein the DNA sequence that encodes a randomized polypeptide library encodes a common N-terminal stabilization sequence. 
     
     
         27 . A host cell configured to express:
 (a) a first fusion protein comprising a DNA-binding moiety;   (b) a second fusion protein comprising a gene activating moiety;   (c) a plurality of cytotoxic reporters, wherein expression of the cytotoxic reporters is under control of a DNA-binding sequence specific for the DNA-binding moiety; and   (d) a nucleotide sequence comprising a nucleotide sequence encoding a polypeptide of 60 or fewer amino acids, wherein the mRNA comprises a 3′ UTR 3′ to the nucleotide sequence encoding polypeptide, and wherein the nucleotide sequence encoding the polypeptide further encodes an N-terminal sequence for peptide stabilization.   
     
     
         28 . The host cell of  claim 27 , wherein the host cell is fungal or bacterial. 
     
     
         29 . The host cell of  claim 27 , wherein the host cell is a haploid yeast cell. 
     
     
         30 . The host cell of  claim 27 , wherein the host cell is a diploid yeast cell. 
     
     
         31 . The host cell of  claim 27 , wherein the host cell expresses a peptide cyclase or N-methylase that cyclizes or methylates the polypeptide of 60 or fewer amino acids and allowing for screening of cyclized or methylated versions of the polypeptide of 60 or fewer amino acids. 
     
     
         32 . The host cell of  claim 31 , wherein the host cell expresses a peptide cyclase and the polypeptide of 60 or fewer amino acids is expressed as a ribosomally synthesized and post-translationally modified (RiPP) prepropeptide. 
     
     
         33 . A plasmid vector, comprising a nucleotide encoding: a first fusion protein comprising a DNA-binding moiety and a second fusion protein comprising a gene activating moiety; 
     
     
         34 . A library of plasmid vectors, each plasmid vector comprising:
 (a) a DNA sequence encoding a different peptide sequence operably linked to a first switchable promoter; and   (b) a DNA sequence encoding a cytotoxic reporter under control of a second switchable promoter.   
     
     
         35 . A kit comprising the plasmid vector of  claim 33 . 
     
     
         36 . The kit of  claim 35 , further comprising the library of plasmid vectors according to  claim 34 .