Cyclic Peptide Inhibitors of the MLH1-MSH3 Interaction as a Potential Therapeutic Strategy for Huntington's Disease

Components of the DNA mismatch repair (MMR) pathway modulate somatic CAG-repeat expansion at the huntingtin (HTT) locus and alter the age of clinical motor diagnosis and progression of Huntington's disease (HD). MSH3, which encodes a subunit of MutSβ (MSH2-MSH3), has been identified as a genetic modifier of age at onset of multiple clinical phenotypes in people with HD (PwHD). MutSβ interacts with MutL endonucleases (MutLα, MLH1-PMS2 and MutLγ MLH1-MLH3) in a ternary complex with DNA to initiate MMR. Disruption of the MLH1-MSH3 protein-protein interaction (PPI) represents a potential therapeutic strategy to inhibit MMR and limit somatic CAG-repeat expansion. Leveraging the MLH1 Interaction Protein (MIP) Box sequence and the structural information of the hMLH1 C-terminal domain (CTD) bound to an MSH3-derived peptide (21 QAVLSRFFQ 29), we designed and optimized potent linear and cyclic peptides targeting the MLH1-MSH3 interface. Dimerization of the optimized cyclic peptide (compound 52) enhanced binding affinity, achieving double-digit nanomolar potency in a MutSβ:MutLα:DNA (MLH1-MSH3-DNA) ternary complex assay in the presence of 100 μM of ATP (compound 52 IC50 = 87 nM, compound 56 IC50 = 32 nM). Evaluation in an in vitro MMR DNA repair assay using cellular nuclear extracts from MMR-proficient and-deficient cell lines demonstrated that our lead compound (56) effectively inhibits MutSβ/MutLα-dependent DNA repair in human cells. These results highlight cyclic peptide-mediated disruption of MLH1-MSH3 as a promising approach to modulate MMR activity and possibly mitigate somatic CAG-repeat expansion in HD.