ABSTRACT
Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , NIMA-Interacting Peptidylprolyl Isomerase/antagonists & inhibitors , NIMA-Interacting Peptidylprolyl Isomerase/metabolism , Animals , Antineoplastic Agents/chemistry , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Survival/drug effects , Cell Transformation, Neoplastic/genetics , Crystallography, X-Ray , Cysteine/metabolism , Drug Design , Enzyme Inhibitors/metabolism , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , NIMA-Interacting Peptidylprolyl Isomerase/chemistry , NIMA-Interacting Peptidylprolyl Isomerase/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Protein Conformation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
Several DNA-binding proteins, such as topoisomerases, helicases and sliding clamps, have a toroidal (i.e. ring) shape that topologically traps DNA, with this quality being essential to their function. Many DNA-binding proteins that function, for example, as transcription factors or enzymes were shown to be able to diffuse linearly (i.e. slide) along DNA during the search for their target binding sites. The protein's sliding properties and ability to search DNA, which often also involves hopping and dissociation, are expected to be different when it encircles the DNA. In this study, we explored the linear diffusion of four ring-shaped proteins of very similar structure: three sliding clamps (PCNA, ß-clamp, and the gp45) and the 9-1-1 protein, with a particular focus on PCNA. Coarse-grained molecular dynamics simulations were performed to decipher the sliding mechanism adopted by these ring-shaped proteins and to determine how the molecular properties of the inner and outer ring govern its search speed. We designed in silico variants to dissect the contributions of ring geometry and electrostatics to the sliding speed of ring-shaped proteins along DNA. We found that the toroidal proteins diffuse when they are tilted relative to the DNA axis and able to rotate during translocation, but that coupling between rotation and translocation is quite weak. Their diffusion speed is affected by the shape of the inner ring and, to a lesser extent, by its electrostatic properties. However, breaking the symmetry of the electrostatic potential can result in deviation of the DNA from the center of the ring and cause slower linear diffusion. The findings are discussed in light of earlier computational and experimental studies on the sliding of clamps.
