ABSTRACT
The design of potent Pin1 inhibitors has been challenging because its active site specifically recognizes a phospho-protein epitope. The de novo design of phosphate-based Pin1 inhibitors focusing on the phosphate recognition pocket and the successful replacement of the phosphate group with a carboxylate have been previously reported. The potency of the carboxylate series is now further improved through structure-based optimization of ligand-protein interactions in the proline binding site which exploits the H-bond interactions necessary for Pin1 catalytic function. Further optimization using a focused library approach led to the discovery of low nanomolar non-phosphate small molecular Pin1 inhibitors. Structural modifications designed to improve cell permeability resulted in Pin1 inhibitors with low micromolar anti-proliferative activities against cancer cells.
Subject(s)
Benzimidazoles/pharmacology , Carboxylic Acids/pharmacology , Enzyme Inhibitors/pharmacology , Peptidylprolyl Isomerase/antagonists & inhibitors , Phosphates/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Catalytic Domain/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/metabolism , Structure-Activity RelationshipABSTRACT
The P21-activated kinases (PAK) are emerging antitumor therapeutic targets. In this paper, we describe the discovery of potent PAK inhibitors guided by structure-based drug design. In addition, the efflux of the pyrrolopyrazole series was effectively reduced by applying multiple medicinal chemistry strategies, leading to a series of PAK inhibitors that are orally active in inhibiting tumor growth in vivo.
Subject(s)
Antineoplastic Agents/chemical synthesis , Pyrazoles/chemical synthesis , Pyrroles/chemical synthesis , p21-Activated Kinases/antagonists & inhibitors , Administration, Oral , Amides/chemical synthesis , Amides/pharmacokinetics , Amides/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Carbamates/chemistry , Carbamates/pharmacokinetics , Carbamates/pharmacology , Crystallography, X-Ray , Dogs , Humans , Hydrogen Bonding , Mice , Models, Molecular , Molecular Conformation , Permeability , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrroles/pharmacokinetics , Pyrroles/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Despite abundant evidence that aberrant Rho-family GTPase activation contributes to most steps of cancer initiation and progression, there is a dearth of inhibitors of their effectors (e.g., p21-activated kinases). Through high-throughput screening and structure-based design, we identify PF-3758309, a potent (K(d) = 2.7 nM), ATP-competitive, pyrrolopyrazole inhibitor of PAK4. In cells, PF-3758309 inhibits phosphorylation of the PAK4 substrate GEF-H1 (IC(50) = 1.3 nM) and anchorage-independent growth of a panel of tumor cell lines (IC(50) = 4.7 +/- 3 nM). The molecular underpinnings of PF-3758309 biological effects were characterized using an integration of traditional and emerging technologies. Crystallographic characterization of the PF-3758309/PAK4 complex defined determinants of potency and kinase selectivity. Global high-content cellular analysis confirms that PF-3758309 modulates known PAK4-dependent signaling nodes and identifies unexpected links to additional pathways (e.g., p53). In tumor models, PF-3758309 inhibits PAK4-dependent pathways in proteomic studies and regulates functional activities related to cell proliferation and survival. PF-3758309 blocks the growth of multiple human tumor xenografts, with a plasma EC(50) value of 0.4 nM in the most sensitive model. This study defines PAK4-related pathways, provides additional support for PAK4 as a therapeutic target with a unique combination of functions (apoptotic, cytoskeletal, cell-cycle), and identifies a potent, orally available small-molecule PAK inhibitor with significant promise for the treatment of human cancers.
Subject(s)
Cell Proliferation/drug effects , Models, Molecular , Neoplasms/metabolism , Pyrazoles/pharmacology , Pyrroles/pharmacology , Signal Transduction/drug effects , p21-Activated Kinases/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Crystallography , Guanine Nucleotide Exchange Factors/metabolism , Humans , Neoplasms/drug therapy , Phosphorylation/drug effects , Pyrazoles/chemistry , Pyrazoles/metabolism , Pyrroles/chemistry , Pyrroles/metabolism , Rho Guanine Nucleotide Exchange FactorsABSTRACT
Pin1 is a member of the cis-trans peptidyl-prolyl isomerase family with potential anti-cancer therapeutic value. Here we report structure-based de novo design and optimization of novel Pin1 inhibitors. Without a viable lead from internal screenings, we designed a series of novel Pin1 inhibitors by interrogating and exploring a protein crystal structure of Pin1. The ligand efficiency of the initial concept molecule was optimized with integrated SBDD and parallel chemistry approaches, resulting in a more attractive lead series.