ABSTRACT
Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Cell Cycle Proteins/antagonists & inhibitors , Cell Proliferation/drug effects , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , HCT116 Cells , Humans , Models, Molecular , Molecular Structure , Structure-Activity Relationship , Valosin Containing ProteinABSTRACT
VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
Subject(s)
Acetanilides/pharmacology , Adenosine Triphosphatases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Benzothiazoles/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Neoplasms/drug therapy , Neoplasms/pathology , Acetanilides/chemistry , Adenosine Triphosphatases/metabolism , Allosteric Regulation/drug effects , Antineoplastic Agents/chemistry , Benzothiazoles/chemistry , Cell Cycle Proteins/metabolism , Cell Death/drug effects , Cell Line, Tumor , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Molecular Structure , Neoplasms/metabolism , Structure-Activity Relationship , Valosin Containing ProteinABSTRACT
Valosine containing protein (VCP), also known as p97, is a member of AAA ATPase family that is involved in several biological processes and plays a central role in the ubiquitin-mediated degradation of misfolded proteins. VCP is an ubiquitously expressed, highly abundant protein and has been found overexpressed in many tumor types, sometimes associated with poor prognosis. In this respect, VCP has recently received a great deal of attention as a potential new target for cancer therapy. In this paper, the discovery and structure-activity relationships of alkylsulfanyl-1,2,4-triazoles, a new class of potent, allosteric VCP inhibitors, are described. Medicinal chemistry manipulation of compound 1, identified via HTS, led to the discovery of potent and selective inhibitors with submicromolar activity in cells and clear mechanism of action at consistent doses. This represents a first step toward a new class of potential anticancer agents.
