A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N 2 ,N 4 -dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.apoptosis | autophagy | unfolded protein response T he AAA (ATPase associated with diverse cellular activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (1) and mice (2). p97 was first linked to the ubiquitin-proteasome system (UPS) through its role in the turnover of ubiquitin−β-galactosidase fusion proteins via the "ubiquitin fusion degradation" (UFD) pathway (3). Since then, p97 has been shown to play a critical role in the degradation of misfolded membrane and secretory proteins (4) and has also been linked to a broad array of cellular processes, including Golgi membrane reassembly (5), membrane transport (6), regulation of myofibril assembly (7), cell division (8), formation of protein aggregates (9), and autophagosome maturation (10, 11). The broad range of cellular functions for p97 is thought to derive from its ability to unfold proteins or disassemble protein complexes, but the detailed mechanism of how p97 works and is linked to specific cellular processes remains largely unknown.The structure of p97 comprises three domains: an N-terminal domain that recruits adaptors/substrate specificity factors, followed by two ATPase domains, D1 and D2 (12, 13). p97 monomers assemble to form a homohexamer that is thought to provide a platform for transduction of chemical activity into mechanical force that is applied to substrate proteins. The D1 domain mediates hexamerization (14) and has very low ATPase activity (15). Most of the ATPase activity is contributed by the D2 domain, which is thought to underlie p97's function as a mechanochemical transducer (16).The mechanochemical activity of p97 is linked to substrate proteins by an array of 13 UBX (ubiquitin regulatory X) domain adapters that bind the N-terminal domain of p97 (17), as well as the non-UBX domain adaptors Ufd1 and Npl4 (18). The functions and mechanisms of action of these different p97-adaptor complexes remain poorly u...
A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (16-(R), (R)-N-(4-(tert-butyl)phenyl)-N-(2-(tert-butylamino)-2-oxo-1-(pyridin-3-yl)ethyl)furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, 16-(R) is a non-covalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multi-component Ugi reaction was utilized to rapidly explore structure activity relationships within S1′, S1, and S2 enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with 16-(R) was instrumental in guiding subsequent rounds of chemistry optimization. 16-(R) provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a non-covalent mechanism of action.
Herein we report the discovery and SAR of a novel series of SARS-CoV 3CLpro inhibitors identified through the NIH Molecular Libraries Probe Production Centers Network (MLPCN). In addition to ML188, ML300 represents the second probe declared for 3CLpro from this collaborative effort. The X-ray structure of SARS-CoV 3CLpro bound with a ML300 analog highlights a unique induced-fit reorganization of the S2-S4 binding pockets leading to the first sub-micromolar non-covalent 3CLpro inhibitors retaining a single amide bond.
Virtually all transcription factors partner with coactivators that
recruit chromatin remodeling factors and interact with the basal transcription
machinery. Coactivators have been implicated in cancer cell proliferation,
invasion and metastasis, including the p160 steroid receptor coactivator (SRC)
family comprised of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3
(AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent
candidate molecular targets for new chemotherapeutics. Here we report on the
results of a high throughput screening effort which identified the cardiac
glycoside bufalin as a potent small molecule inhibitor for SRC-3 and SRC-1.
Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer
cell growth at nanomolar concentrations. When incorporated into a nanoparticle
delivery system, bufalin was able to reduce tumor growth in a mouse xenograft
model of breast cancer. Our work identifies bufalin as a potentially
broad-spectrum small molecule inhibitor for cancer.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.