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Identification and characterization of the first small molecule inhibitor of MDMX.

ABSTRACT: The p53 pathway is disrupted in virtually every human tumor. In approximately 50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and small peptides have been developed that bind MDM2 in the p53-binding pocket and displace the p53 protein, leading to p53-mediated cell cycle exit and apoptosis. To date, peptide inhibitors of MDMX have been developed, but no small molecule inhibitors have been reported. We have developed biochemical and cell-based assays for high throughput screening of chemical libraries to identify MDMX inhibitors and identified the first MDMX inhibitor SJ-172550. This compound binds reversibly to MDMX and effectively kills retinoblastoma cells in which the expression of MDMX is amplified. The effect of SJ-172550 is additive when combined with an MDM2 inhibitor. Results from a series of biochemical and structural modeling studies suggest that SJ-172550 binds the p53-binding pocket of MDMX, thereby displacing p53. This lead compound is a useful chemical scaffold for further optimization of MDMX inhibitors that may eventually be used to treat pediatric cancers and various adult tumors that overexpress MDMX or have similar genetic lesions. When combined with selective MDM2 inhibitors, SJ-172550 may also be useful for treating tumors that express wild-type p53.

SUBMITTER: Reed D

PROVIDER: S-EPMC2856285 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Identification and characterization of the first small molecule inhibitor of MDMX.

Reed Damon D Shen Ying Y Shelat Anang A AA Arnold Leggy A LA Ferreira Antonio M AM Zhu Fangyi F Mills Nicholas N Smithson David C DC Regni Catherine A CA Bashford Donald D Cicero Samantha A SA Schulman Brenda A BA Jochemsen Aart G AG Guy R Kiplin RK Dyer Michael A MA

The Journal of biological chemistry 20100115 14

The p53 pathway is disrupted in virtually every human tumor. In approximately 50% of human cancers, the p53 gene is mutated, and in the remaining cancers, the pathway is dysregulated by genetic lesions in other genes that modulate the p53 pathway. One common mechanism for inactivation of the p53 pathway in tumors that express wild-type p53 is increased expression of MDM2 or MDMX. MDM2 and MDMX bind p53 and inhibit its function by distinct nonredundant mechanisms. Small molecule inhibitors and sm ...[more]

PMID: 20080970

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Project description:Rabies virus (RABV) causes a severe and fatal neurological disease, but morbidity is vaccine preventable and treatable prior to the onset of clinical symptoms. However, immunoglobulin (IgG)-based rabies postexposure prophylaxis (PEP) is expensive, restricting access to life-saving treatment, especially for patients in low-income countries where the clinical need is greatest, and does not confer cross-protection against newly emerging phylogroup II lyssaviruses. Toward identifying a cost-effective replacement for the IgG component of rabies PEP, we developed and implemented a high-throughput screening protocol utilizing a single-cycle RABV reporter strain. A large-scale screen and subsequent direct and orthogonal counterscreens identified a first-in-class direct-acting RABV inhibitor, GRP-60367, with a specificity index (SI) of >100,000. Mechanistic characterization through time-of-addition studies, transient cell-to-cell fusion assays, and chimeric vesicular stomatitis virus (VSV) recombinants expressing the RABV glycoprotein (G) demonstrated that GRP-60367 inhibits entry of a subset of RABV strains. Resistance profiling of the chemotype revealed hot spots in conserved hydrophobic positions of the RABV G protein fusion loop that were confirmed in transient cell-to-cell fusion assays. Transfer of RABV G genes with signature resistance mutations into a recombinant VSV backbone resulted in the recovery of replication-competent virions with low susceptibility to the inhibitor. This work outlines a tangible strategy for mechanistic characterization and resistance profiling of RABV drug candidates and identified a novel, well-behaved molecular probe chemotype that specifically targets the RABV G protein and prevents G-mediated viral entry.IMPORTANCE Rabies PEP depends on anti-RABV IgG, which is expensive and in limited supply in geographical areas with the highest disease burden. Replacing the IgG component with a cost-effective and shelf-stable small-molecule antiviral could address this unmet clinical need by expanding access to life-saving medication. This study has established a robust protocol for high-throughput anti-RABV drug screens and identified a chemically well-behaved, first-in-class hit with nanomolar anti-RABV potency that blocks RABV G protein-mediated viral entry. Resistance mapping revealed a druggable site formed by the G protein fusion loops that has not previously emerged as a target for neutralizing antibodies. Discovery of this RABV entry inhibitor establishes a new molecular probe to advance further mechanistic and structural characterization of RABV G that may aid in the design of a next-generation clinical candidate against RABV.

Dataset Information

Identification and characterization of the first small molecule inhibitor of MDMX.

Publications

Identification and characterization of the first small molecule inhibitor of MDMX.

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