Discovery, Design And Biological Evaluation Of Protein-Protein Interaction Inhibitors

Gene regulation, immune response, signal transduction, cellular architecture andmany biological processes are involved in the protein-protein interactions. Severalprotein-protein interactions have been proven to be valid drug targets, such as p53-MDM2and Keap1-Nrf2.The p53-MDM2is an important target of cancer. The p53, which is a tumorsuppressor, is activated by inhibiting p53-MDM2interaction. There are several candidatestargeting p53-MDM2interaction have advanced into Phase I clinical trials. Nrf2plays acrucial role in celluar oxidative stresses.Nrf2is able to escape Keap1-mediated degradation, then translocate to the nucleus,and activate antioxidant response element-dependent gene expression of a series ofantioxidative proteins and phase II enzymes to protect the cells. However, by now there areno potent compounds targeting Keap1-Nrf2interaction published yet.In the present dissertation, a series of novel p53-MDM2and Keap1-Nrf2protein-protein interaction inhibitors were identified and rationally designed bystructure-based drug design. Biogical activity and mechanism of action of these inhibitorswere also evaluated.1. Discovery, design and biological evaluation of pyrrolidonederivatives as novel inhibitors of p53-MDM2protein-proteininteractionStructure-based virtual screening (SBVS) was applied to discover structurally diversep53-MDM2inhibitors. From25compounds selected by virtual screening from the Specsdatabase, a total of9compounds were found to be p53-MDM2inhibitors. Compound A5and A9were the most potent ones which had the Kivalues of780nM and570nM,respectively. Further optimization of compound A5was peroformed to obtain a series ofpyrrolidone derivatives. The inhibitory activity, in vitro and in vivo antitumor activity wereevaluated to found more than30highly potent novel small molecules. Interestingly,compound A41was proven to be a potent MDM2/MDMX dual inhibitor. Twodiastereoisomers of compound A60showed quite different results in the p53-MDM2binding assay. In addition, compound A41and A60a could reduce tumor growth with low toxicity and good tolerance. At300mg/kg,52.53%tumor growth inhibition of compoundA60a was achieved.2. Discovery, design and biological evaluation ofpyrrolo[3,4-c]pyrazole derivatives as novel dual inhibitors ofp53-MDM2protein-protein interaction and NF-κB pathwayAseries of novel pyrrolo[3,4-c]pyrazole derivatives were then rationally designed andsynthesized as the first-in-class inhibitors of p53-MDM2interaction and NF-κB pathway.Most of the newly designed compounds were identified to possess nanomolar p53-MDM2inhibitory activity. Four compounds (5j,5q,5s and5u) effectively inhibited tumor growthin the A549xenograft model. Among them, compound5q produced a tumor growthinhibitionof52.43%with low toxicity and good tolerance. Further pharmacokinetic studyrevealed that compound5q exhibited excellent oral bioavailability (72.9%).Moreover, compound5s suppressed NF-κB activation and released p53in A549lungcancer cells.Biochemical assay for the kinases also supported the fact that thepyrrolo[3,4-c]pyrazole compounds directly inhibited the IKKs of the NF-κBpathway.Further biological study and molecular modeling characterized the mechanism ofaction of the two enantiomers of pyrrolo[3,4-c]pyrazole compound5s. These twoenantiomers respectively inhibit p53MDM2interaction and the NF κB pathway. Inaddition, the two enantiomers had some synergistic effect in vivo. This study provides apreliminary strategy to help design of such a probe molecule with dual efficacy in cancertherapy.3. Discovery and biological evaluation of novel Keap1-Nrf2protein-protein interaction inhibitorsStructure-based virtual screening (SBVS) was also applied to discover structurallydiverse Keap1-Nrf2inhibitors. From65compounds selected by virtual screening from theSpecs database, a total of9compounds were found to be Keap1-Nrf2inhibitors.Compound S47was the most potent one with an inhibitory activity of2.9μM, which wascomparable to that of the positive control. Hit-based substructure search (HBSS) wasapplied to discover other derivatives of selected three classes of hits and a preliminary SAR was also concluded.Elevencompounds showed excellent protective effect in PC12cells, which were muchbetter than that of the positive control. Further biological study mechanistically supportedcompound S47could interrept Keap1-Nrf2interaction in cells. Then the Nrf2was releasedand translocated into nucleus, which could activate the expressions of downstream targetgenes.