Design,Synthesis And Biological Evaluation Of USP28 And MAT2A Inhibitors

Ubiquitination is one of the well-known post-translational protein modifications.It plays crucial roles in multiple physiological processes by regulating the balance of proteins.Ubiquitin-specific proteases(USPs)28 plays an important role in tumor development by enhancing the stability of various tumor-related proteins such as cMyc,Notch1,LSD1,and has become a potential target for the development of anticancer drugs.The development of potent USP28 inhibitors is still in primitive research stage.our collaborating researchers previously reported the discovery of Vismodegib as a USP28 inhibitor by screening a commercially available drug library and further obtained the cocrystal structure of Vismodegib and USP28.Based on the cocrystal structure,modifications were made to the structure of Vismodegib in order to enhance its activity and selectivity.Elaborative SARs exploration was carried out to afford much more potent USP28 inhibitors than Vismodegib.In the ubiquitin-specific protease family,there are many members with similar structures.It is necessary to evaluate the selectivity of the active compound to the inhibition of USP28 enzyme activity.The representative compounds 2-23,2-26 and 2-27 bearing high potency on USP28 showed high selectivity over USP2,USP7,USP8,USP9 x,UCHL3 and UCHL5.However,due to the high sequence similarity between USP25 and USP28,all of the three compounds could also inhibit the enzymatic activity of USP25 at the similar level.The detailed cellular assay and colony formation assay suggested that compounds 2-23,2-26 and 2-27 could cause cytotoxicity in both human colorectal cancer and lung squamous carcinoma cells and significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.Further immunoblotting analysis indicated that compounds 2-23,2-26 and 2-27 could dose-dependently down-regulated the cellular level of c-Myc through ubiquitin-proteasome system.Examination of Bcl-2 protein levels showed that anti-cancer effects could mainly be attributed to their inhibition on USP28 but not involving the Hedgehog-Smoothened pathway.Thus,our work provided a series of novel and potent USP28 inhibitors derived from Vismodegib and may contribute to the development of USP28 inhibitors.Synthetic lethality refers to the simultaneous perturbation of two genes leading to cell death.Thus,specific killing of tumor cells can be achieved for the purpose of precision treatment of tumors by inhibiting a synthetic lethal partner of a particular mutated gene in the tumor with less impact on normal cells.Methionine adenosyltransferase 2A(MAT2A)is a synthetic lethal target of methylthioadenosine phosphorylase(MTAP)deleted tumors.When MTAP is depleted in tumor cells,such as when the enzymatic activity of MAT2 A is inhibited by MAT2 A inhibitors,synthetic lethal effects are produced.This ultimately leads to the blockage of protein substrate methylation catalyzed by PRMT5,resulting in defects in gene expression,DNA replication,and genome integrity,resulting in tumor Cell-specific apoptosis.Structural optimization was performed based on MAT2 A inhibitors compound 28 reported by Astra Zeneca combining with the characteristics of MAT2 A protein,we designed and synthesized a series of novel structure compounds,detailed SARs exploration was carried out to afford several compounds 3-2,3-6,3-9,and 3-14 to 3-15 with comparable molecular and cellular activity to AG-270.The detailed cellular assay suggested that 3-14 and 3-15 showed higher selectivity for HAP1/MTAP-/-than control compounds AG-270(R = 15.3,13.7 vs 4.5).Pharmacokinetic evaluation will also be carried out for the compounds with good cell activity.The above bioactivity evaluation results indicated that the designed dimer inhibitors still have great development prospects,which will contribute to the development of MAT2 A inhibitors in the future.