Screening And Evaluation Of The Protein Phosphatase PPM1B Inhibitors

The reversible phosphorylation of proteins is a major mechanism of cellular functions,and it regulates almost all aspects of cell life,such as cell growth and differentiation,muscle contraction,metabolism,gene regulation,etc.Protein kinases and phosphatases are regulators of the reversible phosphorylation,which have become potential drug targets,as the abnormal phosphorylation is related to many diseases,such as cancer,Alzheimer disease,chronic inflammation,diabetes,etc.Recently,many protein kinases-targeted drugs have been entered clinic or clinical trials,but the development and utilization of phosphatases-targeted drugs is still in exploratory stage.The aims of our research are to discover and explore novel protein phosphatase inhibitors,and promote the development of phosphatases-targeted drug library.Protein phosphatases can be divided into three famlilies due to their substrates specificity:serine/threonine protein phosphatases(PSPs),tyrosine protein phosphatases(PTPs),and dual specificity protein phosphatases(DUSPs).PSPs can be divided into the PPP family and PPM family.There are at least 17 members in PPM family,including PPM1A,PPM1B(Protein Phosphatase,Mg2+/Mn2+Dependent,1 B),etc.PPM1B could be activated by Mg2+/Mn2+,which is widely distributed in tissues.It has been confirmed that PPM1B could play important roles to regulate cell cycle progress and stress signaling.However,there have been no specific PPM1B inhibitors discovered yet.It has remarkable scientific significance to search specific PPM1B inhibitors,which contributes to recognize PPM1B biological functions,find its interaction proteins and develop new drugs.In this work,182 compounds were screened for the inhibitory effect on PPM1B,including natural products and compounds synthesized based on natural products in our group.The results showed that 7 compounds had good inhibitory effects on PPM1B(IC50<10?M).And the compound 1(HN252)could specifically inhibit PPM 1B and PPM1 A.The compound 1(HN252)could competitively inhibit PP1MB(Ki=0.52?M)and non-competitively inhibit PPM1A(Ki=0.48?M).We found that the inhibitory activities of HN252 on the mutants(R33K and K170A)are significantly weaker than the wild type of PPM1B.which suggested that HN252 inhibit PPM IB by being bound to Arg33 and Lys170 residues of PPM1B.We observed that HN252 could up-regulate the phosphoiylation level of protein serine/threonine sites and the phosphorylation level of the substrate of PPM1B-AMPK alpha subunit in different cells.We inferred that HN252 increased the phosphoiylation level of the substrates of PPM 1B by inhibiting the activities of PPM1B in cells.We used antibody array technology to study the cellular process influenced by HN252 in HL7702 cells.The results showed that HN252 could influence more than 100 biological process such as cell proliferation,immune response process,etc and promoted the cell proliferation in MIN6 cells.Finally,we studied the function of HN252 in vivo.The results suggested that HN252 is a low toxicity compound.In summary,we found the first compound HN252 which could inhibit the PPM1B and PPM1A with efficiency and specificity.HN252 could inhibit PPM1B in vitro by being bound to the Arg33 and Lys170 residues of PPM IB.HN252 could influence the protein phosphorylation levels in cells,promote the cell proliferation in MIN6 cells and have no toxic or side effects in vivo.Our research results provide ideas for exploring new functions of PPM1B and designing specific PPM1B inhibitors.