Study On The Molecular Mechanism Of Allosteric Inhibitor-mediated Kinase Conformation

Protein kinases play an important regulatory role in many important physiological activities in the organism,such as cell proliferation,differentiation,metabolism,aging,and death.They are also important targets for the development of new drugs,especially anti-tumor agents.The combination of specific inhibitors and protein kinases to regulate or block kinase activity is beneficial to the treatment of many diseases such as cancer.At present,the development of highly effective selective inhibitor drugs targeting protein kinases has become a hot spot in new drug design and development area.Kinase inhibitors are classified into reversible inhibitors and irreversible inhibitors according to the way they interact with kinases,among which irreversible inhibitors can covalently bound to kinase proteins.According to whether occupying the ATP binding site,they can be divided into ATP-competitive inhibitors and allosteric inhibitors;The ATP-competitive inhibitors are widely investigated in the medicinal area,and while allosteric inhibitors have attracted much interest in recent years.Previously,researchers often focused on studying the pharmacological effects of certain inhibitors on specific kinases,or designing potent inhibitors to target specific kinases.In this work,we carry out comprehensive database survey and systematic conformational analysis of structureknown complexes of protein kinases with various allosteric inhibitors,aiming to obtain the general molecular insight into the inhibitor-mediated kinase allosterism through an exhaustive analysis of existing allosteric inhibitor–kinase target complex crystal structure data.We perform a large-scale screening and comprehensive survey against nearly 100,000 crystal structure data in the protein structure database(PDB)to identify thousands of small-molecule inhibitor-bound kinase structures,which are then one-byone examined manually to classify the inhibitor-binding mode and pose in kinase structure;we also employ molecular graphics tools to observe the intermolecular interaction of these allosteric inhibitor ligands with kinase receptors as well as the configuration,property and characteristics of inhibitor-binding pocket,to determine the location of binding pocket,and to analyze amino acid type and composition around the pocket.Consequently,totally 107 small-molecule allosteric inhibitors are identified from the PDB database,which can be divided into two broad categories according to the types of target kinases,namely tyrosine-specific and serine/threonine-specific kinases,which can be further classified into 6 and 8 sub-categories,respecitvely.Through molecular graphics inspection,it is initially observed that the binding pockets of allosteric inhibitors can be sorted into five types according to their location in kinase domain: the first four are partially overlapped with or spatially vicinal to the kinase active site;they can directly influence the kinase active site and substrate binding through local structural components such as C-helix and P-loop,whereas the last one is far away from the active site;it indirectly affects theactive site by changing the global conformation of the whole kinase domain.Therefore,the inhibitor-induced allosterism can be considered as local and global regulations.In order to deeply understand the intermolecular interactions between allosteric inhibitors and protein kinases as well as the strategies that inhibitors adopt to change kinase conformation,we also retrieve the structures of the same kinases that are not bound with allosteric inhibitor ligands or only complexed with ATP-competitive inhibitor ligands.These structures are termed as allosteric inhibitor-free counterparts;they are then superposed onto their corresponding allosteric inhibitor-bound kinase structures by using least backbone squares fitting approach,and the conformational difference between these allosteric kinase structures and their free counterparts is examined in details,particularly paying attention to the effect of allosteric regulation on inhibitor-binding pocket and kinase active site.On this basis,the molecular mechanism of kinase conformational change and activity inhibition induced by allosteric inhibitor binding is explored systematically.It is revealed that the type-I allosteric inhibitor partially occupies the ATPbinding site and does not significantly alter the global conformation of kinase domain.This is because it recognizes kinase active conformation in the manner of DFG-in and then regulates the local structure of kinase active site,which can also partially prevents the entry of ATP molecules into the active site.Therefore,it can be considered as an inhibition type spanning between allosteric regulation and ATP competition,or called partial allosteric inhibitor.Type-II allosteric inhibitor binds to the kinase conformation in inactive DFG-out form,which can largely alter local configuration around the kinase active site.When it binds to the region vicinal to the active site,the kinase P-loop moves from N-lobe toward C-lobe and C-helix is shifted outside of C-lobe,which promotes the expansion of kinase active site to define an additional inhibitor-binding pocket.Type-III allosteric inhibitor influence kinase active site mainly through the displacement of C-helix nearby the active site,which is similar to type II-allosteric inhibitor.Type-IV allosteric inhibitor addresses conformational change on the C-lobe of kinase domain,which reshapes the exquisite state of amino acid residues that indirectly affect the fine structural characteristics of kinase active site.V-type allosteric inhibitor works synergistically by combining two or more inhibitor molecules targeting different regions of one kinase domain,which often has a significant effect on the whole conformation of kinase domain as well as the structural configuration of kinase active site.Furthermore,we also employ some structural bioinformatics tools to empirically optimize the kinase structures without allosteric inhibitor binding,in order to obtain their theoretical apo kinase conformation counterparts,which are also compared with corresponding allosteric inhibitor-free crystal structures,indicating a nonessential difference between them.Based on above findings this study not only gives a comprehensive understanding of the conformation,classification and allosterism of allosteric kinase inhibitors as well as their interactions and binding modes to protein kinase receptors,but also reveals some new short-range and long-range strategies adopted by allosteric inhibitors to directly or indirectly influence kianse active site and substrate binding.We also present a systematic classification of existing allosteric inhibitors according to their binding location in kinase domain and systematically explore the molecular mechanism of kinase allosteric inhibition at structural level exerted by different types of allosteric inhibitors.This work would be helpful for the development of new allosteric kinase inhibitor drugs in future.