Research On The Allosteric Mutation Effect And Allosteric Inhibitor Development Of RhoA

RhoA is a typical small GTPase which cycles between an active GTP-bound and an inactive GDP-bound state.As a molecular switcher finely controlled by multiple regulators,RhoA plays pivotal roles in many physiological processes including cytoskeleton reorganization,cell migration and invasion.Most recently,it has been reported that RhoA mutations frequently occur in a wide variety of human cancers.However,due to the highly dynamic structure and complex functional regulation of RhoA,the research on the structure and function of RhoA mutant s remains in its infancy,which limits the development of therapeutic approaches to the diseases associated with RhoA mutation.Besides recurrent gene mutations,abnormal overexpression of RhoA is common in a variety of malignant tumors,which actively accelerates the malignant tumor process.Therefore,the chemical regulator targeting wild-type and mutant RhoA have high potential to become drug candidates,the development of which is of great significance.Given the micromolar GTP/GDP concentration in cells and potent binding affinity of RhoGTPases for GTP and GDP,however,it is difficult to find molecules targeting nucleotide-binding pocket.Moreover,there are few stable or tractable pockets on the surface of RhoGTPases except substrate-binding pocket,which makes RhoA become a well-known“untargetable”protein and retards the development of chemical regulator targeting RhoA.In order to treat RhoA-related diseases,it is necessary and urgent to establish a novel strategy to overcome the above-mentioned bottleneck issues.Due to the high-frequency of RhoA^C16R mutation in T-cell leukemia/lymphoma（ATL）,we investigated the effect of C16R mutation on the structure and function of RhoA in this study.A series of biochemical and cell biological experiments showed that C16R mutation promoted the interaction of RhoA with various RhoGEFs,increased the GDP/GTP exchange rate,but reduced the binding capacity for GDP/GTP,weakened the interaction of RhoA with downstream effectors,and consequently inhibited RhoA signaling pathway in cells.In order to elucidate the inactivation mechanism of RhoA^C16R mutation,we determined the crystal structures of RhoA^C16R,RhoA^C16Land RhoA^C16R,the latter two mutants of which also are common in ATL.It was found that the replacement of cysteine at position 16 was able to induce the long-range conformational changes in the switch I and switch II region of RhoA,enlarge substrate-binding pocket and disrupt the H-bonds between protein and GTP/GDP.These results shed light on the inactivation mechanism of RhoA^C16R mutation from the point of view of structure.Furthermore,the finding that the active site of RhoA is sensitive to allosteric effect indicated that besides substrate-binding pocket,the pocket away from active site could be used as targeting site for the development of chemical regulator targeting RhoA,if this pocket has allosteric communication with active site of RhoA.Based on the above results that the functional sites of RhoA were sensitive to distal conformational changes,we performed molecular dynamics simulations of wild-type and mutant RhoA,and identified a potential compound-binding pocket in the structural dynamics of wild-type RhoA,which locates around Cys107 on the surface of“untargetable”RhoA,far from active sites.This pocket was subsequently named as“CLoc K”.Using a combined strategy of covalent docking and RhoA activity assay,we discovered a covalent regulator DC-Rhoin targeting to the identified pocket,which was able to significantly inhibit the ac tivity of RhoA in vitro.The results of crystal structure determination,MS,NMR and biochemical assays showed that DC-Rhoin was able to covalently bind to Cys107 of RhoA,and consequently stabilize the compound-binding pocket which transiently emerged in the structural dynamics of RhoA.As results,the fragment spanning from 67th to 74th residue of RhoA was induced to be in the disordered conformation,which disrupted the interaction between RhoA and RhoGEF or RhoGDI,with little effect on the binding of RhoA to RhoGAP.These result indicated that the identified DC-Rhoin-binding pocket was an allosteric site,and DC-Rhoin was the first allosteric inhibitor targeting RhoA.Moreover,it was found that DC-Rhoin was highly selective for Rhofamily proteins over other proteins in the Ras superfamily,which was determined by the conservative and specific cysteine at position 107.In order to improve the cellular activity of inhibitor,we designed a series of DC-Rhoin derivatives.After biological activity screening,we obtained DC-Rhoin04,the derivative with most potent cellular activity.It was found that 5μmol/L DC-Rhoin04 was enough to block RhoA signaling pathway,and consequently inhibit the migration,invasion,proliferation and stress fiber formation of MDA-MB-231 cells.The results of knockdown and rescue assays showed that the inhibition of RhoA downstream pathway,cell migration and invasion by DC-Rhoin04 mainly depended on the covalently binding of DC-Rhoin04 to Cys107of RhoA.In conclusion,using multidisciplinary approaches covering structural biology,biochemistry and cell biology,we successfully elucidated the effect of RhoA^C16Rmutation and its underlying mechanism,found the important roles of allosteric effect in the structure and function of RhoA,identified the first allosteric pocket on the surface of RhoA,and developed the first covalent allosteric inhibitor targeting the"untargetable"protein RhoA.This study will provide new strategies for clarifying the pathogenic mechanism of RhoA mutation and developing innovative drugs targeting RhoA,laying the foundation for successful treatment of RhoA-related diseases in future.