Virtual Screening,Activity Evaluation And Molecular Simulation Study Of HPK1 Inhibitors

Tumor immunotherapy has opened up a new era of solid tumor treatment.Tumor immunotherapy is based on the patient’s own immune system,by enhancing the body’s anti-tumor immune response,counteracting the tumor escape mechanism to kill the tumor;Tumor immunotherapy has few side effects and obvious therapeutic effect.Over the past decade,immune checkpoint inhibitors have been widely used in clinical treatment.However,due to the heterogeneity of cancer or the complexity of the evolving tumor microenvironment,a large proportion of cancer patients continue to exhibit primary and acquired resistance to checkpoint inhibitor monotherapy.The overall treatment response rate is limited to a few cases and tumor types,and only a few patients can benefit in the long term.In addition,immune checkpoint inhibition is also limited to negative regulators present on the surface of T cells,which limits their utility.Therefore,there is a need to develop new strategies to restore and enhance T-cell activity.A large number of pharmaceutical companies are focusing on the development of intracellular small molecule immunomodulators or synergistic intracellular immunotherapeutic targets to combat tumors.Hematopoietic progenitor kinase 1（HPK1）is one of the representatives of such intracellular immunomodulators.HPK1 is a member of the Ste20 serine/threonine kinase mitogen-activated protein kinase kinase kinase kinase（MAP4K）family,also known as MAP4K1.It is mainly expressed in hematopoietic cells（such as T cells,B cells,neutrophils,dendritic cells and macrophages）and plays a negative regulatory role in T cells and B cells.Inhibition of HPK1 can evade the immunosuppressive tumor microenvironment,and HPK1 is a very valuable target in immunooncology.So far,no HPK1 inhibitors have been successfully marketed,so it is of great significance to find novel HPK1inhibitors.In this paper,computer-aided drug design is used as a means to promote the research of HPK1 inhibitors from the perspectives of screening novel small molecule inhibitors based on docking-based virtual screening methods,exploring the mechanism of action of hit compounds,optimizing hit compounds,selective inhibition mechanism of HPK1 kinase,and selective inhibition mechanism of HPK1chiral inhibitors.The first chapter summarizes the structure,function,related signaling pathways,related diseases of HPK1,and the research progress of HPK1 inhibitors.Then the molecular docking,virtual screening,conventional molecular dynamics simulation,umbrella sampling molecular dynamics simulation,adaptive bias force simulation and free energy perturbation molecular dynamics simulation methods used in this paper are introduced.In the second chapter,a new type of HPK1 inhibitor was found by virtual screening method based on molecular docking combined with kinase inhibition experiment.After three precision screening of Glide,39 compounds were selected from the Chemdiv database for kinase inhibition experiments.The IC₅₀ values of the five compounds ranged from 2.93±0.09μM to 14.27±1.07μM.The most active compounds M074-2865 and Sunitinib were combined with HPK1 kinase binding sites.It can be seen that the main site difference between Sunitinib and M074-2865 with HPK1 is that the tail of Sunitinib reaches the solvent front,while M074-2865 is almost all in the pocket.This may be the reason for the difference in inhibitory activity between Sunitinib and M074-2865 against HPK1.Subsequently,the binding mechanism of the compound M074-2865 and the positive control Sunitinib with HPK1 kinase was studied by conventional molecular dynamics simulation.The interaction mechanism of M074-2865 and Sunitinib with HPK1 kinase was summarized by combining complex system equilibrium analysis,binding free energy analysis,residue energy decomposition,hydrogen bond analysis and binding mode analysis.The results showed that the inhibitor occupied the ATP binding pocket and formed hydrogen bond interaction with Glu92 and Cys94 in the hinge region.The hydrogen bond interaction with the hinge region is the key to the high inhibitory activity of the inhibitor.Finally,the amide part of the benzocaprolactam in the M074-2865 molecule is considered to be opened to make the tail of the M074-2865molecule reach the solvent frontier.We designed and synthesized a total of 16compounds of anilines,amides,sulfonamides,and aliphatic amines.Among each compound,the compound containing piperazine had the highest inhibition rate at 50μM,the IC₅₀ of three piperazine-containing compounds M2,M8 and M12 were 1.63μM,3.76μM and 2.13μM,respectively,indicating that piperazine plays an important role in the inhibition of HPK1,and these molecules can be further modified to better HPK1 inhibitors.In the third chapter,the reasons for the selectivity difference between HPK1 and JAK1 by two indazole inhibitors（XHS and XHV）were studied by conventional molecular dynamics simulation and umbrella sampling molecular dynamics simulation.The binding differences of inhibitors（XHS and XHV）to HPK1 and JAK1were studied by conventional molecular dynamics simulation and residue energy decomposition.The results showed that the biggest difference in the binding of inhibitor XHS to two kinases was the difference in salt bridge interaction between piperazine and HPK1/Asp101 and JAK1/Glu966.The para-piperazine in the inhibitor XHV extends to the solvent frontier,the salt bridge interaction disappears,and the energy contribution differences of HPK1/Ala96,Gly97,Asp101,Ala154 and JAK1/Ser961,Gly962,Glu966,Gly1020 make XHV have moderate selectivity to these two kinases.Subsequently,umbrella sampling molecular dynamics simulations show that the difference in PMF during the dissociation of the inhibitor XHS from HPK1 and JAK1 is due to the different interaction between the piperazine part and the kinase salt bridge.The difference in PMF during the dissociation of the inhibitor XHV from HPK1 and JAK1 is due to the different hydrophobic interactions between the2-fluoro-6-methoxyphenyl group and the piperazine group and the kinase.The difference in the inhibitory activity of the two inhibitors on HPK1 kinase is mainly due to the extension of the para-piperazine in the ligand XHV to the solvent frontier,while the piperazine in the ligand XHS changes direction and interacts with Asp101.In addition,the binding of U-type inhibitor XHS to HPK1 is better than that of JAK1,which is controlled by the salt bridge between piperazine and the carboxyl group on Asp101.The results of this study will provide useful suggestions for the rational development of better selective HPK1 inhibitors.In the fourth chapter,the reasons for the different inhibitory activities of（1R,3S）-ADFC and（1S,3R）-ADFC were explained.Firstly,we obtained the complex structure of two inhibitors with HPK1 kinase by molecular docking.The results of molecular dynamics simulation,residue energy decomposition and hydrogen bond analysis showed that the difference in the binding of HPK1 to（1R,3S）-ADFC and（1S,3R）-ADFC was due to the contribution of these residues Glu62,Ala154 and Asp155,mainly due to the formation of stable hydrogen bonds between the formamide carbonyl group in（1R,3S）-ADFC and the skeleton NH of Asp155,the hydrogen bonds between the formamide amino group and the side chain carbonyl group of Glu62,and the van der Waals interaction between the methyl and Ala154.The subsequent adaptive bias force（ABF）simulation shows that the dissociation paths of（1R,3S）-ADFC and（1S,3R）-ADFC are different,and the energy required for dissociation of（1R,3S）-ADFC is higher than that of（1S,3R）-ADFC.Our results will help to design new potential HPK1 inhibitors.Chapter 5:Through the calculation in Chapter 3,it is concluded that the hydrogen bond interaction or salt bridge interaction between the U-shaped skeleton structure inhibitor and the Asp101 in the solvent region can improve the inhibitory activity and selectivity of the inhibitor to HPK1.Therefore,in this work,we used the crystal structure of the U-shaped inhibitor binding HPK1 as the glide generation structure,and the hydrogen bonds of Glu92 and Cys94 were limited during the glide generation process.The ability of the Glide molecular docking method to distinguish HPK1inhibitors from non-inhibitors with three precisions was evaluated.After Glide SP and XP precision screening,21 compounds were selected from the Chemdiv database for ADP-Glo kinase assay.The IC₅₀ of compound F091-0167 was 17.77±1.54μM.Next,we searched the homologues of F091-0167 and finally purchased four compounds to determine their inhibitory activity and analyze their docking binding mode.The IC₅₀values of F091-0818,F091-1013 and F091-0819 were 2.74±0.55μM,13.87±1.87μM and 1.55±0.43μM,respectively.Next,molecular docking,molecular dynamics simulation,scaffold hopping and free energy perturbation methods were used to optimize the structure of the hit compound F091-0819 from the theoretical level.The results show that the molecules F7,F8,F7-3,F7-12 have great potential to become efficient inhibitors of HPK1,which lays a good foundation for the subsequent design and optimization.