The Inhibitory Mechanism Of Pyrrolopyrimidine Derivatives As Inhibitors Of P21-activated Kinase Through Molecular Dynamics Simulation

The p21-activated kinase 4(PAK4)is widely found in eukaryotes and is expressed in all human tissues.PAK4 can signal downstream in and out of cells and is involved in a number of processes including cell growth,induction of apoptosis,cell proliferation and senescence.However,studies have shown that PAK4 is prone to overexpression,and its overexpression is associated with a variety of cancers.The inhibition of PAK4 expression has a clear effect on the treatment of cancer;therefore,research on PAK4 inhibitors has been increasing in enthusiasm in recent years.Pyrrolopyrimidines have attracted the attention of a wide range of chemical researchers since their initial discovery.Pyrrolo [2,3-d]pyrimidine structure,with antibacterial,antiviral,and anticancer properties,is an important component of many bioactive substances and is more commonly found in some natural products and drugs.Experimental results have shown that pyrrolopyrimidine,as the parent nucleus of organic small molecules,can act as an inhibitor of PAK4,with a relatively significant inhibitory effect on PAK4 and no cytotoxicity.The inhibitory effect on PAK4 was significantly different depending on the substituent on the parent nucleus of the inhibitor.However,the experimental method can only obtain the magnitude multiples of the difference in inhibitory ability between inhibitors,but it is unable to analyze the specific reasons for this result.In order to explain the obvious differences in the inhibitory effects of inhibitors with different substituents on PAK4,the binding modes and inhibition mechanisms of four 7H-pyrrolo [2,3-d] pyrimidine competitive inhibitors of PAK4 were investigated at the molecular level by means of molecular docking calculations,molecular dynamics simulations,and binding free energy calculations in this paper.The results showed that the four inhibitors interacted with the hinge region of the enzyme(Glu396-Gly400),the β-folded sheet region(β1:Ile325-Gly330,β2: Gly333-Thr339,β3: Leu346-Asp353,β7:Ile446-Thr449),and residues with charged side chains around the4-substituents(Lys350,Glu366,Asp405,Asp444,Asp458,Arg589,and Arg591)had strong interactions.The interactions between inhibitors and hinge residues were the strongest,dominated by hydrogen bonds,which were the main reason for the inhibitory abilities of inhibitors.The hydrogen bonds on the hinge made the parent nucleus and 2-substituents of the inhibitors less susceptible to flipping,effectively ensuring stable binding of the inhibitors.The 4-substituents of the inhibitors were bound to their surrounding side-chain charged residues mainly through electrostatic interactions.The terminal amino of inhibitor 5n was different from the other three inhibitors,which can cause the hydrogen bond formed by the surrounding residues or the enhancement of electrostatic interaction,so it had the strongest inhibitory ability among the four inhibitors.The halogen atoms on the 2-substituents of the inhibitors 5h,5g,and 5e were different and had different charges on the atoms,which could cause differences in the position of the 2-phenylene ring and affect its interaction with residues in the hinge region.The differences in the halogen atoms also affected,to some extent,the orientation of the4-imino and consequently its affinity for the surrounding charged residues.The combined result of these changes resulted in inhibitor 5e having the weakest inhibitory abilities of the four inhibitors.In summary,the structures of the inhibitor substituents,the charge distribution,and the orientation of the charged groups have a strong influence on the inhibitory abilities.Our study explained some of the reasons for the differences in inhibitory ability of inhibitors,which can provide some theoretical basis for the design of PAK4 competitive inhibitors in the future.