【作者】 杜娟；

【导师】 王嵩；

【作者基本信息】 吉林大学 ， 物理化学， 2023， 硕士

【摘要】 p21活化激酶4(PAK4)广泛地存在于真核生物中,在人体的所有组织中均有表达。PAK4可以在细胞内外将信号传递给下游,参与了细胞生长、诱导凋亡、细胞增殖和衰老等多个过程。但研究显示PAK4容易过表达,其过表达与多种癌症相关。抑制PAK4的表达对治疗癌症有明显的作用,所以近年来对PAK4抑制剂的研究热度不断增加。吡咯并嘧啶类化合物自发现之初起就引起了广大化学研究者的注意。吡咯并[2,3-d]嘧啶结构具有抗菌、抗病毒、抗癌等功效,是很多生物活性物质的重要组成部分,在一些天然产物和药物中较为常见。有实验结果显示,吡咯并嘧啶作为母核的有机小分子可作为PAK4的抑制剂,对PAK4有比较明显的抑制效果并且没有细胞毒性。抑制剂母核上取代基的不同对PAK4的抑制效果有明显差异。然而,实验的方法只能得到抑制剂间抑制能力相差的大小倍数,却无法对这种产生的结果进行具体的原因分析。为了解释带有不同取代基的抑制剂对PAK4抑制效果有明显差异的原因,本文采用理论计算的手段,通过分子对接计算、分子动力学模拟配合结合自由能计算的方法,从分子水平上研究了PAK4的四种7H-吡咯并[2,3-d]嘧啶衍生物类竞争型抑制剂的结合模式和抑制机理。计算结果表明,四个抑制剂与酶的铰链区(Glu396-Gly400),β折叠片区(β1:Ile325-Gly330,β2:Gly333-Thr339,β3:Leu346-Asp353,β7:Ile446-Thr449)以及4-取代基周围带有电荷化侧链的残基(Lys350,Glu366,Asp405,Asp444,Asp458,Arg589和Arg591)有较强的相互作用。抑制剂与铰链区残基的相互作用最强,以氢键为主,是抑制剂具有抑制能力的主要原因。铰链上的氢键使抑制剂的母核和2-取代基不易发生翻转,有效地保证了抑制剂的稳定结合。抑制剂的4-取代基与其周围侧链电荷化的残基主要通过静电相互作用结合。抑制剂5n的末端氨基与其它三个抑制剂不同,会造成周围残基与其形成的氢键或静电作用的增强,因此是四个抑制剂中抑制能力最强的。抑制剂5h,5g和5e的2-取代基上的卤素原子不同,原子上所带电荷数也不同,这会造成2-苯环位置上的差异,并影响其与铰链区残基的相互作用。卤素原子的不同还会一定程度上影响4-亚氨基的方向,并因此影响其与周围电荷化残基的亲和力。这些变化综合的结果导致抑制剂5e在四种抑制剂中抑制能力最弱。综上所述,抑制剂取代基的结构、电荷分布和带电基团的方向对抑制能力影响较大。我们的研究阐释了抑制剂抑制能力差异的部分原因,可以为之后PAK4竞争型抑制剂的设计提供一些理论依据。更多还原

【Abstract】 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.更多还原