硝基作为锌离子结合基团的含锌金属蛋白水解酶抑制剂的研究
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摘要
含锌金属蛋白水解酶广泛存在于各类组织中,在众多的生理过程中担当重要角色。含锌金属蛋白水解酶不同于其它蛋白酶的特征是其活性部位含有本质上起催化作用的锌离子。含锌金属蛋白水解酶抑制剂的研究可以为如肿瘤的侵袭和转移、炎症、高血压等多种疾病的治疗提供新型的药物。
对已有的含锌金属蛋白酶抑制剂结构进行分析发现,几乎所有的含锌金属蛋白酶抑制剂都由具有与酶活性中心相匹配的骨架,锌离子结合基团(zinc bindinggroup,ZBG)等组成。选择合适的ZBG是设计高活性抑制剂的关键。为了得到选择性更高,药物代谢药物动学特性更好的含锌金属蛋白酶抑制剂,研究新型的ZBG成为热点。一种方法是以羧肽酶A(carboxypeptidase A,CPA)和嗜热菌蛋白酶(thermolysin,TLN)为模型开发新型的ZBG,因为CPA和TLN的催化部位结构与多种含锌金属蛋白水解酶如血管紧张素转化酶(angiotensinconverting enzyme,ACE)、基质金属蛋白酶(matrix metallo proteinase,MMPs)类似,而CPA和TLN的抑制剂的设计相对简单,合成方便。另外的一种方法则是基于化学模拟的手段,不考虑侧链的设计,单纯地考查各种对锌离子具有螯合能力的小分子对含锌金属蛋白水解酶的催化锌离子的结合能力。
在2-苄基-3-甲基亚磺酰基丙酸的四个异构体对羧肽酶A的抑制活性毫摩尔级结果的工作基础上,寻找一种既能与CPA中的活性部位的锌离子配位,又能与CPA中的Arg-127和Glu-270的胍基有氢键关系的ZBG,成为本学位论文的研究重点。
硝基是羧基电子等排体,而且其键长和键角非常接近羧基,苄基丁二酸是羧肽酶A的有效的可逆性抑制剂,用硝基取代羧基有可能得到卓越的抑制剂,因此,本学位论文用羧肽酶A和嗜热菌蛋白酶为模型开发探讨硝基作为ZBG的可行性。
本学位论文探索了合成的2-硝基-3-苯基丙酸方法(PNP),发现±-2-硝基-3-苯基丙酸甲酯和±-2-硝基-3-苯基丙酸叔丁酯水解均得到2-苯基-1-硝基乙烷。本文用中间体3-硝基丙酸甲酯,低温烷基化,反应得到2位取代的3-硝基丙酸甲酯,在碘化锂存在的条件下进行水解生成4-甲基-2-(硝基甲基)-戊酸(MNP)、2-(硝基甲基)-4-戊烯酸(NMP)和2-苄基-3-硝基丙酸(BNP)。其中用2-苄基-3-硝基丙酸叔丁酯经TFA中水解也可以获得2-苄基-3-硝基丙酸(BNP)。本文也考察了几种合成光学纯的2-苄基-3-硝基丙酸的方法。发现以光学纯的2-苄基-3-羟基丙酸作为起始原料经四步反应生成光学纯的2-苄基-3-硝基丙酸是一条有效的合成路线。
采用Dixon法进行了CPA活性测试。BNP的三种构型即±、L、D三种形式,抑制常数分别是0.7,0.15和68μM。L-BNP其抑制作用是D-BNP的400倍。BNP的结合能力与L-苄基丁二酸(L-BSA)及HCON(OH)-β-Phe相当。BNP的L-型异构体抑制活性强于其对映体,这同CPA底物的立体专一性特点是一致的。简单的构效关系研究发现,引入烯丙基和异丁基的化合物4-甲基2-(硝基甲基)-戊酸(MNP)和2-(硝基甲基)-4-戊烯酸(NMP)对CPA的抑制活性降低,可能是它们与酶CPA的契合度差。
用microdialysis rod进行了CPA的单晶培养。收集得到分辨率为1.7(?)的单晶数据。单晶空间群是P2_1。结构数据保存在蛋白质数据库,(pdb ID:2RFH)。CPA的所有残基和BNP最后的模型在20-1.70(?)范围内被定义,R-factor是0.195,R-free是0.227。CPA·L-BNP复合物的结构表明硝基以非对称的O,O'-双齿方式与CPA中活性部位的锌离子螯合配位(2.10(?)和3.06(?)),显示出syn的立体化学。L-BNP中硝基部分的两个氧原子(O_3和O_4)与Glu-270中的羧基的氢原子和Arg-127中胍基的氢原子成氢键,距离分别是2.60(?)和2.87(?)。分子对接使用Auodock 4.0,探讨了L-BNP与CPA活性部位的结合方式。分子模型清楚看到L-BNP的nitrite与CPA活性部位的锌离子是以双齿配位的方式结合的,锌离子和氧之间的距离分别是1.84和1.91(?),但L-BNP中硝基中的两个氧与Glu-270和Arg-127之间的氢键没有观察到。
TLN底物的结构N-[3-(2-Furyl)acryloyl]-Gly-Leu-NH_2在水解中具有过渡态的结构特征,所以本论文设计的抑制剂考虑并想考察这一特点。设计的TLN抑制剂保留了天然底物的骨架、疏水性基团、酰胺端基,硝基引入到底物结构中的水解部位。合成了3-苯基-2-硝基丙酸甲酯(NPPM)和3-苯基-2-硝基丙酰胺(NPPA)、光学纯的2-苄基-3-硝基丙酸甲酯(BNPM)和2-苄基-3-硝基丙酰胺(BNPA)等一系列含有硝基的TLN抑制剂。硝基和烷氧羰基的吸电子作用,使NPPM和NPPA易发生外消旋化,所以放弃了光学纯的NPPM和NPPA的合成。以消旋的和光学纯的BNP为起始原料,用混合酸酐法,在四氢呋喃中用浓氨水合成相应的BNPA。以苯丙氨酸为起始原料,Kornblum条件合成了±-2-硝基-3-苯基丙酸甲酯。NPPM氨水中氨解得到NPPA。光学活性的2-苄基-3-羟基丙酸的经甲酯化、羟基的甲磺酸酯活化、溴化锂作溴化剂的取代反应、用Amberlite IRA 900(NO_2)发生Kornblum反应得到了光学纯的2-苄基-3-硝基丙酸甲酯。
采用改进的Dixon法进行了TLN活性测试。L-BNPM和L-BNPA都是毫摩级的TLN抑制剂。其中抑制活性最强的是L-BNPA,它的K_i是7.6μM,其活性强于已知高效的含反式异羟肟酸TLN抑制剂L-HCONOH-β-Leu-NH_2(K_i=53μM)。L-BNPA抑制活性强于L-BNPM,这可能是L-BNPA中的酰胺化合物中氮原子上的氢与肽结构骨架中肽键中的羰基氧原子(Asn 112)可以形成氢键的缘故。而L-BNPM中的酯不能形成这样的氢键。±-NPPA(K_i=684μM)的抑制TLN的活性也强于±-NPPM(K_i=2400μM)。
分子对接使用Auodock 4.0,探讨了L-BNPA与TLN活性部位的结合方式。分子模型清楚看到L-BNPA的nitrite与TLN活性部位的锌离子是以双齿配位的方式结合的,锌离子和氧之间的距离分别是1.74(?)和2.05(?),其中的酰胺中氮原子上的氢与TLN中的残基Asn-112中的羰基之间存在氢键(2.02(?),3.29(?))。
所合成的新化合物均经核磁共振碳谱和氢谱,红外光谱,质谱和元素分析表征。
本文成功开发了一个新型的ZBG:硝基。尽管硝基作为平面基团,但是含有硝基的CPA抑制剂L-BNP却展现和四面体过渡态类似的结构特征。这种新的赝过渡态类抑制剂在含锌金属蛋白水解酶中报道尚不多见,对今后的新型的含锌金属蛋白水解酶抑制剂的研究有一定的启发意义。
Zinc proteases,which are differentiated from other proteases by having a catalytically essential zinc ion at the active site,constitute an important class of proteolytic enzymes.They are widely throughout found in a variety of tissues and play key roles in numerous physiological processes,ranging from pathogenic infections to high blood pressure,and even cancer.The most studied drug design targets are angiotensin-converting enzyme(ACE),matrix metalloproteases(MMPs), etc.Carboxypeptidase A(CPA) and Thermolysin(TLN) are a leading prototypical enzyme for zinc proteases.Development of design principles of inhibitors for CPA and TLN would bear special importance because they can be translated to zinc proteases of medicinal value,leading to the discovery of potential lead compounds for drug development.
Inhibitor design for most metalloproteases therefore has focused on small inhibitors capable of interacting with the prime side pockets,a peptidomimetic backbone and incorporating a zinc-binding group(ZBG).The general strategy of incorporating a ZBG,such as a carboxylic acid, sulfhydryl,phosphinate,hydroxamate,or formylhydroxylamine,for the designed inhibitors has proven to be highly successful.The exploration and discovery of more potent and selective ZBGs is required.There are two paths to elucidate drug-metalloprotein interactions by using bioinorganic model compounds screen for ZBG or examination of novel ZBG for use in enzyme inhibitor.CPA and TLN were among the first proteins to have their structures unraveled by X-ray crystallography and they have been studied extensively by biochemical and structural means.Thus,both CPA and TLN serve as prototypes for zinc proteases.
Being an isostere of the carboxylate,the nitro group also shows a variety of coordinative fashions to the metal ions.Compared to the wide applications of the carboxylate as the ZBG in the inhibition of zinc proteases,to the best of our knowledge,the utilization of a nitro group as the ZBG has been rarely explored,is not found in zinc proteases inhibitors.
Nitro-based compounds were designed rationally as inhibitors for CPA. Because±-2-benzyl-3-nitropropanoic acid(BNP) inhibits CPA most potently,the two optically active forms of BNP to explore the stereochem'istry associated with the inhibition were then synthesized. Introduction of the nitro group was achieved by the reaction of halogen-NO_2 exchange using Amberlite IRA-900 resin in the nitrite form.BNP can be obtained through treatment of the ester with lithium iodide in refluxing anhydrous ethyl acetate.Inhibitory activities against CPA were evaluated and their inhibitory constant values in Dixon method using O-(trans-p-chlorocinnamoyl)-L-3-phenylacctate(Cl-CPL) as substrate.In CPA inhibitory assays L-BNP showed potent inhibitory activity with the K_i value of 0.15μM.Its enantiomer was shown to be much less potent(K_i= 68μM).As a contrast,2-nitro-3-phenylpropaonic acid(PNP), 4-methyl-2-(nitromethyl)pentanoic acid(MNP) and(2-nitromethyl)-4-pentenoic acid(NMP) were also designed and synthesized.It can be found that MNP inhibits the CPA-catalyzed hydrolysis more potently than NMP, which should be ascribed to the preference of CPA to the substrate having a large hydrophobic side chain.Introduction of an aromatic ring further augmented the binding affinity of the inhibitors to CPA,which may be resulted from the additional 'edge-to-face' interactions of the benzyl side chain of BNP with aromatic enzyme residues.
To explore the binding mode of L-BNP at the active site of CPA, CPA-L-BNP crystals for X-ray diffraction were then obtained by soaking the enzyme crystals into the solution containing racemic BNP and determined at a resolution of 1.7(?)(PDB ID code:2RFH).Single crystals grew in the P2_1 space group with one molecule in the asymmetric unit.The final model including all residues of CPA and L-BNP was refined in the 20-1.70(?).The nitro group of L-BNP acts as an asymmetric bidentate ligand of the zinc ion in an O,O'-chelation mode(2.10 and 3.06(?)).the two oxygen atoms(03 and 04) of the nitro moiety in L-BNP are engaged in hydrogen bonding with one of the carboxylate hydrogen atoms in Glu-270 and one of the guanidinium hydrogen atoms in Arg-127 with the distances of 2.60 and 2.87(?),respectively.To evaluate the metal binding interactions using AutoDock program,the nitro group in the BNP do coordinate to the zinc ion at the active site of CPA in an O,O'-bidentate fashion,but the hydrogen bonds are missing.
In spite of being not a tetrahedron species,the nitrite in CPA-L-BNP complex exhibited all above the structurally essential features of the transition state.In this sense,the nitro group should be reasonably regarded as a new ZBG,L-BNP as a pseudotransition-state analog inhibitor for CPA.
A series of inhibitors that bear nitro moiety have been evaluated as inhibitors for thermolysin.Optically form of 2-benzyl-3-nitropro panoic acid methyl ester(BNPM) and 2-benzyl-3-nitropropionamide(BNPA), racemic 2-nitro-3-phenylpropionic acid methyl ester(NPPM) and 2-nitro-3-phenylpropionamide(NPPA) have been synthesized and evaluated as inhibitors for TLN.Inhibitory activities of the synthesized nitro compound derivatives for thermolysin were estimated at pH 7.5 by the Dixon method's using 2-Furylacryloyl-Gly-Leu-NH_2 as substrate.The most potent inhibitor is L-BNPA having the K_i value of 7.6μM,it binds TLN better than the existing potent inhibitors bearing reversed hydroxamate such as L-HCONOH-β-Leu-NH_2(K_i= 53μM).The molecular modeling study for TLN and L-BNPA complex suggested that the two oxygen atoms of the nitrite also bind the zinc ions in a O,O'-bidentate fashion.Amides are more potent than esters,suggesting that the hydrogen atom on the amide nitrogen is possibly involved in the formation of hydrogen bond with backbone peptide carbonyl oxygen atom.
On the basis of these results it may be concluded that the nitro-bearing zinc protease inhibitors are a new type of pseudo-transition state analogue inhibitors,although the nitro group is not a tetrahedron species,it also showed the similar binding modes as transition-state analog.Nitro group is a valuable moiety that can be useful as active site zinc coordination functionality in designing inhibitors for CPA and TLN.The novel design protocol may potentially be employed in designing inhibitors effective against zinc proteases such as ACE and MMP,generating inhibitors of therapeutic applications.
对已有的含锌金属蛋白酶抑制剂结构进行分析发现,几乎所有的含锌金属蛋白酶抑制剂都由具有与酶活性中心相匹配的骨架,锌离子结合基团(zinc bindinggroup,ZBG)等组成。选择合适的ZBG是设计高活性抑制剂的关键。为了得到选择性更高,药物代谢药物动学特性更好的含锌金属蛋白酶抑制剂,研究新型的ZBG成为热点。一种方法是以羧肽酶A(carboxypeptidase A,CPA)和嗜热菌蛋白酶(thermolysin,TLN)为模型开发新型的ZBG,因为CPA和TLN的催化部位结构与多种含锌金属蛋白水解酶如血管紧张素转化酶(angiotensinconverting enzyme,ACE)、基质金属蛋白酶(matrix metallo proteinase,MMPs)类似,而CPA和TLN的抑制剂的设计相对简单,合成方便。另外的一种方法则是基于化学模拟的手段,不考虑侧链的设计,单纯地考查各种对锌离子具有螯合能力的小分子对含锌金属蛋白水解酶的催化锌离子的结合能力。
在2-苄基-3-甲基亚磺酰基丙酸的四个异构体对羧肽酶A的抑制活性毫摩尔级结果的工作基础上,寻找一种既能与CPA中的活性部位的锌离子配位,又能与CPA中的Arg-127和Glu-270的胍基有氢键关系的ZBG,成为本学位论文的研究重点。
硝基是羧基电子等排体,而且其键长和键角非常接近羧基,苄基丁二酸是羧肽酶A的有效的可逆性抑制剂,用硝基取代羧基有可能得到卓越的抑制剂,因此,本学位论文用羧肽酶A和嗜热菌蛋白酶为模型开发探讨硝基作为ZBG的可行性。
本学位论文探索了合成的2-硝基-3-苯基丙酸方法(PNP),发现±-2-硝基-3-苯基丙酸甲酯和±-2-硝基-3-苯基丙酸叔丁酯水解均得到2-苯基-1-硝基乙烷。本文用中间体3-硝基丙酸甲酯,低温烷基化,反应得到2位取代的3-硝基丙酸甲酯,在碘化锂存在的条件下进行水解生成4-甲基-2-(硝基甲基)-戊酸(MNP)、2-(硝基甲基)-4-戊烯酸(NMP)和2-苄基-3-硝基丙酸(BNP)。其中用2-苄基-3-硝基丙酸叔丁酯经TFA中水解也可以获得2-苄基-3-硝基丙酸(BNP)。本文也考察了几种合成光学纯的2-苄基-3-硝基丙酸的方法。发现以光学纯的2-苄基-3-羟基丙酸作为起始原料经四步反应生成光学纯的2-苄基-3-硝基丙酸是一条有效的合成路线。
采用Dixon法进行了CPA活性测试。BNP的三种构型即±、L、D三种形式,抑制常数分别是0.7,0.15和68μM。L-BNP其抑制作用是D-BNP的400倍。BNP的结合能力与L-苄基丁二酸(L-BSA)及HCON(OH)-β-Phe相当。BNP的L-型异构体抑制活性强于其对映体,这同CPA底物的立体专一性特点是一致的。简单的构效关系研究发现,引入烯丙基和异丁基的化合物4-甲基2-(硝基甲基)-戊酸(MNP)和2-(硝基甲基)-4-戊烯酸(NMP)对CPA的抑制活性降低,可能是它们与酶CPA的契合度差。
用microdialysis rod进行了CPA的单晶培养。收集得到分辨率为1.7(?)的单晶数据。单晶空间群是P2_1。结构数据保存在蛋白质数据库,(pdb ID:2RFH)。CPA的所有残基和BNP最后的模型在20-1.70(?)范围内被定义,R-factor是0.195,R-free是0.227。CPA·L-BNP复合物的结构表明硝基以非对称的O,O'-双齿方式与CPA中活性部位的锌离子螯合配位(2.10(?)和3.06(?)),显示出syn的立体化学。L-BNP中硝基部分的两个氧原子(O_3和O_4)与Glu-270中的羧基的氢原子和Arg-127中胍基的氢原子成氢键,距离分别是2.60(?)和2.87(?)。分子对接使用Auodock 4.0,探讨了L-BNP与CPA活性部位的结合方式。分子模型清楚看到L-BNP的nitrite与CPA活性部位的锌离子是以双齿配位的方式结合的,锌离子和氧之间的距离分别是1.84和1.91(?),但L-BNP中硝基中的两个氧与Glu-270和Arg-127之间的氢键没有观察到。
TLN底物的结构N-[3-(2-Furyl)acryloyl]-Gly-Leu-NH_2在水解中具有过渡态的结构特征,所以本论文设计的抑制剂考虑并想考察这一特点。设计的TLN抑制剂保留了天然底物的骨架、疏水性基团、酰胺端基,硝基引入到底物结构中的水解部位。合成了3-苯基-2-硝基丙酸甲酯(NPPM)和3-苯基-2-硝基丙酰胺(NPPA)、光学纯的2-苄基-3-硝基丙酸甲酯(BNPM)和2-苄基-3-硝基丙酰胺(BNPA)等一系列含有硝基的TLN抑制剂。硝基和烷氧羰基的吸电子作用,使NPPM和NPPA易发生外消旋化,所以放弃了光学纯的NPPM和NPPA的合成。以消旋的和光学纯的BNP为起始原料,用混合酸酐法,在四氢呋喃中用浓氨水合成相应的BNPA。以苯丙氨酸为起始原料,Kornblum条件合成了±-2-硝基-3-苯基丙酸甲酯。NPPM氨水中氨解得到NPPA。光学活性的2-苄基-3-羟基丙酸的经甲酯化、羟基的甲磺酸酯活化、溴化锂作溴化剂的取代反应、用Amberlite IRA 900(NO_2)发生Kornblum反应得到了光学纯的2-苄基-3-硝基丙酸甲酯。
采用改进的Dixon法进行了TLN活性测试。L-BNPM和L-BNPA都是毫摩级的TLN抑制剂。其中抑制活性最强的是L-BNPA,它的K_i是7.6μM,其活性强于已知高效的含反式异羟肟酸TLN抑制剂L-HCONOH-β-Leu-NH_2(K_i=53μM)。L-BNPA抑制活性强于L-BNPM,这可能是L-BNPA中的酰胺化合物中氮原子上的氢与肽结构骨架中肽键中的羰基氧原子(Asn 112)可以形成氢键的缘故。而L-BNPM中的酯不能形成这样的氢键。±-NPPA(K_i=684μM)的抑制TLN的活性也强于±-NPPM(K_i=2400μM)。
分子对接使用Auodock 4.0,探讨了L-BNPA与TLN活性部位的结合方式。分子模型清楚看到L-BNPA的nitrite与TLN活性部位的锌离子是以双齿配位的方式结合的,锌离子和氧之间的距离分别是1.74(?)和2.05(?),其中的酰胺中氮原子上的氢与TLN中的残基Asn-112中的羰基之间存在氢键(2.02(?),3.29(?))。
所合成的新化合物均经核磁共振碳谱和氢谱,红外光谱,质谱和元素分析表征。
本文成功开发了一个新型的ZBG:硝基。尽管硝基作为平面基团,但是含有硝基的CPA抑制剂L-BNP却展现和四面体过渡态类似的结构特征。这种新的赝过渡态类抑制剂在含锌金属蛋白水解酶中报道尚不多见,对今后的新型的含锌金属蛋白水解酶抑制剂的研究有一定的启发意义。
Zinc proteases,which are differentiated from other proteases by having a catalytically essential zinc ion at the active site,constitute an important class of proteolytic enzymes.They are widely throughout found in a variety of tissues and play key roles in numerous physiological processes,ranging from pathogenic infections to high blood pressure,and even cancer.The most studied drug design targets are angiotensin-converting enzyme(ACE),matrix metalloproteases(MMPs), etc.Carboxypeptidase A(CPA) and Thermolysin(TLN) are a leading prototypical enzyme for zinc proteases.Development of design principles of inhibitors for CPA and TLN would bear special importance because they can be translated to zinc proteases of medicinal value,leading to the discovery of potential lead compounds for drug development.
Inhibitor design for most metalloproteases therefore has focused on small inhibitors capable of interacting with the prime side pockets,a peptidomimetic backbone and incorporating a zinc-binding group(ZBG).The general strategy of incorporating a ZBG,such as a carboxylic acid, sulfhydryl,phosphinate,hydroxamate,or formylhydroxylamine,for the designed inhibitors has proven to be highly successful.The exploration and discovery of more potent and selective ZBGs is required.There are two paths to elucidate drug-metalloprotein interactions by using bioinorganic model compounds screen for ZBG or examination of novel ZBG for use in enzyme inhibitor.CPA and TLN were among the first proteins to have their structures unraveled by X-ray crystallography and they have been studied extensively by biochemical and structural means.Thus,both CPA and TLN serve as prototypes for zinc proteases.
Being an isostere of the carboxylate,the nitro group also shows a variety of coordinative fashions to the metal ions.Compared to the wide applications of the carboxylate as the ZBG in the inhibition of zinc proteases,to the best of our knowledge,the utilization of a nitro group as the ZBG has been rarely explored,is not found in zinc proteases inhibitors.
Nitro-based compounds were designed rationally as inhibitors for CPA. Because±-2-benzyl-3-nitropropanoic acid(BNP) inhibits CPA most potently,the two optically active forms of BNP to explore the stereochem'istry associated with the inhibition were then synthesized. Introduction of the nitro group was achieved by the reaction of halogen-NO_2 exchange using Amberlite IRA-900 resin in the nitrite form.BNP can be obtained through treatment of the ester with lithium iodide in refluxing anhydrous ethyl acetate.Inhibitory activities against CPA were evaluated and their inhibitory constant values in Dixon method using O-(trans-p-chlorocinnamoyl)-L-3-phenylacctate(Cl-CPL) as substrate.In CPA inhibitory assays L-BNP showed potent inhibitory activity with the K_i value of 0.15μM.Its enantiomer was shown to be much less potent(K_i= 68μM).As a contrast,2-nitro-3-phenylpropaonic acid(PNP), 4-methyl-2-(nitromethyl)pentanoic acid(MNP) and(2-nitromethyl)-4-pentenoic acid(NMP) were also designed and synthesized.It can be found that MNP inhibits the CPA-catalyzed hydrolysis more potently than NMP, which should be ascribed to the preference of CPA to the substrate having a large hydrophobic side chain.Introduction of an aromatic ring further augmented the binding affinity of the inhibitors to CPA,which may be resulted from the additional 'edge-to-face' interactions of the benzyl side chain of BNP with aromatic enzyme residues.
To explore the binding mode of L-BNP at the active site of CPA, CPA-L-BNP crystals for X-ray diffraction were then obtained by soaking the enzyme crystals into the solution containing racemic BNP and determined at a resolution of 1.7(?)(PDB ID code:2RFH).Single crystals grew in the P2_1 space group with one molecule in the asymmetric unit.The final model including all residues of CPA and L-BNP was refined in the 20-1.70(?).The nitro group of L-BNP acts as an asymmetric bidentate ligand of the zinc ion in an O,O'-chelation mode(2.10 and 3.06(?)).the two oxygen atoms(03 and 04) of the nitro moiety in L-BNP are engaged in hydrogen bonding with one of the carboxylate hydrogen atoms in Glu-270 and one of the guanidinium hydrogen atoms in Arg-127 with the distances of 2.60 and 2.87(?),respectively.To evaluate the metal binding interactions using AutoDock program,the nitro group in the BNP do coordinate to the zinc ion at the active site of CPA in an O,O'-bidentate fashion,but the hydrogen bonds are missing.
In spite of being not a tetrahedron species,the nitrite in CPA-L-BNP complex exhibited all above the structurally essential features of the transition state.In this sense,the nitro group should be reasonably regarded as a new ZBG,L-BNP as a pseudotransition-state analog inhibitor for CPA.
A series of inhibitors that bear nitro moiety have been evaluated as inhibitors for thermolysin.Optically form of 2-benzyl-3-nitropro panoic acid methyl ester(BNPM) and 2-benzyl-3-nitropropionamide(BNPA), racemic 2-nitro-3-phenylpropionic acid methyl ester(NPPM) and 2-nitro-3-phenylpropionamide(NPPA) have been synthesized and evaluated as inhibitors for TLN.Inhibitory activities of the synthesized nitro compound derivatives for thermolysin were estimated at pH 7.5 by the Dixon method's using 2-Furylacryloyl-Gly-Leu-NH_2 as substrate.The most potent inhibitor is L-BNPA having the K_i value of 7.6μM,it binds TLN better than the existing potent inhibitors bearing reversed hydroxamate such as L-HCONOH-β-Leu-NH_2(K_i= 53μM).The molecular modeling study for TLN and L-BNPA complex suggested that the two oxygen atoms of the nitrite also bind the zinc ions in a O,O'-bidentate fashion.Amides are more potent than esters,suggesting that the hydrogen atom on the amide nitrogen is possibly involved in the formation of hydrogen bond with backbone peptide carbonyl oxygen atom.
On the basis of these results it may be concluded that the nitro-bearing zinc protease inhibitors are a new type of pseudo-transition state analogue inhibitors,although the nitro group is not a tetrahedron species,it also showed the similar binding modes as transition-state analog.Nitro group is a valuable moiety that can be useful as active site zinc coordination functionality in designing inhibitors for CPA and TLN.The novel design protocol may potentially be employed in designing inhibitors effective against zinc proteases such as ACE and MMP,generating inhibitors of therapeutic applications.
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