靶向特定氨基酸的共价抑制剂研究进展

共价抑制剂通过共价键与靶蛋白结合并对其产生抑制作用。虽然在很长一段时间内因为潜在的脱靶可能性使研究者们望而却步,但随着近年来对共价抑制剂作用机制、亲电弹头与亲核氨基酸性质的深入研究与探索以及有效开发策略的提出与改进,众多共价抑制剂成功上市。其在疾病治疗中的优势也逐渐显现,这使得共价抑制剂成为药物研发的热点。通过对共价抑制剂的结合机制、研究进展、开发策略、优势与挑战进行综述,并对其发展前景与方向进行讨论,以期为新型共价抑制剂的开发提供参考。     

氮唑类抗真菌药物药效构象及其与酶活性位点对接

目的： 研究氮唑类抗真菌药物与其受体蛋白活性位点相互作用机理。方法： 用随机构象搜寻和分子动力学模拟退火法确定15个4种不同类型的氮唑类抗真菌药物最低能量构象;用活性类似物法限定药物分子药效基团之间的距离,搜寻到各化合物药效构象;将各化合物药效构象对接到白色念珠菌羊毛甾醇14α去甲基化酶活性位点中。结果： 4种结构类型的氮唑类药物在酶活性位点中有相似的对接位置;真菌和哺乳动物的活性位点结构特异性的残基分布在F螺旋C端、β6-1和β6-2区中;氮唑类抗真菌药物共同的卤代芳环结构落入相同的疏水空穴中,其中Y132的侧链羟苯基结构可与抑制剂卤代芳环形成电子迁移复合物。结论： 对接结果与已知SAR分析结论相符,阐明了氮唑类药物与活性位点的氨基酸残基作用方式,探讨结构选择性药物的结构需求。     

N-肉豆蔻酰基转移酶家族的进化踪迹分析研究

为了阐明抗真菌药物作用新靶点——n-肉豆蔻酰基转移酶(NMT)活性位点中的关键功能残基分布，指导设计特异性抑制剂,开展了NMT家族的蛋白多元序列联配研究，并构建了蛋白系统进化树。在此基础上，采用进化踪迹分析技术识别得到了NMT活性位点中肉豆蔻酰CoA结合位点、催化反应中心和抑制剂结合位点的重要功能残基。通过对白色念珠菌NMT活性位点中药物结合位点的研究发现，Trp126，Asn175和Thr211是NMT家族的高度保守残基，而且不与已有的抑制剂发生直接的相互作用，因此将是发现新型NMT抑制剂的重要药物结合位点。亚家族特异性残基Pro338，Leu350，Ile352和Ala353可作为已有抑制剂结构优化的重要位点，据此设计的新化合物将有望进一步提高对真菌NMT的选择性。进化踪迹分析结果为进一步阐明NMT结构-功能关系和新型NMT抑制剂类抗真菌药物的设计提供重要信息。     

氮唑类抗真菌药物药效构象及其与酶活性位点对接

目的：研究氮唑类抗真菌药物与其受体蛋白活性位点相互作用机理。方法：用随机构象搜寻和分子动力学模拟退火法确定１５个４种不同类型的氮唑类抗真菌药物最低能量构象;用活性类似物法限定药物分子药效基团之间的距离,搜寻到各化合物药效构象;将各化合物药效构象对接到白色念珠菌羊毛甾醇１４α去甲基化酶活性位点中。结果：４种结构类型的氮唑类药物在酶活性位点中有相似的对接位置;真菌和哺乳动物的活性位点结构特异性的残基分布在Ｆ螺旋Ｃ端、β６１和β６２区中;氮唑类抗真菌药物共同的卤代芳环结构落入相同的疏水空穴中,其中Ｙ１３２的侧链羟苯基结构可与抑制剂卤代芳环形成电子迁移复合物。结论：对接结果与已知ＳＡＲ分析结论相符,阐明了氮唑类药物与活性位点的氨基酸残基作用方式,探讨结构选择性药物的结构需求。     

新型非共价结合拟肽类蛋白酶体抑制剂的合成及活性评价

目的 设计、合成系列非共价结合拟肽类蛋白酶体抑制剂,并对其进行活性评价。方法 根据非共价结合蛋白酶体抑制剂与蛋白酶体的结合特点,采用氨基酸替换、生物电子等排等经典的药物设计方法,选取邻氯苄胺作为化合物的羧基末端基团,同时在肽骨架结构中引入六元环以增强肽类化合物的稳定性,设计并合成了一系列短肽非共价结合类蛋白酶体抑制剂,并通过体外蛋白酶体活性抑制实验评价该类化合物的活性。结果 共合成了8个具有全新结构的二肽和三肽化合物,其结构经¹H-NMR、ESI-MS确证,该类化合物对蛋白酶体具有中等的抑制活性。结论 肽链的长短及氨基末端不同的取代基对化合物的蛋白酶体抑制活性都有影响,8个化合物在体外对蛋白酶体都具有不同程度的抑制活性。本研究丰富了蛋白酶体抑制剂的结构类型,为该类化合物的深入研究奠定了基础。     

化疗保护剂的临床研究

化疗药物因治疗指数较低,可造成正常细胞损伤,产生剂量相关毒性,影响病人的生活质量,也影响药物的疗效。化疗保护剂的作用在于减少正常组织与化疗药物潜在的剂量相关性毒性,提高细胞毒药物的治疗指数。经多次临床验证,含硫亲核物质和金属螯合物是有效的化疗保护剂。１ 亲核硫化物 烷化剂的疗效和毒性是通过形成高反应性的亲电子中间体介导的。后者能与亲核性靶点,如ＤＮＡ的鸟嘌呤Ｎ７位点形成共价结合。亲核硫化物如谷胱甘肽,一旦被还原,将提供另一个亲核靶点,而使活性中间体失活。亲核硫化物也能与其它氧自由基等活性物质结合,…     

德曲妥珠单抗的研制

<正>1概说在经典药物化学中,拼合原理是将履行两种功能的结构元件拼接成一个分子,以赋予化合物优良性质。例如形成双靶标药物的两个药效团拼合,产生协同作用;将亲电性基团连接于配体形成共价结合药物以提高活性和选择性,以及前药设计等。     

Akt抑制剂的研究进展

蛋白激酶B(Akt)是一种丝氨酸/苏氨酸蛋白激酶,属于AGC激酶家族成员,作为磷脂酰肌醇-3激酶(PI3K)的下游靶点,通过磷酸化其下游一系列底物来影响细胞的生长、代谢、凋亡等重要的生命活动.Akt的活性失调或过度表达现象见于多种恶性肿瘤疾病,可导致肿瘤的发生、转移以及耐药性的产生.研究表明,Akt是靶向抗肿瘤药物研发...     

应用分子轨道方法说明药物作用原理

迄今已可较有把握地认为,受体是生物大分子的一部分,并想象除核酸及磷脂外,蛋白质为受体的主要可能结构。在过去的五年中,有些学者曾成功地分离出细胞膜上的受体蛋白,并确定其氨基酸组成。从剂量-效应曲线,可以推论受体与酶的相似性,因此酶动力学的规律也可运用于药物-受体的相互作用上。从X-射线结构研究酶的结构图,发现在酶的表面有氨基酸(如精氨酸、赖氨酸、天冬氨酸、酪氨酸、丝氨酸等)的极性残基,或如苯丙氨酸、色氨酸等的可极化的残基,通过与极性氨基酸功能基团相互作用,底物或抑制物可与酶的活性中心相结合。药物-受体复合物形成的分子作用原理     

有机磷酸酯诱发的迟发性神经病靶标酯酶的老化机制

神经病靶标酯酶的老化被认为是有机磷酸酯诱发迟发性神经病的必需步骤。老化的本质是酶分子中的丝氨酸活性位点共价结合的磷酰基脱烷基化,使得酶不可能再复活,但其过程不同于乙酰胆碱酯酶的老化过程,并受有机磷酸酯的构型和纯度的影响。近来研究表明,不同的有机磷酸酯对其老化的机制不一样,存在着可逆的质子丢失和侧链基团的分子内转移两条途径,并推测Asp1044和Asp1004可能是侧链基团转移的结合位点,然而神经病靶标酯酶的老化机制的完全阐明还需要进一步的分子实验证据。     

Chemical Stability of Insulin. 2. Formation of Higher Molecular Weight Transformation Products During Storage of Pharmaceutical Preparations

Formation of covalent, higher molecular weight transformation (HMWT) products during storage of insulin preparations at 4–45°C was studied by size exclusion chromatography. The main products are covalent insulin dimers (CID), but in protamine-containing preparations the concurrent formation of covalent insulin-protamine (CIP) products takes place. At temperatures 25°C parallel or consecutive formation of covalent oligo- and polymers can also be observed. Rate of HMWT is only slightly influenced by species of insulin but varies with composition and formulation, and for isophane (NPH) preparations, also with the strength of preparation. Temperature has a pronounced effect on CID, CIP, and, especially, covalent oligo- and polymer formation. The CIDs are apparently formed between molecules within the hexameric unit common for all types of preparations and rate of formation is generally faster in glycerol-containing preparations. Compared with insulin hydrolysis reactions (see the preceding paper), HMWT is one order of magnitude slower, except for NPH preparations.To whom correspondence should be addressed at Nove Research Institute, Novo Alle, DK-2880Bagsvaerd, Denmark     

Comparative Analysis of Pharmacophore Features and Quantitative Structure–Activity Relationships for CD38 Covalent and Non‐covalent Inhibitors

In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non‐covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure‐based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure–activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non‐covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non‐bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross‐validated correlation coefficient values (q²) of 0.564 and 0.571, and non‐cross‐validated values (r²) of 0.967 and 0.971, respectively. The CD38 non‐covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non‐covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross‐validated correlation coefficient values (q²) of 0.469 and 0.454, and non‐cross‐validated values (r²) of 0.814 and 0.819, respectively.     

Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis,and Biological Evaluation

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The Molecular Toxicology of Acetaminophen

The history of the development of the analgestic drug acetaminophen is reviewed with an emphasis on the characteristics of its overdose toxicity. The P450-catalyzed oxidation of acetaminophen generates a reactive electrophile that binds covalently to proteins. Involvement of specific P450 enzymes in acetaminophen toxicity can be probed by experiments with knock-out mice. The identification of specific target proteins may help to clarify the mechanism of acetaminophen hepatoxocity.     

STRUCTURAL STUDIES ON YEAST 3-PHOSPHOGLYCERATE KINASE: Linear Arrangement of the CNBr Fragments,Partial Amino Acid Sequence of the Inner Part of the Polypeptide Chain,and Analyses of the N-Terminal Domain of the Protein

The purpose of this work was to contribute to the study of the covalent struc ture of yeast 3-phosphoglycerate kinase. First, we undertook the complete align ment of the four fragments produced by cyanogen-bromide cleavage and which constitute the intact protein; we then established the total amino acid sequence of a 30-residue peptide and the N-terminal sequence of a 65-residue peptide. Second, we analyzed the acetylated state of the protein. The analyses of the acid fraction “P” obtained after digestion of 3-phosphoglycerate kinase by pronase enabled us to determine the N-terminal sequence of this enzyme as N-acetylserylglycine. Third, we isolated, purified and analyzed seven tryptic peptides from a fragment containing 102 amino acids coming from the N-terminal end of the protein. The peptides occupying the N- and C-terminal ends of this fragment were also identified.     

Preclinical Characterization of the FAAH Inhibitor JNJ-42165279

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Covalent Binding Between Bucillamine Derivatives and Human Serum Albumin

Purpose. To clarify the mechanism of covalent binding between human serum albumin (HSA) and drugs containing thiol groups, we studied the interactions between HSA and bucillamine (BA) and its derivatives. Methods. To determine the concentration of HSA-drug conjugate, we used columns of N-methylpyridium polymer cross-linked with ethylene glycol dimethacrylate (4VP-Me), and analyzed the reaction between HSA and B A derivatives kinetically. Following pseudo first-order reaction kinetics, the rate constants of reduction of non-mercaptoalbumin (HNA) to mercaptoalbumin (HMA) (k_a) and formation of HSA-drug conjugate (k_c) were determined. Results. Formation of HSA-drug conjugate was observed only for drugs containing one thiol group. In compound IV, the plots of k_a and k_c against pH were found to be linear. The HSA-drug conjugate was affected by various factors such as pK_a, pH, temparture and the microenviroment of Cys³⁴. The increases in k_a and k_c. against pH were mainly due to the increase in mercaptide ion concentration. Further, fatty acid affected the microenviroment of Cys³⁴, which increased HSA-drug formation. Conclusions. Cys³⁴ located in a crevice on the surface of the protein plays an important role on the formation of HSA-drug conjugate. These results may be useful for elucidating the reaction mechanisms between various proteins and thiol compounds.     

靶向共价键药物的研究进展

药物与靶标的结合是启动药理作用的本源,共价键药物是以共享电子的方式来实现与靶标的结合,其中大多为抗感染、抗肿瘤以及心脑血管、神经系统和代谢类药物。简介共价键药物与非共价键药物的区别以及既往的重磅级共价键药物与靶标的结合特点,分类综述靶向共价键药物的理性设计及与靶标的结合反应。     

Photoreactive derivative of Kunitz's soybean trypsin inhibitor.

The photoreactive arylsulfenyl chloride 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) has been used for the selective modification of tryptophan in Kunitz's soybean trypsin inhibitor (SBTI). The ultraviolet absorption spectrum and amino acid analysis of 2,4-NAPS-SBTI indicated that only one of the two tryptophans (93 or 117) present in SBTI was modified. CNBr cleavage of 2,4-NAPS-SBTI resulted in two fragments 1–114 and 115–181.Amino acid analysis of the two separated fragments showed that only tryptophan 93 underwent modification. 2,4-NAPS-SBTI fully retained its inhibitory activity against trypsin. The photoaffinity labeling of trypsin with 2,4-NAPS-Cl was performed on tritiated trypsin prepared by reacting bovine trypsin with [³H]-succinimidyl propionate. The covalent attachment of 2,4-NAPS-SBTI to the tritiated trypsin after photolysis was demonstrated by exclusion chromatography on Sephadex G-50 in the presence of guanidine hydrochloride.     

Covalent Binding of a Bucillamine Derivative with Albumin in Sera from Healthy Subjects and Patients with Various Diseases

Purpose. To investigate the difference of pharmacokinetics of thiol-containing drugs in various disease states, we studied the covalent binding of SA3786, a bucillamine derivative, with proteins in patient serum compared with that in healthy serum. Methods. Sera from healthy volunteers and patients of various diseases were supplied by the Japanese Red Cross Kumamoto Hospital. For the formation of conjugate experiments, SA3786 was added to a final concentration of 7 × 10^–4M. After 6h incubation at 37°C, HPLC analysis of 5 1 aliquots of each sample was performed using a column of N-methylpyridinium polymer (4VP-Me). Results. The extent of HSA-SA3786 conjugate formation was found to be lower in the sera from healthy volunteers (control) than those from patients of various diseases. Especially high reactivity with SA3786 was observed in sera from rheumatic patients and hepatic patients. With the exception of the fraction of mercaptoalbumin (f_HMA), none of the parameters showed a good correlation with conjugate formation. Conclusions. The parameter f_HMA must be considered to be one of the most important factors in formation of conjugates between plasma protein and thiol compounds. However, other factors may be involved in addition to f_HMA although the nature of these factors is not clear.