Potency variation of small-molecule chymase inhibitors across species

Chymases (EC 3.4.21.39) are mast cell serine proteinases that are variably expressed in different species and, in most cases, display either chymotryptic or elastolytic substrate specificity. Given that chymase inhibitors have emerged as potential therapeutic agents for treating various inflammatory, allergic, and cardiovascular disorders, it is important to understand interspecies differences of the enzymes as well as the behavior of inhibitors with them. We have expressed chymases from humans, macaques, dogs, sheep (MCP2 and MCP3), guinea pigs, and hamsters (HAM1 and HAM2) in baculovirus-infected insect cells. The enzymes were purified and characterized with kinetic constants by using chromogenic substrates. We evaluated in vitro the potency of five nonpeptide inhibitors, originally targeted against human chymase. The inhibitors exhibited remarkable cross-species variation of sensitivity, with the greatest potency observed against human and macaque chymases, with K_i values ranging from ∼0.4 to 72 nM. Compounds were 10-300-fold less potent, and in some instances ineffective, against chymases from the other species. The X-ray structure of one of the potent phosphinate inhibitors, JNJ-18054478, complexed with human chymase was solved at 1.8 Å resolution to further understand the binding mode. Subtle variations in the residues in the active site that are already known to influence chymase substrate specificity can also strongly affect the compound potency. The results are discussed in the context of selecting a suitable animal model to study compounds ultimately targeted for human chymase.     

仓鼠深Ⅱ度烧伤创面愈合过程中糜蛋白酶的表达

背景：肥大细胞糜蛋白酶在烧伤创面表达的研究较少。 目的：观察肥大细胞糜蛋白酶是否存在于烫伤创面以及深Ⅱ度烫伤后不同时间点的表达变化。 方法：应用75 ℃的水接触仓鼠背部皮肤12 s制备深Ⅱ度烫伤创面(病理组织学证实)。分别于烫伤前及烫伤后1，3，7，14 d取烫伤组织进行试验。 结果与结论：实时定量RT-PCR及放射免疫 检测结果显示烫伤后创面组织中糜蛋白酶mRNA表达量和活性均有所增高，在烫伤后第3天最高。表明肥大细胞糜蛋白酶在烧伤创面有所表达，并可能参与了烧伤损伤愈合过程。     

Angiotensin II-Forming Systems in Cardiovascular Diseases

Both the systemic and the tissue renin–angiotensin system (RAS)-are heavily involved in cardiovascular homeostasis, but their excess activation seems to be associated with increased morbidity and mortality in various stages of cardiovascular diseases, since angiotensin-converting enzyme (ACE) inhibitors have been shown to improve hypertension, congestive heart failure, and acute myocardial infarction. A clinical megastudy (ELITE) of elderly patients has recently shown that an AT1 receptor antagonist was superior to an ACE inhibitor for improvement of patients' prognosis. One of the possible mechanisms of this beneficial effect of the AT1 receptor antagonist compared to the ACE inhibitor could be the blockade of all the angiotensin (Ang) II formed not only by ACE but also by alternative pathways.Recent studies have disclosed that chymase, the most abundant Ang II-forming enzyme in human tissues, could be involved in the development of atherosclerosis, the remodeling of the myocardium after infarction or hypertrophy, restenosis after vascular injury, and chronic inflammatory conditions. Kallikrein-dependent Ang II formation also seems to take place under various ischemic conditions, as shown in the ischemic dog heart after ligation of a coronary artery, human leg circulation of patients with arteriosclerosis obliterans, or systemic circulation during a graded exercise. However, detailed mechanisms of non-ACE Ang II-forming enzymes involved in these pathological changes are not known. Current knowledge about ACE and non-ACE Ang II-forming enzymes in cardiovascular diseases are reviewed in this article.     

糜蛋白酶在烟草烟雾诱导的仓鼠肺动脉高压中的作用

目的探讨糜蛋白酶在烟草烟雾所诱导的肺动脉重构和肺动脉高压中的作用。方法将仓鼠暴露在烟草产生的烟雾中（烟雾暴露组,n=6）,4个月后,使用免疫组织化学法、蛋白免疫印迹法、放射免疫学测定、反转录PCR等测定仓鼠肺的形态学和肺组织的生物化学改变。对烟雾暴露组与对照组（n=6）上述指标进行比较。结果长期烟草烟雾暴露使仓鼠右心室收缩压升高,肺小动脉中层细胞肥大,同时肺组织糜蛋白酶活性及合成增加,血管紧张素Ⅱ（AngⅡ）水平升高（与对照组比较,P均﹤0.05）。糜蛋白酶抑素（chymostatin）可以降低烟草诱导的仓鼠肺组织中糜蛋白酶活性的增加和AngⅡ水平,改善肺小动脉的重构程度,降低右心室收缩压,但对仓鼠肺组织中血管紧张素转换酶（ACE）的活性无影响。结论长期烟草烟雾暴露可增加仓鼠肺中糜蛋白酶的活性及表达,激活的糜蛋白酶进一步诱导肺组织AngⅡ形成,这可能是烟草诱导的肺动脉高压发生机制的一部分。因此,糜蛋白酶抑素也许会对吸烟的肺动脉高压患者有益。     

肝组织中Chymase浓度在酒精性肝纤维化中的意义

目的:探讨肝组织中Chynmse浓度在酒精性肝纤维化中的意义.方法:采用酶联免疫吸附实验(ELISA)检测48例酒精性肝纤维化患者肝组织中Chymase浓度,与28例急性肝炎做对比;用免疫组织化学染色法观察Chymase单克隆抗体(mAb)标记的肥大细胞分布情况.结果:酒精性肝纤维化患者肝组织中Chymase浓度为(17.5±4.4)μg/g,明显高于急性肝炎(2.8±1.0)μg/g,P<0.01;免疫组化染色结果,Chymase标记的肥大细胞主要分布于纤维化旺盛的肝细胞周围及中心静脉周围,其分布与纤维化部位相一致,且肝组织中Chymase浓度高的患者,其肝组织内Chymase标记的肥大细胞分布增多.结论:肝组织中Chymase浓度可能与酒精性肝纤维化关系密切.     

心脏糜酶在自发性高血压大鼠心肌纤维化中的作用

目的观察心脏糜酶在自发性高血压大鼠(SHR)心肌组织胶原合成和心肌纤维化中的作用。方法应用病理检查、计算机分析结合逆转录-聚合酶链式反应等方法,检测SHR应用糜酶抑制剂(Chy-Ⅰ)组、SHR(SHR)组及对照组(WKY)组收缩压、心肌胶原容积分数(CVF)、心肌血管周围胶原面积比(PVCA)和心肌糜酶及Ⅰ、Ⅲ型胶原mRNA表达。结果Chy-Ⅰ组心脏CVF、PVCA分别为(26.8±8.7)%和0.4±0.1,SHR组分别为(46.4±7.8)%和1.9±0.9,WKY组为(24.4±10.7)%和0.4±0.1,Chy-Ⅰ组比SHR组明显下降(P<0.01),与WKY组无明显差别(P>0.05)。Chy-Ⅰ组心肌组织Ⅰ、Ⅲ型胶原和糜酶mRNA表达相对含量均明显低于SHR组(P<0.01),与WKY组无明显差别(P>0.05)。应用Chy-Ⅰ后大鼠血压无改变。结论心肌组织糜酶参与胶原的合成,参与细胞外基质的形成和降解,促进自发性高血压大鼠心肌纤维化。     

海洛因中毒者血浆类胰蛋白酶及类糜蛋白酶含量的测定

目的探讨海洛因中毒为代表的非变态反应性过敏反应患者血浆类胰蛋白酶及类糜蛋白酶的含量变化及其机制。方法对照组为22名正常人,实验组1为19例海洛因中毒者,实验组2为20例颅脑外伤患者,实验组3为22例初发急性心肌梗死患者。应用ELISA方法检测对照组和实验各组血浆胰蛋白酶及类糜蛋白酶含量。结果三组实验组与对照组比较血浆胰蛋白酶及类糜蛋白酶含量差异有统计学意义（P〈0.05）,即实验组血浆类胰蛋白酶和类糜蛋白酶含量较对照组均升高,而三组实验组间两两比较,差异无统计学意义（P〉0.05）。结论海洛因中毒、颅脑创伤与心肌梗死者血浆类胰蛋白酶及类糜蛋白酶含量均增加,提示在非变态反应性过敏反应及上述两种疾病患者均伴有肥大细胞脱颗粒或溶解,类胰蛋白酶和类糜蛋白酶含量升高可以作为非变态反应性过敏反应的诊断依据之一,但应排除急性心肌梗死和颅脑损伤,或者联合其他指标综合判断。     

Isolation and characterization of trypsin-like and chymotrypsin-like proteinases from human cholesteatoma

The mast-cell-specific proteolytic enzymes tryptase and chymase were identified in and isolated from cholesteatoma in a ratio similar to that found in human skin. We assume that this ratio reflects a similar distribution of tryptase-containing and tryptase/chymase-containing mast cells in both these tissues. It seems conceivable that mechanisms able to trigger excessive and/or continuous mast cell degranulation in the middle ear might be causative for the formation of cholesteatoma either directly or via primed chronic inflammatory reactions. By their ability to amplify degranulation of mast cells, mast cell proteinases, in particular chymase, may contribute to the chain of events leading to the formation of cholesteatoma.Prof. Dr. Karl Hochstrasser died on 16 August 1993     

Cysteinyl cathepsins and mast cell proteases in the pathogenesis and therapeutics of cardiovascular diseases

The initiation and progression of cardiovascular diseases involve extensive arterial wall matrix protein degradation. Proteases are essential to these pathological events. Recent discoveries suggest that proteases do more than catabolize matrix proteins. During the pathogenesis of atherosclerosis, abdominal aortic aneuryms, and associated complications, cysteinyl cathepsins and mast cell tryptases and chymases participate importantly in vascular cell apoptosis, foam cell formation, matrix protein gene expression, and pro-enzyme, latent cytokine, chemokine, and growth factor activation. Experimental animal disease models have been invaluable in examining each of these protease functions. Deficiency and pharmacological inhibition of cathepsins or mast cell proteases have allowed their in vivo evaluation in the setting of pathological conditions. Recent discoveries of highly selective and potent inhibitors of cathepsins, chymase, and tryptase, and their applications in vascular diseases in animal models and non-vascular diseases in human trials, have led to the hypothesis that selective inhibition of cathepsins, chymases, and tryptase will benefit patients suffering from cardiovascular diseases. This review highlights recent discoveries from in vitro cell-based studies to experimental animal cardiovascular disease models, from protease knockout mice to treatments with recently developed selective and potent protease inhibitors, and from patients with cathepsin-associated non-vascular diseases to those affected by cardiovascular complications.     

慢性阻塞性肺疾病患者痰中肥大细胞和嗜酸粒细胞因子变化及其相关性研究

目的探讨慢性阻塞性肺疾病(COPD)患者类糜蛋白酶(chymase)活性,类胰蛋白酶(tryptase)、白细胞介素8(IL8)、嗜酸粒细胞趋化因子(eotaxin)水平和中性粒细胞(NEU)、嗜酸粒细胞(EOS)计数的相关性及临床意义。方法老年COPD患者73例(重度21例、中度21例、轻度31例),采用双抗体夹心酶联免疫吸附测定,检测诱导痰IL8、eotaxin水平。在UniCAP100全自动体外变应原检测仪上进行tryptase检测,Chymase活性测定使用琥珀酰丙氨酸丙氨酸脯氨酸苯丙氨酸酰苯氨(SAAPP)作为底物,采用酶标仪在410nm连续监测吸光度的变化。结果(1)老年急性加重期COPD患者(重、中、轻)痰tryptase的中位数分别为2840、2150、595ng/L,治疗后各组痰tryptase水平显著下降(分别为1510、920、33ng/L,P均<001)。加重期重度、中度与轻度患者比较差异均有显著性(P均=0)。急性加重期痰IL8、痰eotaxin中位数分别为12998、4549、787ng/L;227、151、74ng/L。重度、中度组痰IL8、eotaxin水平均高于轻度组(P均<001)。治疗后痰IL8、eotaxin中位数分别为10375、3266、679ng/L;79、63、68ng/L。重度、中度患者痰IL8、eotaxin水平均比治疗前显著降低(P均<001)。(2)重、中度COPD患者急性加重期痰chymase活性高于轻度组,黄豆胰蛋白酶抑制剂(SBTI)、α1抗胰蛋白酶(α1A