CN111840303A - 胞质羧肽酶抑制剂及其应用 - Google Patents
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Abstract
本发明涉及胞质羧肽酶抑制剂及其应用。2‑PMPA作为胞质羧肽酶抑制剂的浓度范围为1nM‑100mM；首次开发了CCP家族的高活性抑制剂。与传统M14金属羧肽酶抑制剂相比，2‑PMPA可以高效抑制CCP家族酶的活性；2‑PMPA不仅抑制CCP家族对蛋白质底物，还可以抑制其催化合成多肽和神经肽的活性。通过酶促反应实验确定了2‑PMPA抑制CCP1催化合成多肽的半数抑制浓度(IC50)为0.99μM，且为混合型抑制，其Ki和Ki’分别为0.096μM和0.235μM。2‑PMPA在实验条件下抑制CCP1催化微管蛋白去谷氨酰化的IC50为0.21mM。
Description
技术领域
本发明涉及胞质羧肽酶抑制剂领域，特别是涉及2-(膦酰基甲基)戊二酸(2-PMPA)作为胞质羧肽酶抑制剂的应用。
背景技术
胞质羧肽酶(Cytosolic Carboxypeptidase,CCP)是M14金属羧肽酶的一个亚家族，催化蛋白的翻译后修饰-多聚谷氨酰化的去修饰。多聚谷氨酰化是一种新型的蛋白质翻译后修饰，发生在蛋白质一级结构中谷氨酸的γ－羧基上，通过酰胺键与游离谷氨酸的氨基相连形成支链，支点上的谷氨酸进而与多个谷氨酸以肽键(α－羧基)依次连接，形成长短不一的多聚谷氨酰侧链。多聚谷氨酰化在体内维持动态的平衡，其起始和延长由类微管蛋白酪氨酸连接酶(Tubulin Tyrosin Ligase Like,TTLL)家族的9个成员催化。而胞质羧肽酶的6个家族成员则催化多聚谷氨酰侧链的缩短和消化。
调控多聚谷氨酰化的修饰酶和去修饰酶功能异常时会导致多聚谷氨酰化失衡，从而会引发多种疾病，包括神经退行，视网膜炎症，雄性不育等。例如CCP1突变会导致pcd小鼠(Purkinje cell degeneration，pcd)的浦肯野细胞退变，还会造成新生绵羊四肢瘫痪，最近研究发现婴儿期开始逐渐发生神经退变的人类患者中携带失活的突变CCP1基因。TTLL1的功能缺失导致KIF1A异常靶向所致的囊泡传递受损。TTLL5变异会导致由RPGR(RetinitisPigmentosa GTPase Regulator)低水平谷氨酰化引起的视网膜营养不良。而开发多聚谷氨酰化去修饰酶抑制剂，将为通过干预这一修饰的平衡治疗相应疾病提供可能。根据我们的检索，迄今没有文献报道过针对胞质羧肽酶家族的有效抑制剂。而通用M14金属羧肽酶家族的抑制剂对胞质羧肽酶家族的抑制选择性差，体外实验表明需要至少毫摩尔的浓度(Wu,H.Y.et al.,Astructural and functional analysis of Nna1 in Purkinje celldegeneration(pcd)mice.FASEB J 26,4468–4480,2012)。因此，寻找高效的胞质羧肽酶抑制剂具有重要的研究意义和应用前景。
2-(膦酰甲基)戊烷二酸(2-PMPA)是谷氨酸羧肽酶II(GCPII)的高效抑制剂，但该化合物对其他羧肽酶的活性影响尚未见报道。胞质羧肽酶和谷氨酸羧肽酶II存在很大差异。两者分别属于不同的金属羧肽酶家族:胞质羧肽酶是M14金属羧肽酶的亚家族，而谷氨酸羧肽酶II是M28金属羧肽酶的亚家族；两者的催化机理不同：胞质羧肽酶依赖于一个锌离子，谷氨酸羧肽酶II除了需要两个锌离子外，钙离子和氯离子对其催化活性也是不可或缺的；在功能上胞质羧肽酶催化多聚谷氨酰化的去修饰，而谷氨酸羧肽酶II将大量神经肽N-乙酰天冬氨酰谷氨酸(NAAG)分解为N-乙酰天冬氨酸(NAA)和谷氨酸。
发明内容
本发明首次开发了2-PMPA可以作为胞质羧肽酶家族的高活性抑制剂，为2-PMPA提供一种新用途，同时提供了抑制胞质羧肽酶家族活性的方法。
为了探索和解决现有技术的问题，本发明通过酶促反应分别探究了不同的化合物:1,10-邻菲啰啉(OP),2-苯甲基琥珀酸(BZS),L(+)-2-氨基-4-膦酰基丁酸(L-AP4),2-(膦酰基甲基)戊二酸(2-PMPA)对胞质羧肽酶的抑制效果。通过对比和筛选发现2-PMPA对胞质羧肽酶有良好的抑制效果，并通过对不同底物的探究发现2-PMPA不仅可以抑制胞质羧肽酶家族对蛋白质底物，如催化微管蛋白去谷氨酰化的活性，还可以抑制其催化合成多肽和神经肽去谷氨酰化的活性。
本发明提供了2-PMPA以及包含2-PMPA的组合物作为胞质羧肽酶抑制剂的应用,同时提供了抑制胞质羧肽酶家族活性的方法，并为利用2-PMPA来治疗多聚谷氨酸化失衡引起的相应疾病提供可能。
本发明提供如下技术方案：
第一方面提供2-PMPA作为胞质羧肽酶抑制剂的应用。
优选的，2-PMPA作为胞质羧肽酶抑制剂的浓度范围为1nM-100mM；更加优选的浓度范围为10nM-10mM。
优选的，提供2-PMPA抑制胞质羧肽酶催化合成多肽去谷氨酰化活性的应用。
优选的，提供2-PMPA抑制胞质羧肽酶催化蛋白质底物去谷氨酰化活性的应用。
优选的，提供2-PMPA抑制胞质羧肽酶催化神经肽去谷氨酰化活性的应用。
第二方面提供一种胞质羧肽酶抑制剂组合物，所述组合物中含有2-PMPA。
第三方面提供抑制胞质羧肽酶的方法，其包括使胞质羧肽酶在PBS缓冲溶液，HEPES缓冲溶液或MOPS缓冲溶液中与2-PMPA接触。
第四方面提供2-PMPA在制备用于预防和/或治疗与胞质羧肽酶相关的疾病或病症的药物中的应用。
第五方面提供用于治疗与胞质羧肽酶相关的疾病的药物组合物或药物制剂，其包含2-PMPA。
本发明首次开发了胞质羧肽酶的高活性抑制剂。不同于其他传统M14金属羧肽酶抑制剂，2-PMPA可以高效抑制胞质羧肽酶家族酶的活性。2-PMPA不仅可以抑制胞质羧肽酶家族对蛋白质底物，如催化微管蛋白去谷氨酰化的活性，还可以抑制其催化合成多肽和神经肽去谷氨酰化的活性。通过酶促反应实验确定了2-PMPA抑制CCP1催化合成多肽的半数抑制浓度(IC50)为0.99μM，且为混合型抑制，其Ki和Ki’分别为0.11μM和0.24μM。2-PMPA在实验条件下抑制CCP1催化微管蛋白去谷氨酰化的IC50为0.21mM。
附图说明
图1显示实施例1中不同化合物对重组CCP1催化多肽Biotin-3EG2E去谷氨酰化的抑制效果。
图2显示实施例2中在HEPES缓冲溶液中，2-PMPA对重组CCP1催化多肽Biotin-3EG2E去谷氨酰化的抑制效果。
图3显示实施例2中在MOPS缓冲溶液中，2-PMPA对重组CCP1催化多肽Biotin-3EG2E去谷氨酰化的抑制效果。
图4显示实施例2中2-PMPA对重组CCP4催化多肽Biotin-3EG2E去谷氨酰化的抑制效果。
图5显示实施例3中2-PMPA在不同缓冲溶液体系中对重组CCP1催化微管蛋白去谷氨酰化的半数抑制浓度效果。
图6显示实施例3中2-PMPA对重组CCP4催化微管蛋白去谷氨酰化的抑制效果。
图7显示实施例3中2-PMPA对CCP5细胞裂解液催化微管蛋白去谷氨酰化的抑制效果。
图8显示实施例3中2-PMPA对CCP6细胞裂解液催化微管蛋白去谷氨酰化的抑制效果。
图9显示实施例4中2-PMPA对重组CCP1催化神经肽去谷氨酰化的抑制效果。
具体实施方式
本发明首次记载2-PMPA不仅可以抑制胞质羧肽酶催化蛋白质底物去谷氨酰化的活性，还可以抑制其催化合成多肽和神经肽去谷氨酰化的活性。发明人结合具体实施例对本发明作进一步的说明，但本发明的保护内容不仅限于这些实施例。
2-PMPA抑制胞质羧肽酶的浓度范围是10nM-10mM，缓冲溶液可以是PBS，HEPES或者MOPS。
实施例1
不同化合物对重组CCP1的活性影响
实验中使用试剂的配置：
茚三酮-氯化铬溶液：0.2g茚三酮溶于20mL乙醇和2.5mL乙酸混合溶液中，随后加入0.25mL氯化铬水溶液(5.5mM)。
100μL的反应体系中包含0.6μg纯化的重组小鼠CCP1，40μM多肽biotin-3EG2E，25mM HEPES-K(pH 7.4)，100mM NaCl，1mM 1,10-邻菲啰啉(OP)/2-苯甲基琥珀酸(BZS)/2-PMPA/L(+)-2-氨基-4-膦酰基丁酸(L-AP4)。加热灭活的CCP1的反应作为阴性对照。设置三个平行组于37℃孵育1h。然后加入200μL茚三酮-氯化铬溶液混合均匀后于84℃水浴中加热5分钟终止反应。恢复室温后在酶标507nm下进行吸光度检测。释放的氨基酸使用由已知量的谷氨酸生成的标准曲线进行定量(L-AP4反应采用高效液相色谱法进行定量)。抑制效果如图1所示，不加抑制剂的CCP1活性为百分之百作为阳性对照，加入相同浓度的传统抑制剂OP、BZS和L-AP4后，CCP1的相对活性分别为16％，96％和27％，而2-PMPA几乎可以完全抑制CCP1的活性。
实施例2
(一)2-PMPA抑制CCP1催化多肽Biotin-3EG2E反应
100μL的反应体系中包含0.6μg纯化的重组小鼠CCP1，40μM多肽biotin-3EG2E，25mM HEPES-K(pH 7.4)或10mM MOPS(pH 7.4)，100mM NaCl，以及0，0.01，0.025，0.05，0.5，5或50μM 2-PMPA。加热灭活的CCP1的反应作为阴性对照。设置三个平行组于37℃孵育1h。检测生成谷氨酸的方法和上述相同，在HEPES缓冲溶液中2-PMPA抑制CCP1催化多肽去谷氨酰化效果如图2所示，0.05-50μM的2-PMPA对CCP1催化多肽的活性有抑制效果，经Prism计算IC50＝0.99μM。在MOPS缓冲溶液中2-PMPA抑制CCP1催化多肽效果如图3所示，1μM的2-PMPA对CCP1催化多肽去谷氨酰化抑制效果为52％。
(二)2-PMPA抑制CCP4催化多肽的活性
100μL的反应体系中包含0.6μg纯化的重组小鼠CCP4，40μM多肽Biotin-3EG2E，25mM HEPES-K(pH 7.4)，100mM NaCl，以及0，0.01，0.025，0.05，0.5，5或50μM 2-PMPA。加热灭活的CCP4的反应作为阴性对照。设置三个平行组于37℃孵育1h。检测生成谷氨酸的方法和上述相同，2-PMPA抑制CCP4催化多肽的活性效果如图4所示，0.01-50μM的2-PMPA对CCP4催化多肽的活性有抑制效果，经Prism计算IC50＝0.37μM。
实施例3
2-PMPA抑制CCP催化微管蛋白去谷氨酰化的活性
(一)2-PMPA抑制重组CCP催化微管蛋白去谷氨酰化的活性
1.样品准备
在40μL的反应体系中含有1μg纯化的重组小鼠CCP1或CCP4和2μg猪微管蛋白，以及0,0.1,1,5,10mM 2-PMPA于PBS或HEPES或MOPS缓冲溶液中在37℃下孵育1h。10mM OP在相同反应条件下作为阴性对照。加入10μl 5*SDS-PAGE蛋白上样缓冲液混匀于95℃加热10分钟终止反应。
2.电泳
(1)SDS-PAGE凝胶配制
安装玻璃板，按照表中数值配置8％(w/v)的分离胶溶液和5％(w/v)浓缩胶溶液，并分别灌注：
| 成份 | 8％分离胶溶液(mL) | 5％浓缩胶溶液(mL) |
| ddH<sub>2</sub>O | 9.3 | 6.8 |
| 1.5M Tris-HCl(pH 8.8) | 5 | —— |
| 1.0M Tris-HCl(pH 6.8) | —— | 1.25 |
| 10％SDS | 0.2 | 0.1 |
| 30％丙烯酰胺溶液 | 5.3 | 1.7 |
| 10％过硫酸铵 | 0.2 | 0.1 |
| TEMED | 0.012 | 0.01 |
| 总体积 | 20 | 10 |
(2)上样与电泳
上样：取10μL样品上样于8％聚丙烯酰胺凝胶电泳分离。
电泳：浓缩胶80V，分离胶120V，电泳至溴酚蓝即将跑出胶即可终止电泳。
(3)转膜
使用半干式转膜仪器，将蛋白胶上的蛋白转移到硝酸纤维素膜(NC膜)上，设定转膜电流为100mA，转膜时间为60分钟。
(4)封闭
采用5％脱脂奶粉/TBST于室温封闭30分钟。
(5)孵育一抗
用5％脱脂奶粉/TBST稀释抗体，终浓度为polyE(1:4000，识别大于或等于3个谷氨酸侧链),α-tubulin(EP1332Y，1:5000)。在摇床上4℃过夜孵育。
回收一抗。加入TBST洗涤液，在摇床上摇动洗涤10分钟。吸尽洗涤液后，再加入洗涤液洗涤10分钟。共洗涤3次。
(6)孵育二抗
用5％脱脂奶粉/TBST稀释抗体，二抗donkey anti rabbit终浓度为1:5000。在摇床上室温孵育2h。
回收二抗。加入TBST洗涤液，在摇床上摇动洗涤10分钟。吸尽洗涤液后，再加入洗涤液洗涤10分钟。共洗涤3次。
(7)蛋白检测
将ECL发光液均匀滴加在膜上，用凝胶成像系统检测2-PMPA在不同缓冲溶液体系中对CCP催化蛋白质底物的抑制效果。根据该Western blotting结果中polyE和α-tubulin的抗体免疫反应的灰度比，经Prism计算，2-PMPA在PBS,HEPES和MOPS缓冲溶液体系中抑制CCP1催化微管蛋白去谷氨酰化的IC50分别为0.21mM，0.15mM，0.19mM，如图5所示。2-PMPA抑制CCP4催化微管蛋白的反应结果如图6所示，10mM传统抑制剂OP作为阴性对照，0.1-10mM2-PMPA对CCP4催化微管蛋白去谷氨酰化的活性有明显抑制效果。
(二)2-PMPA抑制含有CCP的细胞裂解液的微管蛋白去谷氨酰化活性
用表达CCP5或CCP6的质粒(氮端含有myc-标签)转染HEK293细胞，培育40小时后，将细胞用预冷的DPBS洗涤两次，并用含有蛋白酶抑制剂和0.2％乙基苯基聚乙二醇(NP-40)的DPBS溶液裂解。在4℃以24,000g转速离心20分钟后，弃沉淀留上清作为CCP5或CCP6细胞裂解液备用。
在40μL的反应体系中含有20μL CCP5或CCP6细胞裂解液和1μg猪微管蛋白，以及0,0.1,1mM 2-PMPA于PBS溶液在37℃下孵育1h。10mM OP在相同反应条件下作为阴性对照。加入10μL5*SDS-PAGE蛋白上样缓冲液混匀于95℃加热10分钟终止反应。2-PMPA抑制含CCP5细胞裂解液的微管蛋白去谷氨酰化活性的检测方法如上所述，Western blotting一抗孵育GT335(1:4000，识别支链上γ－羧基相连的谷氨酸)和anti-myc(1:2000)，二抗孵育goatanti mouse(1:5000)抑制效果如图7所示；2-PMPA对含CCP6细胞裂解液的微管蛋白去谷氨酰化活性抑制效果如图8示，lacZ作为空白对照，不加抑制剂的为阳性对照，10mM传统抑制剂OP作为阴性对照，0.1-1mM 2-PMPA几乎可以完全抑制CCP5和CCP6催化微管蛋白去谷氨酰化的活性。
实施案例4
2-PMPA抑制CCP1催化神经肽N-乙酰天冬氨酰谷氨酸(N-Acetylaspartyl-glutamic acid,NAAG)的反应
100μL的反应体系中包含0.6μg CCP1，0.1mM NAAG，25mM HEPES-K(pH 7.4)，100mMNaCl，以及1μM 2-PMPA，以不加入2-PMPA的反应为阳性对照，加入加热灭活的CCP1的反应作为阴性对照。设置三个平行组于37℃孵育1h。然后加入200μL茚三酮-氯化铬溶液混合均匀后于84℃水浴中加热5分钟终止反应。恢复室温后在酶标507nm下进行吸光度检测。释放的氨基酸使用由已知量的谷氨酸生成的标准曲线进行定量。抑制效果如图9所示，不加抑制剂的作为阳性对照，1μM 2-PMPA对CCP1催化神经肽去谷氨酰化的抑制效果为72％。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开。对于本领域的技术人员而言，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、改进、等同替代等，都应包含在本公开的保护范围之内。
Claims (10)
1.一种胞质羧肽酶抑制剂，其特征是包含2-(膦酰基甲基)戊二酸。
2.2-(膦酰基甲基)戊二酸在制备胞质羧肽酶抑制剂中的应用。
3.一种抑制胞质羧肽酶的方法，其中使所述胞质羧肽酶与2-(膦酰基甲基)戊二酸在抑制所述胞质羧肽酶的条件下接触。
4.如权利要求2所述的方法，其特征在于，抑制所述胞质羧肽酶的缓冲体系不限，优选PBS，HEPES，MOPS；接触2-(膦酰基甲基)戊二酸的浓度为1nM-100mM，优选10nM-10mM。
5.如权利要求2或3所述的方法，其特征在于，所述抑制使胞质羧肽酶催化底物去谷氨酰化的活性降低至少20％，优选50％，更优选80％。
6.一种药物组合物，所述药物组合物包含作为活性成分的2-(膦酰基甲基)戊二酸以及药学上耐受的佐剂和/或赋形剂。
7.如权利要求6所述的药物组合物，其特征在于，还包括一种或多种其他活性成分。
8.如权利要求6或7所述的药物组合物，其特征在于，其中的2-(膦酰基甲基)戊二酸含量不低于20％。
9.如权利要求6或7所述的药物组合物，其特征在于，用于预防性或治疗性治疗多聚谷氨酰化失衡相关的疾病。
10.如权利要求6所述的药物组合物，其特征在于，所述疾病选自：神经退行、视网膜炎症和雄性不育。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023154939A3 (en) * | 2022-02-14 | 2023-09-28 | The Johns Hopkins University | Gcpii inhibition for the treatment of sarcopenia and aging |
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| CN1222078A (zh) * | 1996-06-17 | 1999-07-07 | 吉尔福德医药公司 | 用作naalad酶抑制剂的氧膦基衍生物 |
| BR9711555A (pt) * | 1996-09-27 | 1999-08-24 | Guilford Pharm Inc | Composi-{es de naaladase e m-todos para tratar anomalia de glutamato e provocar atividade neuronal em animais |
| RU2211697C2 (ru) * | 1996-09-27 | 2003-09-10 | Гилфорд Фармасьютикалз Инк. | Способ лечения глутаматных расстройств |
| WO2016112382A2 (en) * | 2015-01-09 | 2016-07-14 | The Johns Hopkins University | Intranasal administration of glutamate carboxypeptidase (gcp-ii) inhibitors |
| EP3424524A2 (en) * | 2017-07-04 | 2019-01-09 | CureVac AG | Cancer rna-vaccine |
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| CN1222078A (zh) * | 1996-06-17 | 1999-07-07 | 吉尔福德医药公司 | 用作naalad酶抑制剂的氧膦基衍生物 |
| BR9711555A (pt) * | 1996-09-27 | 1999-08-24 | Guilford Pharm Inc | Composi-{es de naaladase e m-todos para tratar anomalia de glutamato e provocar atividade neuronal em animais |
| RU2211697C2 (ru) * | 1996-09-27 | 2003-09-10 | Гилфорд Фармасьютикалз Инк. | Способ лечения глутаматных расстройств |
| WO2016112382A2 (en) * | 2015-01-09 | 2016-07-14 | The Johns Hopkins University | Intranasal administration of glutamate carboxypeptidase (gcp-ii) inhibitors |
| EP3424524A2 (en) * | 2017-07-04 | 2019-01-09 | CureVac AG | Cancer rna-vaccine |
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| 刘婧等: "胞浆羧肽酶1的研究进展" * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023154939A3 (en) * | 2022-02-14 | 2023-09-28 | The Johns Hopkins University | Gcpii inhibition for the treatment of sarcopenia and aging |
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