CN116672454A - 一种抗结核感染的小分子化合物 - Google Patents
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Abstract
本发明属于生物技术领域，具体涉及一种抗结核分枝杆菌感染的小分子化合物的筛选及应用，本发明基于HDAC6的特殊结构，确认HDAC6特异性在结核抵抗者巨噬细胞中维持表达，并且证实结核抵抗者人群来源的巨噬细胞高效清除Mtb感染的能力依赖于HDAC6，探索抑制HDAC6降解的蛋白靶点，筛查相应的抑制剂，从而在HDAC6泛素化区域发现了抑制HDAC6的小分子化合物，为治疗结核病提供了一种新思路，还为活动性结核病患者免疫治疗方案奠定了理论基础。
Description
技术领域
本发明属于生物技术领域，具体涉及一种抗结核分枝杆菌感染的小分子化合物的筛选及应用。
背景技术
结核病(tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis，Mtb)引起的全球致死率最高的人类慢性传染病之一，也是全球公共卫生面临的重大挑战之一，还是艾滋病病毒(human immunodeficiency virus，HIV)感染者的“头号杀手”，还是抗菌素耐药相关的主要致死性传染病，在同时感染耐多药结核分枝杆菌的HIV携带者中，死亡率非常高，这种混合感染也是耐多药结核病流行的原因。据世界卫生组织估算，2021年全球新发结核病患者超过1060万，死亡人数超过160万，只有36％的需要治疗的患者得到了治疗，耐药菌株的流行和HIV的合并感染更进一步加剧了全球TB的负担。2021年开始治疗的利福平耐药结核病(rifampicin resistance-tuberculosis，RR-TB)和耐多药结核病(multiple drug resistant tuberculosis，MDR-TB)患者数为45万例和161746例，仅仅覆盖了需要疗的全部患者的1/3，而HIV感染者中有18.7万例死于结核病。研究表明利福平和异烟肼的联合治疗方案对普通结核病患者有很好的治疗效果，但是结核病的完整治疗不仅包括清除处于活跃代谢的结核分枝杆菌，同时需要清除在患者体内处于休眠状态或者慢代谢的结核分枝杆菌，尤其是杀灭后者对于提高结核病治疗效果，降低结核病复发具有重要作用。然而目前治疗结核病的核心药物主要针对活跃代谢的结核分枝杆菌生命周期中的重要环节发挥作用，但是对于潜伏在巨噬细胞内的结核分枝杆菌作用不显著。
研究表明，HDACs在调控病毒感染所诱导的宿主天然免疫反应中发挥重要作用。其中，HDAC6是唯一一个具有两个去乙酰化酶结构域(DD1和DD2)和一个C端锌指泛素结合域(BUZ)的胞质去乙酰化酶，HDAC6存在于细胞质中，在C末端存在2个催化剂结合位点和1个泛素结合区，能够通过去乙酰化酶活性和泛素依赖性机制调节机体多种生物学功能，包括病毒复制、细胞迁移、细胞增殖、分化以及错误折叠蛋白质的降解等。由于HDAC6结构特殊，其具有多种独特的生物学功能，成为多种疾病潜在的治疗靶点，在前期结核抵抗者中的研究，确认HDAC6特异性在结核抵抗者巨噬细胞中维持表达，并且证实结核抵抗者人群来源的巨噬细胞高效清除Mtb感染的能力依赖于HDAC6，为临床上TB患者密切接触者的TB感染和发病风险预测提供了重要新标识，并为靶向宿主的TB治疗提供了新思路(HDAC6 contributesto human resistance a-gainst Mycobacterium tuberculosis infection viamediating innate immune responses.FASEB Journal.2021Nov；35(11):e22009.)。
因此，聚焦基于HDAC6筛选的抗结核新策略研究，探索抑制HDAC6降解的抑制剂，验证HDAC6表达效果及抗结核能力。为制订有效阻断结核病易感人群感染的干预策略，并完善活动性结核病患者免疫治疗方案奠定理论基础。
发明内容
本发明通过研究抗结核患者的巨噬细胞，确认HDAC6特异性在结核抵抗者巨噬细胞中维持表达，并且证实结核抵抗者人群来源的巨噬细胞高效清除Mtb感染的能力依赖于HDAC6，而在HDAC6泛素化区域发现了抑制HDAC6的小分子化合物，为治疗结核病提供了一种新思路。
第一方面，本发明提供一种小分子化合物在制备抑制结核分枝杆菌感染的药物中的应用，所述小分子化合物选自6lc、6b、Lenalidomide、Pomalidomide、Homo-PROTACcereblon degrader 1、VH-032、VH-298的一种或多种。
进一步的，所述小分子化合物优选Lenalidomide、Pomalidomide、VH-032、VH-298的一种或多种。
进一步的，所述小分子化合物与其他活性成分连用，所述其他活性成分是能促进机体抗结核的活性物质和/或是改善结核病症状的药物。
进一步的，所述的结核分枝杆菌感染包括：原发性感染、继发性感染和肺外感染。
第二方面，本发明提供一种小分子化合物在制备抗结核病药物中的应用，所述小分子化合物选自6lc、6b、Lenalidomide、Pomalidomide、Homo-PROTAC cereblon degrader1、VH-032、VH-298的一种或多种。
进一步的，所述小分子化合物优选Lenalidomide、Pomalidomide、VH-032、VH-298的一种或多种。
进一步的，所述小分子化合物与其他活性成分连用，所述其他活性成分是能促进机体抗结核的活性物质和/或是改善结核病症状的药物。
进一步的，所述的治疗结核病的药物为糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、丹剂、混悬剂、散剂、酒剂、配剂、滴剂、注射液、粉针剂、乳膏剂、缓释剂、靶向剂等。
更进一步的，所述治疗结核病的药物的给药方式包括口服、注射、植入、外用、喷雾或吸入。
进一步的，所述的结核病包括但不限于耐药结核病、非耐药结核病、肺结核、肺外结核等。
更进一步的，所述的耐药结核病包括但不限于单耐药结核病、多耐药结核病、耐多药结核病、广泛耐药结核病。
进一步的，所述的肺结核包括原发性肺结核、继发性肺结核、血型播散性肺结核、气管-支气管结核、结核性胸膜炎、菌阴肺结核等。
进一步的，所述的肺外结核包括但不限于肠结核、肾结核、骨关节结核等。
第三方面，本发明提供一种由第一方面所述的小分子化合物的筛选方法，所述方法包括如下步骤：
S1.在HDAC6泛素化区域确定抑制泛素化酶活性的小分子化合物的种类；
S2.建立分枝杆菌感染的细胞系，验证小分子化合物促进HDAC6蛋白表达；
S3.计算小分子化合物抑制结核分枝杆菌的菌落数，并筛选出抑制结核分枝杆菌最好的小分子化合物。
进一步的，所述的结核分枝杆菌感染包括：原发性感染、继发性感染、肺外感染。
进一步的，所述细胞系选自THP-1。
进一步的，所述的结核病包括但不限于耐药结核病、非耐药结核病、肺结核、肺外结核等。
更进一步的，所述的耐药结核病包括但不限于单耐药结核病、多耐药结核病、耐多药结核病、广泛耐药结核病。
进一步的，所述的肺结核包括原发性肺结核、继发性肺结核、血型播散性肺结核、气管-支气管结核、结核性胸膜炎、菌阴肺结核等。
进一步的，所述的肺外结核包括但不限于肠结核、肾结核、骨关节结核等。
附图说明
图1在耻垢分枝杆菌感染24h后，Pomalidomid促进HDAC6表达；
图2在耻垢分枝杆菌感染24h后，Pomalidomid促进胞内耻垢分枝杆菌清除；
图3在BCG(a)和H37Rv(b)感染24h后，Pomalidomid促进HDAC6表达；
图4在BCG(a)和H37Rv(b)感染24h后，Pomalidomid促进胞内BCG和H37Rv的清除。
具体实施方式
实施例1基于HDAC6泛素化区域筛查降解HDAC6的泛素化酶，并进一步确定抑制泛素化酶活性的小分子化合物。
文献查阅，发现Cullin3-SPOP泛素化E3连接酶、CRBN E3泛素连接酶、VHL E3泛素连接酶可以降解HDAC6，进一步查找抑制泛素连接酶活性的抑制剂，结果如表1，本项目筛查了Lenalidomid、Pomalidomide、VH-032和VH-298在Mtb感染下对HDAC6的维持能力。
表1HDAC6泛素化连接酶的抑制剂
2.在耻垢分枝杆菌感染下，Pomalidomide维持HDAC6蛋白表达
(A)人单核细胞白血病细胞系THP-1细胞诱导分化
(1)将THP-1细胞铺入12孔板之前，加入含有10％胎牛血清、100ng/ml佛波酯(PMA)和4mM 1-谷氨酰胺的RPMI1640培养基，然后按照每孔加入1×106个细胞铺板，在细胞培养箱诱导分化12小时。
(B)耻垢分枝杆菌感染诱导分化的THP-1细胞
(1)将耻垢分枝杆菌mc2155在0.05％吐温-80的7H9培养基中培养到OD600＝0.6，进行1ml分装在-80℃冻存。
(2)第二天取出1管冻存的耻垢分枝杆菌进行解冻复苏，在7H9培养基中按照10倍梯度稀释，然后涂7H10琼脂平板上，置于37℃，5％CO2培养3-5天，进行耻垢分枝杆菌CFU计算，从而计算出每管冻存的耻垢分枝杆菌的活菌量。(培养耻垢分枝杆菌)
(3)先取出1管冻存的耻垢分枝杆菌进行解冻复苏，4500rpm低速离心5min，沉淀重悬浮于含10％FBS的RPMI1640培养基中，颠倒混匀，得到耻垢分枝杆菌悬液(培养耻垢分枝杆菌之后的细菌悬液)。
(4)设置感染时间为2小时和24小时，按照MOI＝10进行耻垢分枝杆菌感染，即每孔加入1×107个耻垢分枝杆菌。
(5)设置组别为等体积的DMSO和50μm Pomalidomide、50μm Lenalidomid、100μmVH-032和100μm VH-298组，耻垢分枝杆菌悬液中按照分组分别加入等体积的DMSO和50μmPomalidomide、50μm Lenalidomid、100μm VH-032和100μm VH-298，颠倒混匀(在悬液里面加入含有溶剂的抑制剂)。
(6)在37℃，5％CO2下按照MOI＝10感染诱导分化的THP-1细胞。
(C)收集感染的THP-1细胞提取总蛋白
(1)在感染2小时和24小时后，去掉培养板的培养基后，加入4℃预冷的500μl 1×PBS，轻轻摇晃培养板清洗细胞，然后吸掉PBS。
(2)每个孔加入500μl蛋白裂解液(货号：R0020-100ml，牌子：Solarbio)，在蛋白裂解液使用之前先加入终浓度为1mM的蛋白酶抑制剂PMSF，在冰上裂解10min，吹打混匀后收集裂解液进入1.5ml EP管中，置于-80℃冻存。
(3)感染完毕后，在-80℃冰箱中取出收集的冻存蛋白混合液，在冰上解冻。
(4)解冻完毕后，于4℃下12000rpm离心15-20min(提前开离心机预冷)。将离心后的上清转移倒新的1.5ml离心管中(提前预冷离心管)，置于冰上。
(D)总蛋白含量的测定
(1)从－20℃取出1mg/ml BSA，室温融化后，备用。
(2)按照BSA蛋白浓度测定试剂盒(增强型)制作标准曲线，并测定蛋白浓度。
(3)根据蛋白浓度配制浓度一致的蛋白样品。
(4)加入终浓度为1×的蛋白上样缓冲液，在Heaterblocker上95℃,10min变性蛋白。
(E)HDAC6蛋白含量的测定
(1)按照One-Step PAGE Gel Fast Preparation Kit(12％)说明(货号：E304-01125 gels/0.75mm，牌子：诺唯赞)配制12％分离胶、5％浓缩胶。
(2)上样量为2ug。
(3)电泳：电泳时间先80V，30min后，将电压调为120V约1.5h。电泳至溴酚兰刚跑出即可终止电泳，进行转膜。
(4)转膜：采用湿转的方式进行转膜，用200mA转移3h。
(5)免疫反应：将膜移至含有封闭液的四方形盒子中，室温下脱色摇床上摇动封闭4小时；将HDAC6抗体(#7558，Cell Signaling)按照1：1000在5％BSA中稀释；PVDF膜装于适当大小的一次性自封袋中，修剪至合适大小，用封口机封住三面后，加入适量的抗体溶液，赶出残留气泡后将第四面封好，置于摇床上，在4℃过夜；用1×TBST在室温下脱色摇床上洗三次，每次10min；二抗辣根酶标记山羊抗兔IgG(H+L)(货号：ZB-5301牌子：中杉金桥)按照1：10000在TBST稀释并与膜接触，室温下孵育2h后，用TBST在室温下脱色摇床上洗三次，每次10min，进行化学发光反应。
(6)按照化学显色液高敏型ECL化学发光检测试剂盒(即用型)(E412-01,诺唯赞)说明配制后，均匀加在PVDF膜上，在仪器(cytiva ImageQuant800)上显影。
(F)实验结果分析
在培养基中分别加入等体积的DMSO、50μm Pomalidomide、50μm Lenalidomid、100μmVH-032和100μm VH-298后进行耻垢分枝杆菌，结果表明，相比于其他组，50μmPomalidomide作用组在耻垢分枝杆菌感染24小时后可以促进HDAC6蛋白表达(见图1)。3.在耻垢分枝杆菌感染下，Pomalidomide促进胞内分枝杆菌的清除
(A)人单核细胞白血病细胞系THP-1细胞诱导分化，操作同2.(A)
(B)耻垢分枝杆菌感染诱导分化的THP-1细胞，操作同2.(B)
(C)菌落形成单位计数：
(1)利用上述(B)中感染的2小时和24小时的THP-1细胞，加入含有0.05％SDS的500μl 1×PBS吹打混匀，裂解THP-1细胞10min，收集裂解液。
(2)取100μl裂解液用7H9溶液进行10倍梯度稀释，稀释102、103、104倍时，分别各取100μl涂布在7H10琼脂平板上，置于37℃，5％CO2培养。
(3)培养3-5天进行耻垢分枝杆菌的CFU计数。
(D)实验结果分析
在培养基中分别加入等体积的DMSO、50μm Pomalidomide、50μm Lenalidomid、100μmVH032和100μm VH298后进行耻垢分枝杆菌感染，结果表明，相比于其他组，50μmPomalidomide作用组可以促进胞内耻垢分枝杆菌的清除(见图2)。****P<0.001(two-wayANOVA)。
4.在减毒活疫苗菌株(BCG)和结核分枝杆菌(H37Rv)分枝杆菌感染下，Pomalidomide维持HDAC6蛋白表达
(A)人单核细胞白血病细胞系THP-1细胞诱导分化，操作同2.(A)
(B)减毒活疫苗菌株(BCG)和结核分枝杆菌(H37Rv)分别感染诱导分化的THP-1细胞
(1)将BCG、H37Rv在含10％OADC增菌液和0.05％吐温-80的7H9培养基中培养到OD600＝0.6，约浓度为2.5×107CFU/ml。
(2)设置感染时间为2小时、8小时和24小时，按照MOI＝5进行BCG、H37Rv感染，即每孔加入5×106个BCG或H37Rv。
(3)将含BCG、H37Rv培养液在4500rpm低速离心5min，沉淀重悬浮于含10％FBS的RPMI1640培养基中，颠倒混匀，得到BCG、H37Rv悬液。
(4)设置组别为空白对照组、等体积的DMSO和浓度为50μm Pomalidomide组，在等体积的DMSO和浓度为50μm Pomalidomide组中，BCG和H37Rv悬液中分别加入等体积的DMSO和浓度为50μm Pomalidomide，颠倒混匀。
(4)在37℃，5％CO2下按照MOI＝5感染诱导分化的THP-1细胞。
(C)收集感染的THP-1细胞提取总蛋白，操作同2.(C)
(D)总蛋白含量的测定，操作同2.(D)
(1)从－20℃取出1mg/ml BSA，室温融化后，备用。
(2)按照BSA蛋白浓度测定试剂盒(增强型)制作标准曲线，并测定蛋白浓度。
(3)根据蛋白浓度配制浓度一致的蛋白样品。
(4)加入终浓度为1×的蛋白上样缓冲液，在Heaterblocker上95℃,10min变性蛋白。
(E)HDAC6蛋白含量的测定，操作同2.(E)
(F)实验结果分析
在培养基中分别加入等体积的DMSO、浓度为50μm Pomalidomide后进行(a)BCG、(b)H37Rv感染，结果表明，相比于其他组，50μm Pomalidomide作用组在BCG和H37Rv感染24小时后可以促进HDAC6蛋白表达(见图3)。
5.在BCG和H37Rv感染下，Pomalidomide促进胞内分枝杆菌的清除
(A)人单核细胞白血病细胞系THP-1细胞诱导分化，操作同2.(A)
(B)BCG和H37Rv感染诱导分化的THP-1细胞，操作同4.(B)
(C)菌落形成单位计数：
(1)利用上述(B)中感染的2小时、8小时和24小时的THP-1细胞，加入含有0.05％SDS的500μl 1×PBS吹打混匀，裂解THP-1细胞10min，收集裂解液。
(2)取100μl裂解液用7H9溶液进行10倍梯度稀释，稀释102、103、104倍时，分别各取100μl涂布在7H10琼脂平板上，置于37℃，5％CO2培养。
(3)培养3-4周进行，进行BCG和H37Rv的CFU计数。
(D)实验结果分析
在空白对照组、等体积的DMSO和50μm Pomalidomide组后进行(a)BCG、(b)H37Rv感染，结果表明，相比于其他组，50μm Pomalidomide作用组可以促进胞内BCG和H37Rv的清除(见图4)。**P<0.01，***P<0.001(two-way ANOVA)。
Claims (10)
1.一种小分子化合物在制备抑制结核分枝杆菌感染的药物中的应用，所述小分子化合物选自6lc、6b、Lenalidomide、Pomalidomide、Homo-PROTACcereblon degrader1、VH-032、VH-298的一种或多种。
2.如权利要求1所述的小分子化合物在制备抑制结核分枝杆菌感染的药物中的应用，其特征在于，所述小分子化合物选自Lenalidomide、Pomalidomide、VH-032、VH-298的一种或多种。
3.如权利要求1所述的小分子化合物在制备抑制结核分枝杆菌感染的药物中的应用，其特征在于，所述小分子化合物与其他活性成分连用，所述其他活性成分是能促进机体抗结核的活性物质和/或是改善结核病症状的药物。
4.一种小分子化合物在制备抗结核病药物中的应用，所述小分子化合物选自6lc、6b、Lenalidomide、Pomalidomide、Homo-PROTACcereblondegrader1、VH-032、VH-298的一种或多种。
5.如权利要求4所述的小分子化合物在制备抗结核病药物中的应用，其特征在于，所述小分子化合物选自Lenalidomide、Pomalidomide、VH-032、VH-298的一种或多种。
6.如权利要求5所述小分子化合物在制备抗结核病药物中的应用，其特征在于，所述的治疗结核病的药物为糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、丹剂、混悬剂、散剂、酒剂、配剂、滴剂、注射液、粉针剂、乳膏剂、缓释剂或靶向剂。
7.如权利要求6所述小分子化合物在制备抗结核病药物中的应用，其特征在于，所述治疗结核病的药物的给药方式包括口服、注射、植入、外用、喷雾或吸入。
8.如权利要求7所述，其特征在于，所述的结核病包括但不限于耐药结核病、非耐药结核病、肺结核或肺外结核。
9.如权利要求8所述小分子化合物在制备抗结核病药物中的应用，其特征在于，所述的耐药结核病包括但不限于单耐药结核病、多耐药结核病、耐多药结核病或广泛耐药结核病。
10.一种如权利要求1所述的小分子化合物的筛选方法，所述方法包括如下步骤：
S1.在HDAC6泛素化区域确定抑制泛素化酶活性的小分子化合物的种类；
S2.建立分枝杆菌感染的细胞系，验证小分子化合物促进HDAC6蛋白表达；
S3.计算小分子化合物抑制结核分枝杆菌的菌落数，并筛选出抑制结核分枝杆菌最好的小分子化合物；所述细胞系选自THP-1。
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| CN117462552B (zh) * | 2023-12-27 | 2024-05-17 | 深圳国家感染性疾病临床医学研究中心 | 依曲韦林和/或其衍生物在制备抗结核药物中的应用 |
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