CN116459240A - Kl-2及其衍生物用于制备抗细菌感染药物中的应用 - Google Patents
Kl-2及其衍生物用于制备抗细菌感染药物中的应用 Download PDFInfo
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- CN116459240A CN116459240A CN202310502305.1A CN202310502305A CN116459240A CN 116459240 A CN116459240 A CN 116459240A CN 202310502305 A CN202310502305 A CN 202310502305A CN 116459240 A CN116459240 A CN 116459240A
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Abstract
本发明提供了KL‑2及其衍生物用于制备抗细菌感染药物中的应用,所述KL‑2及其衍生物的结构如式(1)所示,其中,X为F或Br;所述KL‑2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用,所述KL‑2及其衍生物与外排泵抑制剂苯丙氨酸‑精氨酸‑β‑萘胺联合具有抑制革兰氏阴性菌生长的作用。本发明的技术方案公开了KL‑2的新用途,KL‑2对包括革兰氏阳性菌临床分离株表现出抑菌活性,其联合外排泵抑制剂PAβN后对革兰氏阴性菌表现出抑菌活性,而且能够抑制金黄色葡萄球菌和粪肠球菌生物被膜的形成,并清除金黄色葡萄球菌和粪肠球菌已形成的生物被膜。
Description
技术领域
本发明属于医药技术领域,尤其涉及SEC inhibitor KL-2(以下简称为KL-2)及其衍生物用于制备抗细菌感染药物中的应用。
背景技术
近年来随着抗生素的广泛使用,临床院感防控面临严峻的耐药形势,细菌耐药性的增加已经成为全球公共卫生问题。ESKAPE病原体包括屎肠球菌(Enterococcusfaecium)、金黄色葡萄球菌(Staphylococcus aureus)、肺炎克雷伯菌(Klebsiellapneumoniae)、鲍曼不动杆菌(Acinetobacter baumannii)、铜绿假单胞菌(Pseudomonasaeruginosa)和肠杆菌属(Enterobacter spp),这六种病原菌是导致医院感染做最容易产生耐药性的细菌,对多种抗生素产生了耐药性。随着多重耐药菌株的不断增多,传统的抗生素治疗已经失效,导致医疗负担和患者死亡率的上升。耐药菌株的流行导致医疗资源的大量消耗,同时给患者带来了更高的医疗风险和更长的治疗时间。
生物被膜是导致细菌耐药性的一个重要因素。生物被膜是一种由微生物在物体表面形成的复杂的生物膜结构。生物被膜中的细菌通常处于高密度状态,并具有良好的适应性和生存能力。这些细菌之间通过细胞外多聚物、纤维素和蛋白质等分泌物质相互联系形成复杂的网络结构。这种结构可以在外部环境的变化中保持相对稳定,从而提供了一种良好的保护和藏身之地,使细菌得以抵御环境压力和抗生素的攻击。此外,生物被膜的形成还可以阻碍抗生素进入细胞内部,从而减少细胞内药物的浓度,降低抗生素的杀菌效果。生物被膜内还可能存在着一些特殊的耐药性基因和耐药性质体,这些基因和质体可以在细菌群体中快速传播,从而导致整个微生物群体产生耐药性。因此,生物被膜的形成可以导致细菌耐药性的产生和传播。细菌形成生物被膜进一步可导致临床抗感染治疗失败。因此,开发新型抗生素对于破坏生物被膜、消除细菌耐药性具有重要意义。
发明内容
针对以上技术问题,本发明公开了KL-2及其衍生物用于制备抗细菌感染药物中的应用,这是KL-2的一种新药物用途,通过实验发现,KL-2对包括金黄色葡萄球菌、粪肠球菌、屎肠球菌、表皮葡萄球菌等革兰氏阳性菌等临床分离株表现出抑菌活性,以及联合外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺(Phe-Arg-β-naphthylamide,PAβN)后对鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌和铜绿假单胞菌等革兰氏阴性菌表现出抑菌活性,KL-2的衍生物KL-3的抑菌活性与KL-2相当,但是细胞毒性比KL-2更低。为基于KL-2的抗菌药物的开发以及针对革兰氏阴性菌的联合用药提供参考依据。
对此,本发明采用的技术方案为:
KL-2及其衍生物用于制备抗细菌感染药物中的应用,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;X为F时,即式(2),为KL-2,其CAS编号为900308-51-2。X为Br时,即式(3),为KL-2的衍生物KL-3。所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用,所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
超延伸复合物(SEC)是通过释放RNA聚合酶II(Pol II)及其P-TEFb模块和促进其ELL2亚基的转录过程而实现稳健和高产的转录所必需的。SEC功能异常可导致包括癌症在内的多种人类疾病。SEC抑制剂KL-2是一种拟肽先导化合物,是一种有效的特异性SEC抑制剂,可破坏SEC支架蛋白AFF4和P-TEFb之间的相互作用,导致启动子-近端暂停位点Pol II释放受损,并降低平均转录延伸速率。SEC抑制剂KL-2对AFF4-CCNT1相互作用具有剂量依赖性抑制作用,Ki为1.50μM。目前为止未见KL-2的抗菌活性的相关报道。
作为本发明的进一步改进,所述革兰氏阳性菌为金黄色葡萄球菌、粪肠球菌、屎肠球菌、表皮葡萄球菌或肺炎链球菌中的至少一种,所述革兰氏阴性菌为鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌中的至少一种。
经过大量的实验研究,KL-2对多种革兰氏阳性菌包括金黄色葡萄球菌、粪肠球菌、屎肠球菌、表皮葡萄球菌等表现出较佳的抑菌活性。KL-2对100株革兰氏阳性菌的最低抑菌浓度(minimum inhibitory concentration,MIC)在0.5-4μg/mL,值得注意的是,对20株临床分离的耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的MIC≤4μg/mL,KL-2联合外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺(Phe-Arg-β-naphthylamide,PAβN)后对鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌和铜绿假单胞菌等革兰氏阴性菌表现出抑菌活性,生长曲线分析显示KL-2在较低浓度(1/16×MIC)时可显著延缓金黄色葡萄球菌和粪肠球菌的生长。结晶紫染色显示KL-2在亚抑菌浓度可显著抑制金黄色葡萄球菌和粪肠球菌生物被膜的形成,同时在4×MIC浓度时可以清楚金葡菌已形成的生物被膜。此外,KL-2对哺乳动物细胞的毒性较小,对脐静脉内皮细胞HUEVC、人肝星形细胞LX-2以及人肾上皮细胞系293T增殖的毒性较小。而KL-2衍生物KL-3对革兰氏阳性菌的MIC与KL-2相似,但是细胞毒性显著低于KL-2。这些结果表明,KL-2及其衍生物在临床抗细菌感染治疗中具有潜在应用价值。
作为本发明的进一步改进,KL-2在处理体系中的浓度为不小于0.25μg/mL。进一步的,所述KL-2在处理体系中的浓度为0.25~16μg/mL。
作为本发明的进一步改进,所述KL-2及其衍生物与外排泵抑制剂PaβN联合后对革兰氏阴性菌具有抑制细菌生长和生物被膜形成的作用。
作为本发明的进一步改进,所述药物为药物组合物或制剂。进一步的,所述药物为注射剂、片剂、丸剂、胶囊、悬浮剂、颗粒剂、喷剂或乳剂。
本发明还公开了KL-2及其衍生物用于制备抑制细菌的涂料中的应用,所述涂料用于医疗器械的表面,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;其中,X为F或Br;X为F时,即式(2),为KL-2,其CAS编号为900308-51-2。X为Br时,即式(3),为KL-2的衍生物KL-3。所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用。
作为本发明的进一步改进,所述涂料中,所述KL-2的浓度为不小于0.25μg/mL。
作为本发明的进一步改进,所述涂料中包含KL-2或其衍生物和外排泵抑制剂PaβN。所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
本发明还公开了KL-2及其衍生物用于制备抗菌剂的应用,所述KL-2及其衍生物的结构如式(1)所示
其中,X为F或Br;
所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用。
作为本发明的进一步改进,所述抗菌剂中包含KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
一种药物组合物,其包括KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
本发明公开了KL-2及其衍生物用于制备抗细菌感染药物组合物中的应用,其包括KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
与现有技术相比,本发明的有益效果为:
本发明的技术方案公开了KL-2的一种医药新用途,KL-2对包括金黄色葡萄球菌、粪肠球菌、屎肠球菌、表皮葡萄球菌等革兰氏阳性菌等临床分离株表现出抑菌活性,以及联合外排泵抑制剂PAβN后对鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌和铜绿假单胞菌等革兰氏阴性菌表现出抑菌活性,而且能够抑制金黄色葡萄球菌和粪肠球菌生物被膜的形成,清除金黄色葡萄球菌和粪肠球菌已形成的生物被膜。这些结果提示KL-2具备治疗临床细菌感染的可能性。且MIC浓度下KL-2对哺乳动物细胞包括人脐静脉内皮细胞HUEVC、人肝星形细胞LX-2以及人肾上皮细胞系293T的毒性较小,适宜临床使用。可见,KL-2及其衍生物KL-3在临床抗细菌感染治疗中具有潜在应用价值。
附图说明
图1是本发明实施例添加亚抑菌浓度KL-2后对革兰氏阳性菌金黄色葡萄球菌和粪肠球菌的生长曲线,其中(a)~(f)分别为金黄色葡萄球菌实验室常用菌株SA113、USA300,临床MRSA株YUSA139、YUSA145,粪肠球菌实验室菌株OG1RF,粪肠球菌临床分离株EF16C51实验结果,图中MIC代表最低抑菌浓度。
图2是本发明实施例KL-2抑制革兰氏阳性菌粪肠球菌和金黄色葡萄球菌生物被膜形成分析,图中,(a)为结晶紫染色分析KL-2对粪肠球菌OG1RF和金黄色葡萄球菌USA300生物被膜形成影响的照片,(b)为结晶紫染色定量分析KL-2对粪肠球菌生物被膜形成的影响,(c)为结晶紫染色定量分析KL-2对甲氧西林敏感金黄色葡萄球菌(methicillin-susceptible Staphylococcus aureus,MSSA)生物被膜形成的影响,(d)为结晶紫染色定量分析KL-2对MRSA生物被膜形成的影响,图中MIC代表最低抑菌浓度,E.faecalis代表粪肠球菌。
图3是本发明实施例的激光共聚焦显微镜观察下不同浓度KL-2抑对金黄色葡萄球菌临床株USA300生物被膜形成的影响,图中,(a)为对照组生物被膜,(b)为1/4×MIC KL-2处理后的YUSA145生物被膜,(c)为1/2×MIC KL-2处理后的YUSA145生物被膜,图中标尺为5μM。
图4是本发明实施例的KL-2清除粪肠球菌和金黄色葡萄球菌成熟生物被膜结果,图中,(a)为不同浓度KL-2对粪肠球菌OG1RF和金黄色葡萄球菌USA300成熟生物被膜影响的结晶紫染色结果,(b)为结晶紫染色定量分析KL-2对粪肠球菌和金黄色葡萄球菌成熟生物被膜的影响,(c)激光共聚焦显微镜观察下4×MIC KL-2抑对金黄色葡萄球菌临床株USA300成熟生物被膜的影响,图中MIC代表最低抑菌浓度,图中标尺为5μM。
图5是本发明实施例的不同浓度KL-2及其衍生物KL-3对人脐静脉内皮细胞HUEVC、人肝星形细胞LX-2以及人肾上皮细胞系293T细胞增殖毒性检测结果。图中,(a)~(c)分别为不同浓度KL-2对HUEVC、人LX-2以及293T细胞增殖毒性检测结果,(d)~(f)分别为不同浓度KL-2衍生物KL-3对HUEVC、人LX-2以及293T细胞增殖毒性检测结果。
具体实施方式
下面对本发明的较优的实施例作进一步的详细说明。
实施例1
1.1菌株来源
本实施例中使用的100株革兰氏阳性菌(包括20株MRSA,20株MSSA,20株粪肠球菌,20株屎肠球菌和20株表皮葡萄球菌)和8株革兰氏阴性菌(包括鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌和大肠埃希菌各2株)临床株收集于医院不同住院患者或者由本实验室保存的实验菌株。所有临床菌株都通过Phoenix 100自动微生物鉴定***鉴定,继代培养后采用基质辅助激光解吸电离/飞行时间质谱(MALDI-TOF-MS)对所有菌株进行重新鉴定。质量控制菌株金黄色葡萄球菌ATCC29213购自ATCC菌株库。
1.2主要仪器和试剂
微量移液器,Phoenix-100全自动细菌鉴定/药敏***,全自动生物质谱检测***IVD MALDI Biotyper(德国布鲁克公司),全自动生长曲线分析仪,CAMHB培养基,TSB培养基,激光共聚焦显微镜FV3000。定制化合物库、CCK-8试剂盒、KL-2、PAβN,结晶紫,LIVE/DEADBacLightTM荧光染料,葡萄糖,96孔细胞培养板。
本实施例中,采用96孔板,进行KL-2对金黄色葡萄球菌和粪肠球菌的生长影响实验,具体步骤为:
取过夜培养菌液(金黄色葡萄球菌ATCC25923和粪肠球菌OG1RF)用比浊杯调整浊度至0.5麦氏(菌量约为1.0-1.5×108cfu/mL)。取菌液同CAMHB培养基1:200稀释后加入96孔板,每行12孔,每孔200uL。添加KL-2稀释至50uM,第12孔加入200uL CAMHB培养基设为阴性对照。37℃培养24小时后观察结果,以肉眼不能看见细菌生长的孔中添加的化合物被认为具有潜在的抑菌活性。
除此之外,还分别以添加TH-257、SJ572403、GW806742X、DS-1001b、AZ191、ChX710等作为对比例,37℃培养24小时后观察结果,以肉眼不能看见细菌生长的孔中添加的化合物被认为具有潜在的抑菌活性。
本实施例中,添加50uM KL-2的金黄色葡萄球菌ATCC25923和粪肠球菌OG1RF在24小时后培养基都表现为澄清,未观察到细菌有生长,600nm处的吸光度(OD600)都小于0.1。而添加TH-257、SJ572403、GW806742X、DS-1001b、AZ191、ChX710的都仍有细菌有生长。可见KL-2对金黄色葡萄球菌和粪肠球菌具有潜在的抑菌活性。
实施例2
本实施例中,采用微量肉汤稀释法检测KL-2对100株临床分离革兰氏阳性菌株(包括20株MSSA、20株MRSA、20株粪肠球菌、20株屎肠球菌和20株表皮葡萄球菌)的最低抑制浓度MIC,具体步骤为:
取过夜培养菌液用比浊杯调整浊度至0.5麦氏(菌量约为1.0-1.5×108cfu/mL)。取菌液同CAMHB培养基1:100稀释后加入96孔板,每行12孔,KL-2设置10个梯度孔(64,32,16,8,4,2,1,0.5,0.25,0.125μg/mL),第11孔加入200uL上述菌液设为阳性对照,第12孔加入200uL CAMHB培养基设为阴性对照。37℃培养18小时后观察结果,以肉眼不能看见菌液沉淀的药物浓度孔计算为MIC值。
本实施例中,KL-2对金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、屎肠球菌等革兰氏阳性菌的MIC值统计结果如表1所示,可见KL-2对多种革兰氏阳性菌具有较为广谱的抑菌活性,MIC值主要分布在4μg/mL到64μg/mL之间,其中对表皮葡萄球菌的抑制效果最佳,MIC50为4μg/mL。
表1常见革兰氏阳性菌对KL-2的MIC值分布
注:MIC:最小抑菌浓度;MSSA:甲氧西林敏感金黄色葡萄球菌;MRSA:耐甲氧西林金黄色葡萄球菌;S.epidermidis:表皮葡萄球菌;E.faecalis:粪肠球菌;E.faecium:屎肠球菌;n为所测菌株数量。MIC50:抑制50%受试菌生长所需的药物浓度;MIC50:抑制90%受试菌生长所需的药物浓度.
实施例3
KL-2对革兰氏阳性菌的生长曲线影响实验。
为了验证KL-2是否能够抑制革兰氏阳性菌的生长,我们使用不同亚抑菌浓度的KL-2分别处理,4株金黄色葡萄球菌(SA113、USA300、YUSA139和YUSA145)和2株粪肠球菌(OG1RF和EF16C51),在不同时间点检测其600nm波长吸光度(OD600)值,具体步骤为:取过夜培养菌液用胰蛋白胨大豆肉汤(TSB)培养基稀释1000倍后加入96孔板,添加不同浓度(1/16×,1/8×,1/4×,1/2×和1×MIC)KL-2后置于全自动生长曲线分析仪中,每间隔1小时测定OD600吸光值,以检测培养上清液中浮游菌含量,孵育温度为37℃,绘制生长曲线。
生长曲线分析如图1所示,可见在即使在1/16×MIC浓度下,KL-2也会显著所有金黄色葡萄球菌和粪肠球菌浮游菌的生长,随着浓度升高,细菌生长也会相应的滞后。而浓度达到1×MIC时,金黄色葡萄球菌和粪肠球菌在10小时内的生长完全被抑制,这些结果初步表明KL-2具有作为抗细菌感染药物的潜力。
实施例4
本实施例采用结晶紫法,研究KL-2对粪肠球菌和金黄色葡萄球菌株的抑制生物被膜形成活性,具体步骤为:
采用结晶紫染色法检测生物被膜量的变化,重复3次,每次设3个复孔,取平均值为最终检测结果。操作步骤简述如下,取过夜菌液用含2%葡萄糖的TSBG培养基稀释1000倍于96孔板中,每组3个复孔,添加不同亚抑菌浓度KL-2,以不添加菌液的空白培养基作为阴性对照,溶剂DMSO为阳性对照。于37℃培养箱中孵育24小时后。轻柔吸弃培养液,用无菌PBS冲洗3次,室温下晾干。甲醇固定15分钟,1%结晶紫染液染色15分钟,无菌水冲洗3次至对照孔为无色,室温下晾干。每孔加入200μL无水乙醇溶解,震荡1分钟于酶标仪测定570nm吸光值。
结晶紫染色结果如图2(a)所示,不同亚抑菌浓度KL-2对粪肠球菌OG1RF和金黄色葡萄球菌USA300生物被膜的形成有显著的抑制效果。对生物被膜结晶紫染色结果半定量分析,结果如图2(b)、2(c)和2(d)所示,当KL-2为1/4×MIC时,全部粪肠球菌和金黄色葡萄球菌株形成的生物被膜生物量都显著减少。当KL-2浓度在1/2×MIC时,所测12株菌中有11株的生物被膜形成被抑制50%以上,表明KL-2能抑制粪肠球菌和金黄色葡萄球菌生物膜的形成。
实施例5
本实施例采用荧光染色和激光共聚焦显微镜分析方法,研究KL-2的抑制生物被膜形成活性,具体步骤为:
取过夜金黄色葡萄球菌液用含2%葡萄糖的TSBG培养基稀释1000倍于细胞培养皿中,添加不同亚抑菌浓度KL-2,以不添加菌液的培养基作为对照。于37℃培养箱中孵育24小时后。轻柔吸弃培养液,用无菌PBS冲洗3次,用LIVE/DEAD BacLightTM荧光染料进行染色。LIVE/DEAD BacLightTM荧光染料包含两种不同的核酸染料,可将质膜完整的活细菌与质膜不完整的死细菌快速区分开来。其中碘化丙啶(PI)用于检测膜渗透性,当其通过被破坏的细胞膜进入细菌与核酸结合时,荧光强度会增加。1小时后激光共聚焦显微镜观察处理后的USA300形成的生物被膜并拍照。
激光共聚焦显微镜观察对照组USA300形成的生物膜结果如图3(a)所示,USA300可形成致密的生物被膜,厚度约15μm左右;如图3(b)和(c)所示发现KL-2处理后的金黄色葡萄球菌USA300生物膜的红色荧光细菌的比例显著升高,且形成的生物被膜厚度显著变薄,说明KL-2对金黄色葡萄球菌生物被膜的形成有较佳的抑制效果。
实施例6
本实施例采用结晶紫法,研究KL-2对粪肠球菌和金黄色葡萄球菌株已形成生物被膜的清除活性,具体步骤为:
采用结晶紫染色法检测生物被膜量的变化,重复3次,每次设3个复孔,取平均值为最终检测结果。操作步骤简述如下,取过夜菌液用含2%葡萄糖的TSBG培养基稀释1000倍于96孔板中,每组3个复孔,于37℃培养箱中孵育24小时后形成成熟生物膜。倒掉浮游菌,PBS情绪一遍,加入新鲜的添加不同浓度KL-2的TSBG培养基,以不添加KL-2的培养基作为对照。于37℃培养箱中继续孵育24小时后。轻柔吸弃培养液,用无菌PBS冲洗3次,室温下晾干。甲醇固定15分钟,1%结晶紫染液染色15分钟,无菌水冲洗3次至对照孔为无色,室温下晾干。每孔加入200μL无水乙醇溶解,震荡1分钟于酶标仪测定570nm吸光值。
结晶紫染色结果如图4(a)所示,不同浓度KL-2对粪肠球菌OG1RF和金黄色葡萄球菌USA300已形成的成熟生物被膜有显著的清除抑制效果。对生物被膜结晶紫染色结果半定量分析,结果如图4(b)所示,当KL-2为4×MIC时,全部粪肠球菌和金黄色葡萄球菌株形成的生物被膜生物量都在KL-2处理后显著减少,6株中有5株菌株的生物被膜被清除50%以上,表明KL-2能清除粪肠球菌和金黄色葡萄球菌已形成的生物被膜。
实施例7
本实施例为微量肉汤稀释法分析KL-2与外排泵抑制剂PAβN对8株革兰氏阴性菌(包括鲍曼不动杆菌、铜绿假单胞菌、肺炎克雷伯菌和大肠埃希菌各2株)的联合抑菌作用,具体步骤为:取过夜培养菌液用比浊杯调整浊度至0.5麦氏(菌量约为1.0-1.5×108cfu/mL)。分别用微量肉汤稀释法检测KL-2、PaβN的MIC。取菌液同CAMHB培养基1:100稀释后加入96孔板,每行12孔,KL-2和PaβN设置10个梯度孔(128,64,32,16,8,4,2,1,0.5,0.25μg/mL),第11孔加入200uL上述菌液设为阳性对照,第12孔加入200uL CAMHB培养基设为阴性对照。进一步的,分析KL-2与PAβN联合用药抑菌效果,检测添加32μg/mL的外排泵抑制剂PaβN之后测量KL-2的MIC,KL-2参考上述设置10个梯度孔。37℃培养18小时后观察结果,以肉眼不能看见菌液沉淀的药物浓度孔计算为MIC值。
MIC值结果如表2所示,PaβN和KL-2对8株革兰氏阴性菌的MIC都大于128μg/mL,基本都没有抑菌活性,然而添加32μg/mL的PaβN后KL-2对所有革兰氏阴性菌都表现出抑菌活性,MIC在4μg/mL和32μg/mL之间,表明KL-2对革兰氏阴性菌没有抑菌活性是因为外排泵作用KL-2在革兰氏阴性菌内部无法积累,浓度较低引起的,将来可通过与外排泵抑制剂联合用药对革兰氏阴性菌发挥抑菌活性。
表2 KL-2与PAβN对革兰氏阴性菌联合抑菌活性MIC(μg/mL)分析结果
Bacterial isolates | KL-2 | PaβN | KL-2+PaβN |
E.coli ATCC25922 | >128 | >128 | 4 |
E.coli Eco2221 | >128 | >128 | 4 |
A.baumannii AB2201 | >128 | >128 | 16 |
A.baumannii AB2201 | >128 | >128 | 8 |
P.aeruginosa ATCC27853 | >128 | >128 | 32 |
P.aeruginosa PA2243 | >128 | >128 | 32 |
K.pneumonia K2044 | >128 | >128 | 8 |
K.pneumonia KP2202 | >128 | >128 | 16 |
注:E.coli:大肠埃希菌;A.baumannii:鲍曼不动杆菌;P.aeruginosa:铜绿假单胞菌;K.pneumonia:肺炎克雷伯菌。
实施例8
本实施例中,采用微量肉汤稀释法检测KL-2衍生物KL-3对革兰氏阳性菌株(包括4株金黄色葡萄球菌和4株粪肠球菌)的MIC,具体步骤为:
取过夜培养菌液用比浊杯调整浊度至0.5麦氏(菌量约为1.0-1.5×108cfu/mL)。取菌液同CAMHB培养基1:100稀释后加入96孔板,每行12孔,KL-2和KL-3设置10个梯度孔(64,32,16,8,4,2,1,0.5,0.25,0.125μg/mL),第11孔加入200uL上述菌液设为阳性对照,第12孔加入200uL CAMHB培养基设为阴性对照。37℃培养18小时后观察结果,以肉眼不能看见菌液沉淀的药物浓度孔计算为MIC值。
本实施例中,KL-3对金黄色葡萄球菌和粪肠球菌的MIC值统计结果如表3所示,可见KL-3与KL-2抑菌活性相当,MIC值主要分布在0.5μg/mL到4μg/mL之间。
表3 KL-2衍生物KL-3对金黄色葡萄球菌和粪肠球菌抑菌活性MIC(μg/mL)结果
Bacterial isolates | KL-2 | KL-3 |
S.aureus ATCC25923 | 1 | 2 |
S.aureus USA300 | 1 | 1 |
S.aureus YUSA139 | 2 | 1 |
S.aureus YUSA145 | 1 | 1 |
E.faecalis OG1RF | 1 | 1 |
E.faecalis EF16C51 | 4 | 2 |
E.faecalis EF16C152 | 1 | 1 |
E.faecalis EF16C166 | 2 | 1 |
注:S.aureus:金黄色葡萄球菌;E.faecalis:粪肠球菌。
实施例7
本实施例为KL-2及其衍生物KL-3对人脐静脉内皮细胞HUEVC、人肝星形细胞LX-2以及人肾上皮细胞系293T细胞的细胞增殖影响实验,具体步骤为:
在96孔板中配制100μL的人脐静脉内皮细胞HUEVC、人肝星形细胞LX-2或人肾上皮细胞系293T细胞毒性细胞悬液。将培养板放在培养箱预培养24小时(37℃,5%CO2)。向培养板加入10μL不同浓度的KL-2及其衍生物KL-3,设置7个梯度孔(32,16,8,4,2,1,0.5μg/mL)。将培养板在培养箱孵育24小时,向每孔加入10μL CCK-8溶液。将培养板在培养箱内孵育1-4小时。用酶标仪测定在450nm处的吸光度。然后通过吸光度计算细胞活力。
CCK-8试剂盒,是一种基于WST-8(化学名:2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐)的广泛应用于细胞增殖和细胞毒性的快速高灵敏度检测试剂盒。其工作原理为:在电子耦合试剂存在的情况下,可以被线粒体内的脱氢酶还原生成高度水溶性的橙黄色的甲臜产物(formazan)。颜色的深浅与细胞的增殖成正比,与细胞毒性成反比。使用酶标仪在450nm波长处测定OD值,间接反映活细胞数量。LTX-315对HUEVC、LX-2和293T细胞毒性的结果如图5所示,可见,KL-2及其衍生物KL-3的细胞毒性较小,在浓度低于4μg/mL以内的KL-2及其衍生物KL-3对几种细胞的增殖没有显著影响,高于其对所有检测的全部革兰阳性菌临床株的MIC值,且衍生物KL-3的细胞毒性浓度高于其母体KL-2,说明两者都具有潜在的临床抗感染应用潜力,且KL-3的毒副作用可能更少。
上述所有的实验均使用GraphPad Prism 8.0软件进行数据处理及绘制图像。P<0.05被认为具有统计学差异。
对比例
在上述实施例的基础上,本对比例选择SEC inhibitor KL-1(KL-1)为研究对象,KL-1的结构式如下:
按照实施例2的步骤进行实验,结果如下:
本实施例中,采用微量肉汤稀释法检测KL-2结构类似物KL-1(CAS编号为1373422-53-7)对多种革兰阳性细菌(金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、屎肠球菌)的MIC。KL-1对金黄色葡萄球菌、表皮葡萄球菌、粪肠球菌、屎肠球菌等革兰氏阳性菌的MIC值统计结果如表3所示,可见KL-1对多种革兰氏阳性菌同样有抑菌活性,MIC值主要分布在2μg/mL到16μg/mL之间,抑菌效果显著劣于KL-2。
表4常见革兰氏阳性菌对KL-1的MIC值分布
注:MIC:最小抑菌浓度;MSSA:甲氧西林敏感金黄色葡萄球菌;MRSA:耐甲氧西林金黄色葡萄球菌;S.epidermidis:表皮葡萄球菌;E.faecalis:粪肠球菌;E.faecium:屎肠球菌;n为所测菌株数量。MIC50:抑制50%受试菌生长所需的药物浓度;MIC50:抑制90%受试菌生长所需的药物浓度.
通过上述实验结果可见,KL-2对革兰氏阳性菌表现出较佳的抗菌活性,此外,KL-2表现出较佳的抗生物被膜活性,能够显著抑制金黄色葡萄球菌和粪肠球菌临床株生物被膜的形成,并且能够清除金黄色葡萄球菌和粪肠球菌已形成的生物被膜。MIC浓度的KL-2对人多种细胞的增殖没有显著的影响,具有较低的细胞毒性。KL-2的衍生物KL-3对革兰氏阳性菌的抑菌活性与KL-2相当,细胞毒性进一步降低,这些结果提示KL-2具备治疗革兰氏阳性菌感染的可能性。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
Claims (10)
1.KL-2及其衍生物用于制备抗细菌感染药物中的应用,其特征在于:所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用,所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
2.根据权利要求1所述的KL-2及其衍生物用于制备抗细菌感染药物中的应用,其特征在于:所述革兰氏阳性菌为金黄色葡萄球菌、粪肠球菌、屎肠球菌、表皮葡萄球菌或肺炎链球菌中的至少一种,所述革兰氏阴性菌为鲍曼不动杆菌、肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌中的至少一种。
3.根据权利要求2所述的KL-2及其衍生物用于制备抗细菌感染药物中的应用,其特征在于:所述KL-2在处理体系中的浓度为不小于0.25μg/mL。
4.根据权利要求2所述的KL-2及其衍生物用于制备抗细菌感染药物中的应用,其特征在于:所述药物为注射剂、片剂、丸剂、胶囊、悬浮剂、颗粒剂、喷剂或乳剂。
5.KL-2及其衍生物用于制备抑制细菌的涂料中的应用,其特征在于:所述涂料用于医疗器械的表面,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用。
6.根据权利要求5所述的KL-2及其衍生物用于制备抑制细菌的涂料中的应用,其特征在于:所述涂料中,所述KL-2的浓度为不小于0.25μg/mL。
7.根据权利要求5所述的KL-2及其衍生物用于制备抑制细菌的涂料中的应用,其特征在于:所述涂料中包含KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
8.KL-2及其衍生物用于制备抗菌剂的应用,其特征在于:所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物具有抑制革兰氏阳性菌生长和生物被膜形成的作用。
9.根据权利要求8所述的KL-2及其衍生物用于制备抗菌剂的应用,其特征在于:所述抗菌剂中包含KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
10.一种药物组合物,其特征在于:其包括KL-2或其衍生物和外排泵抑制剂PaβN,所述KL-2及其衍生物的结构如式(1)所示,
其中,X为F或Br;
所述KL-2及其衍生物与外排泵抑制剂苯丙氨酸-精氨酸-β-萘胺联合具有抑制革兰氏阴性菌生长的作用。
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