Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Mechanism for the paradoxical inhibition and stimulation of calcineurin by the immunosuppresive drug tacrolimus (FK506)

We examined the paradoxical inhibition and stimulation of calcineurin, the calcium-activated protein phosphatase, using the drug FK506 (tacrolimus) which acts as a complex together with its binding protein; the complex is designated here as FKC. We reproduced FKC inhibition with RIIp, a phosphorylated peptide substrate, and FKC stimulation with p-nitrophenylphosphate (pNPP) as substrate. The presence of RIIp in the pNPP assay caused inhibition. Yet, under these conditions, FKC still stimulated pNPP dephosphorylation to the same extent. The effects of Mn2+ were strikingly different for the two substrates when calcineurin was measured under otherwise identical conditions: Mn2+ stimulated pNPP dephosphorylation several fold, but only stimulated RIIp dephosphorylation by about 50%. When Pi was used as product inhibitor, FKC stimulation, but not calmodulin stimulation, was attenuated. We conclude that FKC enhances substrate binding to the enzyme. This would lead to inhibition with RIIp, known to bind calcineurin tightly, but stimulation with pNPP, known to bind calcineurin weakly. The result not only resolves the paradox but also elucidates the mechanism of action for this class of immunosuppressive drugs.     

他克莫司联合黄葵胶囊治疗难治性膜性肾病的临床疗效

目的:探讨他克莫司联合黄葵胶囊治疗难治性膜性肾病疗效及安全性。方法:选取2014年3月-2015年10月我院收治的60例难治性膜性肾病患者,按随机数字表法分为观察组和对照组各30例,对照组给予泼尼松治疗,观察组在此基础上增加他克莫司联合黄葵胶囊口服,两组均治疗6个月,观察两组临床疗效,检测并对比两组治疗前后尿蛋白(uPRO)、血清白蛋白(sALB)、血清肌酐(sCr)、血谷丙转氨酶(sALT)、肿瘤坏死因子α(TNF-α)、转化生长因子β1(TGF-β1)以及不良反应情况。结果:观察组的有效率高于对照组(P0.05);治疗后两组uPRO、TNF-α、TGF-β1均明显降低,sALB、sCr明显升高(P0.05),且观察组uPRO、TNF-α、TGF-β1低于对照组(P0.05);两组不良反应发生率比较差异无统计学意义(P0.05)。结论:他克莫司联合黄葵胶囊治疗难治性膜性肾病具有较好的疗效,降低肾功能损伤,不良反应低,值得临床推广。     

药学监护对肝肾移植术后他克莫司浓度的个体内变异性影响研究

目的:评估药学监护(PhC)对剂量校正全血他克莫司(TAC)谷浓度,免疫抑制治疗依从性及临床预后的影响。方法:纳入238例肝肾移植接受者,将患者随机分为PhC(n=119)组与对照组(n=119),从肝肾移植术后3天到90天,PhC组接受由药剂师提供的PhC治疗,对照组由医护人员进行标准治疗,在移植后第1、3、5、和7天检测并比较患者校正全血TAC低谷浓度,并计算个体内变异性变异系数(CV)。比较两组患者巴塞尔坚持免疫抑制药物治疗量表(BAASIS)评分与临床预后。结果:PhC和对照组CV比较差异无统计学意义(31.5±12.4%vs 32.6±16.2%,P=0.674);预定义TAC目标浓度范围内患者比例无显著差异(均P0.05);第28天(16.7%vs.25.8%,P=0.135)与第90天(27.1%vs.24.6%,P=0.457)时,PhC与对照组的依从性差患者所占的百分率比较,差异无统计学意义。两组患者临床预后比较,差异无统计学意义(均P0.05)。结论:肝肾脏移植后3个月内,应用药学监护治疗与剂量校正全血TAC谷浓度的个体内变异性降低、依从性或早期移植患者预后无关。     

他克莫司对HaCaT细胞NF-κB表达的影响

目的：探讨他克莫司对HaCaT细胞中NF-κB分子表达的影响。方法：培养人HaCaT细胞,分别与10μmol·1^-1和50μmol·1^-1他克莫司溶液共孵育后,通过RT-PCR和Western-blot方法,分别从基因和蛋白水平,观察NF-κB分子表达的变化。结果：经10μmol·1^-1和50μmol·1^-1的他克莫司（FK506）溶液孵育后,HaCaT细胞中NF-κB分子表达明显低于对照组,同时体现一定的剂量效应,50μmol·1^-1组的表达水平更低。结论：他克莫司可以剂量依赖性地抑制HaCaT细胞中NF-κB的分子表达。     

他克莫司和卡泊三醇软膏治疗四肢斑块状银屑病的疗效和安全性分析

目的:探讨他克莫司和卡泊三醇软膏治疗四肢斑块状银屑病的疗效和安全性。方法:选取2011年4月至2013年8月于我院诊治的84例四肢斑块状银屑病患者,将患者随机分为A组和B组,每组各42例,分别采用他克莫司和卡泊三醇软膏治疗。评定PASI以及疗效指数,并对用药过程中患者的不良事件进行观察记录。结果:A、B两组患者治疗后各时间点PASI评分与治疗前相比较均显著降低,差异有统计学意义(P0.05);但同一时间组间对比,差异并无统计学意义(P0.05)。A组银屑病患者的总有效率为64.29%,与B组的69.05%相比,差异无统计学意义(P0.05)。结论:他克莫司与卡泊三醇软膏治疗四肢斑块状银屑病均安全有效,且二药相比,疗效相当。     

玉屏风颗粒联合他克莫司对原发性肾病综合征患儿肾功能、免疫功能以及Th1/Th2细胞平衡的影响

目的:探讨玉屏风颗粒联合他克莫司对原发性肾病综合征(PNS)患儿肾功能、免疫功能以及Th1/Th2细胞平衡的影响。方法:选取2017年1月～2019年6月期间我院收治的PNS患儿97例,根据随机数字表法将患者分为对照组(n=48)和研究组(n=49),对照组患儿给予他克莫司治疗,研究组在对照组的基础上联合玉屏风颗粒治疗,比较两组患儿疗效、肾功能指标[尿素氮(BUN)、血肌酐(Scr)、免疫功能指标[CD3+、CD4+/CD8+、免疫球蛋白G(Ig G)、免疫球蛋白A(Ig A)]以及Th1/Th2细胞平衡因子[Th1细胞分泌的白细胞介素-2(IL-2)、转化生长因子-β1(TGF-β1)及Th2细胞分泌的白介素-6(IL-6)、白介素-10(IL-10)],记录两组治疗期间不良反应发生情况。结果:研究组治疗9个月后的临床总有效率为91.84%(45/49),高于对照组的75.00%(36/48)(P0.05)。两组治疗9个月后Scr、BUN均下降,且研究组低于对照组(P0.05)。两组治疗9个月后CD3+、CD4+/CD8+、Ig G、Ig A均升高,且研究组高于对照组(P0.05)。两组不良反应发生率比较无差异(P0.05)。两组治疗9个月后TGF-β1、IL-2、IL-10、IL-6均下降,且研究组低于对照组(P0.05)。结论:玉屏风颗粒联合他克莫司治疗PNS患儿,疗效显著,可有效改善患儿肾功能、免疫功能以及Th1/Th2细胞平衡,且不增加不良反应发生率,安全可靠。     

他克莫司乳膏联合治疗白癜风的疗效评价

目的:通过比较自体表皮移植术联合他克莫司和传统自体表皮移植术治疗白癜风的疗效,明确他克莫司乳膏对白癜风复色率的影响。方法:回顾性分析自体表皮移植术联合他克莫司和传统自体表皮移植术治疗的稳定期白癜风患者共90例,比较其均匀复色时间和疗效。结果:表皮移植术联合他克莫司组起效时间显著性短于单一表皮移植术组(P<0.05),1月、3月的显效率和痊愈率均明显高于单一表皮移植术组(P<0.05)。单一表皮移植术组颈部的疗效相比于头面部、躯干部、四肢更佳(P<0.05),表皮移植术联合0.1%他克莫司软膏外用治疗(相对于单一表皮移植术)可显著性提高位于四肢的皮损的疗效(P<0.05)。相较于单一表皮移植术而言,表皮移植术联合他克莫司对提高节段型和局限型白癜风疗效显著(P<0.05)。然而,他克莫司联合治疗可提高散发型和肢端型白癜风的疗效,但无统计学意义(P>0.05)。表皮移植术联合他克莫司组和单一表皮移植术组中,男性疗效均高于女性,但无显著性差异(P>0.05)。结论:自体表皮移植术联合0.1%他克莫司软膏治疗白癜风的疗效优于单一自体表皮移植术,他克莫司可有效提高白癜风复色。     

Liquid chromatography-mass spectrometry characterization of FK506 biosynthetic intermediates in Streptomyces clavuligerus KCTC 10561BP

The development of an efficient analytical method for the reliable detection and identification of the biosynthetic intermediates found in microbial cultures, which usually produce complex intermediates of the metabolites of interest, is essential for further biosynthetic investigations. This study developed a simple and highly selective method for detecting the biosynthetic intermediates involved in the FK506 pathway of Streptomyces clavuligerus KCTC 10561BP involving a cleanup procedure using a solid-phase extraction technique to provide reliable extraction of FK506-related compounds from a cell culture broth and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to separate and detect the FK506-related intermediates at concentrations as low as 0.2 μg/L in the broth. This method enabled the analytical profiling of the intermediates formed during the biosynthesis of FK506 in this S. clavuligerus strain, which produced FK506 as a main product. Eight FK506 intermediates—FK520, 37,38-dihydroFK506, prolylFK506, 9-decarbonyl-9-hydroxylFK506, 9-deoxoFK506, desmethylFK520, prolylFK520, and 9-deoxoFK520—were identified. This is the first report of the LC-ESI-MS/MS characterization of a wide range of FK506 analogs from a bacterial fermentation broth. The protocol employed in this study may be useful for estimating the structure of the metabolites without the need for a time-consuming isolation process and nuclear magnetic resonance (NMR) spectroscopy.     

A calcineurin antifungal strategy with analogs of FK506

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.