3'-axial CH2 OH substitution on glucopyranose does not increase glycogen phosphorylase inhibitory potency. QM/MM-PBSA calculations suggest why

Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure‐based design pinpointed that the 3′‐position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme’s cofactor, pyridoxal 5′‐phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1‐(β‐d ‐glucopyranosyl)5‐fluorouracil (GlcFU) and its 3′‐CH₂OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with K_i values of 7.9 and 27.1 μm , respectively. X‐ray crystallography revealed that the 3′‐CH₂OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ‐phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics – Poisson–Boltzmann Surface Area (QM/MM‐PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM‐PBSA revealed that the desolvation energy cost for binding of the 3′‐CH₂OH‐substituted glucopyranose derivative out‐weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM‐PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant.     

Effect of geniposide,a hypoglycemic glucoside,on hepatic regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin

Aim： Hepatic glycogen phosphorylase （GP） and glucose-6-phosphatase （G6Pase） play an important role in the control of blood glucose homeostasis and are proposed to be potential targets for anti-diabetic drugs. Geniposide is an iridoid glucoside extracted from Gardenia jasminoides Ellis fruits and has been reported to have a hypoglycemic effect. However, little is known about the biochemical mechanisms by which geniposide regulates hepatic glucose-metabolizing enzymes. The present study investigates whether the hypoglycemic effect of geniposide is mediated by GP or G6Pase. Methods： Type 2 diabetic mice, induced by a high-fat diet and streptozotocin injection, were treated with or without geniposide for 2 weeks. Blood glucose levels were monitored by a glucometer. Insulin concentrations were analyzed by the ELISA method. Total cholesterol （TC） and triglyceride （TG） levels were measured using LabassayTM kits. Activities of hepatic GP and G6Pase were measured by glucose-6-phosphate dehydrogenase-coupled reaction. Real-time RT-PCR and Western blotting were used to determine the mRNA and protein levels of both enzymes. Results： Geniposide （200 and 400 mg/kg） significantly decreased the blood glucose, insulin and TG levels in diabetic mice in a dose-dependent manner. This compound also decreased the expression of GP and G6Pase at mRNA and immunoreactive protein levels, as well as enzyme activity. Conclusion： Geniposide is an effective hypoglycemic agent in diabetic mice. The hypoglycemic effect of this compound may be mediated, at least in part, by inhibiting the GP and G6Pase activities.     

蛋白酶抑制剂联合CRRT对脓毒症患者 FABP3、GPBB水平的影响

目的 探讨蛋白酶抑制剂联合连续肾脏代替治疗(CRRT)对脓毒症患者心型脂肪酸结合蛋白(FABP3)、糖原磷酸化酶同工酶BB(GPBB)水平的影响。方法 选取2019年2月至2020年6月我院收治的脓毒症患者134例,按照随机数字表法分为CRRT组与联合治疗组,每组各67例。其中CRRT组患者进行CRRT治疗,联合治疗组患者进行CRRT联合蛋白酶抑制剂进行治疗。使用流式细胞仪对CD4+、CD8+、CD4+/CD8+指标水平进行测定与比较,采用酶联免疫法检测白介素-8(IL-8)、肿瘤细胞因子-α(TNF-α)、白介素-6(IL-6)及脂肪酸结合蛋白3(FABP3)、糖原磷酸化酶同工酶BB(GPBB)水平,并比较,免疫组化法检测D-乳酸、二胺氧化酶(DAO)水平,并对两组患者治疗疗效进行评价。结果 治疗后,与CRRT组相比,联合治疗组CD4+指标水平与CD4+/CD8+均较高,CD8+指标水平较低(P0.05);与CRRT组相比,联合治疗组IL-8、TNF-α、IL-6水平均较低(P0.05);联合治疗组D-乳酸水平及DAO水平均低于CRRT组(P0.05);与CRRT组相比,联合治疗组FABP3水平较高、GPBB水平较低(P0.05);与CRRT组相比,联合治疗组治疗总有效率较高(P0.05)。结论 使用蛋白酶抑制剂联合CRRT对脓毒症患者进行治疗,能够显著改善患者免疫功能,抑制炎症反应,同时能够降低肠道黏膜损害程度,使得FABP3水平升高、GPBB水平降低,从而减轻脓毒症患者心肌损伤程度。     

胸苷磷酸化酶在肝细胞癌中的表达及临床意义

目的:探讨胸苷磷酸化酶(TP)在肝细胞癌(HCC)的表达和临床意义及在血管生成中的作用。方法:对47例经手术病理证实的HCC病灶进行回顾性分析,观察其临床病理特征,包括肿瘤大小、包膜、子灶、门静脉癌栓、淋巴结转移、坏死等。用免疫组织化学方法检测癌组织中TP和CD34的表达情况,微血管的计数采用Weidner的计数方法。分析评价TP与上述指标的相关性。结果:TP与微血管密度(MVD)明显相关,转移高危组(子灶、门静脉癌栓、淋巴结转移)、包膜欠完整和/或无包膜组和有坏死组TP蛋白表达率均高于转移低危组、包膜完整组和无坏死组(P<0.05)。结论:TP是促进HCC肿瘤血管生成的重要的血管生成因子,并与侵袭转移有关。     

Capecitabine-Based Chemoendocrine Combination as First-Line Treatment for Metastatic Hormone-Positive Metastatic Breast Cancer: Phase 2 Study

BackgroundPreclinical studies have suggested a synergistic effect of tamoxifen and capecitabine in estrogen receptor–positive cell lines. We evaluated the safety and efficacy of first-line chemoendocrine treatment in patients with metastatic breast cancer. Biochemical assessment was performed of serum levels of thymidine phosphorylase enzyme (TP), serum tamoxifen, hydroxytamoxifen, and 5-fluorouracil in relationship to efficacy.Patients and MethodsThis prospective phase 2 interventional study studied patients with estrogen receptor-positive, HER2⁻ metastatic breast cancer who received either tamoxifen/capecitabine or letrozole/capecitabine as first-line treatment. The dose of capecitabine provided at 2000 mg per day continuously as a fixed dose.ResultsForty women with a median age of 49.3 years were enrolled. For the whole study group, median progression-free survival (PFS) was 10 months and median overall survival (OS) was 23.3 months. The overall response rate was 60% and the clinical benefit rate 82.5%. Progesterone receptor positivity was associated with significantly longer PFS (12 vs. 7 months, P = .021). The most frequent adverse events were palmar–plantar erythrodysesthesia (62.5%), fatigue (62.5%), diarrhea (30%), abdominal pain (12.5%), and constipation (10%). Changes in serum level of TP were not correlated to response to treatment, PFS, or OS. Higher serum levels of tamoxifen and hydroxytamoxifen were correlated with higher response rates and longer PFS but not OS.ConclusionChemoendocrine treatment is well tolerated, with no evidence of contradictory effects between the combination components. However, the efficacy data need more validation.     

干扰素-α诱导肾癌细胞胸苷磷酸化酶表达对5-FU化疗敏感性的影响

目的 探讨干扰素-α诱导肾癌细胞胸苷磷酸化酶（thymidine phosphorylase，TP）表达对5-氟尿嘧啶（5-FU）化疗敏感性的影响。方法采用不同浓度的干扰素-α2b处理肾癌细胞株786—0，应用RT—PCR和免疫印迹方法分别检测TP mRNA和TP蛋白水平变化。MTT法检测不同处理组786—0细胞中5-FU的半数抑制浓度（IC50）。建立肾透明细胞癌移植瘤动物模型，观察干扰素-α2b联合5-FU化疗对移植瘤生长的影响，免疫组化检测移植瘤中TP的表达，TUNEL检测肿瘤细胞的凋亡。结果 干扰素-α2b3000和6000IU／ml处理组，TP mRNA表达量（灰度峰值）分别为0．5733±0．0231和0．8233±0．0404，TP蛋白表达量分别为0．6347±0．0719和0．8735±0．0640。干扰素-α对TP的表达有剂量依赖性增强作用（P〈0．01）。在干扰素-α2b作用下，5-FU对786—0半数抑制浓度由（13．9467±3．7140）μmol／L下降至（5．3200±0．1039）μmol／L，化疗敏感性增加（P〈0．01）。联合用药组移植瘤体积为（0．0940±0．0492）cm^3，小于单纯5-FU组的（0．5424±0．1591）cm^3（P〈0．05）。单纯5-FU组和联合用药组移植瘤中肿瘤细胞凋亡指数差异无统计学意义（P〉0．05）。结论 干扰素-α2b诱导的TP增强表达参与了干扰素-α联合5-FU化疗增效作用，TP是干扰素-α2b的靶基因之。     

TS、TP及DPD表达与乳腺癌新辅助化疗的关系

目的 检测乳腺癌标本中胸苷酸合成酶（Thymidylate Synthase,TS）、胸腺嘧啶脱氧核苷磷酸化酶（Thymidine Phosphorylase,TP）、二氟嘧啶脱氢酶（Dihydropyrimidine Dehydrogenase,DPD）表达状况,评价TS、TP、DPD的表达与新辅助化疗效果之间的关系.方法 选择81例可手术的乳腺癌患者,手术前均接受CMF方案新辅助化疗,采用免疫组织化学染色的方法对新辅助化疗前麦默通活检系统切取的乳腺癌组织中TS、TP和DPD的表达进行检测.结果 TS、TP阴性患者的部分缓解率要明显高于阳性患者,而DPD阳性患者的部分缓解率明显高于阴性患者,差异具有统计学意义（P＜0.05）;DPD阴性患者Ⅲ～Ⅳ度毒副反应发生率高于阳性患者（P＜0.05）,而TS、TP的表达水平与化疗所产生的毒副反应之间无明显关系（P＞0.05）.结论 TS、TP及DPD的表达与乳腺癌患者接受CMF方案的新辅助化疗的治疗效果相关,而DPD的表达则与含5-FU类化疗药物所产生的毒副反应相关.     

Crystal structure of Schistosoma purine nucleoside phosphorylase complexed with a novel monocyclic inhibitor

A novel inhibitor of Schistosoma PNP was identified using an “in silico” approach allied to enzyme inhibition assays. The compound has a monocyclic structure which has not been previously described for PNP inhibitors. The crystallographic structure of the complex was determined and used to elucidate the binding mode within the active site. Furthermore, the predicted pose was very similar to that determined crystallographically, validating the methodology. The compound Sm_VS1, despite its low molecular weight, possesses an IC₅₀ of 1.3 μM, surprisingly low when compared with purine analogues. This is presumably due to the formation of eight hydrogen bonds with key residues in the active site E203, N245 and T244. The results of this study highlight the importance of the use of multiple conformations for the target during virtual screening. Indeed the Sm_VS1 compound was only identified after flipping the N245 side chain. It is expected that the structure will be of use in the development of new highly active non-purine based compounds against the Schistosoma enzyme.