Avoiding Drug Resistance by Substrate Envelope-Guided Design: Toward Potent and Robust HCV NS3/4A Protease Inhibitors.
通过底物包膜引导设计避免耐药性: 走向强效和稳健的 HCV NS3/4A 蛋白酶抑制剂。
- 作者列表："Matthew AN","Zephyr J","Nageswara Rao D","Henes M","Kamran W","Kosovrasti K","Hedger AK","Lockbaum GJ","Timm J","Ali A","Kurt Yilmaz N","Schiffer CA
:Hepatitis C virus (HCV) infects millions of people worldwide, causing chronic liver disease that can lead to cirrhosis, hepatocellular carcinoma, and liver transplant. In the last several years, the advent of direct-acting antivirals, including NS3/4A protease inhibitors (PIs), has remarkably improved treatment outcomes of HCV-infected patients. However, selection of resistance-associated substitutions and polymorphisms among genotypes can lead to drug resistance and in some cases treatment failure. A proactive strategy to combat resistance is to constrain PIs within evolutionarily conserved regions in the protease active site. Designing PIs using the substrate envelope is a rational strategy to decrease the susceptibility to resistance by using the constraints of substrate recognition. We successfully designed two series of HCV NS3/4A PIs to leverage unexploited areas in the substrate envelope to improve potency, specifically against resistance-associated substitutions at D168. Our design strategy achieved better resistance profiles over both the FDA-approved NS3/4A PI grazoprevir and the parent compound against the clinically relevant D168A substitution. Crystallographic structural analysis and inhibition assays confirmed that optimally filling the substrate envelope is critical to improve inhibitor potency while avoiding resistance. Specifically, inhibitors that enhanced hydrophobic packing in the S4 pocket and avoided an energetically frustrated pocket performed the best. Thus, the HCV substrate envelope proved to be a powerful tool to design robust PIs, offering a strategy that can be translated to other targets for rational design of inhibitors with improved potency and resistance profiles.IMPORTANCE Despite significant progress, hepatitis C virus (HCV) continues to be a major health problem with millions of people infected worldwide and thousands dying annually due to resulting complications. Recent antiviral combinations can achieve >95% cure, but late diagnosis, low access to treatment, and treatment failure due to drug resistance continue to be roadblocks against eradication of the virus. We report the rational design of two series of HCV NS3/4A protease inhibitors with improved resistance profiles by exploiting evolutionarily constrained regions of the active site using the substrate envelope model. Optimally filling the S4 pocket is critical to avoid resistance and improve potency. Our results provide drug design strategies to avoid resistance that are applicable to other quickly evolving viral drug targets.
: 丙型肝炎病毒 (HCV) 感染全球数百万人，导致可导致肝硬化、肝细胞癌和肝移植的慢性肝病。在过去的几年里，包括 NS3/4A 蛋白酶抑制剂 (PIs) 在内的直接作用抗病毒药物的出现，显著改善了 HCV 感染患者的治疗结果。然而，选择耐药相关置换和基因型之间的多态性可导致耐药性，在某些情况下导致治疗失败。对抗抗性的主动策略是将 PIs 限制在蛋白酶活性位点的进化保守区域内。利用衬底包络设计 PIs 是利用衬底识别约束降低电阻敏感性的合理策略。我们成功地设计了两个系列的 HCV NS3/4A pi 来利用底物包膜中未利用的区域来提高效力，特别是对抗 d168 的耐药相关替换。我们的设计策略在 FDA 批准的 NS3/4A PI grazoprevir 和母体化合物中对临床相关的 D168A 替代均获得了更好的耐药性。晶体学结构分析和抑制试验证实，最佳填充底物包膜对于提高抑制剂效力同时避免耐药性至关重要。具体来说，增强 S4 口袋中的疏水性包装并避免能量受挫口袋的抑制剂表现最好。因此，HCV 底物包膜被证明是设计稳健 PIs 的有力工具，提供了一种可以转化为其他靶点的策略，用于合理设计具有改善效力和耐药性特征的抑制剂。重要性尽管取得重大进展，丙型肝炎病毒 (HCV)仍然是一个主要的健康问题，全世界有数百万人感染，每年有数千人因由此产生的并发症而死亡。最近的抗病毒药物组合可以达到> 95% 的治愈，但是晚期诊断、低获得治疗以及由于耐药性而导致的治疗失败仍然是阻止病毒根除的障碍。我们报道了通过利用底物包络模型开发活性位点的进化约束区域，合理设计了两个系列的 HCV NS3/4A 蛋白酶抑制剂，改善了抗性谱。最佳填充 S4 口袋对于避免耐药性和提高效力至关重要。我们的研究结果提供了避免耐药性的药物设计策略，适用于其他快速发展的病毒药物靶点。
METHODS:BACKGROUND:Opioids are often prescribed for pain in cirrhosis and may increase the risk of hepatic encephalopathy (HE). AIMS:To assess the association between opioids and HE in patients with well-compensated cirrhosis. METHODS:We used the IQVIA PharMetrics (Durham, NC) database to identify patients aged 18-64 years with cirrhosis. We excluded patients with any decompensation event from 1 year before cirrhosis diagnosis to 6 months after cirrhosis diagnosis. Over the 6 months after cirrhosis diagnosis, we determined the duration of continuous opioid use and classified use into short term (1-89 days) and chronic (90-180 days). We assessed whether patients developed HE over the subsequent year (ie 6-18 months after cirrhosis diagnosis). We used a landmark analysis and performed multivariable Cox proportional hazards regression to assess associations between opioid use and HE, adjusting for relevant confounders. RESULTS:The cohort included 6451 patients with compensated cirrhosis, of whom 23.3% and 4.7% had short-term and chronic opioid prescriptions respectively. Over the subsequent year, HE occurred in 6.3% patients with chronic opioid prescriptions, 5.0% with short-term opioid prescriptions and 3.3% with no opioid prescriptions. In the multivariable model, an increased risk of HE was observed with short-term (adjusted hazard ratio, HR 1.44, 95% CI 1.07-1.94) and chronic opioid prescriptions (adjusted HR 1.83, 95% CI 1.07-3.12) compared to no opioid prescriptions. CONCLUSION:In this national cohort of privately insured patients with cirrhosis, opioid prescriptions were associated with the risk of incident HE. Opioid use should be minimised in those with cirrhosis and, when required, limited to short duration.
METHODS:BACKGROUND AND AIMS:Cirrhosis is characterized by extensive fibrosis of the liver and is a major cause of liver-related mortality. Cirrhosis is partially heritable but genetic contributions to cirrhosis have not been systemically explored. Here, we carry out association analyses with cirrhosis in two large biobanks and determine the effects of cirrhosis associated variants on multiple human disease/traits. METHODS:We carried out a genome-wide association analysis of cirrhosis as a diagnosis in UK BioBank (UKBB; 1088 cases vs. 407 873 controls) and then tested top-associating loci for replication with cirrhosis in a hospital-based cohort from the Michigan Genomics Initiative (MGI; 875 cases of cirrhosis vs. 30 346 controls). For replicating variants or variants previously associated with cirrhosis that also affected cirrhosis in UKBB or MGI, we determined single nucleotide polymorphism effects on all other diagnoses in UKBB (PheWAS), common metabolic traits/diseases and serum/plasma metabolites. RESULTS:Unbiased genome-wide association study identified variants in/near PNPLA3 and HFE, and candidate variant analysis identified variants in/near TM6SF2, MBOAT7, SERPINA1, HSD17B13, STAT4 and IFNL4 that reproducibly affected cirrhosis. Most affected liver enzyme concentrations and/or aspartate transaminase-to-platelet ratio index. PheWAS, metabolic trait and serum/plasma metabolite association analyses revealed effects of these variants on lipid, inflammatory and other processes including new effects on many human diseases and traits. CONCLUSIONS:We identified eight loci that reproducibly associate with population-based cirrhosis and define their diverse effects on human diseases and traits.
METHODS:BACKGROUND:Spontaneous bacterial peritonitis (SBP) is a serious complication in patients with liver cirrhosis. In recent years, it has been postulated that the rate of multidrug-resistant organisms (MDROs) is increasing, especially in nosocomial SBP patients. Aim of the present work was to investigate this hypothesis and its possible clinical consequences. MATERIALS AND METHODS:One hundred and three culture-positive patients between 2007 and 2014 were compared with 81 patients between 2015 and 2017, to study the change of microbiological profiles and their clinical consequences. The cirrhosis patients with bacterascites requiring treatment were included as well. RESULTS:The most prevalent Gram-negative bacteria isolated from ascites were Enterobacterales (31.6%) and in Gram-positive pathogens Staphylococci (22.8%). There was a significant increase in MDROs (22.3% ICU 40.7%, P = .048), accompanied by an increased incidence of sepsis (from 21.4% to 37.0%, P = .021), hepatorenal syndrome (from 40.8% to 58.0%, P = .007) and the need of catecholamine therapy (from 21.4% to 38.8%, P = .036). Nosocomial origin correlated with higher MDRO proportion, more complications and lower antimicrobial susceptibility rates in 12 commonly used antibiotics. MDROs were confirmed as an isolated predictor for inpatient mortality and complications in multivariable logistic regression. CONCLUSIONS:The feeling in clinical practice that MDROs have increased in the last 11 years could be confirmed in our study in Munich, Germany. Nosocomial SBP correlated with significantly higher MDRO rates (nearly 50%) and complication rates. In our opinion, an antibiotic combination with comprehensive effect should be taken into account in nosocomial SBP patients in this region.