Development of Antiviral Therapy of HIV-1 Infection

HIV-1感染抗病毒治疗的进展

• 批准号: 8554020
• 负责人: Hiroaki Mitsuya
• 金额: $ 75.59万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8554020
• 关键词: AIDS therapy; Acquired Immunodeficiency Syndrome; Adverse effects; Aniline; Anti-Retroviral Agents; Antiviral Agents; Antiviral Therapy; Benzamides; Binding; Biological; Biological Assay; Blood; CCR5 gene; CXCR4 gene; Chronic; Clinical; Clinical Trials; Collaborations; Complex; Computational algorithm; Computer Simulation; Crystallography; DNA polymerase gamma; Data; Deoxyadenosines; Development; Diarrhea; Disease; Dose; Drug Kinetics; Drug resistance; Enzymes; Epidemic; Excision; Goals; HIV; HIV-1; Highly Active Antiretroviral Therapy; Human; Immune; Immunologics; Individual; Infection; Inflammation; Japan; Kinetics; Knowledge; Lead; Libraries; Ligands; Macaca; Macaca mulatta; Malignant Neoplasms; Mediating; Metabolism; Mitochondrial DNA; Molecular Models; Multi-Drug Resistance; Mutate; Nucleosides; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacodynamics; Physiological; Plasma; Polymerase; Population; Preparation; Protease Inhibitor; Reporting; Resistance; Rest; Reverse Transcriptase Inhibitors; SIV; Schedule; Scientist; Screening procedure; Simian Acquired Immunodeficiency Syndrome; Staging; Structure; Structure-Activity Relationship; Symptoms; Syndrome; Testing; Therapeutic; Thymidine; Time; Tissues; Toxic effect; Transcript; Universities; Variant; Vertebral column; Viral; Viral Genes; Viral Load result; Viremia; Virus; Virus Receptors; Weight Gain; analog; antiretroviral therapy; base; cost; design; dideoxythymidine; experience; functional group; inhibitor/antagonist; insight; molecular modeling; mutant; novel; pre-clinical; prevent; professor; restoration; theories; virtual; wasting

1. Characterization of the profiles of EFdA for potential clinical development.A number of individuals who received currently available nucleoside reverse transcriptase inhibitors (NRTIs) have developed resistant HIV-1 variants. However, there are only a few NRTIs that are on the pipeline as of today. The development of novel NRTIs, which are more potent and less toxic, and prevent or delay the emergence of resistant HIV-1 variants, is needed. We have previously identified 4?-ethynyl-substituted-2-halo-2?-deoxynucleosides, which potently inhibit the replication of various existing multi-NRTI-resistant HIV-variants in collaboration with scientists of Yamasa Corporation, Chiba, Japan. From such 4?-ethynyl congeners, we have chosen 4?-ethynyl-2-fluoro-2?-deoxyadenosine (EFdA) for further preclinical and clinical development, based on our findings on the potency and favorable pharmacokinetic/pharmacodynamic profiles potentially allowing once daily (QD) dosing schedule. In the period covered by this report, in order to ask the propensity of EFdA to allow the development of HIV-1 resistance to EFdA as compared to TDF, FTC, and another 4?-ethynyl NRTI, 4?-ethynyl-2?,3?-dideoxythymidine (Ed4T), we have started selection of HIVNL4-3 and a mixture of 11 multi-drug-resistant HIV-1 clinical isolates (HIV11MIX) as starting virus populations. We have already obtained a favorable resistance selection profile with EFdA, showing that the emergence of EFdA-resistant variants is significantly delayed compared to those of TDF and 4?-ethynyl-2?,3?-dideoxy-didehydro-thymidine (Ed4T) that is presently in early clinical trials. We also conducted pre-steady-state kinetics to examine the interactions of human mitochondrial DNA polymerase gamma with EFdA to assess the potential for EFdA toxicity in collaboration with Professor Anderson of Yale University. It was found that polymerase gamma incorporated EFdA-TP 4,300-fold less efficiently than its physiologic counterpart dATP, with an excision rate similar to ddATP. These data demonstrate that EFdA is a poor polymerase gamma substrate, suggesting minimal polymerase gamma-mediated toxicity of EFdA. In collaboration with Professor Michael Parniak of University of Pittsburgh, we administered EFdA (2 mg/kg, QD, subcutaneously) to 2 rhesus macaques that had shown end-stage SAIDS (chronic diarrhea, wasting, and persistent high plasma viral loads) at the time of initiation of EFdA treatment. EFdA showed excellent antiviral potency, bringing SIV viremia to undetectable level. SAIDS symptoms disappeared within 4 weeks of treatment and both macaques gained weight and showed no detectable adverse effects during 174 days on EFdA treatment. These data strongly suggest that EFdA is a promising candidate with a possibility of QD dosing schedule and less adverse effects than other NRTIs as a therapeutic for individuals harboring multidrug-resistant HIV-1 variants.2. Identification of novel protease inhibitors active against wild-type and multi-drug-resistant HIV-1 variants including DRV-resistant HIV-1 variants.We continued collaborative efforts with Professor Ghosh of Purdue University and identified more than 10 potent protease inhibitors and PDIs. Such novel agents include GRL-02031, GRL-216, GRL-0519, GRL-1398, and GRL-015-11 (Aoki, Ghosh, and Mitsuya: mansucript in preparation). Structural analysis was also performed for selected compounds using molecular modeling and x-ray crystallographic approaches.Analysis of crystal structure of a mutant HIV-1 protease isolated from a multi-drug-resistant clinical HIV-1 isolate (PRMDR-A02) in complex with GRL-0519 in comparison with crystal structures of APV and DRV with PRMDR-A02 was performed to possibly explain the greater potency of GRL-0519. We found that GRL-0519 is involved in maximum number of contacts with the MDR protease among the three compounds tested. Taken together, the bis-THF and tris-THF moieties as the P2 functional groups synergistically boost the potency of DRV and GRL-0519, respectively, explaining their greater inhibition profile compared to that of APV. Structure-function studies of two DRV analogs, one containing carboxybenzene (GRL007-11) and the other benzamide (GRL008-11) as the P2' moiety revealed that the GRL008-11 is at least >30-fold more potent against HIV-1NL4-3 than GRL007-11. GRL008-11 shows relatively greater number of overall contacts, explaining its greater potency.3. Study of the structure-activity relationship studies using various biological/antiviral assays and macromolecular crystallography. In this project, we used DRV structure as a lead template since it has a unique feature of direct polar contacts with the protease backbone, and new compounds were designed. Typically, the P2' aniline moiety of DRV was replaced with other moieties such as carboxybenzene and benzamide generating GRL-007-11 and GRL008-11, respectively. These two novel PIs were to be examined for their interactions with wild-type protease.Crystal structure of GRL-0519 in complex with PRMDR-A02 provided new insights regarding the binding and inhibition profiles of this novel compound. We co-crystallized various potent novel compounds in complex with PRMDR-A02 to better understand their potency. We also investigated the structural profiles of interactions of various novel PIs (DVR, TPV, and newly generated PIs etc.) with mutated protease molecules derived from various highly resistant HIV-1 variants such as DRV-resistant variants (HIV8MIXP51, HIVV32I/L33F/I54M/V82I, and HIVV32I/L33F/I54M/I84V), TPV-resistant HIV-1 variants (HIV11MIXP10, HIVL24M, HIVL33F, and HIVL33I), and DRV-resistant clinical HIV-1 variants.

1。对潜在临床发育的EFDA曲线的表征。当前收到目前可用的核苷逆转录酶抑制剂（NRTI）的个体已经开发了抗HIV-1变体。但是，到目前为止，只有几个NRTI在管道上。 需要新型NRTI的发展，这些NRTI更有效，毒性更少，并且需要或延迟或延迟抗性HIV-1变体的出现。 我们以前已经确定了4个？-Ethynyl-subsubSttestured-2-Halo-2？ - 脱氧核苷，它们有效地抑制了与日本Chiba Yamasa Corporation的科学家合作的各种现有的多NRT-NRTI-NRTI-NRTI-NRTI-NRTI-NRTI-NRTI-NRTI-NRTI-NRT-VARIANT。 从这样的4个？-Ethynyl同类物中，我们选择了4个？-Ethynyl-2-fluoro-2？-Deoxyadenosine（EFDA），以取得进一步的临床前和临床开发，基于我们对效力和有利的药物代动力学/药物学特征的发现，潜在地允许一次潜在地允许每日（QD）给药时间表。 在本报告所涵盖的期间，为了要求EFDA的倾向与TDF，FTC和另外4个？-Ethynyl NRTI相比，允许对EFDA的HIV-1耐药性发展，4？ethynyl-2？ 3？ - 二氧基甲胺（ED4T），我们已经开始选择HIVNL4-3和11种多药耐药性HIV-1临床分离株（HIV11MIX）的混合物作为开始病毒群体。 我们已经通过EFDA获得了有利的抗药性选择曲线，表明与TDF和4？-Ethynyl-2？，3？ - 二维氧基 - 羟基甲酰胺（ED4T）相比，EFDA抗性变体的出现显着延迟。目前正在早期临床试验中。我们还进行了稳态的态动力学，以检查人线粒体DNA聚合酶与EFDA的相互作用，以评估与耶鲁大学安德森教授合作的EFDA毒性的潜力。已经发现，聚合酶伽马含量的EFDA-TP比其生理性对应物的DATP效率低4,300倍，其切除率与DDATP相似。 这些数据表明，EFDA是一种差的聚合酶γ底物，表明最小的聚合酶γ介导的EFDA毒性。与匹兹堡大学的迈克尔·帕尼亚克（Michael Parniak）教授合作，我们管理EFDA（2毫克/千克，QD，皮下），到2个恒河猕猴，在终点上表现出了终点（慢性腹泻，浪费，浪费，持久性血浆病毒载荷） EFDA治疗开始的时间。 EFDA表现出极好的抗病毒效力，使SIV病毒血症达到无法检测到的水平。表示症状在治疗后的4周内消失，两种猕猴的体重增加，在174天对EFDA治疗中没有可检测到的不良反应。这些数据强烈表明，与其他NRTI相比，EFDA是具有QD给药时间表的有前途的候选人，对于具有抗多药HIV-1变体的个体，与其他NRTI相比，与其他NRTI相比，EFDA可能具有QD给药。2。鉴定了针对野生型和多药耐药的HIV-1变体的新型蛋白酶抑制剂，包括抗DRV的HIV-1变体。我们继续与普渡大学的Ghosh教授继续合作，并确定了10多种有效的蛋白酶抑制剂和PDIS。 这种新颖的药物包括GRL-02031，GRL-216，GRL-0519，GRL-1398和GRL-015-11（Aoki，Ghosh和Mitsuya：准备中的Mansucript）。还使用分子建模和X射线晶体学方法对所选化合物进行了结构分析。突变体HIV-1蛋白酶的晶体结构从与多药抗性的临床HIV-1分离株分离出来，与复杂的复杂的多药临床HIV-1分离株（PRMDR-A02）分离。与PRMDR-A02的APV和DRV的晶体结构相比，GRL-0519可能解释了GRL-0519的较高效力。我们发现GRL-0519参与了测试的三种化合物中与MDR蛋白酶的最大接触次数。综上所述，BIS-THF和TRIS-THF部分作为P2官能团分别提高了DRV和GRL-0519的效力，与APV相比，解释了它们更大的抑制作用。两个DRV类似物的结构功能研究，一个含羧酸（GRL007-11）和另一个苯甲胺（GRL008-11），因为P2'部分显示GRL008-11至少对HIV-1NL4的效力至少30倍-3比GRL007-11。 GRL008-11显示了相对较大的总体接触数量，从而解释了其更大的效力3。使用各种生物/抗病毒测定法和大分子晶体学研究结构活性关系研究。在这个项目中，我们使用DRV结构作为铅模板，因为它具有与蛋白酶主链直接触点的独特功能，并设计了新化合物。通常，DRV的P2'Aniline部分分别被其他部分替换为羧基苯苯和苯甲酰胺产生的GRL-007-11和GRL008-11。这两个新型的PI与野生型蛋白酶的相互作用进行了检查。与PRMDR-A02复杂的GRL-0519的晶体结构为这种新型化合物的结合和抑制谱提供了新的见解。我们共结合了与PRMDR-A02复杂化的各种有效的新颖化合物，以更好地了解它们的效力。我们还研究了各种新型PI（DVR，TPV和新生成的PI等）相互作用的结构曲线，这些突变蛋白酶分子来自各种高度抗性HIV-1变体，例如DRV耐药性变体（HIV8MIXP51，HIV8MIXP51，HIVVV32I/L332I/L332I/L332I/L332I/L332I/L332I/L332I/L332I/L332I/L332I/L332i/L332i/L332i/l332i/l332i/l332i/l332i/l332i/l332i/l332i/l33f/l332i/l33f/ I54M/V82I和HIVV32I/L33F/I54M/i84v），TPV耐药HIV-1变体（HIV11MIXP10，HIVL24M，HIVL33F和HIVL33F和HIVL33i），以及DRV抗性的HIV-HIV-HIV-HIV-hiv-1 variants。