Development of Antiviral Therapy of HIV-1 Infection

HIV-1感染抗病毒治疗的进展

基本信息

批准号：
9154253
负责人：
Hiroaki Mitsuya
金额：
$ 51.12万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/9154253
关键词：
Acquired Immunodeficiency Syndrome Active Sites Adverse effects Amides Amino Acids Amprenavir Annual Reports Anti-Retroviral Agents Antiviral Agents Antiviral Therapy Atazanavir Binding Binding Sites Blood Blood - brain barrier anatomy Brain Clinical Collaborations Data Developing Countries Development Disease Drug resistance Epidemic HIV HIV-1 HIV-associated neurocognitive disorder Hydrogen Hydrogen Bonding Hydroxyl Radical Immune Immunologics In Vitro Individual Infection Inflammation Inhibitory Concentration 50 Integrase Inhibitors Laboratories Ligands Lopinavir Malignant Neoplasms Modeling Molecular Models Multi-Drug Resistance Nitrogen Oxygen Patients Penetration Peptide Hydrolases Pharmaceutical Preparations Population Principal Investigator Property Protease Inhibitor Recombinants Regimen Reporting Resistance Resistance development Rest Retrovirology Reverse Transcriptase Inhibitors Ritonavir Structure Sulfonamides Surface Syndrome Therapeutic Therapeutic Agents Time Tissues Toxic effect Universities Variant Vertebral column Viral antiretroviral therapy base cost cytotoxicity design drug resistant virus experience human GHR protein molecular modeling novel pre-clinical restoration tetrahydrofuran

项目摘要

1. Identification of novel protease inhibitors active against wild-type and multi-drug-resistant HIV-1 variants including DRV-resistant HIV-1 variants that potentially have a favorable CNS penetration. In the period of this Annual Report, we designed, synthesized, and identified GRL-0739, a novel non-peptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor containing tricycle (cyclohexyl-bis-THF) and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.0019 - 0.0036 mM) with minimal cytotoxicity (CC50: 21.0 mM). GRL-0739 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to 5 mM concentration of ritonavir or atazanavir (EC50: 0.035 - 0.058 mM). GRL-0739 was also highly active against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, and HIV-2ROD. The development of resistance against GRL-0739 was substantially delayed compared to APV. The effects of non-specific binding of human serum proteins on GRL-0739's antiviral activity were insignificant. In addition, GRL-0739 showed a favorable blood-brain barrier (BBB) penetration property as assessed using a novel in vitro BBB model. Molecular modeling demonstrated that the tricyclic ring and methoxybenzene of GRL-0739 have a larger surface and seem to make greater van der Waals contact with protease than in the case of darunavir. The present data demonstrate that GRL-0739 has favorable features as a highly active therapeutic with potentially favorable CNS-penetration capability for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants and that the newly generated cyclohexyl-bis-THF moiety with methoxybenzene should be critical for the strong binding of GRL-0739 to HIV-1 protease substrate binding site and should have a potential to confer highly favorable anti-HIV-1 potency in designing novel protease inhibitors with greater CNS-penetration profiles. We also identified GRL-09510, a novel non-peptidic HIV-1 protease inhibitor containing a unique polycyclic ring as P2 moiety and a sulfonamide isostere, which is highly active against laboratory HIV-1 strains and primary clinical isolates (EC50: 0.0014 - 0.0028 mM) with minimal cytotoxicity (CC50: 39.0 mM). GRL-09510 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to 5 mM concentrations of atazanavir, lopinavir, or amprenavir (EC50: 0.0037 - 0.0048 mM). GRL-09510 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, and against HIV-2ROD. The development of resistance against GRL-09510 was substantially delayed compared to that of APV, or integrase inhibitor, raltegravir. Crystallographic analysis demonstrated that the polycyclic ring (P2 moiety) of GRL-09510 has strong hydrogen bond interactions with protease active-site amino acids. Moreover, it has a larger surface and makes greater van der Waals contacts with protease than in the case of darunavir. The data on GRL-09510 demonstrate that the compound has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants. The structure of the newly designed GRL-09510 is unique, suggesting that the polycyclic-P2 moiety confers highly desirable anti-HIV-1 potency in designing novel protease inhibitors. 2. The C3-substituted tetrahydropyrano-tetrahydofuran-derived protease inhibitors (PIs) exert potent inhibition of the replication of HIV-1 variants highly resistant to various PIs including darunavir. In this subproject, we identified three non-peptidic HIV-1 protease inhibitors (PIs), GRL-015, GRL-085, and GRL-097, which contain a tetrahydropyrano-tetrahydrofuran (Tp-THF) with a hydroxyl moiety at the P2site of the compounds. The three compounds showed potent antiviral activity against a wild-type laboratory HIV-1 strain with 50% inhibitory concentrations (IC50s) of 3.0 to 49 nM and minimal cytotoxicity, with 50% cytotxic concentrations (CC50) for GRL-015, -085, and -097 of 80, 100, and 100 mM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected in vitro with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multi-drug resistant HIV-1 variants (HIVMDR). Most importantly, a highly darunavir (DRV)-resistant HIV-1 variant, HIV-1DRVRP51, notably less susceptible to DRV over 1,000 times compared to wild-type HIV-1, whereas the three compounds remained active to HIV-1DRVRP51 only with 6.8- to 68-fold reduction. Moreover, in vitro development of drug resistant HIV-1 variants against the three compounds were comparable to or delayed compared to that of DRV, especially highly drug resistant viruses against GRL-085 and -097 were not obtained even by using two different kinds of DRV-resistant HIV-1s as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen-bonds with the polar hydrogen atoms associated with the backbone amide nitrogen atoms of active-site amino acids (Asp29 and Asp30) of HIV-1 protease in a similar fashion to that of bis-THF of DRV. Additionally, strong hydrogen bonding between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings warrant that the three compounds be further studied as possible therapeutic agents for treating individuals harboring wild-type and/or HIVMDR.

1. 鉴定对野生型和多重耐药 HIV-1 变体（包括可能具有有利 CNS 渗透性的 DRV 耐药 HIV-1 变体）具有活性的新型蛋白酶抑制剂。在本年度报告期间，我们设计、合成并鉴定了GRL-0739，一种新型非肽人类免疫缺陷病毒1型（HIV-1）蛋白酶抑制剂，含有三环（环己基双THF）和磺酰胺等排体，对实验室 HIV-1 菌株和初级临床分离株具有高度活性 (EC50: 0.0019 - 0.0036 mM)，且作用极小细胞毒性（CC50：21.0 mM）。 GRL-0739 阻断由高达 5 mM 浓度的利托那韦或阿扎那韦选择的 HIV-1NL4-3 变体的感染性和复制（EC50：0.035 - 0.058 mM）。 GRL-0739 对从患者体内分离出的多重耐药性临床 HIV-1 变体（长期抗逆转录病毒治疗后不再对现有抗病毒方案做出反应）和 HIV-2ROD 也具有高度活性。与 APV 相比，GRL-0739 耐药性的产生大大延迟。人血清蛋白的非特异性结合对 GRL-0739 抗病毒活性的影响微不足道。此外，使用新型体外 BBB 模型评估，GRL-0739 显示出良好的血脑屏障 (BBB) 渗透特性。分子模型表明，GRL-0739 的三环和甲氧基苯具有更大的表面，并且似乎比达芦那韦与蛋白酶有更大的范德华接触。目前的数据表明，GRL-0739 具有作为高活性治疗剂的有利特征，具有潜在有利的 CNS 渗透能力，用于治疗感染野生型和/或多药耐药性 HIV-1 变异体的患者，并且新生成的环己基-带有甲氧基苯的双 THF 部分对于 GRL-0739 与 HIV-1 蛋白酶底物结合位点的强结合至关重要，并且应该具有赋予高度有利的抗 HIV-1 效力的潜力设计具有更强中枢神经系统渗透能力的新型蛋白酶抑制剂。我们还鉴定了 GRL-09510，一种新型非肽 HIV-1 蛋白酶抑制剂，含有独特的多环作为 P2 部分和磺酰胺等排体，对实验室 HIV-1 菌株和初级临床分离株具有高度活性（EC50：0.0014 - 0.0028） mM），具有最小的细胞毒性（CC50：39.0 mM）。 GRL-09510 阻断由高达 5 mM 浓度的阿扎那韦、洛匹那韦或安普那韦选择的 HIV-1NL4-3 变体的感染性和复制（EC50：0.0037 - 0.0048 mM）。 GRL-09510还保持了对从长期抗逆转录病毒治疗后对现有抗病毒方案不再有反应的患者中分离出的多重耐药临床HIV-1变异体和HIV-2ROD的强大抗病毒活性。与 APV 或整合酶抑制剂拉替拉韦相比，GRL-09510 耐药性的产生大大延迟。晶体分析表明，GRL-09510 的多环（P2 部分）与蛋白酶活性位点氨基酸具有强氢键相互作用。此外，与达芦那韦相比，它具有更大的表面，并与蛋白酶进行更大的范德华接触。 GRL-09510 的数据表明，该化合物具有治疗感染野生型和/或多药耐药性 HIV-1 变异体的患者的理想特性。新设计的GRL-09510的结构是独特的，这表明多环-P2部分在设计新型蛋白酶抑制剂时赋予了非常理想的抗HIV-1效力。 2. C3 取代的四氢吡喃四氢呋喃衍生蛋白酶抑制剂 (PI) 可有效抑制对包括达芦那韦 (darunavir) 在内的各种 PI 具有高度耐药性的 HIV-1 变体的复制。在这个子项目中，我们鉴定了三种非肽 HIV-1 蛋白酶抑制剂 (PI)：GRL-015、GRL-085 和 GRL-097，它们含有在 P2 位点具有羟基部分的四氢吡喃并四氢呋喃 (Tp-THF)的化合物。这三种化合物对野生型实验室 HIV-1 毒株表现出有效的抗病毒活性，50% 抑制浓度 (IC50) 为 3.0 至 49 nM，细胞毒性极小，GRL-015、-085、-085 的细胞毒浓度 (CC50) 为 50%和-097分别为80、100和100 mM。所有三种化合物均能有效抑制体外筛选出的高度 PI 耐药性 HIV-1 变异体的复制，其中每种变异体均采用目前可用的 PI 和从携带多重耐药性 HIV-1 变异体 (HIVMDR) 的患者中获得的重组临床 HIV-1 分离株。最重要的是，与野生型 HIV-1 相比，高度抗达芦那韦 (DRV) 的 HIV-1 变体 HIV-1DRVRP51 对 DRV 的敏感性明显降低了 1,000 倍以上，而这三种化合物对 HIV-1DRVRP51 的敏感性仅为 6.8 - 减少 68 倍。此外，针对这三种化合物的耐药HIV-1变体的体外开发与DRV相当或延迟，特别是即使使用两种不同的DRV也没有获得针对GRL-085和-097的高度耐药病毒抗性 HIV-1 作为起始人群。在结构分析中，GRL-015、-085和-097的Tp-THF与HIV-1活性位点氨基酸（Asp29和Asp30）的主链酰胺氮原子相关的极性氢原子显示出强氢键。 1 蛋白酶的方式与 DRV 的双 THF 类似。此外，新发现了 Tp-THF 的羟基部分和 Gly48 的羰基氧原子之间的强氢键。目前的研究结果表明，这三种化合物需要进一步研究，作为治疗携带野生型和/或 HIVMDR 个体的可能治疗剂。