Development of Antiviral Therapy of HIV-1 Infection

HIV-1感染抗病毒治疗的进展

• 批准号: 6947464
• 负责人: Hiroaki Mitsuya
• 金额: --
• 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/6947464
• 关键词: AIDS therapy; HIV infections; RNA directed DNA polymerase; SCID mouse; antiAIDS agent; cytokine receptors; drug resistance; drug screening /evaluation; gene mutation; genetic strain; human immunodeficiency virus 1; human subject; human therapy evaluation; multidrug resistance; nucleoside analog; patient oriented research; pharmacogenetics; pharmacokinetics; protease inhibitor; reverse transcriptase inhibitors; virus protein; zidovudine

In this report period, we carried on our long-standing research line, development of anti human immunodeficiency virus (HIV) drugs. We particularly focused two major research areas relating to the treatment of HIV-1 infection. (1) We identified several groups of novel agents active against a wide spectrum of HIV including multi-drug-resistant HIV variants. (2) We also attempted to elucidate the mechanism(s) of the emergence of HIV-1 variants resistant to multi nucleoside reverse transcriptase inhibitors (HIV-1MDR). We designed, synthesized, and identified UIC-94017 (TMC114), a novel nonpeptidic HIV-1 protease inhibitor (PI) containing a 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) and a sulfonamide isostere, which is extremely potent against laboratory HIV-1 strains and primary clinical isolates (IC50: 0.003 mM; IC90: 0.009 mM) with minimal cytotoxicity (CC50: 74 mM in CD4+ MT-2 cells). UIC-94017 blocked the infectivity and replication of each of HIV-1NL4-3 variants exposed to and selected by up to 5 mM concentration of saquinavir, indinavir, nelfinavir, or ritonavir (IC50: 0.003 - 0.029 mM), although it was less active to HIV-1NL4-3 selected by amprenavir (IC50: 0.22 mM). UIC-94017 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents. Structural analyses revealed that the close contact of UIC-94017 with the main chains of the protease active site amino acids (Asp-29 and Asp-30) is important for its potency and wide-spectrum activity against multi-PI-resistant HIV-1 variants. Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for treatment of infection with primary and multi-PI-resistant HIV-1. It is of note that a patent application for the identification and characterization of UIC 94017 and its analogs have been filed and licensed to Johnson and Johnson/Tibotec and UIC 94017 is currently undergoing phase IIa clinical trials in Europe. We previously reported that a series of novel low molecular weight spirodiketopiperazine derivatives including a prototypic E913 (Mr. 484). In this report period, we designed, synthesized, and identified a far more potent CCR5 inhibitor, designated AK602. AK602 exhibited a high affinity to CCR5 (KD values of 3 nM), exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates including multi-drug resistant HIV-1MDR (IC50 values of 0.2-0.6 nM), and potently blocked HIV-1-gp120/CCR5 binding. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors including TAK779 and SCH-C, preserved RANTES and MIP-1b binding to CCR5+ cells and their functions, while TAK779 and Sch-C fully blocked the CC-chemokines/CCR5 interactions. When intraperitoneally administered twice daily to R5 HIV-1JRFL-infected hu-periphearl blood mononuclear cells-transplanted, IL-2Rg-chain-knocked-out, non-obese diabetes-SCID mice, AK602 blocked CD4+ cell decrease and suppressed plasma HIV viremia by 2 logs. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection. We also investigated the mechanism by which the Q151M mutation in reverse transcriptase (RT) requiring a two base change (CAGATG) develops, which confers multi-dideoxynucleoside resistance on HIV-1 and attempted to elucidate the reason for the relatively lengthy period of time required for its emergence under therapy with multiple nucleoside RT inhibitors (NRTIs). We compared the fitness of various infectious clones including two putative intermediates (HIV-1Q151K(AAG) and HIV-1Q151L(CTG)) for HIV-1Q151M(ATG), in relation to their sensitivity to drugs. Propagation assays and competitive HIV-1 replication assays were employed to evaluate viral fitness of various clones in relation to drug sensitivity of such clones to zidovudine and didanosine. Steady-state kinetic constants of recombinant RTs were also determined. The data showed that HIV-1Q151M most likely develops through a poorly replicating HIV-1Q151L, however, it also possibly occurs through two concurrent base changes. The data explained the mechanism by which HIV-1Q151M emerges after long-term chemotherapy with NRTIs. The emergence of HIV variants resistant to multiple anti-HIV agents represents a major cause of "treatment failure"in those receiving antiviral therapy of AIDS. The virological significance of amino acid (AA) insertions near the Gag protein cleavage sites in multi-PI resistant HIV variants is not known. When we newly generated full-sized infectious molecular clones using HIV isolates obtained from heavily drug-experienced/therapy-failed AIDS patients, of such 6 full-sized infectious clones generated, 4 clones were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites in addition to the known multiple AA substitutions within the protease (PR). The competitive HIV replication assay proved that such Gag-inserts mostly compromised the replication of wild-type HIV, whereas the primary clones containing inserts replicated significantly better than those without the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type PR was impaired by the inserts, whereas that by mutant PR was substantially improved by the inserts but was greatly hindered without them. Thus we conclude that the inserts seen in the proximity of the Gag cleavage sites in highly multi PI resistant HIV variants restore the otherwise compromised enzymatic activity of mutant PR, enabling the multi-PI-resistant HIV variants to remain replication-competent, another newly identified mechanism of the acquisition of HIV drug resistance to protease inhibitors.

在本报告期间，我们进行了长期的研究系，抗人免疫缺陷病毒（HIV）药物的发展。我们特别关注与HIV-1感染治疗有关的两个主要研究领域。 （1）我们确定了几组新型药物对广泛的艾滋病毒的活性，包括多药耐药的艾滋病毒变体。 （2）我们还试图阐明HIV-1变体的出现机制，该变体具有抗多核苷逆转录酶抑制剂（HIV-1MDR）的抗性。 我们设计，合成和鉴定了UIC-94017（TMC114），这是一种新型的非肽HIV-1蛋白酶抑制剂（PI），其中含有3（r），3A（r），3A（s），6A（r）bis-bis-bis-trahydrofuranyylyylyylyyylane（bis-thf）（bis-thf）和磺酰胺ISOSTORE，它对实验室HIV-1菌株和原代临床分离株（IC50：0.003 mm; IC90：0.009 mm）具有极大的有效性，具有最小的细胞毒性（CC50：74 mm在CD4+ MT-2细胞中）。 UIC-94017阻止了暴露于并通过高达5 mm浓度的Saquinavir，Indinavir，Nelfinavir或Ritonavir或Ritonavir或Ritonavir（IC50：0.003-0.029 mm）暴露并选择的HIV-1NL4-3变体的感染性和复制。由Amprenavir（IC50：0.22 mm）选择的HIV-1NL4-3。 UIC-94017也对从没有反应的患者中分离出对现有抗病毒药物的多种抗病毒治疗剂的多种抗临床HIV-1变种也有效。结构分析表明，UIC-94017与蛋白酶活性位点氨基酸（ASP-29和ASP-30）的主要链的密切接触对于其效力和针对多PI-耐药HIV-1的宽光谱活动很​​重要变体。 Considering the favorable pharmacokinetics of UIC-94017 when administered with ritonavir, the present data warrant that UIC-94017 be further developed as a potential therapeutic agent for treatment of infection with primary and multi-PI-resistant HIV-1.值得注意的是，已对UIC 94017及其类似物的识别和表征进行了专利申请，并将其许可给Johnson和Johnson和Johnson/Tibotec以及UIC 94017目前正在欧洲进行IIA期临床试验。 我们先前报道说，一系列新型的低分子量螺旋哌嗪衍生物，包括原型E913（484先生）。在本报告期间，我们设计，合成并确定了更有效的CCR5抑制剂，该抑制剂指定为AK602。 AK602表现出对CCR5的高亲和力（KD值为3 nm），对广泛的实验室和主要R5 HIV-1分离株（包括多药抗性HIV-1MDR（IC50值为0.2-0.6 nm））和原发性R5 HIV-1分离株发挥了有效的活性。有力阻止HIV-1-GP120/CCR5结合。尽管AK602的抗HIV-1活性比其他先前发表的CCR5抑制剂（包括TAK779和SCH-C）的抗HIV-1活性要大得多，但保留了与CCR5+细胞及其功能结合的MIP-1B，而TAK779和SCH-C完全阻止了CC--趋化因子/CCR5相互作用。腹膜内每天两次对R5 HIV-1JRFL感染的Hu- periphearl血液单核细胞转移，IL-2RG-链敲出，非肥胖，非肥胖的糖尿病小鼠，AK602阻止CD4+细胞降低的CD4+ CD4+ CD4+细胞HIV Virasma Hiv Viremia， 2个日志。药代动力学研究表明，啮齿动物的口服生物利用度有利。这些数据需要进一步发展AK602作为HIV-1感染的潜在治疗方法。 我们还研究了逆转录酶中Q151M突变（RT）中需要进行两次基本变化（CAGATG）的机制，该机制赋予了HIV-1上多氧化核苷的耐药性，并试图阐明所需的相对较长时间的原因由于其在多个核苷RT抑制剂（NRTI）中的治疗中出现。我们比较了各种感染性克隆的适应性，包括两个推定的中间体（HIV-1Q151K（AAG）和HIV-1Q151L（CTG））对HIV-1Q151M（ATG）（ATG），与它们对药物的敏感性有关。采用了传播测定和竞争性HIV-1复制测定法来评估各种克隆在此类克隆对齐多夫丁和迪地糖苷的敏感性方面的病毒适应性。还确定了重组RT的稳态动力学常数。数据表明，HIV-1Q151M最有可能通过复制较差的HIV-1Q151L而发展，但是，它也可能通过两次并发基础变化而发生。数据解释了与NRTIS长期化疗后HIV-1Q151M出现的机制。 抗多种抗HIV药物的HIV变体的出现代表了接受艾滋病抗病毒治疗的人“治疗失败”的主要原因。尚不清楚氨基酸（AA）在GAG蛋白裂解位点附近的氨基酸（AA）插入的病毒学意义。当我们使用从高度药物经验/治疗的艾滋病患者中获得的HIV分离株新生成全尺寸的传染性分子克隆时，生成的6种全尺寸感染性克隆的患者被发现具有独特的插入（TGN，SQVN，AQVN，AQQQA） ，SRPE，APP和/或PTAPPA）附近P17/P24和P1/P6 GAG裂解位点，除了蛋白酶（PR）中已知的多个AA取代。竞争性艾滋病毒复制测定法证明，这种插入的插入大部分损害了野生型艾滋病毒的复制，而包含插入材料的主要克隆比没有插入物的插入物要好得多。 Western印迹分析表明，插入物损坏了野生型PR的GAG蛋白的加工，而突变PR的插入片段可被插入片段大大改善，但没有它们而受到很大的阻碍。 因此，我们得出的结论是，在高度多PI的耐PI抗HIV变体中，在插科打裂解位点的接近性中看到的插入物恢复了突变体PR的原本受损的酶促活性对蛋白酶抑制剂抗HIV耐药性的获取机制。