Development of Antiviral Therapy of HIV-1 Infection

HIV-1感染抗病毒治疗的进展

• 批准号: 9556765
• 负责人: Hiroaki Mitsuya
• 金额: $ 71.24万
• 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/9556765
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Adverse event; Anti-Retroviral Agents; Antiviral Agents; Antiviral Therapy; Atazanavir; Binding; Brain; Cerebrospinal Fluid; Charge; Clinic; Clinical; Complex; Data; Dental crowns; Detection; Development; Diarrhea; Dimerization; Dose; FDA approved; Failure; Fluorine; Gastrointestinal Diseases; General Population; Genetic; HIV resistance; HIV therapy; HIV-1; HIV-1 drug resistance; HIV-2; HIV-associated neurocognitive disorder; Height; Hyperbilirubinemia; Immunologics; Impairment; In Vitro; Individual; Infection; Inflammation; Integrase; Ions; Life Expectancy; Ligands; Lopinavir; Neuraxis; Neuropsychology; Patients; Penetration; Peptide Hydrolases; Pharmaceutical Preparations; Plasma; Process; Protease Inhibitor; Rattus; Regimen; Reporting; Resistance; Resistance development; Reverse Transcriptase Inhibitors; Safety; Sampling; Serious Adverse Event; Site; Specificity; Spectrometry, Mass, Electrospray Ionization; Structure; Tenofovir; Treatment Failure; Update; Variant; Viral; Viral Load result; antiretroviral therapy; base; benzothiazole; cytotoxicity; dimer; experience; improved; in vivo; indexing; individual patient; inhibitor/antagonist; monomer; mortality; novel; pharmacophore; prototype; sexual HIV transmission; virology

Since our first report of darunavir (DRV) in 2003, we continued optimization based on the structure of DRV, seeking novel protease inhibitors (PIs) that are more potent against a variety of existing multi-PI-resistant HIV-1 variants with greater safety, do not permit or substantially delay the emergence of HIV-1 variants resistant to the very PIs, and favorably penetrate into the CNS, and identified GRL-142. GRL-142 contains newly generated pharmacophores such as an unprecedented 6-5-5 ring-fused crown-like tetrahydropyranofuran as the P2-ligand (Crn-THF), P1-bis-fluoro-benzyl (bis-Fbz), and P2'-cyclopropyl-amino-benzothiazole (Cp-Abt). GRL-139, a prototype to GRL-142, structurally resembles DRV but contains the Crn-THF moiety instead of the bis-THF of DRV, and exerts comparable antiviral activity against wild-type HIV-1 (HIVNL4-3) as compared to DRV. However, GRL-139 failed to block the replication of three highly DRV-resistant HIV-1 variants (HIVDRVRs) that were selected by propagating in the presence of increasing concentrations of DRV and are highly resistant to all presently clinically available PIs including DRV and nucleos(t)ide-reverse-transcriptase inhibitors such as tenofovir (TDF). By contrast, GRL-036 also resembles DRV but has the Cp-Abt moiety and shows an improved anti-HIV-1 profile, more effectively blocking the replication of HIVDRVRs than DRV. GRL-121 contains both Crn-THF and Cp-Abt moieties and more effectively blocked the replication of HIVNL4-3 by about 10-fold compared to DRV. GRL-121 more potently suppressed the replication of all three HIVDRVRs. Interestingly, the addition of two fluorine atoms to GRL-121, generating GRL-142 further strengthened the activity against HIVNL4-3 achieving an IC50 value as low as 0.019 nM compared to the 9 FDA-approved PIs of which IC50s range from 3.2 to 330 nM. GRL-142 had a much improved cytotoxicity profile with a selectivity index (CC50/IC50) as high as 2,473,684. GRL-142 also highly potently blocked the replication of all three HIVDRVRs by factors of 27-83 compared to GRL-121. Notably, the IC50 value of GRL-142 against HIVDRVRP51 (1.2 nM), the most multi-PI/NRTI-resistant HIVDRVR, was 3-fold lower than that of DRV against HIVNL4-3 (3.2 nM). We further examined the activity of GRL-142 against two HIV-2 strains and found that GRL-142 also exerts highly potent antiviral activity against the HIV-2 strains examined. We further examined the activity of five PIs including GRL-121 and -142 against seven resistant HIV-1 variants, which we had previously selected in vitro with each of the seven FDA-approved PIs (invitroHIVPIRs). Most of the seven variants were significantly less susceptible to two PIs, lopinavir (LPV) and ATV, that have presently been relatively well used in clinics. DRV also failed to effectively block most of the seven variants with IC50 value fold-differences ranging from 2- to 86-fold. However, GRL-121 showed extremely potent activity against all the seven variants examined, presenting IC50 values ranging 0.0018 to 0.13 nM. The activity of GRL-121 against all of the seven variants was significantly more potent than that against HIVNL4-3. Surprisingly, GRL-142 showed even further more potent activity against the seven variants with IC50 values of 0.0000019 nM (1.9 fM) to 0.015 nM. The activity of GRL-142 against the variants was also significantly more potent than that against HIVNL4-3. We have previously demonstrated that PR monomer subunits initially interact at the active site interface, generating unstably dimerized PR subunits, and subsequently the termini interface interactions occur, completing the dimerization process. DRV binds in the proximity of the active site interface of PR and blocks PR subunits dimerization. Therefore, we asked whether GRL-142 binds to monomer subunits using electrospray ionization mass spectrometry (ESI-MS). As shown in Figure 2C, the ESI-MS spectra of PR containing D25N substitution (PRD25N), which was folded in the presence of drugs revealed five peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,900. Since +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z=2164.75 for PRD25N), the greatest peak detected at m/z 2164.75 was determined to represent two forms, a PR monomer and PR dimer. Thus, the five peaks represent a monomer, two dimers, and two monomer+dimers. When unfolded PRD25N was re-folded in the presence of DRV, six additional significant peaks appeared, three for monomer+DRV, and three for dimer+DRV. When the same PRD25N was re-folded in the presence of GRL-142, six significant peaks appeared, representing three for monomer+GRL-142, and three for dimer+GRL-142. Each of the six additional peaks seen with GRL-142 appeared greater than those seen with DRV. When compared with the heights of the dimer+monomer peak rendered 1.0, the average height of the three peaks of DRV-bound monomers and that of the three peaks of GRL-142-bound monomers were 0.046 and 0.312, respectively; and the average height of the three peaks with DRV-bound dimers and that of the three peaks with GRL-142-bound dimers were 0.060 and 0.188, respectively. These data suggest that GRL-142 more tightly bound to monomers by 6.78-fold and to dimers by 3.13-fold than DRV and at least in part explain the reason GRL-142 much more strongly blocked PR dimerization than DRV. Persistent HIV-1 replication and inflammation in the CNS, which can occur even in patients receiving cART with an undetectable plasma viral load, is most likely responsible for HAND. Hence, we finally quantified GRL-142 concentrations in plasma, cerebrospinal fluid (CSF), and brain of rats (n=2) and compared those figures with those of DRV obtained under the same conditions. When DRV was perorally administered at a dose of 5 mg/kg together with RTV (8.33 mg/kg), the Cmax was achieved around 90 min after the PO administration. The DRV concentrations determined in 15 and 90 min after the peroral administration turned out to be 0.595 microM and 0.847 microM in plasma; 0.00100 microM and 0.00116 microM in CSF; and 0.0110 microM and 0.0157 microM in brain, respectively. The plasma concentrations of DRV were much greater than the IC50 value (3.2 microM); however, concentrations in CSF were lower than the IC50 value and those in brain were slightly above the DRV IC50 value but still substantially lower than the DRV IC95 value of DRV (0.3 microM), suggesting that DRV likely fail blocking the replication of HIV-1 in the CNS. By contrast, when GRL-142 was perorally administered at a dose of 5 mg/kg together with RTV (8.33 mg/kg), the Cmax was achieved around 360 min after the PO administration. Plasma samples collected 60 and 360 min after the PO administration contained 0.189 microM GRL-142 and 0.974 microM, respectively. CSF samples contain below detection levels and 0.000532 microM); brain contained 0.00724 microM and 0.0326 microM in 60 and 360 min, respectively. Since the IC50 and IC95 values of GRL-142 are 19 pM and 0.28 nM, GRL-142 concentrations in brain are calculated to be 1,882-fold greater than the IC50 value of GRL-142 and 114-fold greater than IC95 of GRL-142, while 562-fold lower than CC50 of GRL-142. These data strongly suggest that GRL-142 would potently block the infection and replication of HIV-1 in the brain.

自2003年首次报道达芦那韦（DRV）以来，我们根据DRV的结构不断优化，寻找对现有多种多重PI耐药的HIV-1变种更有效、更安全的新型蛋白酶抑制剂（PI） ，不允许或实质上延迟对PI具有抗药性的HIV-1变体的出现，并有利地渗透到CNS中，并鉴定了GRL-142。 GRL-142 含有新生成的药效团，例如前所未有的 6-5-5 环稠合冠状四氢吡喃呋喃作为 P2-配体 (Crn-THF)、P1-双氟苄基 (bis-Fbz) 和 P2' -环丙基-氨基-苯并噻唑(Cp-Abt)。 GRL-139 是 GRL-142 的原型，结构上类似于 DRV，但包含 Crn-THF 部分而不是 DRV 的双 THF，并且与野生型 HIV-1 (HIVNL4-3) 相比，对野生型 HIV-1 (HIVNL4-3) 具有类似的抗病毒活性。驾驶室。然而，GRL-139 未能阻止三种高度 DRV 抗性的 HIV-1 变体 (HIVDRVR) 的复制，这些变体是通过在 DRV 浓度不断增加的情况下繁殖而选择的，并且对所有目前临床可用的 PI（包括 DRV 和核）具有高度抗性(t)ide-逆转录酶抑制剂，例如替诺福韦 (TDF)。相比之下，GRL-036 也类似于 DRV，但具有 Cp-Abt 部分，并显示出改进的抗 HIV-1 特性，比 DRV 更有效地阻止 HIVDRVR 的复制。 GRL-121同时含有Crn-THF和Cp-Abt部分，与DRV相比，更有效地阻断HIVNL4-3的复制约10倍。 GRL-121 更有效地抑制所有三种 HIVDRVR 的复制。有趣的是，在 GRL-121 中添加两个氟原子，生成 GRL-142，进一步增强了针对 HIVNL4-3 的活性，与 FDA 批准的 9 种 PI（其 IC50 范围为 3.2 至 330）相比，IC50 值低至 0.019 nM纳米。 GRL-142 的细胞毒性显着改善，选择性指数 (CC50/IC50) 高达 2,473,684。与 GRL-121 相比，GRL-142 还可以高效阻断所有三种 HIVDRVR 的复制，阻断效果为 27-83 倍。值得注意的是，GRL-142针对HIVDRVRP51（对PI/NRTI耐药性最强的HIVDRVR）的IC50值（1.2 nM）比DRV针对HIVNL4-3（3.2 nM）的IC50值低3倍。我们进一步检查了 GRL-142 对两种 HIV-2 毒株的活性，发现 GRL-142 对所检查的 HIV-2 毒株也具有高效的抗病毒活性。我们进一步检查了包括 GRL-121 和 -142 在内的 5 种 PI 对 7 种耐药 HIV-1 变异体的活性，这些变异体是我们之前在体外与 FDA 批准的 7 种 PI 中的每一种 (invitroHIVPIR) 一起选择的。七种变体中的大多数对洛匹那韦 (LPV) 和 ATV 两种 PI 的敏感性明显较低，这两种药物目前在临床中使用相对较好。 DRV 也未能有效阻断七种变体中的大多数，IC50 值倍数差异范围为 2 至 86 倍。然而，GRL-121 对所检测的所有七种变体均表现出极强的活性，IC50 值范围为 0.0018 至 0.13 nM。 GRL-121 针对所有七种变体的活性均明显强于针对 HIVNL4-3 的活性。令人惊讶的是，GRL-142 对七种变体表现出更有效的活性，IC50 值为 0.0000019 nM (1.9 fM) 至 0.015 nM。 GRL-142 针对变异体的活性也明显强于针对 HIVNL4-3 的活性。我们之前已经证明PR单体亚基最初在活性位点界面相互作用，产生不稳定的二聚化PR亚基，随后发生末端界面相互作用，完成二聚化过程。 DRV 结合在 PR 活性位点界面附近并阻止 PR 亚基二聚化。因此，我们使用电喷雾电离质谱 (ESI-MS) 询问 GRL-142 是否与单体亚基结合。如图 2C 所示，含有 D25N 取代 (PRD25N) 的 PR 在药物存在下折叠，其 ESI-MS 谱显示在质荷比 (m/z) 为 1,500 的范围内有五个带不同电荷离子的峰-2,900。由于带+5电荷的单体离子和带+10电荷的二聚体离子具有相同的m/z（对于PRD25N，m/z=2164.75），因此确定在m/z 2164.75处检测到的最大峰代表两种形式，PR单体和PR二聚体。因此，五个峰代表一个单体、两个二聚体和两个单体+二聚体。当未折叠的 PRD25N 在 DRV 存在下重新折叠时，出现了六个额外的显着峰，三个为单体+DRV，三个为二聚体+DRV。当相同的PRD25N在GRL-142存在下重新折叠时，出现六个显着的峰，代表单体+GRL-142的三个峰，二聚体+GRL-142的三个峰。使用 GRL-142 观察到的另外六个峰中的每一个似乎都比使用 DRV 观察到的峰更大。与二聚体+单体峰的高度1.0相比，DRV结合单体的三个峰的平均高度和GRL-142结合单体的三个峰的平均高度分别为0.046和0.312； DRV结合二聚体的三个峰的平均高度和GRL-142结合二聚体的三个峰的平均高度分别为0.060和0.188。这些数据表明GRL-142与单体的结合比DRV更紧密6.78倍，与二聚体的结合比DRV更紧密3.13倍，并且至少部分解释了GRL-142比DRV更强烈地阻断PR二聚化的原因。即使在接受 cART 且血浆病毒载量无法检测到的患者中，中枢神经系统中持续的 HIV-1 复制和炎症也可能发生，这很可能是导致 HAND 的原因。因此，我们最终量化了大鼠（n=2）血浆、脑脊液（CSF）和大脑中GRL-142的浓度，并将这些数据与相同条件下获得的DRV数据进行比较。当 DRV 以 5 mg/kg 的剂量与 RTV (8.33 mg/kg) 一起口服给药时，PO 给药后约 90 分钟达到 Cmax。口服给药后15分钟和90分钟测定的DRV血浆浓度分别为0.595μM和0.847μM；脑脊液中 0.00100 microM 和 0.00116 microM；大脑中分别为 0.0110 microM 和 0.0157 microM。 DRV的血浆浓度远高于IC50值（3.2μM）；然而，CSF中的浓度低于IC50值，脑中的浓度略高于DRV IC50值，但仍远低于DRV的DRV IC95值（0.3μM），表明DRV可能无法阻止HIV-1的复制在中枢神经系统中。相比之下，当GRL-142以5 mg/kg的剂量与RTV (8.33 mg/kg)一起口服给药时，PO给药后约360分钟达到Cmax。 PO 给药后 60 分钟和 360 分钟收集的血浆样品分别含有 0.189 microM GRL-142 和 0.974 microM。 CSF 样本含量低于检测水平（0.000532 microM）； 60 分钟和 360 分钟内，大脑中分别含有 0.00724 microM 和 0.0326 microM。由于GRL-142的IC50和IC95值为19 pM和0.28 nM，因此计算出脑中GRL-142浓度比GRL-142的IC50值高1,882倍，比GRL-142的IC95高114倍，而比 GRL-142 的 CC50 低 562 倍。这些数据强烈表明 GRL-142 可以有效阻止 HIV-1 在大脑中的感染和复制。