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.

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是GRL-142的原型，在结构上类似于DRV，但包含CRN-THF部分而不是DRV的BIS-THF，并且与DRV相比，针对野生型HIV-1（HIVNL4-3）发挥了可比的抗病毒活性。但是，GRL-139未能阻止三种高度抗DRV的HIV-1变体（HIVDRVR）的复制，这些变体（HIVDRVR）是通过在增加浓度的DRV的情况下传播而选择的，并且对所有目前临床上可用的PI都具有高度抗性，包括DRV和NucleOS（包括IDE反复转移酶抑制剂），例如Tenofovir（Tdffovir）（TDFFOVIR）。相比之下，GRL-036也类似于DRV，但具有CP-ABT部分，并且显示出改进的抗HIV-1曲线，比DRV更有效地阻止了HIVDRVR的复制。 GRL-121既包含CRN-THF和CP-ABT部分，并且更有效地将HIVNL4-3的复制量与DRV相比约为10倍。 GRL-121更有效地抑制了所有三个HIVDRVR的复制。有趣的是，与HIVNL4-3的GRL-121中添加了两个氟原子，与HIVNL4-3产生的活性进一步增强了IC50值低至0.019 nm的活性，而IC50S的9个IC50范围为3.2至330 Nm。 GRL-142的细胞毒性轮廓有了很大改善，其选择性指数（CC50/IC50）高达2,473,684。与GRL-121相比，GRL-142还高度有效地阻止了所有三个HIVDRVR的复制。值得注意的是，GRL-142对HIVDRVRP51（1.2 nm）的IC50值，最多的PI/NRTI耐药性HIVDRVR，比HIVNL4-3（3.2 nm）低3倍。我们进一步研究了GRL-142对两种HIV-2菌株的活性，发现GRL-142还针对所检查的HIV-2菌株发挥了高度有效的抗病毒活性。我们进一步研究了五个PI的活性，包括GRL-121和-142对七个抗性HIV-1变体的活性，我们以前在体外选择了七个FDA批准的PI（InvitroHivpirs）。七种变体中的大多数明显不太容易受到两个PIS，洛皮那韦（LPV）和ATV的影响，这些PIS目前在诊所中使用了相对较好的使用。 DRV还无法有效阻止七个变体中的大多数，IC50值折叠率在2-至86倍之间。然而，GRL-121对所有检查的七个变体显示出极有效的活性，呈现为0.0018至0.13 nm的IC50值。 GRL-121对所有七个变体的活性都比HIVNL4-3的活性明显高得多。令人惊讶的是，GRL-142对七个IC50值的七种变体显示了进一步的有效活性，其值为0.0000019 nm（1.9 fm）至0.015 nm。 GRL-142对变体的活性也比对HIVNL4-3的活性更有效。我们先前已经证明，PR单体亚基最初在活动位点接口上相互作用，生成不稳定的二聚PR亚基，然后终端接口相互作用发生，完成二聚过程。 DRV在PR的活动位点界面的接近度和阻断PR亚基二聚体的接近度中结合。因此，我们询问GRL-142是否使用电喷雾电离质谱法（ESI-MS）与单体亚基结合。如图2C所示，在存在药物的情况下折叠的含D25N取代的PR的ESI-MS光谱（PRD25N）显示出五个不同电荷离子的质量/电荷比（M/Z）的五个峰为1,500-2,900。由于带有+5带电的单体离子和+10带电的二聚体离子具有相同的m/z（m/z = 2164.75，PRD25N）确定在m/z 2164.75时检测到的最大峰值表示两种形式，一种PR单体和PR二聚体。因此，五个峰代表单体，两个二聚体和两个单体+二聚体。当展开的PRD25N在存在的存在下重新折叠时，出现了六个明显的峰，单体+DRV的三个，三个用于二聚体+DRV。当在GRL-142的存在下重新折叠相同的PRD25N时，出现了六个显着峰，代表单体+GRL-142的三个，而二聚体+GRL-142的三个。 GRL-142看到的六个额外峰中的每个峰似乎都比DRV看到的峰更大。与二聚体+单体峰的高度相比，渲染1.0时，DRV结合单体的三个峰的平均高度和GRL-142结合的单体的三个峰的平均高度分别为0.046和0.312。三个峰的平均高度和GRL-142绑定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更紧密地与单体紧密绑定6.78倍，二聚体比DRV的二倍比DRV倍数3.13倍，至少在某种程度上解释了GRL-142的原因与DRV相比，GRL-142更加强大的PR二聚化。 CNS中持续的HIV-1复制和炎症，即使在接受无法检测到的血浆病毒载量的手推车的患者中也可能发生，这也很可能是手动的原因。因此，我们最终将大鼠的血浆，脑脊液（CSF）和大脑（n = 2）的GRL-142浓度定量，并将这些数字与在相同条件下获得的DRV进行了比较。当DRV与RTV（8.33 mg/kg）一起以5 mg/kg的剂量多剂量施用时，在PO给药后90分钟内实现了CMAX。吞噬施用后15和90分钟确定的DRV浓度被证明为0.595 microm，血浆中的Microm为0.847 microm； CSF中的0.00100 microm和0.00116 microm;和0.0110 microM和0.0157 microm在大脑中。 DRV的血浆浓度远大于IC50值（3.2 microm）；但是，CSF的浓度低于IC50值，大脑中的浓度略高于DRV IC50值，但仍大大低于DRV的DRV IC95值（0.3 microM），这表明DRV可能会失败阻断CNS中HIV-1的复制。相比之下，当GRL-142与RTV（8.33 mg/kg）一起以5 mg/kg的剂量多剂量给药时，在PO给药后约360分钟就可以实现CMAX。 PO给药后收集了60和360分钟的血浆样品分别包含0.189 microm GRL-142和0.974 microm。 CSF样品包含以下检测水平和0.000532 microm）；大脑在60和360分钟内分别包含0.00724 microm和0.0326 microm。由于GRL-142的IC50和IC95值为19 pm，脑中的GRL-142浓度比GRL-142的IC50值高1,882倍，而GRL-142的IC50值大，而GRL-142的IC95倍，而GRL-142的IC95值比GRL-142的IC95高。这些数据强烈表明，GRL-142会有力阻止大脑中HIV-1的感染和复制。