Study of HIV Protease Dimerization (PD) and Identification of PD Inhibitors

HIV蛋白酶二聚化(PD)的研究及PD抑制剂的鉴定

• 批准号: 8937957
• 负责人: Hiroaki Mitsuya
• 金额: $ 11.38万
• 依托单位: DIVISION OF BASIC 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/8937957
• 关键词: Active Sites; Affinity; Amino Acid Substitution; Amino Acids; Binding; Biological Assay; C-terminal; Charge; Data; Data Reporting; Data Set; Dimerization; Dissociation; Excision; FDA approved; Fluorescence Resonance Energy Transfer; Gagging; Genes; HIV; HIV Protease; HIV-1; In Vitro; Individual; Intervention; Ions; Isotopes; Minor; Molecular; Molecular Conformation; Mutate; NMR Spectroscopy; Nelfinavir; Peptide Hydrolases; Pharmaceutical Preparations; Play; Process; Property; Protease Inhibitor; Protein Precursors; RNA-Directed DNA Polymerase; Recombinants; Reporting; Resistance; Role; Saquinavir; Scanning; Spectrometry, Mass, Electrospray Ionization; System; Translations; Viral; base; beta pleated sheet; clinical application; dimer; gag Gene Products; inhibitor/antagonist; intermolecular interaction; monomer; mutant; pol Gene Products

The ESI-MS spectra of PRWT and PRD25N revealed four peaks of differently charged ions in the range of mass/charge ratio (m/z) of 1,500-2,500. Since +5 charged monomer ion and +10 charged dimer ion have the same m/z (m/z=2164.51 as calculated with their average mass in the case of PRD25N), the greatest peak detected at m/z 2164.51 was determined to represent two forms, a PR monomer and PR dimer, thus being [PRD25N]5+ and [2PRD25N]10+. In the present study, we designatee a monomer and a dimer ion of PRX as [PRX]Y and [2PRX]Y, respectively, where X denotes an amino acid substitution(s) and Y denotes a charge of ion. To determine whether the detected ions represented monomers and/or dimers, we examined multiply-charged isotopologue clusters of PRD25N using the Solarix FT-ICR MS (Bruker Daltonics) and analyzed the difference in m/z ratios of two adjacent isotope peaks because monomer and dimer PRD25N ions with the same m/z show different peak values in order of their charges. We also constructed three mutated protease species containing amino acid substitutions at the active site (PRD29N, PRT26A, and PRR87K), a C-terminus-truncated mutant (PR1-C95A), and the mutant including L97A and F99A substitutions (PR97/99). Two PRD29N dimer ions ([2PRD29N]11+ and [2PRD29N]9+) were detected, while no dimer ions were detected with PRT26A and PRR87K. Additional analyses of the isotopologue ion peaks with PRT26A and PRR87K confirmed the absence of dimer ions. Importantly, two PR1-C95A dimer ions ([2PR1-C95A]11+ and [2PR1-C95A]9+) were identified although PR1-C95A monomer ion ([PR1-C95A]6+) was found to be a major peak. Two PR97/99 dimer ions were also detected. Considering that [PRWT]5++[2PRWT]10+ representing monomers+dimers was found to be a major peak together with a minor peak of [PRWT]6+, the PR1-C95A and PR97/99 species had a significantly reduced but persistent ability to dimerize in comparison to PRWT. Taken together, the data strongly suggest that the protease dimerization process consists of two distinct steps: (i) initial albeit weak intermolecular interactions occurring in the active site interface, constructing unstable or transient dimers and (ii) subsequent interactions in the termini interface, resulting in the complete and tighter protease dimerization. To regard the thermal stability of PRWT and various mutated species, we employed the differential scanning fluorimetry (DSF). The order of thermal stability was PRWT PRD25N PRD29N PR97/99 PRT26A PRR87K PR1-C95A (Tm; 53.37 52.18 51.02 48.22 48.12 47.02 44.46 C, respectively). The difference in Tm values between PRD25N and PRD29N (1.16 C) was less than the difference between PRD25N and PR1-C95A (7.62 C), indicating that in terms of thermal stability, PRD25N is closer to PRD29N compared to the most unstable PR1-C95A. Thus, PRD29N monomer subunits are likely to interact at the active site interface and subsequently at the termini interface, forming stable dimers. The DSF data, however, showed that Tm value of PR97/99 (48.22 C) was quite low compared to that of PRWT (53.37 C) and PRD29N (51.02 C), suggesting that PR97/99 dimers are likely to be unstable. The Tm value of PR1-C95A was further lower (44.46 C), suggesting that PR1-C95A dimers are also likely to be unstable. Taken the ESI-MS and DSF results together, one can say that the present ESI-MS assay detects both unstable (transient) and stable dimers. Furthermore, DSF data indicated that the stability of PR97/99 and PR1-C95A dimers were lower than that of PRD25N dimer (dimer dissociation constant; KD=1.3 microM), suggesting KD value of unstable or transient dimers were higher than 1.3 microM (21). We previously reported that DRV unhibit not only proteolytic activity but also PR dimerization, while two FDA-approved anti-HIV-1 drugs, saquinaqvir (SQV) and nelfinavir (NFV), showed no dimerization inhibition activity as examined with the FRET-based HIV-1 expression system. To analyze the mechanism of the PR dimerization inhibition by DRV, we therefore examined the binding properties of DRV, SQV and NFV with PRWT. The ESI-MS spectrum of PRWT without drugs showed four peaks derived from differently charged ions, [PRWT]6+, [2PRWT]11+, [PRWT]5++[2PRWT]10+, and [2PRWT]9+. In the presence of DRV, four additional peaks appeared, ([PRWT+DRV]6+, [2PRWT+DRV]10+, [PRWT+DRV]5+, and [2PRWT+DRV]9+). Additional analysis of the isotopologue ion peaks with PRD25N in the presence of DRV confirmed the identity of monomer and dimer ions. On the other hand, the binding of SQV to PRWT yielded only two additional peaks, [2PRWT+SQV]10+ and [2PRWT+SQV]9+, indicating that SQV binds only to PRWT dimers, not to monomers. In the presence of NFV, as in the case of SQV, two additional peaks, [2PRWT+NFV]10+ and [2PRWT+NFV]9+, were identified. The relatively weak intensity of SQV- and NFV-bound PRWT dimers is presumably due to their relatively low binding affinity to PRWT. Taken together, these data clearly indicate that SQV and NFV bind to PRWT dimers but not to monomers and DRV inhibits PR dimerization by binding to PR monomers in a one-to-one molar ratio. Highly DRV-resistant HIV-1 isolates we generated in vitro had acquired a unique combination of 4 amino acid substitutions (V32I/L33F/I54M/I84V) and DRV had decreased its binding to PR monomers containing such 4 amino acid substitutions. We, therefore, examined whether such 4 amino acid substitutions altered the binding profiles of DRV with PR using ESI-MS. The ESI-MS spectrum of PR32/33/54/84 re-folded in the absence of DRV showed four peaks derived from 5 differently charged ions [PR32/33/54/84]6+, [2PR32/33/54/84]11+, [PR32/33/54/84]5++[2PR32/33/54/84]10+, and [2PR32/33/54/84]9+. However, in the presence of DRV, only a substantially low peak representing DRV-bound PR32/33/54/84 dimers or [2PR32/33/54/84+DRV]10+ was detected at m/z 2230.05 and no DRV-bound PR monomers were detected. Thus, it seems that the loss of binding affinity to PR32/33/54/84 in the monomeric form is greater than that in the dimeric form.. Finally, we asked if DRV had an ability to bind to the PR precursor protein, Gag-Pol polyprotein, which is produced through the frameshifting process in the Gag-encoding gene translation and subsequently maturates following the duly excision through autoproteolysis. To examine the DRV binding to the PR precursor protein, a transframe precursor form of PR containing D25N substitution, TFR-PRD25N, was constructed. In the absence of drugs, TFR-PRD25N generated [TFR-PRD25N]10+, [TFR-PRD25N]9+, [TFR-PRD25N]8+, and [TFR-PRD25N]7+, indicating that TFR-PRD25N failed to dimerize, in line with the NMR data reported by Ishima and her colleagues. In the presence of DRV, two additional peaks [TFR-PRD25N+DRV]8+ and [TFR-PRD25N+DRV]9+ appeared, indicating that DRV bound to the TFR-PRD25N monomers. It has been shown that the addition of C-terminus 4 AAs (PISP) to TFR-PR increases thermal stability of DRV-bound TFR-PR. In the present study, we generated TFR-PRD25N-7AA, which contained additional seven N-terminus AAs of reverse transcriptase (7AA; PISPIET) at the C-terminus of TFR-PRD25N. The ESI-MS revealed that TFR-PRD25N-7AA formed dimers, suggesting that the addition of the seven AAs allowed TFR-PRD25N-7AA to dimerize probably by giving TFR-PRD25N-7AA proper conformation. The ESI-MS then showed that DRV binds to both TFR-PRD25N-7AA monomers and dimmers. These results strongly suggest that the loss of dimerization ability of TFR-PRD25N resulted in the loss of DRV's dimer binding. The present data set should represent the first demonstration of the two-step PR dimerization dynamics and the mechanism of dimerization inhibition by DRV.

PRWT和PRD25N的ESI-MS光谱显示，质量/电荷比（M/Z）的1,500-2,500的质量/电荷比（M/Z）的四个峰。由于+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 = 2164.51，在prd25n的情况下使用其平均质量计算出来），因此确定在m/z 2164.51中检测到的最大峰值表示表示。两种形式，一种PR单体和PR二聚体，因此为[PRD25N] 5+和[2PRD25N] 10+。在本研究中，我们将PRX的单体和二聚体分别指定为[Prx] Y和[2PRX] Y，其中X表示氨基酸取代（S），Y表示离子的电荷。为了确定检测到的离子是否代表单体和/或二聚体，我们使用Solarix FT-ICR MS（Bruker Daltonics）检查了PRD25N的倍数型同位素簇，并分析了两个相邻同位素峰的M/Z比率差异具有相同M/Z的二聚体PRD25N离子在其电荷顺序显示出不同的峰值。我们还构建了三种突变的蛋白酶种类，该蛋白酶在活性部位（PRD29N，PRT26A和PRR87K），C-末端截断的突变体（PR1-C95A）以及包括L97A和F99A替代品（PR97/99）在内的突变体（PR1-C95A）（PR1-C95A）（PR1-C95A）（PR97/99） 。检测到两个PRD29N二聚体离子（[[2prd29n] 11+和[2prd29n] 9+），而未检测到PRT26A和PRR87K的二聚体离子。对具有PRT26A和PRR87K的同位素离子峰的其他分析证实了没有二聚体离子。重要的是，发现两个PR1-C95A二聚体离子（[[2PR1-C95A] 11+和[2PR1-C95A] 9+），尽管发现PR1-C95A单体离子（[PR1-C95A] 6+）是一个主要峰。还检测到两个PR97/99二聚体离子。考虑到代表单体+二聚体的[PRWT] 5 ++ [2PRWT] 10+是一个主要峰，与[PRWT] 6+的次要峰一起，PR1-C95A和PR97/99种的峰值显着降低，但与PRWT相比，持续的二聚体能力。综上所述，数据强烈表明蛋白酶二聚过程由两个不同的步骤组成：（i）初始的弱分子间相互作用，发生在活动位点接口中，构造了不稳定或瞬态二聚体，以及（ii）在末端界面中的随后相互作用，导致了。在完整而紧密的蛋白酶二聚化中。为了考虑PRWT和各种突变物种的热稳定性，我们采用了差异扫描荧光法（DSF）。热稳定性的顺序为PRWT PRD25N PRD29N PR97/99 PRT26A PRR87K PR1-C95A（TM; 53.37 52.18 51.02 48.22 48.22 48.12 47.02 44.02 44.46 C，分别为）。 PRD25N和PRD29N（1.16 C）之间TM值的差异小于PRD25N和PR1-C95A（7.62 C）之间的差异，这表明与最不稳定的PRD29N相比，PRD25N在热稳定性方面更接近PRD25N 。因此，PRD29N单体亚基很可能在活动位点接口上，然后在末端接口处相互作用，从而形成稳定的二聚体。但是，DSF数据表明，与PRWT（53.37 C）和PRD29N（51.02 C）相比，PR97/99（48.22 C）的TM值非常低，这表明PR97/99二聚体可能不稳定。 PR1-C95A的TM值进一步较低（44.46 C），这表明PR1-C95A二聚体也可能不稳定。将ESI-MS和DSF的结果一起处理，可以说当前的ESI-MS分析检测不稳定（瞬态）和稳定二聚体。此外，DSF数据表明PR97/99和PR1-C95A二聚体的稳定性低于PRD25N二聚体（二聚体解离常数； KD = 1.3 microM）的稳定性，这表明不稳定或瞬态二聚体的KD值高于1.3 microM（21 ）。我们先前曾报道过，DRV不仅违反了蛋白水解活性，而且还没有PR二聚化，而两种FDA批准的抗HIV-1药物Saquinaqvir（SQV）和Nelfinavir（NFV）均未显示出与基于FRET HIV的HIV所检查的二聚体抑制活性-1表达系统。为了分析DRV抑制PR二聚化的机理，因此我们研究了DRV，SQV和NFV与PRWT的结合特性。没有药物的PRWT的ESI-MS光谱显示出来自不同带电的离子的四个峰[PRWT] 6+，[2PRWT] 11+，[PRWT] 5 ++ [2PRWT] 10+和[2PRWT] 9+。在存在DRV的情况下，出现了另外四个峰，（[[PRWT+DRV] 6+，[2PRWT+DRV] 10+，[PRWT+DRV] 5+和[2PRWT+DRV] 9+）。在存在DRV的情况下，对具有PRD25N的同位素离子峰的附加分析证实了单体和二聚体离子的身份。另一方面，SQV与PRWT的结合仅产生两个额外的峰，[2PRWT+SQV] 10+和[2PRWT+SQV] 9+，表明SQV仅与PRWT二聚体结合，而不是与单体。在存在NFV的情况下，与SQV一样，确定了另外两个峰，[2PRWT+NFV] 10+和[2PRWT+NFV] 9+。 SQV和NFV结合的PRWT二聚体的强度相对较弱，可能是由于它们对PRWT的结合相对较低。综上所述，这些数据清楚地表明SQV和NFV与PRWT二聚体结合，但与单体不结合，DRV通过以一对一的摩尔比与PR单体结合来抑制PR二聚体。我们在体外产生的高度抗性HIV-1分离株获得了4种氨基酸取代（V32I/L33F/I54M/I84V）的独特组合，DRV降低了其与含有这种4氨基酸替代的PR单体的结合。因此，我们检查了使用ESI-MS与PR的DRV的结合分布是否改变了这种4种氨基酸的替代。在没有DRV的情况下重折的Pr32/33/54/84的ESI-MS光谱显示出源自5个不同电荷离子的四个峰[PR32/33/54/84] 6+，[2PR32/33/54/84/84 ] 11+，[PR32/33/54/84] 5 ++ [2PR32/33/54/84] 10+和[2PR32/33/54/84] 9+。但是，在存在DRV的情况下，仅在M/z 2230.05和无DRV-检测到了大量代表DRV结合的PR32/33/54/84二聚体或[2PR32/33/54/84+ DRV] 10+的较低峰。检测到绑定的PR单体。因此，看来单体形式中与PR32/33/54/84的结合亲和力的丧失大于二聚体形式的结合亲和力。最后，我们问DRV是否具有与PR前体蛋白结合的能力，GAG -pol多蛋白，是通过gag编码基因翻译中的帧速率过程产生的，随后通过自动蛋白解切除后成熟。为了检查与PR前体蛋白的DRV结合，构建了含有D25N取代的PR的转换前体形式TFR-PRD25N。在没有药物的情况下，TFR-PRD25N产生[TFR-PRD25N] 10+，[TFR-PRD25N] 9+，[TFR-PRD25N] 8+和[TFR-PRD25N] 7+与Ishima及其同事报告的NMR数据一致。在存在DRV的情况下，出现了两个附加峰[TFR-PRD25N+ DRV] 8+和[TFR-PRD25N+ DRV] 9+，表明DRV与TFR-PRD25N单体结合。已经表明，在TFR-PR中添加C端4 AA（PISP）增加了DRV结合的TFR PR的热稳定性。在本研究中，我们产生了TFR-PRD25N-7AA，其中包含七个逆转录酶（7AA; pispiet）在TFR-PRD25N的C末端的N-末端AA。 ESI-MS揭示了TFR-PRD25N-7AA形成二聚体，这表明添加七个AAS允许TFR-PRD25N-7AA可能通过给出TFR PRD25N-7AA适当的构型来二聚体。然后，ESI-MS表明DRV与TFR-PRD25N-7AA单体和调光器都结合。这些结果强烈表明，TFR-PRD25N二聚化能力的丧失导致DRV二聚体结合的丧失。目前的数据集应代表两步PR二聚动力学的首次演示以及DRV二聚化抑制的机理。