Study of Structures of CCR5 and Its Interactions with CCR5 Inhibitors

CCR5的结构及其与CCR5抑制剂相互作用的研究

• 批准号: 9556454
• 负责人: Hiroaki Mitsuya
• 金额: $ 5.94万
• 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/9556454
• 关键词: AMD3100; Active Sites; Affinity; Alanine; Amino Acids; Antiviral Agents; Binding; Binding Sites; Biological Assay; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell fusion; Cells; Chemicals; Clinical; Complex; Coupled; Crystallization; Data; Databases; Disease; Docking; Event; FDA approved; G-substrate; GTP-Binding Proteins; Glutamic Acid; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Receptors; HIV-1; Human; Hydrogen Bonding; Individual; Infection; Intervention; Libraries; Ligands; Molecular; Molecular Conformation; Names; Patients; Pharmacologic Substance; Phenols; Phenotype; Play; Positioning Attribute; Proteins; RANTES; Reporting; Research; Role; Shapes; Specificity; Stromal Cell-Derived Factor 1; Structure; Testing; Therapeutic; Therapeutic Index; Viral; Virus; Virus Replication; base; chemokine receptor; cytotoxicity; design; env Gene Products; glycoprotein CX 1; improved; inhibitor/antagonist; member; novel; receptor; screening; simulation; small molecule; small molecule inhibitor; virtual

We searched a general screening library from ChemBridgeTM, which has more than six hundred thousand molecules, to select molecules for assays as potential inhibitors of CXCR4. Previous studies have shown that determination of molecular similarity plays a critical role in analyzing large compound databases in chemical and pharmaceutical research. Therefore, we determined the shape similarity of the molecules, from the ChemBridgeTM library to IT1t, a molecule that binds with high affinity to CXCR4 and demonstrates anti-HIV-1 activity. Seven-hundred-fifty-three (753) unique molecules with one-thousand-five (1,005) configurations/conformations had a shape Tanimoto coefficient of at least 0.70. The binding mode and interactions of these molecules with CXCR4 were determined by molecular docking to the crystal structure of CXCR4 to determine their possible binding modes. Since it had been determined that CXCR4 transmembrane residues Asp97, Tyr116, Asp262, Glu288, Phe292 and ECL2 residues Phe174, Ala175, Asp182, Asp187, Arg188, and Tyr190 appeared to be important for the cell fusion event, we hypothesized that molecules that bound around the active site determined in the crystal structure, and formed hydrogen bond interactions with at least two of these residues are likely to competitively inhibit the interactions of CXCR4 with gp120. Our candidate molecule selection was based on shape similarity to a known inhibitor (IT1t) and putative binding mode and interactions with residues that were determined to be important for the HIV-1-gp120-elicited cell fusion event with CXCR4. Based on the hypothesis described above, we selected sixteen compounds (named CX1 to CX16) for biological assays from ChemBridgeTM general screening library. We asked whether the sixteen selected compounds bound to CXCR4 by blocking the intracellular Ca2+ mobilization induced by SDF-1alpha, whose primary receptor is CXCR4. One of the compounds, described as CX6 blocked the SDF-1alpha-induced Ca2+ mobilization in Molt4 cells with an EC50 value of 92 nM. In the same assay, AMD3100 showed inhibition of SDF-1alpha-induced Ca2+ mobilization with an EC50 value of around 10 nM. Two other compounds, CX11 and CX13, both piperidinylethanamine (PEA) derivatives like CX6, blocked SDF-1alpha-induced Ca2+ mobilization in Molt4 cells with EC50 values of 161 and 149 nM, respectively. None of the other compounds bound to CXCR4. Of note, all three PEA derivatives (CX6, CX11 and CX13) failed to block the Ca2+ mobilization induced with RANTES, whose receptor is CCR5, indicating that these compounds did not bind to CCR5. When we asked if these three compounds had agonistic effects to induce Ca2+ mobilization in CXCR4+ cells, none induced Ca2+ mobilization, suggesting that CX6, CX11, and CX13 were antagonists of CXCR4. In summary, the above data strongly suggest that the PEA derivatives bound to CXCR4 with specificity and were antagonists of CXCR4. The fusion event elicited by the interactions of HIV viral envelope glycoprotein with CXCR4 enables the virus to gain entry to cells, eventually leading to viral replication. The sixteen compounds were selected through docking simulations suggesting that they bound to a potential orthosteric binding site of CXCR4. These compounds formed hydrogen bonds to at least two amino acid residues most likely to be important for the fusion event, and thereby have the potential to competitively inhibit the fusion event. We thus determined if the compounds were actually able to inhibit the interactions of the HIV-1NL4-3 envelope protein with CXCR4, and to block the fusion event in the HIV-1-gp120-elicited cell-cell fusion assays with the wild-type CXCR4. Both CX6 and CX11 blocked the fusion with EC50 values of 1.9 microM and 7.9 microM. Moreover, none of the compounds including CX6 and CX11 inhibited the fusion event as examined with the HIV-1-gp120-elicited cell-cell fusion assays using the cells expressing the wild-type CCR5-derived from HIV-1BaL (R5-HIV-1). This suggested that CX6 and CX11 inhibited the fusion event associated with CXCR4 but not with CCR5. CX6 had an excellent shape Tanimoto overlap coefficient of 0.73. The imidazothiazole ring of IT1t overlays with the cyclopentylpiperidinyl group of CX6. The interactions of the identified hit compound (CX6) with CXCR4 were deduced by molecular docking. The phenol group of CX6 interacted with Glu-32 located in the N-terminus of CXCR4. Asp97 has been shown to be important for the binding of AMD070 as well as for CXCR4-gp120-elicited fusion. Glu288 is important for the fusion event as it is shown that substitution of Glu288 with alanine results in loss of the CXCR4-gp120-elicited fusion. Glutamic acid at position 6 in TM7 is a highly conserved amino acid residue in chemokine receptors and has been demonstrated to be important for the binding of non-peptidic ligands to chemokine receptors. Our current study suggests that Glu288 is an important residue to target for rational structure based design and discovery of CXCR4 inhibitors. We determined the anti-HIV-1 activity and cytotoxicity of CX6 with the MTT assay using X4-HIV-1NL4-3 and MT4 as target cells. CX6 exerted substantial activity against HIV-1NL4-3 and HIV-1LAI, both of which are X4-HIV-1 strains, with EC50 values of 1.5 and 3.0 microM, respectively. The cytotoxicity CC50 of CX6 was 58 microM with a therapeutic index of 39. CX6 blocked the fusion event as examined in the fusion inhibition assay using HIV-1NL-4-3-derived envelope protein. In a Ca2+-flux inhibition assay, in which SDF-1alpha-elicited Ca2+-flux levels are determined in the presence or absence of a potential small molecule inhibitor, CX6 was found to block the flux at an EC50 value of 92 nM. However, as expected, CX6 failed to block the infectivity and replication of an R5-HIV-1 (HIV-1BaL)(EC50: 10 microM) as examined in an anti-HIV assay using HIV-1BaL and MAGI cells. CX11 and CX13 similarly exerted activity against X4-HIV-1NL4-3 and X4-HIV-1LAI, but failed to block the infectivity and replication of R5-HIV-1BaL.

我们从 ChemBridgeTM 中检索了一个通用筛选文库，该文库包含超过 60 万个分子，以选择作为 CXCR4 潜在抑制剂进行分析的分子。先前的研究表明，分子相似性的确定在化学和药物研究中分析大型化合物数据库中起着至关重要的作用。因此，我们确定了 ChemBridgeTM 库中的分子与 IT1t 的形状相似性，IT1t 是一种与 CXCR4 高亲和力结合并具有抗 HIV-1 活性的分子。具有一千五 (1,005) 种构型/构象的七百五十三 (753) 个独特分子的形状谷本系数至少为 0.70。通过与CXCR4晶体结构的分子对接来确定这些分子与CXCR4的结合模式和相互作用，以确定它们可能的结合模式。由于已经确定 CXCR4 跨膜残基 Asp97、Tyr116、Asp262、Glu288、Phe292 和 ECL2 残基 Phe174、Ala175、Asp182、Asp187、Arg188 和 Tyr190 似乎对细胞融合事件很重要，因此我们假设结合在周围的分子晶体结构中确定的活性位点，并形成与这些残基中的至少两个的氢键相互作用可能竞争性地抑制CXCR4与gp120的相互作用。我们的候选分子选择是基于与已知抑制剂 (IT1t) 的形状相似性、推定的结合模式以及与残基的相互作用，这些残基被确定对 HIV-1-gp120 引发的与 CXCR4 的细胞融合事件很重要。基于上述假设，我们从ChemBridgeTM通用筛选库中选择了16个化合物（命名为CX1至CX16）用于生物测定。我们询问这 16 种选定的化合物是否通过阻断 SDF-1α 诱导的细胞内 Ca2+ 动员而与 CXCR4 结合，SDF-1α 的主要受体是 CXCR4。其中一种化合物 CX6 可阻断 Molt4 细胞中 SDF-1α 诱导的 Ca2+ 动员，EC50 值为 92 nM。在同一测定中，AMD3100 显示出对 SDF-1α 诱导的 Ca2+ 动员的抑制作用，EC50 值约为 10 nM。另外两种化合物 CX11 和 CX13 都是哌啶乙胺 (PEA) 衍生物，如 CX6，可阻断 Molt4 细胞中 SDF-1α 诱导的 Ca2+ 动员，EC50 值分别为 161 和 149 nM。其他化合物均未与 CXCR4 结合。值得注意的是，所有三种 PEA 衍生物（CX6、CX11 和 CX13）均未能阻断 RANTES（其受体为 CCR5）诱导的 Ca2+ 动员，表明这些化合物不与 CCR5 结合。当我们询问这三种化合物是否具有诱导 CXCR4+ 细胞中 Ca2+ 动员的激动作用时，没有任何化合物诱导 Ca2+ 动员，表明 CX6、CX11 和 CX13 是 CXCR4 的拮抗剂。总之，上述数据强烈表明PEA衍生物与CXCR4特异性结合并且是CXCR4的拮抗剂。 HIV病毒包膜糖蛋白与CXCR4相互作用引发的融合事件使病毒能够进入细胞，最终导致病毒复制。通过对接模拟选择了 16 种化合物，表明它们与 CXCR4 的潜在正构结合位点结合。这些化合物与至少两个对融合事件最可能重要的氨基酸残基形成氢键，从而具有竞争性抑制融合事件的潜力。因此，我们确定这些化合物是否确实能够抑制 HIV-1NL4-3 包膜蛋白与 CXCR4 的相互作用，并阻止 HIV-1-gp120 引发的细胞与细胞融合测定中与野生型的融合事件CXCR4。 CX6 和 CX11 均阻断融合，EC50 值为 1.9 microM 和 7.9 microM。此外，包括 CX6 和 CX11 在内的化合物均未抑制融合事件，如使用表达源自 HIV-1BaL (R5-HIV- 1).这表明CX6和CX11抑制与CXCR4相关的融合事件，但不抑制与CCR5相关的融合事件。 CX6拥有出色的外形谷本重叠系数为0.73。 IT1t 的咪唑并噻唑环与 CX6 的环戊基哌啶基重叠。通过分子对接推断了所鉴定的命中化合物 (CX6) 与 CXCR4 的相互作用。 CX6 的酚基团与位于 CXCR4 N 末端的 Glu-32 相互作用。 Asp97 已被证明对于 AMD070 的结合以及 CXCR4-gp120 引发的融合很重要。 Glu288 对于融合事件很重要，因为显示用丙氨酸取代 Glu288 会导致 CXCR4-gp120 引发的融合丧失。 TM7中6位的谷氨酸是趋化因子受体中高度保守的氨基酸残基，并已被证明对于非肽配体与趋化因子受体的结合非常重要。我们目前的研究表明，Glu288 是基于合理结构的 CXCR4 抑制剂设计和发现的重要靶标残基。我们使用 X4-HIV-1NL4-3 和 MT4 作为靶细胞，通过 MTT 测定测定了 CX6 的抗 HIV-1 活性和细胞毒性。 CX6 对 HIV-1NL4-3 和 HIV-1LAI 表现出显着的活性，这两种病毒都是 X4-HIV-1 毒株，EC50 值分别为 1.5 和 3.0 microM。 CX6 的细胞毒性 CC50 为 58 microM，治疗指数为 39。在使用 HIV-1NL-4-3 衍生的包膜蛋白进行的融合抑制测定中检测到，CX6 阻断了融合事件。在 Ca2+-通量抑制测定中，在存在或不存在潜在小分子抑制剂的情况下测定 SDF-1α 引发的 Ca2+-通量水平，发现 CX6 以 92 nM 的 EC50 值阻断通量。然而，正如预期的那样，在使用 HIV-1BaL 和 MAGI 细胞的抗 HIV 测定中检测到，CX6 未能阻断 R5-HIV-1 (HIV-1BaL)(EC50：10 microM) 的感染性和复制。 CX11和CX13同样对X4-HIV-1NL4-3和X4-HIV-1LAI发挥活性，但未能阻断R5-HIV-1BaL的感染性和复制。