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: 8937962
• 负责人: Hiroaki Mitsuya
• 金额: $ 28.45万
• 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/8937962
• 关键词: AMD3100; Active Sites; Affect; Affinity; Alanine; Amino Acid Substitution; Amino Acids; Antiviral Agents; Binding; Binding Sites; Bioavailable; Biological Assay; Biological Availability; CCR5 gene; CXCR4 gene; Cell fusion; Cells; Charge; Clinical; Complex; Computer software; Cyclic Peptides; Data; Databases; Disulfide Linkage; Docking; Drug Kinetics; Event; FDA approved; G-Protein-Coupled Receptors; HIV; HIV Envelope Protein gp120; HIV-1; Hydrogen Bonding; Infection; Inhibitory Concentration 50; Lead; Libraries; Ligands; Molecular; Molecular Conformation; Names; Nitrogen; Oral; Peptides; Phenols; Proteins; Publishing; Receptor Activation; Relative (related person); Reporting; Safety; Sequence Alignment; Shapes; Side; Site-Directed Mutagenesis; Specificity; Stromal Cell-Derived Factor 1; Structure; T140 peptide; Therapeutic; Transmembrane Domain; analog; base; carboxylate; design; disulfide bond; env Gene Products; extracellular; glycoprotein CX 1; improved; inhibitor/antagonist; interest; mutant; novel; piperidine; receptor; screening; simulation; small molecule; therapeutic target

We hypothesized that small molecules that bind to amino acid residues in the orthosteric binding site of CXCR4 would likely inhibit the CXCR4-gp120 interactions. To determine these residues, fifty-six amino acid residues in the extracellular and transmembrane regions of CXCR4 were selected for introducing amino acid substitution(s) and the HIV-1-gp120-elicited cell fusion levels were determined and compared to that of wild type CXCR4. The significance of some of the residues selected for substitution are as follows: Asp133, Arg134, and Tyr135 in TM-3 are the conserved DRY motif in various GPCRs and are reportedly important in triggering ligand-induced conformational changes that lead to receptor activation. The second extracellular loop (ECL2) is known to be important for the structure and function of CXCR4, CCR5 and other GPCRs; therefore, several residues in ECL2 were selected. Cys109 located in the extracellular region of TM3 forms a disulfide bond with Cys186 in ECL2. This disulfide linkage is conserved for class-A GPCRs. When we determined the changes in the magnitude of cell-cell fusion levels with wild type or mutant CXCR4, 11 amino acid(s) substitutions (D97A, Y116A, F174A, A175F, D182A, D187A, R188A, Y190A, D262A, E288A, and F292A) resulted in a substantial reduction in the fusion level by more than 30%. It is noteworthy that there are a number of negatively charged acidic residues (D97A, D182A, D187A, D262A, and E288A) whose substitution significantly decreased gp120 fusion. These acidic residues may interact with the basic residues of gp120 to affect co-receptor selectivity. We analyzed the crystal structures of CXCR4 to understand the interactions and orientation of the residues whose substitution adversely impacted the interactions of CXCR4 with the HIV-1-envelope protein. Six amino acid substitutions (F174A, A175F, D182A, D187A, R188A, and Y190A) that impacted the fusion event were identified in or near ECL2, strongly suggesting that this region as a whole probably affects fusion more than any other loop or transmembrane region of CXCR4. Site-directed mutagenesis studies have suggested that Asp262 is important for the binding of AMD3100. Glu288 (TM7) has a hydrogen bond with the side chain of Tyr116 (TM3) and an intra-helix hydrogen bond with Phe292 (TM7), and these three residues form part of the binding pocket within the transmembrane domain. The side chain of Asp97 forms part of the binding pocket for small molecules whereas the carboxylate side chain of Asp182 is oriented towards the extracellular region and away from the binding pocket located inside the transmembrane domain. Substitution of residues in TM5 or ECL3 did not affect the fusion event in the assay. We determined the shape similarity of the molecules, from the ChemBridge library to IT1t. The highest shape similarity Tanimoto coefficient to IT1t from the database was 0.85. Seven hundred fifty three (753) unique molecules with 1005 configurations/conformations had shape Tanimoto coefficient of at least 0.70. The binding mode and interactions of these molecules with CXCR4 were determined by molecular docking using Glide (version 5.6, Schrodinger, LLC). To avoid any issues that may arise through using conformations generated by Omega with Glide docking, Ligprep was used to generate molecular configurations and conformations for docking, and it generated 14,226 of those. These configurations/conformations were docked 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. Of note, we did not use any energy-based or empirical scoring functions for estimating the relative affinity for the selection of compounds. Based on the hypothesis described above, we selected sixteen compounds (named CX1 to CX16) for biological assays from ChemBridge general screening library (the minimum purity of the selected compounds was 90% or greater). Three most interesting compounds were piperidinylethanamine derivatives. When we asked whether the selected compounds bind to CXCR4 by blocking the intracellular Ca2+ mobilization induced by SDF-1alpha, the data strongly suggest that the PEA derivatives bind to CXCR4 with specificity and are antagonists of CXCR4. Subsequently, we newly synthesized CX6, and more than twenty PEA derivatives in high purity. The sixteen compounds were selected through docking simulations suggesting that they bound to the a potential orthosteric binding site of CXCR4, 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 IC50 values of 1.9 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), suggesting that CX6 and CX11 inhibit the fusion event associated with CXCR4 but not with CCR5. When we examined the three dimensional shape overlay of CX6 with IT1t, as determined by ROCS (version 3.0.0, OpenEye Scientific Software, the molecules 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 following amino acid residues of CXCR4 were seen to form the active site for the binding of CX6. Thus, the molecule CX6 bound in the active site predominantly formed by residues from transmembranes 1, 2, 3, 7, and ECL2. As expected, no amino acid residues of TM4, TM5, and TM6 had interactions with CX6. The nitrogens of both piperidine groups were determined to be protonated. For CX6, the protonated nitrogen of cyclopentylpiperidinyl formed hydrogen bond interactions with Glu-288 and the protonated nitrogen of piperidinylethanamine formed hydrogen bond interactions with Asp97. 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. In sequence alignment, the residue corresponding to Glu288 of CXCR4 is Glu283 of CCR5. Therefore, E283 for CCR5 should be important for the binding of CCR5 antagonist aplaviroc and its analogs, in line with previously published results. Indeed, the substitution of E283 of CCR5 resulted in loss of the CCR5-gp120-elicited fusion event, as previously described.

我们假设在CXCR4的正常结合位点与氨基酸残基结合的小分子可能会抑制CXCR4-GP120相互作用。为了确定这些残基，选择了CXCR4外细胞外和跨膜区域中的56个氨基酸残基，并确定了引入氨基酸取代的氨基酸取代，并确定了HIV-1-GP120释放的细胞融合水平，并与野生型的细胞融合水平进行了比较。 CXCR4。某些用于取代的残基的意义如下：TM-3中的ASP133，ARG134和Tyr135是各种GPCR中保守的干式基序，据报道对于触发配体诱导的构象变化而言至关重要，导致受体激活。已知第二个细胞外环（ECL2）对于CXCR4，CCR5和其他GPCR的结构和功能很重要。因此，选择了ECL2中的几个残基。位于TM3细胞外区域的Cys109在ECL2中与Cys186形成二硫键。该二硫键是为A类GPCR保留的。当我们确定用野生型或突变型CXCR4或11氨基酸替代的细胞细胞融合水平的变化时（D97A，Y116A，F174A，A175F，D182A，D182A，D187A，D187A，R188A，R188A，R188A，Y190A，Y190A，D262A，D262A，e2888a，and E2888a，and E2888a，and E2888a，and E2888a，and E2888a等F292a）导致融合水平的大幅度降低了30％以上。值得注意的是，有许多带负电荷的酸性残基（D97A，D182A，D187A，D262A和E288A）的取代大大降低了GP120融合。这些酸性残基可能与GP120的基本残基相互作用，以影响受体的选择性。我们分析了CXCR4的晶体结构，以了解替代的残基的相互作用和取向，其替代物对CXCR4与HIV-1-螺旋蛋白的相互作用产生了不利影响。在ECL2或附近发现了六个氨基酸取代（F174A，A175F，D182A，D187A，R188A和Y190A），在ECL2或附近发现了融合事件，这强烈表明该区域的整体可能比其他任何LOOP或跨膜区域都更大的融合影响CXCR4。位置定向的诱变研究表明，ASP262对于AMD3100的结合很重要。 GLU288（TM7）与Tyr116（TM3）的侧链（TM3）和与PHE292（TM7）的螺旋内氢键具有氢键，这三个残基构成了跨膜结构域内结合袋的一部分。 ASP97的侧链构成了小分子的结合袋的一部分，而ASP182的羧酸盐侧链则定向细胞外区域，并远离跨膜结构域内部的结合口袋。在TM5或ECL3中取代残基不会影响测定中的融合事件。我们确定了从Chembridge库到IT1T的分子的形状相似性。数据库对IT1T的最高形状相似性系数为0.85。具有1005个构型/构象的七百三十三个（753）独特的分子的形状系数至少为0.70。这些分子与CXCR4的结合模式和相互作用是通过使用Glide（版本5.6，Schrodinger，LLC）来确定的。为了避免通过使用欧米茄（Omega）与滑行对接产生的构象出现的任何问题，使用Ligprep来生成分子构型和对接的构象，并生成14,226个。将这些构型/构象停靠在CXCR4的晶体结构上，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，ASP187，ASP187，ARG188和Tyr190似乎对细胞融合事件显得很重要，因此，我们的糖尿病是重要的。在晶体结构中确定的活性位点，并与至少两个残基形成氢键相互作用可能会竞争抑制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）和假定结合模式的形状相似性以及与CXCR4与HIV-1-GP120引起的细胞融合事件非常重要的残基相互作用。值得注意的是，我们没有使用任何基于能量的或经验评分函数来估计化合物选择的相对亲和力。基于上述假设，我们从Chembridge通用筛选文库中选择了16种化合物（称为CX1至CX16）（称为CX1至CX16）（所选化合物的最低纯度为90％或更高）。三种最有趣的化合物是哌啶基乙胺衍生物。当我们询问所选化合物是否通过阻断由SDF-1Alpha诱导的细胞内Ca2+动员来结合CXCR4时，数据强烈表明，PEA衍生物具有特异性，并且是CXCR4的拮抗剂。随后，我们新合成CX6，并具有高度纯度的二十多种豌豆衍生物。通过对接模拟选择了16种化合物，这表明它们与CXCR4的潜在正常结合位点结合，形成至少两个氨基酸残基最有可能对融合事件很重要的氢键，从而有可能竞争性抑制融合事件。因此，我们确定化合物是否实际上能够抑制HIV-1NL4-3包膜蛋白与CXCR4的相互作用，并在HIV-1-GP120引用的细胞融合中阻断融合事件与野生型的融合事件CXCR4。 CX6和CX11都以1.9和7.9 microM的IC50值阻止了融合。此外，使用HIV-1-GP120引诱的细胞融合测定法检查了包括CX6和CX11在内的任何化合物都抑制了融合事件，该化合物使用表达野生型CCR5源自HIV-HIV-1BAL的细胞（R5-HIV--1BAL（R5-HIV-） 1），表明CX6和CX11抑制与CXCR4相关的融合事件，但与CCR5无关。 When we examined the three dimensional shape overlay of CX6 with IT1t, as determined by ROCS (version 3.0.0, OpenEye Scientific Software, the molecules had an excellent shape Tanimoto overlap coefficient of 0.73. The imidazothiazole ring of IT1t overlays with the cyclopentylpiperidinyl group of CX6与CXCR4相互作用（CX6）是通过分子对接推导的。由跨膜1、2、3、7和ECL2的残基形成，环戊烯基的质子氮与GLU-288形成氢键相互作用，哌啶基乙胺的质子化氮与ASP97相互作用的CX6与GLU-32相互作用。 ASP97已被证明对于AMD070以及CXCR4-GP120引起的融合至关重要。 GLU288对于融合事件很重要，因为它表明替代丙氨酸的GLU288会导致CXCR4-GP120诱导的融合的损失。在序列比对中，与CXCR4的GLU288相对应的残基为CCR5的GLU283。因此，对于CCR5而言，E283对于CCR5拮抗剂Aplaviroc及其类似物的结合至关重要，与先前发表的结果一致。实际上，如前所述，CCR5的E283的取代导致CCR5-GP120引起的融合事件的损失。