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: 8763348
• 负责人: Hiroaki Mitsuya
• 金额: $ 9.89万
• 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/8763348
• 关键词: Affinity; Amino Acid Substitution; Amino Acids; Anti-Retroviral Agents; Antiviral Agents; Binding; Biological; Biological Assay; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell fusion; Cells; Clinical; Clinical Trials; Collaborations; Complex; Coupled; Cyclopentane; Data; Development; Disease; Docking; Dose; Evaluation; FDA approved; GTP-Binding Proteins; HIV; HIV Envelope Protein gp120; HIV-1; Human; Hydrogen Bonding; Individual; Infection; Inhibitory Concentration 50; Intervention; Japan; Lead; Libraries; Ligand Binding; Literature; Manuscripts; Maps; Modeling; Molecular; Molecular Conformation; Molecular Models; Patients; Pharmacologic Substance; Phase; Phenotype; Preparation; Proteins; Publishing; Regimen; Relative (related person); Reporting; Resistance; Shapes; Site-Directed Mutagenesis; Specificity; Stromal Cell-Derived Factor 1; Structural Models; Structure; Structure-Activity Relationship; Tablets; Testing; Therapeutic; Transmembrane Domain; Treatment Failure; United States; Universities; Variant; Viral; Viremia; analog; chemokine; design; inhibitor/antagonist; insight; liver injury; member; molecular dynamics; molecular modeling; molecular shape; novel; piperidine; prevent; professor; receptor; scaffold; screening; simulation; small molecule; virtual

1. Determination of binding mode of newly developed CCR5 inhibitors with CCR5 and elucidation of structure-activity relationships. We previously examined the interactions between CCR5 and CCR5 inhibitors containing the SDP scaffold, AK530 and AK317, both of which were docked into the hydrophobic cavity located between the upper transmembrane domain and ECL2 of CCR5. The data showed the lipophilic potential mapped on the binding cavity of CCR5 and relative binding modes of AK530 and AK317. Molecular dynamics simulations for inhibitor-unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants. We continued the design, synthesis, and evaluation of different CCR5 inhibitors in collaboration with Professor Ghosh of Purdue University. We initially started from a published CCR5 antagonist from the literature, and built structural models of its interaction with CCR5. Over the last five years, the design, synthesis and biological evaluation of more than 100 inhibitors has been carried out and several novel and potent inhibitors of CCR5 have been discovered in our study. Identified CCR5 antagonists include GRL-117, GRL-10007, and GRL-10018, which proved to be active against R5-HIV-1 with antiretroviral IC50 values of 0.6 nM, 1.4 nM and 2.9 nM, respectively. We built structural models of such newly identified CCR5 inhibitors to gain insight to the mechanism of potency of such inhibitors. In a model of the interaction of GRL117, GRL117 binds in a cavity formed within the transmembrane helices and ECL2. The models suggest that GRL117 has polar interactions with Y37, C178, K191, and T195. Y37 is located in transmembrane-1 (TM-1), K191 and T195 are located in TM-5. C178 is located in ECL2 and is highly conserved amongst class-A GPCRs. These residues are also important for gp120 fusion, and for the binding of APL and other CCR5 inhibitors. We postulated that interactions of inhibitors with CCR5 residues that are important for gp120 fusion may cause conformation change in CCR5, and may represent a salient molecular mechanism enabling allosteric inhibition. The interactions of GRL117 with such residues might be responsible for its potent antiretroviral IC50 of 0.6 nM. 2. Study of impacts of amino acid substitutions on CD4/gp120-CXCR4-induced fusion and identification of lead CXCR4 inhibitors. We attempted to examine the impacts of amino acid substitution on CD4/gp120-CXCR4-induced fusion by introducing amino acid substitutions into human wild-type CXCR4 (CXCR4WT) and determined the changes in fusion levels, compared to the fusion activity of CXCR4WT (referenced as 100%). We found that amino acid substitutions D97A, D262A, and E288A located in different transmembrane domains resulted in substantial loss of fusion activity. These residues have also been shown to be important for the binding of different CXCR4 inhibitors. As expected, various amino acid substitutions in ECL2, such as A175F, D182A, D187A, R188A and Y190A also resulted in compromised fusion. The availability of the crystal structure of CXCR4 with a small molecule inhibitor greatly aided our efforts to identify lead CXCR4 inhibitors. Out of an initial selection of 16 molecules with virtual screening of 622,000 different molecules, we identified cyclopentane-piperidine analogues as having anti-HIV activity (Das, Maeda, Hayashi, and Mitsuya: manuscript in preparation). 3. Optimization and development of potent CXCR4 inhibitors specific to CXCR4. CX-6 and other derivatives that inhibited HIV replication in MTT assay also blocked the biding of SDF-1alpha to CXCR4, suggesting that the derivatives inhibit HIV replication through CXCR4. Hence, we started collaboration with Professor Ghosh in optimizing the CX-6 scaffold. As of now, the group of Dr. Ghosh has synthesized more than 100 derivatives as candidates of CXCR4 inhibitors and all molecules were examined for their anti-HIV activity using MTT assay with X4-HIV (HIV-1NL4-3). Among them, 60 molecules that have cyclopentane-piperidine scaffold were tested and several compounds had decent anti-HIV activity with IC50 values of less than 1 micro M. We also started to synthesize molecules that have some other scaffolds as core structures. After optimization, we have identified several potent CXCR4 inhibitors that have tetrahydroquinoline scaffolds as a core structure. Among them, GRL-13004CX was most potent with IC50 value of 0.2 nM in MTT assay, followed by GRL-12066CX (IC50: 3 nM) and GRL-13006 (IC50: 3 nM). We also identified several compounds that have IC50 values less than 100 nM. GRL-13004CX and other derivatives were examined for other anti-HIV assays. They showed activity in MAGI assay and cell-cell fusion assay using X4-HIV-1 or an X4-HIV envelope (gp120), but they were not active when R5-HIV was used for MAGI assay, suggesting that these compounds are active only for X4-HIV-1. The ability of chemokine binding inhibition was also tested and it was found that all tested compounds inhibited the SDF-1alpha binding to CXCR4. From these findings, we concluded that the GRL-13004CX and other derivatives are CXCR4-spesific anti-HIV inhibitors. Moreover, it is considered that the potency of GRL-13004CX is the strongest among all CXCR4 inhibitors currently reported.

1。确定具有CCR5的新开发的CCR5抑制剂的结合模式和结构活性关系的阐明。我们先前检查了含有SDP支架，AK530和AK317的CCR5和CCR5抑制剂之间的相互作用，它们都停靠在CCR5的上跨膜域和ECL2之间的疏水腔中。数据显示，映射在CCR5的结合腔以及AK530和AK317的相对结合模式上的亲脂势。抑制剂unbound CCR5的分子动力学模拟显示跨膜残基Y108，E283和Y251之间的氢键相互作用，这对于HIV-1-GP120/SCD4复杂结合和HIV-1融合至关重要。数据不仅应帮助描述抑制剂结合后CCR5的动力学，而且还有助于设计CCR5抑制剂，这些抑制剂对HIV-1更有效，并防止或延迟抗性HIV-1变体的出现。我们继续与普渡大学的戈什教授合作，继续对不同CCR5抑制剂的设计，合成和评估。我们最初是从文献中发表的CCR5拮抗剂开始的，并建立了与CCR5相互作用的结构模型。在过去的五年中，已经对100多种抑制剂进行了设计，合成和生物学评估，并且在我们的研究中发现了几种新型和有效的CCR5抑制剂。鉴定出的CCR5拮抗剂包括GRL-117，GRL-10007和GRL-10018，事实证明，抗逆转录病毒IC50值分别为0.6 nm，1.4 nm和2.9 nm，对R5-HIV-1具有活性。我们建立了这种新鉴定的CCR5抑制剂的结构模型，以了解这种抑制剂的效力机理。在GRL117相互作用的模型中，GRL117在跨膜螺旋和ECL2内形成的空腔中结合。这些模型表明GRL117与Y37，C178，K191和T195具有极性相互作用。 Y37位于Transmmbrane-1（TM-1），K191和T195位于TM-5中。 C178位于ECL2中，在A类GPCR中是高度保守的。这些残基对于GP120融合以及APL和其他CCR5抑制剂的结合也很重要。我们假设抑制剂与CCR5残基对GP120融合很重要的相互作用可能会导致CCR5的构象变化，并且可能代表具有变构抑制的显着分子机制。 GRL117与此类残基的相互作用可能导致其有效的抗逆转录病毒IC50为0.6 nm。 2。研究氨基酸取代对CD4/GP120-CXCR4诱导的融合的影响以及铅CXCR4抑制剂的鉴定。我们试图通过将氨基酸取代引入人类野生型CXCR4（CXCR4WT）中，以检查氨基酸取代对CD4/GP120-CXCR4诱导的融合的影响，并确定与CXCR4WT的融合活性相比，并确定了融合水平的变化（为100％）。我们发现，位于不同跨膜结构域中的氨基酸取代D97A，D262A和E288A导致融合活性的大量丧失。这些残基也已被证明对于不同CXCR4抑制剂的结合至关重要。正如预期的那样，ECL2中的各种氨基酸取代，例如A175F，D182A，D187A，R188A和Y190A，也导致融合受损。用小分子抑制剂的CXCR4晶体结构的可用性极大地帮助了我们识别铅CXCR4抑制剂的努力。在最初选择16个分子和622,000个不同分子的16个分子的选择中，我们确定环戊烷 - 彼得丁丁类似物具有抗HIV活性（DAS，Maeda，Hayashi和Mitsuya：制备中的手稿）。 3。优化和开发CXCR4特定的有效CXCR4抑制剂。在MTT分析中抑制HIV复制的CX-6和其他衍生物也阻止了SDF-1Alpha对CXCR4的竞标，这表明衍生物通过CXCR4抑制HIV复制。因此，我们开始与Ghosh教授合作，以优化CX-6脚手架。截至目前，Ghosh博士已经合成了100多个衍生物，因为使用X4-HIV（HIV-1NL4-3）检查了CXCR4抑制剂的候选和所有分子的抗HIV活性。其中，测试了60个具有环戊烷 - 二副氨酸支架的分子，并且几种化合物具有不错的抗HIV活性，IC50值小于1微米。我们还开始合成具有其他脚手架作为核心结构的分子。优化后，我们确定了几种具有四氢喹啉支架作为核心结构的有效CXCR4抑制剂。其中，GRL-13004CX在MTT分析中最有效，IC50值为0.2 nm，其次是GRL-12066CX（IC50：3 nm）和GRL-13006（IC50：3 nm）。我们还确定了几种具有小于100 nm的IC50值的化合物。检查了GRL-13004CX和其他衍生物的其他抗HIV分析。他们使用X4-HIV-1或X4-HIV包膜（GP120）显示了在MAGI分析和细胞细胞融合测定中的活性，但是当使用R5-HIV用于MAGI测定时，它们并不活跃，这表明这些化合物仅活性对于X4-HIV-1。还测试了趋化因子结合抑制的能力，发现所有测试化合物都抑制了SDF-1Alpha与CXCR4的结合。从这些发现中，我们得出的结论是，GRL-13004CX和其他衍生物是CXCR4-Spesific抗HIV抑制剂。此外，认为GRL-13004CX的效力是目前报道的所有CXCR4抑制剂中最强的。