Design of inhibitors targeted to the CD4 binding site on HIV-1 gp120

描述(申请人提供)：HIV-1进入宿主细胞是由包膜糖蛋白gp120与宿主细胞受体CD4结合介导的。Gp120-CD4-17b复合体的结构表明，gp120上的CD4结合位点(CD4BS)位于一个疏水空腔中，称为“Phe43空腔”，Phe43CD4在那里进行关键的疏水接触。对这些结构的研究也证实了Arg59CD4和Asp368gp120之间存在重要的静电相互作用。最近报道了几种有效的广谱中和抗体(BNAbs)的类似结构模仿。Gp120的CD4BS残基的高度保守性以及它们在结合CD4和CD4BS靶向的bNAbs中的作用证实了该位点是一个有希望的治疗和预防干预的靶点。尽管HIV-1包膜糖蛋白序列、糖基化和构象灵活性有显著的多样性，但gp120必须保留与CD4结合所需的保守结构域。然而，保存完好的CD4结合位点在设计预防HIV-1进入的药物时尚未充分发挥其潜力，这将是我们研究的重点。我们首次发现了两种针对gp120上CD4BS的小分子抑制剂NBD-556和NBD-557。这些分子表现出前所未有的能力，可以诱导gp120的构象变化，类似于CD4的构象变化，从而发挥激动剂的作用。最近，我们解开了NBD-556与gp120核心的复合体的结构，证实了NBD-556与gp120的空腔结合。我们利用这种结构设计了具有更好的抗HIV-1活性的新线索。然而，这些先导化合物显示出了CD4激动剂的特性。在进一步的研究中，我们证实了一种新设计的先导化合物nbd-11021是一种cd4拮抗剂。此外，最近的一份报告显示，bNAb PGV04靶向CD4BS，但诱导gp120发生不被17b抗体识别的明显构象变化。因此，PGV04不会引起辅受体结合部位的暴露，这表明它是一种CD4拮抗剂，而不是激动剂。我们假设，包括NBD-11021在内的先导化合物可以通过结合PGV04与gp120的独特结合特征来优化，以产生一种更有效和更有选择性的具有CD_4拮抗剂特性的新型艾滋病毒-1进入抑制剂。拟议研究的目标将通过三个高度整合的具体目标来实现。在AIM-1中，将通过结构指导设计和药物化学来优化引线。在AIM-2中，将测量热力学和动力学性质，如热、熵、开和关速率和结合亲和力(Kd)，以及它们的抗病毒效力和毒性。新型缓蚀剂的X-射线结构 在与gp120的复合体中将被测定。在AIM-3中，将研究作用机制并选择抗药性突变体。这些研究的数据将被用于设计有效的下一代进入抑制剂，这些抑制剂可能会逃脱耐药性，并具有临床意义。长期目标是开发一种新型的HIV-1进入抑制剂，用于治疗Pre 临床和临床研究。

DESCRIPTION (provided by applicant): The entry of HIV-1 into host cells is mediated by the binding of the envelope glycoprotein gp120 to the host cell receptor CD4. The structures of the gp120-CD4-17b complex indicate that the CD4 binding site (CD4BS) on gp120 is located in a hydrophobic cavity, termed "Phe43 cavity" where Phe43CD4 makes critical hydrophobic contacts. Studies of these structures also confirmed an important electrostatic interaction between Arg59CD4 and Asp368gp120. Similar structural mimicry of several potent broad neutralizing antibodies (bNAbs) has recently been reported. The highly conserved nature of the residues in the CD4BS of gp120 and their role in binding to CD4 and CD4BS targeted bNAbs validates this site as a promising target for therapeutic and prophylactic intervention. Despite th remarkable diversity of the HIV-1 envelope glycoprotein sequence, glycosylation and conformational flexibility, gp120 must retain conserved domains required for binding to CD4. However, the well-conserved CD4 binding site has yet to be exploited to its full potential as a target in designing drugs to prevent HIV-1 entry, which will be the focus of our research. We were the first to identify two small molecule inhibitors, NBD-556 and NBD-557, which target the CD4BS on gp120. These molecules show unprecedented ability to induce conformational changes in gp120 similar to that of CD4; thereby, acting as agonists. Recently, we solved the structure of NBD-556 in complex with the gp120 coree, confirming that NBD-556 binds to the cavity of gp120. We used this structure to design new leads with improved anti-HIV-1 activity. However, these lead compounds showed CD4 agonist properties. In additional studies, we confirmed that a newly designed lead, NBD-11021, is a CD4 antagonist. Moreover, a recent report showed that the bNAb PGV04 targets the CD4BS but induces distinct conformational changes in gp120 that are not recognized by the 17b antibody. PGV04 thus does not induce exposure of the coreceptor binding site, suggesting that it is a CD4 antagonist not an agonist. We hypothesize that lead compounds, including NBD-11021, can be optimized by incorporating the distinct binding features of PGV04 with gp120 to produce a more potent and selective new class of HIV-1 entry inhibitors with CD4 antagonist properties. The goals of the proposed studies will be accomplished by three highly integrated specific aims. In Aim-1, the leads will be optimized through structure-guided design and medicinal chemistry. In Aim-2, thermodynamic and kinetic properties such as enthalpy, entropy, on- and off-rates and binding affinity (KD) will be measured as well as their antiviral potency and toxicity. The x-ray structures of new inhibitors in complex with gp120 will be determined. In Aim-3, mechanism of action will be studied and the resistant mutants of the inhibitors will be selected. The data from these studies will be used in designing potent next-generation entry inhibitors that will potentially escape resistance and be clinically relevant. The long-term goal is to develop a new class of HIV-1 entry inhibitors for pre clinical and clinical studies.