基于多价态结合理论及高效合成筛选技术发现新型HIV-1 gp120三聚体抑制剂

HIV-1 envelope glycoprotein gp120 is displayed as a trimeric complex on the surface of virion and infected T-cells. Binding of gp120 to the CD4 receptor is the first step in the HIV-1 infectious cycle, making it a typical and validated multivalent drug target to prevent HIV-1 entry to cells. Yet no drugs against gp120 have been approved by the US FDA to date. Therefore, there is a critical need to develop novel drugs against this target. The goal of this proposal is to take advantage of the structural symmetry of multimeric gp120 protein, a rarely explored property, to arrive eventually at structurally complementary multidentate gp120 ligands as novel HIV-1 entry inhibitors with ultra-high affinity and specificity, using a multidisciplinary approach (multivalent concept, fragment-based assembly using bioorthogonal reactions combined with in situ screening, structure-based design and comprehensive medicinal chemistry). In this project, the conserved CD4 binding site (and the adjacent allosteric sites) on HIV-1 trimeric gp120 was selected as target for drug design to discover highly specific and effective HIV-1 entry inhibitors. Using the representative gp120 inhibitors as model “Binders”, then the multivalent inhibitors were created, in which three gp120 binders were tethered to scaffold component by a “Linker” of varied length. By the hierarchical multiple-filter (binding affinity, drug-like profiles) virtual database searching strategy to predict the favorable molecules from a large collection of triazoles, the top-ranking compounds will be selected and be dissected into a set of alkyne-containing scaffolds and azide-containing binders fragments. Then, a library of hundreds of triazoles between diverse alkynes and azides are rapidly constructed using CuAAC or SPAAC reaction and evaluated their anti-HIV activity and toxicity studies using a cell culture assay without purification (in situ screening) to find hit molecules. From the preliminary inhibition data, the most potent library members were individually re-synthesized in large-scale for complete characterization and further identification of their mechanism of action to find high-quality lead compounds. Subsequently, the binding modes of selected lead compounds will be investigated by X-ray co-crystal structure of the multivalent inhibitors and the trimeric gp120 complex. This work will develop the key technology that discovery of Polymeric proteins regulate molecules using compounds library construction and in situ screening, which contribute to discovery of multi-valent gp120 inhibitors.

HIV-1 gp120已成为抗艾滋病药物研究的重要靶标。本项目基于前期gp120抑制剂研究基础，根据结构生物学研究进展及多价态结合理论，以HIV-1 gp120对称三聚体作为药物设计靶标，提出“利用gp120三聚体的对称性，可设计超高亲和力和特异性的多价态配体，导致gp120完全失活”的假说。为验证该假说，选取具有开发前景的抑制剂为优势片段（Binder），运用基于生物正交反应的高效合成及原位筛选技术，构建Binder-Linker-Scaffold片段组合库并进行细胞水平抗病毒及毒性评价，发现苗头化合物；经作用机制研究（时间-过程实验、细胞融合、gp120亲和力、gp120-CD4结合）、结构生物学研究以及多轮循环结构优化发现活性显著和靶标明确的先导化合物。同时，形成运用化合物库构建及原位筛选发现多聚体蛋白调控分子的关键技术，为发现靶向gp120的多价态抗艾滋病药物奠定基础。

尽管目前已有30余种抗艾滋病药物被批准上市，但由于病毒自身的遗传异质性和基因组的高度变异性导致的HIV-1耐药问题严重影响这些药物的临床疗效。因此，病毒耐药问题是抗艾滋病药物治疗面临的严峻挑战。本研究一方面瞄准具有潜力的新靶标HIV-1 gp120，以苯基草酰胺类化合物为先导，基于其与靶标的共晶结构分析对其进行系统的结构改造，发现活性优于先导NBD556（EC50 = 3.61 μM）的化合物LL-2（EC50 = 1.9 μM）与LL-12（EC50 = 3.27 μM）；此外，本研究以以在HIV-1生命周期中发挥重要作用的逆转录酶为靶标，，基于结构生物学新进展以及高效抗耐药性的药物设计策略对其进行精准药物设计，发现了多个对HIV-1野生株和常见突变株均具有纳摩尔活性的先导化合物，经初步的成药性发现了4个具有良好成药性质的抗HIV-1候选药物。此外，对化合物16b进行了结构生物学研究，为基于共晶结构的精准药物设计奠定了结构生物学基础。.在本项目的资助下，在药物化学主流期刊共发表了标注的相关SCI论文11篇，影响因子均大于5，其中有6篇发表在J Med Chem上；授权中国发明专利3项，申请PCT专利3项。项目负责人康东伟2022年获得了国家自然科学基金面上项目（编号：82273773）与山东省优秀青年项目（编号：ZR2020YQ61）的资助、第一届中国博士后创新创业大赛优胜奖（2021）等。此外，培养了多博士研究生1名与硕士研究生2名。

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