Abstract 4: Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4

Abstract Introduction: CXCR4 is a member of chemokine receptor and G protein coupled receptor (GPCR) families. Its interaction with the chemotactic ligand stromal cell-derived factor 1 (SDF-1 or CXCL12) plays important roles in physiological and pathological processes. Recent studies have showed that the blockade of CXCL12-CXCR4 interaction by small molecules can be used in several clinical applications, including sensitizing cancer cells to chemotherapy, mobilizing hematopoietic stem cells (HSCs) to the blood for HSC collection, and inhibiting the tumor metastasis. To date, AMD3100 (Plerixafor) is the only clinically approved drug used in combination with G-CSF for mobilizing HSCs. Here, we report the discovery and development of a new class of highly potent small molecule CXCR4 inhibitors which can strongly antagonize CXCL12-CXCR4 interaction and mobilizing HSCs. Methods: Based on the crystal structures of CXCR4 showing a major and minor subpockets, we applied a fragment-based combinatorial design (FBCD) strategy to design a new class of hybrid molecules to recognize these two subpockets. A series of designed compounds were chemically synthesized and tested in a panel of biological assays including competitive CXCR4 binding, chemotaxis, calcium mobilization and CXCR4 internalization to characterize their biological activities. Furthermore, site-directed mutagenesis coupled with molecular docking simulation was carried out to determine the binding modes of the most potent compounds. Results: More than 30 compounds were synthesized and characterized. Among them, compound HFX51116 showed the most potent CXCR4 binding activity (IC50 = 12.2 nM), which was more potent than AMD3100 tested in parallel (IC50 = 325.3 nM). Furthermore, HFX51116 displayed remarkable inhibition of calcium mobilization (IC50 = 1.478 nM) and chemotaxis activity (IC50 = 252.7 nM), both of which are better than those of AMD3100. In addition, mutational studies and molecular docking simulation suggested that HFX51116 recognizes the minor subpocket mainly and major subpocket partially. Conclusions: Our findings have demonstrated that HFX51116 is a promising novel lead compound for developing a new class of small molecule therapeutics targeting CXCR4. Citation Format: Xiong Fang, Qian Meng, Xiao Fang, Siyu Zhu, Yazi Huang, Jing An, Yan Xu, Ziwei Huang. Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4.