以溶酶体Heparanase介导自噬通路为靶标设计、合成硫酸肝素类抑制剂及其逆转骨髓瘤化疗耐药研究

Drug resistance to multiple myeloma prevails clinically, and its resistance is closely related with high expression of heparanse (HPSE) induced by chemotherapy. In the past, HPSE inhibitors restrained mainly HPSE function of extracellular matrix to block angiogenesis and tumor metastasis, completely neglecting the inhibition of HPSE within intracellular to result in HPSE function to "escape" that affecting tumor resistance because of structure limitation. In our preliminary work, we have prepared thioglycoside-containing heparan sulfates as HPSE inhibitors, and it proved that they could reverse drug resistant RPMI8226 cell via enhanced autophagy mediated by lysosome HPSE. To guarantee targeting and penetration of intracellar HPSE inhibitors, in this project lysosome HPSE mediated autophagy pathways is taken as an therapeutic target, new heparan sulfate-like HPSE inhibitors will be designed and synthesized to reverse chemoresistant cells. Specially speaking, we will use HPSE-based enzymolysis mechanism as a guide and disaccharide repeating unit of heparan sulfate as a lead structure, together with attachment of biocompatible, fat-soluble and non-toxic fragments or carriers to HPSE inhibitors using unique sulfur-containing structure, favoring crossing cell membrane. Chemoenzymatic approach will be adopted to synthesize HPSE inhibitors, and 14C isotope and Cy5 fluorescence will be used to trace their transmembrane process and intracellular distribution respectively, and specificity of HPSE inhibitors will be evaluated with mammal HPSE and antithrombin as enzymatic models, and ultimately myeloma cells with high expression of HPSE and its in vivo models were applied to evaluate their reversal ability of resistance. Additionally, mechanism of drug resistance was also explored. In summary, this project will not only open an avenue to develop a new generation of HPSE inhibtiors, also provide a novel direction and mechanism for reversing clinical drug resistance.

化疗诱导肝素酶（Heparanase, HPSE）高表达是多发性骨髓瘤产生耐药的主要原因，目前HPSE抑制剂因结构所限无法穿膜入胞，主要抑制胞外基质中HPSE，阻止肿瘤血管形成与转移，而HPSE经溶酶体加工活化后诱导自噬增强可能导致产生耐药性，我们前期制备得到的与溶酶体HPSE诱导自噬增强密切有关的硫代糖苷硫酸肝素类HPSE抑制剂，能有效逆转RPMI8226细胞耐药作用。为增加硫酸肝素类HPSE抑制剂的入胞能力与胞内靶向性，以便深入研究胞内HPSE诱导自噬与耐药相关性，本项目拟以溶酶体HPSE介导自噬通路为靶标，基于HPSE酶解作用机制设计新型逆转耐药硫酸肝素类HPSE抑制剂，采用酶化学方法合成，以高表达HPSE骨髓瘤细胞及其体内模型评价其逆转耐药能力，并探讨其逆转HPSE诱导自噬耐药作用机制，为研发新一代HPSE抑制剂另辟蹊径，也为逆转临床耐药研究提供新机制和新方向。

化疗诱导肝素酶（Heparanase, HPSE）高表达是多发性骨髓瘤产生耐药的主要原因，目前HPSE抑制剂因结构所限无法穿膜入胞，主要抑制胞外基质中HPSE，阻止肿瘤血管形成与转移，而HPSE经溶酶体加工活化后诱导自噬增强可能导致产生耐药性。本项目以溶酶体HPSE介导自噬通路为靶标，基于HPSE酶解作用机制设计新型逆转耐药硫酸肝素类HPSE抑制剂，采用酶化学方法合成，以HPSE高表达的骨髓瘤细胞及其体内模型评价其逆转耐药能力，并探讨其逆转HPSE诱导自噬耐药作用机制。通过项目实施，构建高质量的哺乳源HPSE表达制备系统、HPSE抑制剂WST-1荧光高通量筛选平台以及慢病毒转染HPSE高表达RPMI-8226细胞株，制备4个含巯基或氧亚甲基的葡萄糖胺重要糖构建模块，设计合成19个入胞硫酸肝素类HPSE抑制剂。构效关系发现，硫原子或氧亚甲基取代糖苷中氧原子增强硫酸肝素对HPSE耐受性，高硫酸化能强化其对HPSE抑制作用，还原端引入亲脂性片段如棕榈酸、胆酸等有利于其穿膜入胞。候选化合物HSAI9能抑制体内外HPSE高表达骨髓瘤RPMI-8226生长转移，协同逆转体内外骨髓瘤细胞对硼替佐米耐药。Cy5.0标记HSAI9具有体内肿瘤靶向性，与体外相应的骨髓瘤细胞摄取趋势一致。逆转耐药机制研究发现，HPSE过表达增强自噬标志蛋白LC3-B的表达，形成更多自噬小体，加剧骨髓瘤细胞耐药，抑制HPSE，LC3-B显著降低，但其不完全消失，暗示HSAI9部分逆转骨髓瘤因自噬途径引发耐药，项目取得研究结果为研发新一代HPSE抑制剂另辟蹊径，也为逆转临床耐药研究提供新机制和新方向。