二聚多羥基吡咯啶作為酵素穩定劑並應用於法布瑞氏症

Abstract

如何設計小分子作為特定的酵素穩定劑是化學和化學生物學中的挑戰之一。 在我們以前的研究中，糖的類似物或亞氨基糖有潛力能藉由與糖苷酶活性位點的結合而抑制因不利的培養基或環境（例如pH值或溫度變化）引發的蛋白質變性。 眾所周知，包括溶酶體α-半乳糖苷酶A（α-Gal A）在內的幾種糖苷酶均顯示為同源二聚體。因此，我們提出了開發通過透過合適連結的二聚小分子成為下一代新型蛋白質穩定劑的假設。 法布瑞氏症（Fabry disease）是一種遺傳的溶酶體代謝性疾病，肇因於於溶酶體α-半乳糖苷酶A（α-Gal A）的活性不足，導致一些醣神經胺醇脂 （glycosphingolipid），特別是globotriaosylceramide（Gb3）在患者細胞內積聚。利用基因重組之人類α-半乳糖苷酶A（rh-α-Gal A）的酵素替代療法（enzyme replacement therapy, ERT）是法布瑞氏症的標準治療方法，儘管大多數患者都採用這種方式進行治療，但由於成本高昂和的不穩定，ERT受到了嚴重限制。因此使蛋白質藥物在療程中穩定的新策略或新的治療方法是迫切需要的。本文提出了將基因重組之人類α-半乳糖苷酶A與小分子酵素穩定劑共同施用的策略以提昇ERT的療效並降低其成本。 在本文中，我們想展示一種應用於新一代酵素穩定劑開發的新策略，即在適當連接單元的兩端與兩個活性位點結合劑結合。作為該概念的證明，我們基α-半乳糖苷酶A固有的同源二聚性質和治療重要性將其選作為我們的研究目標。因此，以具保護基的鏡像純環硝酮為起點，設計並製備了六個帶有不同長度連接單元的雙-多羥基化吡咯啶，其中通過路易斯酸促進的不對稱斯特雷克反應（Strecker reaction）將α-氨基腈（α-aminonitrile）部分引入吡咯啶骨架的C-2位為關鍵轉換，後續通過肽鍵（amide bond）的形成將連接單元的兩端與兩個受保護的亞氨基糖的末端一級胺進行接合，然後全去保護以生成我們所需的分子。這些分子通過基於熒光的熱變性試驗用於穩定性研究。幸運的是，令人鼓舞的結果支持了我們的假設。 我們也通過細胞層級的生物試驗對這些雙亞氨基糖進行了進行評估，其結果顯示基因重組之人類α-半乳糖苷酶A以及法布瑞氏症相關之突變α-半乳糖苷酶A能被有效的二聚酵素穩定劑顯著穩定。在與二聚穩定劑12-B和重組之人類α-半乳糖苷酶A的共同給藥下，觀察到法布瑞氏症患者來源的細胞中酵素活性比僅以重組之人類α-半乳糖苷酶A給藥的組別增強3.4倍。同樣，我們也觀察到了以二聚酵素穩定11-B，12-B和13-B分別對法布瑞氏症患者來源的淋巴細胞進行給藥後，酵素活性提高了9倍，代表二聚酵素穩定劑也可作為藥理伴護小分子（pharmacological chaperones）。

How to design small molecules as specific enzyme stabilizers is one of the challenges in chemistry and chemical biology. In our previous works, synthetic sugar mimics or iminosugars binding to the active site of glycosidases, might potentially suppress the protein denaturation resulting from environment stress such as pH value or temperature change. As we know, several glycosidases including lysosomal α-galactosidase A are homo-dimeric; therefore, our hypothesis is whether it is feasible to develop dimeric small molecules linked by a proper spacer to become the next generation of new protein stabilizers or not. Fabry disease is a lysosomal metabolic disorder due to insufficient enzyme activity of lysosomal α-galactosidase A, resulting in the accumulation of globotriaosylceramide (Gb3) in patient cells. Enzyme replacement therapy (ERT) with recombinant human α-Gal A (rh-α-Gal A) is the standard treatment for FD. Although most patients are being treated in this way, ERT is severely limited due to the high cost and circulatory unstableness of protein drugs. Therefore, a new strategy for enzyme stabilization during treatment is urgently needed. Herein a cost-cutting strategy was put forward to enhance the efficacy of ERT by co-administration of rh-α-Gal A with enzyme stabilizers. In this thesis, we would like to demonstrate a new strategy for developing the next-generation enzyme stabilizers by conjugating at the both ends of the proper spacer with two individual active-site binders. As a proof of this concept, α-Gal A was selected as our target protein due to its intrinsic homodimeric property and therapeutic importance. Hence, six bis-polyhydroxylated pyrrolidines bearing PEG spacers with a variant length were designed and prepared from the enantiopure protected cyclic nitrone as the starting point. Notably, the α-aminonitrile moiety was introduced at C-2 position of pyrrolidine scaffold via Lewis acid-promoted asymmetric Strecker reaction as the key transformation. The terminal free amine of the protected iminosugars were conjugated at the both ends of the linker through the amide bond formation followed by global deprotection to generate our desired molecules. These molecules were applied for the stabilization study by a fluorescence-based thermal denaturation assay; fortunately, the encouraging results supported our hypothesis to be a new type protein stabilizer. Next, the co-adminstration of qualified candidates with rh-α-Gal A, compared with the treatment of enzyme only, was further investigated in the cell-based study. The best hit, 12-B, revealed a 3.4-fold enhancement of enzyme activity toward Fabry W162X fibroblasts. For the investigation of chaperone effect, the single treatment of individual 11-B, 12-B, or 13-B to Fabry N215S lymphocytes showed a 9-fold enhancement of enzyme activity.