發展選擇性SGLT2抑制劑之非放射性細胞篩選平台

Abstract

第二型糖尿病是目前最普遍的代謝性疾病，它會伴隨許多的併發症，像是冠狀動脈心臟性疾病、視網膜病變、神經性病變和腎臟病變…等，也造成一定的死亡率。糖尿病人口遍及全球，預計在2030年之前全世界將有3.66億人口患有第二型糖尿病。一直以來，高血糖被視為糖尿病併發症的高風險因子，如何控制血糖值也是治療糖尿病的主要目標。除了傳統使用胰島素來控制血糖，目前已開發出利用抑制腎小管再吸收葡萄糖的機轉，將體內多餘糖份經由尿液排出，進而達到控制血糖的化合物: 選擇性SGLT2抑制劑。 目前已有許多可能具選擇性抑制SGLT2活性的化合物被合成出來，因此需要一個篩選系統來做初步的篩選，以減少藥物開發的時間和成本。輻射性標定受質(14C-AMG: 14C-methyl-α-D-glucopyranoside)對於SGLT有專一性，而且具有和D-glucose接近的動力學參數，因此它是目前最普遍用來篩選此類化合物和研究SGLT的受質。但操作放射性物質的實驗具有一定的危險性和成本考量的問題，因此我們嘗試建立一個不具放射活性的細胞篩選平台，利用螢光標定受質(1-NBDG: 1-[N-(7-nitrobenz-2-oxz-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose)和短暫表達hSGLT1之COS-7細胞株與穩定表達hSGLT2之CHO-K1細胞株來進行化合物抑制葡萄糖攝取的實驗，進而篩選出選擇性SGLT2抑制劑。 在篩選化合物之前，我們先由已知的SGLT受質D-glucose和AMG (methyl-α-D-glucopyranoside)與1-NBDG在短暫表達hSGLT1的COS-7細胞中作競爭，確認1-NBDG確實為SGLT的受質。另外，在短暫表達hSGLT1的COS-7細胞中分析1-NBDG對於hSGLT1的動力學參數(Km)與D-glucose和AMG比較，發現1-NBDG的Km值(0.55 ± 0.01 mM)相對於AMG (0.40 ± 0.05 mM)而言，更接近D-glucose (0.51 ± 0.02 mM)。加上由非選擇性SGLT抑制劑(Phlorizin)在使用14C-AMG和1-NBDG為受質下對hSGLT1之IC50得到相同(0.11 μM)的結果認為，1-NBDG與14C-AMG在我們所建立的系統中具有相互取代性。接著，藉由選擇性SGLT2抑制劑(Dapagliflozin)在本系統中所求出對於hSGLT1與hSGLT2的IC50值(T1: 880 nM, T2: 1.86 nM)與已發表的結果比較，發現結果相符，進而確認本系統對於篩選選擇性SGLT2抑制劑的可行性。因此我們就進行了化合物的篩選(化合物由梁碧惠博士實驗室所提供)，發現其中有三個化合物對於hSGLT2具有選擇性抑劑的活性。 由實驗結果證明我們成功地建立選擇性SGLT2抑制劑之非放射性細胞篩選平台，此系統除了可經由量化的結果來判斷化合物的活性外，也可藉由肉眼來觀察螢光受質1-NBDG在細胞中的攝取; 除此之外，也可用來作為其他表達SGLT1與SGLT2的細胞株的研究工具。

Nowadays, type 2 diabetes mellitus (T2DM) is the most common metabolic disease, which leads to many complications, including, retinopathy, nephropathy, neuropathy and coronary heart disease, which are also associated with considerable morbidity and mortality. The incidence of T2DM is increasing at an alarming rate worldwide, which may affect a projected 366 million individuals by the year 2030. Meanwhile, ‘hyperglycemia’ is considered as the major risk factor for the complications. How to control the blood glucose level seems to be the priority for treating the patients. Besides the traditional use of insulin, there is a novel mechanism for lowering the blood glucose level by blocking the renal glucose reabsorption, which excretes the extra glucose from body, using the selective SGLT2 inhibitor. There are many compounds synthesized for the development of selective SGLT2 inhibitors; therefore, a screening platform is needed ahead for decreasing the time and cost of the drug development. An isotope-labeled substrate (14C-AMG: 14C-methyl-α-glucopyranoside) is a specific substrate for SGLT, and has a Km value close to that of D-glucose, so it is currently the most common used in compound screening and in the studies of SGLT; however, operating a radioactive experiment is quite risky and the substrate is relatively expensive. We sought to establish a non-radioactive cell-based screening platform, using a fluorescent glucose analogue (1-NBDG: 1-[N-(7-nitrobenz-2-oxz-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose) as a substrate for SGLTs, transiently transfected COS-7 cells expressing hSGLT1 and a CHO-K1 stable clone expressing hSGLT2 to conduct the glucose uptake assay to screen for selective SGLT2 inhibitors. A non-radioactive cell-based screening platform for selective SGLT2 inhibitor has been established successfully. We could not only measure the potency of the compounds by quantitative data, but also observe the uptake of the fluorescent substrate, 1-NBDG, visually. Moreover, 1-NBDG might be a helpful tool to study cells expressing SGLTs.