Steuber Holger, Zentgraf Matthias, Podjarny Alberto, Heine Andreas, Klebe Gerhard
Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
J Mol Biol. 2006 Feb 10;356(1):45-56. doi: 10.1016/j.jmb.2005.10.067. Epub 2005 Nov 10.
The crystal structure of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase, the first enzyme of the polyol pathway, has been determined to 1.43 angstroms and 0.95 angstroms resolution. The ternary complex of inhibitor, cofactor and enzyme has been obtained by soaking of preformed crystals. Supposedly due to low solubility in the crystallisation buffer, in both structures the inhibitor shows reduced occupancy of 74% and 46% population, respectively. The pyridazinone head group of the inhibitor occupies the catalytic site, whereas the chloro-benzofuran moiety penetrates into the opened specificity pocket. The high-resolution structure provides some evidence that the pyridazinone group binds in a negatively charged deprotonated state, whereas the neighbouring His110 residue most likely adopts a neutral uncharged status. Since the latter structure is populated by the ligand to only 46%, a second conformation of the C-terminal ligand-binding region can be detected. This conformation corresponds to the closed state of the specificity pocket when no or only small ligands are bound to aldose reductase. The two conformational states are in good agreement with frames observed along a molecular dynamics trajectory describing the transition from closed to open situation. Accordingly, both geometries, superimposed in the averaged crystal structure, correspond to snapshots of the ligand-bound and the unbound state. Isothermal titration calorimetry has been applied to determine the binding constants of the investigated pyridazinone in comparison to the hydantoin sorbinil and the carboxylate-type inhibitors IDD 594 and tolrestat. The pyridazinone exhibits a binding affinity similar to those of tolrestat and sorbinil, and shows slightly reduced affinity compared to IDD 594. These studies elucidating the binding mode and providing information about protonation states of protein side-chains involved in binding of this novel class of inhibitors establish the platform for further structure-based drug design.
已确定一种新型磺酰基哒嗪酮抑制剂与多元醇途径的首个酶醛糖还原酶形成的复合物的晶体结构，分辨率分别为1.43埃和0.95埃。通过将预形成的晶体浸泡获得抑制剂、辅因子和酶的三元复合物。据推测，由于在结晶缓冲液中的溶解度较低，在这两种结构中抑制剂的占有率分别降低至74%和46%。抑制剂的哒嗪酮头部基团占据催化位点，而氯代苯并呋喃部分则深入到开放的特异性口袋中。高分辨率结构提供了一些证据，表明哒嗪酮基团以带负电荷的去质子化状态结合，而相邻的His110残基最可能处于中性不带电状态。由于后一种结构中配体的占有率仅为46%，因此可以检测到C端配体结合区域的第二种构象。这种构象对应于当没有或只有小配体与醛糖还原酶结合时特异性口袋的关闭状态。这两种构象状态与描述从关闭到开放状态转变的分子动力学轨迹中观察到的帧很好地一致。因此，叠加在平均晶体结构中的两种几何形状分别对应于配体结合状态和未结合状态的快照。已应用等温滴定量热法来确定所研究的哒嗪酮与乙内酰脲类索比尼尔以及羧酸盐型抑制剂IDD 594和托瑞司他相比的结合常数。哒嗪酮表现出与托瑞司他和索比尼尔相似的结合亲和力，与IDD 594相比亲和力略有降低。这些阐明结合模式并提供有关参与这类新型抑制剂结合的蛋白质侧链质子化状态信息的研究为进一步基于结构的药物设计奠定了平台。
