Mahajan Sumit S, Hou Liming, Doneanu Catalin, Paranji Rajan, Maeda Dean, Zebala John, Atkins William M
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
J Am Chem Soc. 2006 Jul 5;128(26):8615-25. doi: 10.1021/ja061766n.
Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.
二聚体谷胱甘肽S-转移酶（GSTs）是包括癌症在内的多种疾病的药理学靶点。由于其底物选择性重叠，很难实现同工型特异性。在此，我们展示了二价GST抑制剂的效用及其通过组合接头设计进行的优化。制备了一个组合文库，其中二肽接头从中央支架（双-3,5-氨基甲基苯甲酸，AMAB）对称发出，连接两个依他尼酸部分，并通过迭代去卷积针对同工型GSTA1-1和GSTP1-1进行解码。该文库产生了高亲和力的GSTA1-1选择性抑制剂（选择性为70-120倍），且抑制剂与GSTA1-1二聚体的化学计量比为1:1。其中一种抑制剂的饱和转移差异（STD）核磁共振显示，接头结构为（Asp-Gly-AMAB-Gly-Asp），对GSTA1-1的解离常数（K(D)） = 42 nM，表明Asp-Gly接头与GSTA1-1紧密相互作用，但与P1-1不相互作用。氢/氘交换质谱用于绘制蛋白质结合位点，结果表明亚基间裂隙和底物结合位点内的肽段受到抑制剂保护，不与溶剂发生交换。提出了抑制剂结合方向的模型，该模型与蛋白质裂隙和抑制剂接头之间的静电互补性一致，是同工型选择性和高亲和力的来源。结果证明了组合或“非理性”接头设计在优化二价抑制剂方面的效用。
