Structural studies of molecular cancer targets and drug development

目前，我们正在开展一项多学科合作，以开发有效的和特异性的人类Chk2激酶抑制剂。我们在这个项目中的作用是确定Chk2与小分子抑制剂配合物的共晶结构。筛选技术部利用来自Open Repository library的10万多个化合物筛选Chk2的新抑制剂，鉴定出一种双胍腙，NSC 109555 （4,4- diacetyldiphenylu脲-双胍腙）作为Chk2的先导化合物，IC50为240 nM。该化合物的生化特性证实它是一种可逆的竞争性抑制剂，靶向Chk2的ATP结合袋。NSC 109555对20多种激酶的初始激酶谱分析表明，NSC 109555对Chk2具有高选择性。为了阐明NSC 109555和其他类似化合物抑制Chk2的分子基础，我们开始了与这些小分子共结晶酶的努力。为此，我们克隆了Chk2的催化结构域，表达为His6-MBP融合蛋白，并对其进行了内部纯化。我们成功地用分子置换法测定了nsc109555与Chk2催化结构域配合物在2.07分辨率下的共晶结构。晶体结构证实了抑制剂以细长的方式与Chk2的atp结合口袋结合，但重要的是，结合模式与分子模型预测的不同。NSC10955通过末端鸟酰腙部分与Glu273的氢键以及抑制剂的主链羰基与Glu302和Met304之间的水介导氢键锚定在活性位点上。随后由普罗维德制药公司合成了一系列NSC 109555类似物，其中包括四种不同类型的结构修饰。这些修饰的目的是实现化合物的去对称化，并扫描鸟酰腙、烷基和芳基上的各种取代基。我们已经能够从每个结构类中确定至少一种化合物的共晶结构，其中最有效的是PV1019 （IC50 = 15 nM, 2.07）。我们成功地确定了Chk2与PV1019配合物的共晶结构，为进一步优化该化合物的结构提供了机会。PV1019通过鸟酰腙部分与Glu273的直接氢键和2-硝基吲哚与Glu302和Met304的直接氢键与Chk2的活性位点结合。我们的电子密度图还揭示了几个有序的水分子在PV1019与Glu308和Glu302的羰基和酰胺主链之间形成水介导的氢键的活性位点中的关键作用。共晶结构的一个值得注意的特征是PV1019的甲基官能团正上方存在一个疏水腔，这与相关的Chk1激酶略有不同。在Chk2中，这个空腔完全由疏水残基组成，而在Chk1中有一个极性亲水残基Asn59 （Chk2中的Leu277）。我们提出，通过优化与该腔体的结合，有可能提高Chk2抑制剂的特异性。目前的研究重点是基于邻甲基的官能团取代来合成类似物，以优化抑制剂与该空腔之间的相互作用。Provid根据PV1019结合模式的结构特征合成了三种新的类似物，并成功地将其中两种化合物与Chk2共结晶；PV1322 （IC50 = 370 nM, 1.90）和PV1162 （IC50 = 12 nM, 2.2）。第三种类似物PV1352特别有希望，因为它已被证明比PV1019有效约5倍。我们最近收到了这种化合物，正试图将其与Chk2共结晶。Chk2与PV1162配合物共晶结构的测定也提供了新的细节。PV1162是通过将PV1019的2-硝基吲哚基改变为5-甲氧基吲哚，并在PV1019的甲基上附着一个异丙基片段以填充疏水腔而制成的。Chk2与PV1162共晶表明，吲哚环已经翻转，从而改善了吲哚NH基团与活性位点的氢键网络。另外，加到甲基上的异丙基现在可以紧贴在疏水腔中。该化合物将作为开发新的类似物的指南，其中我们研究了吲哚环上的附加取代基以及甲基上的附加疏水取代基。关于癌症相关分子靶点的其他合作项目正处于早期发展阶段。这些靶点包括人RSK2激酶、人MyD88蛋白和几种B-Zip转录因子。在所有这些情况下，合作者可以使用小分子抑制剂与蛋白质共结晶。

We are currently involved in a multidisciplinary collaboration to develop potent and specific inhibitors of human Chk2 kinase. Our role in this project is to determine co-crystal structures of Chk2 in complex with small molecule inhibitors. Using a library of over 100,000 compounds from the Open Repository Library to screen for novel inhibitors of Chk2, the Screening Technologies Branch identified a bis-guanylhydrazone, NSC 109555 (4,4-diacetyldiphenylurea-bis(guanylhydrazone), as a lead compound with an IC50 of 240 nM for Chk2. Biochemical characterization of this compound confirmed that it is a reversible and competitive inhibitor that targets the ATP binding pocket of Chk2. Initial kinase profiling of NSC 109555 against a panel of over 20 kinases demonstrated high selectivity of NSC 109555 for Chk2. To illuminate the molecular basis of Chk2 inhibition by NSC 109555 and other similar compounds, we initiated an effort to co-crystallize the enzyme with these small molecules. To this end, the catalytic domain of Chk2 was cloned, expressed as a His6-MBP fusion protein and purified to homogeneity in-house. We succeeded in determining the co-crystal structure of NSC 109555 in complex with the catalytic domain of Chk2 at 2.07 resolution by molecular replacement. The crystal structure confirmed that the inhibitor binds to the ATP-binding pocket of Chk2 in an elongated fashion, but, importantly, the mode of binding was different that what had been predicted by molecular modeling. NSC10955 is anchored to the active site via hydrogen bonding of one terminal guanylhydrazone moiety to Glu273 and water mediated hydrogen bonds between a backbone carbonyl of the inhibitor with Glu302 and Met304. A series of NSC 109555 analogs were subsequently synthesized by Provid Pharmaceuticals that included four different classes of structural modifications. The goals of the modifications were to achieve desymmetrization of the compound and scan a variety of substituents on the guanylhydrazone, alkyl, and aryl moieties. We have been able to determine co-crystal structures for at least one compound from each structural class, the most potent of which was PV1019 (IC50 = 15 nM, 2.07 ). Our success in determining the co-crystal structure of Chk2 in complex with PV1019 presented an opportunity for further structure-based optimization of the compound. PV1019 binds to the active site of Chk2 via direct hydrogen bonds of the guanylhydrazone moiety with Glu273 and the 2-nitro-indole with Glu302 and Met304. Our electron density maps also revealed the key roles of several well-ordered water molecules in the active site that form water-mediated hydrogen bonds between the carbonyl and amide backbone of PV1019 and Glu308 and Glu302. A noteworthy feature of the co-crystal structure is the presence of a hydrophobic cavity directly above the methyl functional group of PV1019 which differs slightly from the related Chk1 kinase. In Chk2, this cavity is composed entirely of hydrophobic residues, whereas in Chk1 there is one polar, hydrophilic residue, Asn59 (Leu277 in Chk2). We proposed that it might be possible to improve the specificity of the Chk2 inhibitor by optimizing the binding to this cavity. Current efforts are now focused on synthesizing analogs based on functional group substitutions on the neighboring methyl group next to the guanylhydrazone moiety which projects towards this cavity with the goal of optimizing the interactions between the inhibitor and this cavity. Provid has synthesized three new analogs based on structural characterization of the binding mode of PV1019 and we have successfully co-crystallized two of these compounds with Chk2; PV1322 (IC50 = 370 nM, 1.90 ) and PV1162 (IC50 = 12 nM, 2.2 ). A third analogue, PV1352, is especially promising, as is it has been shown to be approximately 5-fold more potent than PV1019. We have recently received this compound and are attempting to co-crystallize it with Chk2. The determination of the co-crystal structure of Chk2 in complex with PV1162 has also provided new details. PV1162 was created by altering the 2-nitro-indole group of PV1019 to a 5-methoxy-indole and an isopropyl moiety was attached to the methyl group of PV1019 in an effort to fill the hydrophobic cavity. The co-crystal of Chk2 with PV1162 reveals that the indole ring has now flipped over, thereby improving the hydrogen bonding network of the indole NH group with the active site. Additionally, the isopropyl group that was added to the methyl group now fits snuggly into the hydrophobic cavity. This compound will serve as a guide for the development of new analogs in which we examine additional substituents on the indole ring as well as additional hydrophobic substituents on the methyl group. Additional collaborative projects on cancer-related molecular targets are at an earlier stage of development. These targets include human RSK2 kinase, human MyD88 protein, and several B-Zip transcription factors. In all of these cases, small molecule inhibitors are available from collaborators to co-crystallize with the proteins.