Structural studies of molecular cancer targets and drug development

我们目前正在进行多学科合作，以开发有效和特异性的人Chk 2激酶抑制剂。我们在这个项目中的作用是确定Chk 2与小分子抑制剂复合的共晶结构。 使用来自Open Repository Library的超过100，000种化合物的库来筛选Chk 2的新型抑制剂，筛选技术分支鉴定了双鸟苷酰腙，NSC 109555（4，4-二乙酰基二苯基脲-双（鸟苷酰腙））作为先导化合物，其对Chk 2的IC 50为240 nM。该化合物的生物化学表征证实，它是一种可逆的竞争性抑制剂，靶向Chk 2的ATP结合口袋。NSC 109555针对一组超过20种激酶的初始激酶谱显示NSC 109555对Chk 2的高选择性。 为了阐明NSC 109555和其他类似化合物抑制Chk 2的分子基础，我们开始努力使酶与这些小分子共结晶。为此，克隆了Chk 2的催化结构域，表达为His 6-MBP融合蛋白，并在内部纯化至均一。我们成功地确定了NSC 109555与Chk 2催化结构域复合物的共晶结构，分辨率为2.07。晶体结构证实，抑制剂结合的ATP结合口袋的Chk 2在一个拉长的方式，但重要的是，结合的模式是不同的，已经预测的分子建模。NSC 10955通过一个末端鸟苷酰腙部分与Glu 273的氢键和抑制剂的骨架羰基与Glu 302和Met 304之间的水介导的氢键锚定到活性位点。随后由Provid Pharmaceuticals合成了一系列NSC 109555类似物，包括四种不同类型的结构修饰。修改的目标是实现化合物的去对称化，并扫描鸟苷酰腙、烷基和芳基部分上的各种取代基。我们已经能够确定每个结构类别中至少一种化合物的共晶结构，其中最有效的是PV 1019（IC 50 = 15 nM，2.07）。 我们在确定Chk 2与PV 1019复合物的共晶结构方面的成功为该化合物的进一步基于结构的优化提供了机会。PV 1019通过鸟苷酰腙部分与Glu 273的直接氢键以及2-硝基-吲哚与Glu 302和Met 304的直接氢键与Chk 2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活性位点中的关键作用，这些活性位点在PV 1019和Glu 308和Glu 302的羰基和酰胺骨架之间形成水介导的氢键。共晶结构的一个值得注意的特征是在PV 1019的甲基官能团正上方存在疏水空腔，其与相关的Chk 1激酶略有不同。在Chk 2中，这个空腔完全由疏水残基组成，而在Chk 1中有一个极性亲水残基Asn 59（Chk 2中的Leu 277）。我们提出，通过优化与该空腔的结合，有可能提高Chk 2抑制剂的特异性。目前的努力现在集中在合成类似物的基础上的功能基团取代的邻甲基旁边的鸟苷酰腙部分，其项目朝着这个空腔的目标是优化抑制剂和这个空腔之间的相互作用。Provid基于PV 1019结合模式的结构表征合成了三种新的类似物，我们成功地将其中两种化合物与Chk 2共结晶; PV 1322（IC 50 = 370 nM，1.90）和PV 1162（IC 50 = 12 nM，2.2）。第三种类似物PV 1352特别有希望，因为它已被证明比PV 1019效力高约5倍。我们最近收到了这种化合物，并试图将其与Chk 2共结晶。Chk 2与PV 1162复合物的共晶结构的测定也提供了新的细节。通过将PV 1019的2-硝基-吲哚基团改变为5-甲氧基-吲哚并将异丙基部分连接到PV 1019的甲基基团以填充疏水空腔来产生PV 1162。Chk 2与PV 1162的共晶体显示吲哚环现在已经翻转，从而改善了吲哚NH基团与活性位点的氢键网络。此外，添加到甲基基团的异丙基基团现在紧密地配合到疏水空腔中。该化合物将作为指导新的类似物的开发，其中我们检查吲哚环上的其他取代基以及甲基上的其他疏水取代基。

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.