We previously discovered first-in-class multitargeted 5-substituted pyrrolo[3,2-d]pyrimidine antifolates that inhibit serine hydroxymethyltransferase 2 (SHMT2), resulting in potent in vitro and in vivo antitumor efficacies. In this report, we present crystallographic structures for SHMT2 in complex with an expanded series of pyrrolo[3,2-d]pyrimidine compounds with variations in bridge length (3–5 carbons) and the side chain aromatic ring (phenyl, thiophene, fluorine-substituted phenyl, and thiophene). We evaluated structural features of the inhibitor-SHMT2 complexes and correlations to inhibitor potencies (i.e., K_is), highlighting conserved polar contacts and identifying 5-carbon bridge lengths as key determinants of inhibitor potency. Based on the analysis of SHMT2 structural data, we investigated the impact of mutation of Tyr105 in SHMT2 kinetic analysis and studies with HCT116 cells with inducible expression of wild-type and Y105F SHMT2. Increased enzyme inhibition potency by the pyrrolo[3,2-d]pyrimidine inhibitors with Phe105 SHMT2 accompanied an increased growth inhibition of Phe105-expressing HCT116 cells compared to wild-type SHMT2. Pyrrolo[3,2-d]pyrimidine inhibitors with polyglutamate modifications were evaluated for potencies against SHMT2. We determined the crystal structures of SHMT2 in complex with our lead antifolate AGF347 lacking L-glutamate, or as a diglutamate and triglutamate, for comparison with parent AGF347. These data provide the first insights into the influence of antifolate polyglutamylation on SHMT2:inhibitor interactions. Collectively, our results provide new insights into the critical structural determinants of SHMT2 binding by pyrrolo[3,2-d]pyrimidine inhibitors as novel antitumor agents, as well as the first structural characterization of human SHMT2 in complex with polyglutamates of an SHMT2-targeted antifolate.

中文翻译：

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5-取代吡咯并[3,2-d]嘧啶抗叶酸抑制剂与人丝氨酸羟甲基转移酶 2 复合物的结构表征

我们之前发现了一流的多靶点 5-取代吡咯并[3,2- d ]嘧啶抗叶酸剂，可抑制丝氨酸羟甲基转移酶 2 (SHMT2)，从而在体外和体内产生有效的抗肿瘤功效。在本报告中，我们展示了 SHMT2 与一系列扩展的吡咯并[3,2- d ]嘧啶化合物形成的复合物的晶体结构，这些化合物具有不同的桥长度（3-5 个碳）和侧链芳环（苯基、噻吩、氟） -取代的苯基和噻吩）。我们评估了抑制剂-SHMT2 复合物的结构特征以及与抑制剂效力的相关性（即K _i s），强调保守的极性接触并确定 5 碳桥长度作为抑制剂效力的关键决定因素。基于SHMT2结构数据的分析，我们研究了Tyr105突变对SHMT2动力学分析的影响，并用诱导表达野生型和Y105F SHMT2的HCT116细胞进行研究。与野生型 SHMT2 相比，吡咯并[3,2- d ]嘧啶抑制剂与 Phe105 SHMT2 的酶抑制效力的增加伴随着表达 Phe105 的 HCT116 细胞的生长抑制的增加。评估了具有聚谷氨酸修饰的吡咯并[3,2- d ]嘧啶抑制剂针对SHMT2的效力。我们确定了SHMT2与缺乏L-谷氨酸或作为二谷氨酸和三谷氨酸的主要抗叶酸剂AGF347复合的晶体结构，以与亲本AGF347进行比较。这些数据首次深入了解抗叶酸剂聚谷氨酰化对 SHMT2：抑制剂相互作用的影响。总的来说，我们的结果为吡咯并[3,2- d ]嘧啶抑制剂作为新型抗肿瘤药物结合SHMT2的关键结构决定因素提供了新的见解，以及人SHMT2与SHMT2靶向聚谷氨酸盐复合物的首次结构表征抗叶酸剂。