Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
Department of Chemistry, University of Missouri, Columbia, Missouri, USA.
Protein Sci. 2024 Jul;33(7):e5072. doi: 10.1002/pro.5072.
Δ-pyrroline-5-carboxylate reductase isoform 1 (PYCR1) is the last enzyme of proline biosynthesis and catalyzes the NAD(P)H-dependent reduction of Δ-pyrroline-5-carboxylate to L-proline. High PYCR1 gene expression is observed in many cancers and linked to poor patient outcomes and tumor aggressiveness. The knockdown of the PYCR1 gene or the inhibition of PYCR1 enzyme has been shown to inhibit tumorigenesis in cancer cells and animal models of cancer, motivating inhibitor discovery. We screened a library of 71 low molecular weight compounds (average MW of 131 Da) against PYCR1 using an enzyme activity assay. Hit compounds were validated with X-ray crystallography and kinetic assays to determine affinity parameters. The library was counter-screened against human Δ-pyrroline-5-carboxylate reductase isoform 3 and proline dehydrogenase (PRODH) to assess specificity/promiscuity. Twelve PYCR1 and one PRODH inhibitor crystal structures were determined. Three compounds inhibit PYCR1 with competitive inhibition parameter of 100 μM or lower. Among these, (S)-tetrahydro-2H-pyran-2-carboxylic acid (70 μM) has higher affinity than the current best tool compound N-formyl-l-proline, is 30 times more specific for PYCR1 over human Δ-pyrroline-5-carboxylate reductase isoform 3, and negligibly inhibits PRODH. Structure-affinity relationships suggest that hydrogen bonding of the heteroatom of this compound is important for binding to PYCR1. The structures of PYCR1 and PRODH complexed with 1-hydroxyethane-1-sulfonate demonstrate that the sulfonate group is a suitable replacement for the carboxylate anchor. This result suggests that the exploration of carboxylic acid isosteres may be a promising strategy for discovering new classes of PYCR1 and PRODH inhibitors. The structure of PYCR1 complexed with l-pipecolate and NADH supports the hypothesis that PYCR1 has an alternative function in lysine metabolism.
Δ-吡咯啉-5-羧酸还原酶同工酶 1（PYCR1）是脯氨酸生物合成的最后一种酶，催化 NAD（P）H 依赖性的 Δ-吡咯啉-5-羧酸还原为 L-脯氨酸。高 PYCR1 基因表达在许多癌症中观察到，并与患者预后不良和肿瘤侵袭性相关。PYCR1 基因的敲低或 PYCR1 酶的抑制已被证明能抑制癌细胞和癌症动物模型中的肿瘤发生，从而激发抑制剂的发现。我们使用酶活性测定法，对 71 种低分子量化合物（平均分子量为 131Da）对 PYCR1 的文库进行了筛选。通过 X 射线晶体学和动力学测定来验证命中化合物，以确定亲和力参数。对文库进行了人 Δ-吡咯啉-5-羧酸还原酶同工酶 3 和脯氨酸脱氢酶（PRODH）的反筛选，以评估特异性/混杂性。确定了 12 个 PYCR1 和 1 个 PRODH 抑制剂晶体结构。三种化合物以 100μM 或更低的竞争抑制参数抑制 PYCR1。其中，（S）-四氢-2H-吡喃-2-羧酸（70μM）对 PYCR1 的亲和力高于当前最佳工具化合物 N-甲酰基-L-脯氨酸，对人 Δ-吡咯啉-5-羧酸还原酶同工酶 3 的特异性高 30 倍，对 PRODH 的抑制作用可忽略不计。结构-亲和力关系表明，该化合物杂原子的氢键结合对与 PYCR1 结合很重要。与 1-羟乙磺酸结合的 PYCR1 和 PRODH 的结构表明，磺酸基团是羧酸锚的合适替代品。这一结果表明，羧酸类似物的探索可能是发现新的 PYCR1 和 PRODH 抑制剂的一种有前途的策略。与 L-哌啶酸和 NADH 结合的 PYCR1 结构支持了 PYCR1 在赖氨酸代谢中具有替代功能的假设。
