Department of Chemistry, University of Missouri, Columbia, Missouri, USA.
Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
J Biol Chem. 2020 Dec 25;295(52):18316-18327. doi: 10.1074/jbc.RA120.016106. Epub 2020 Oct 27.
Pyrroline-5-carboxylate reductase 1 (PYCR1) catalyzes the biosynthetic half-reaction of the proline cycle by reducing Δ-pyrroline-5-carboxylate (P5C) to proline through the oxidation of NAD(P)H. Many cancers alter their proline metabolism by up-regulating the proline cycle and proline biosynthesis, and knockdowns of PYCR1 lead to decreased cell proliferation. Thus, evidence is growing for PYCR1 as a potential cancer therapy target. Inhibitors of cancer targets are useful as chemical probes for studying cancer mechanisms and starting compounds for drug discovery; however, there is a notable lack of validated inhibitors for PYCR1. To fill this gap, we performed a small-scale focused screen of proline analogs using X-ray crystallography. Five inhibitors of human PYCR1 were discovered: l-tetrahydro-2-furoic acid, cyclopentanecarboxylate, l-thiazolidine-4-carboxylate, l-thiazolidine-2-carboxylate, and -formyl l-proline (NFLP). The most potent inhibitor was NFLP, which had a competitive (with P5C) inhibition constant of 100 μm The structure of PYCR1 complexed with NFLP shows that inhibitor binding is accompanied by conformational changes in the active site, including the translation of an α-helix by 1 Å. These changes are unique to NFLP and enable additional hydrogen bonds with the enzyme. NFLP was also shown to phenocopy the knockdown in MCF10A H-RAS breast cancer cells by inhibiting proline biosynthesis and impairing spheroidal growth. In summary, we generated the first validated chemical probe of PYCR1 and demonstrated proof-of-concept for screening proline analogs to discover inhibitors of the proline cycle.
吡咯啉-5-羧酸还原酶 1（PYCR1）通过氧化 NAD（P）H 将 Δ-吡咯啉-5-羧酸（P5C）还原为脯氨酸，从而催化脯氨酸循环的生物合成半反应。许多癌症通过上调脯氨酸循环和脯氨酸生物合成来改变其脯氨酸代谢，而 PYCR1 的敲低会导致细胞增殖减少。因此，PYCR1 作为一种潜在的癌症治疗靶点的证据越来越多。癌症靶点的抑制剂可用作研究癌症机制的化学探针和药物发现的起始化合物；然而，针对 PYCR1 的验证抑制剂却明显缺乏。为了填补这一空白，我们使用 X 射线晶体学对脯氨酸类似物进行了小规模的重点筛选。发现了五种人 PYCR1 的抑制剂：L-四氢-2-呋喃酸、环戊烷羧酸、L-噻唑烷-4-羧酸、L-噻唑烷-2-羧酸和 -甲酰基 L-脯氨酸（NFLP）。最有效的抑制剂是 NFLP，其与 P5C 的竞争抑制常数为 100μm。与 NFLP 结合的 PYCR1 结构表明，抑制剂结合伴随着活性部位构象的变化，包括 1Å 的α-螺旋平移。这些变化是 NFLP 特有的，并能与酶形成额外的氢键。NFLP 还通过抑制脯氨酸生物合成和损害球体生长来模拟 MCF10A H-RAS 乳腺癌细胞中的 knockdown。总之，我们生成了第一个经验证的 PYCR1 化学探针，并证明了筛选脯氨酸类似物以发现脯氨酸循环抑制剂的概念验证。
