Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín - CONICET (IIBio-UNSAM), Avenida 25 de Mayo y Francia CP (1650), San Martín (Buenos Aires), Argentina.
Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Box 8204, Raleigh, NC 27695, USA.
Bioorg Med Chem Lett. 2021 Jan 15;32:127723. doi: 10.1016/j.bmcl.2020.127723. Epub 2020 Nov 26.
Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (K = 5.5 ± 0.1 μM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC = 17.40 ± 1.055 μM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.
核糖-5-磷酸异构酶 B 型（RPI-B）是戊糖磷酸途径的关键酶，催化核糖-5-磷酸（R5P）和核酮糖-5-磷酸（Ru5P）的异构化。克氏锥虫 RPI-B（TcRPI-B）似乎是一个合适的药物靶点，主要有以下原因：（i）它是必需的（如先前在其他原生动物中所示），（ii）它在迄今为止测序的哺乳动物基因组中没有同源物，（iii）它参与 NADPH 和核苷酸/核酸的产生，这些对寄生虫细胞的存活至关重要。在这项研究中，我们通过 Dixon 法报告了 TcRPI-B 对底物类似物抑制剂 Compound B（K = 5.5 ± 0.1 μM）的竞争性抑制作用。该化合物具有碘乙酰胺部分，易受到亲核攻击，特别是半胱氨酸巯基的攻击。Compound B 被设计为专门针对重要的活性位点残基 Cys-69。通过将 TcRPI-B 与 Compound B 孵育，用胰蛋白酶消化 LC-MS/MS 分析发现 Compound B 与 Cys-69 发生了共价结合。该抑制剂对感染 NIH-3T3 鼠源宿主细胞的 T. cruzi 感染性生命阶段也表现出显著的体外杀锥虫活性（IC = 17.40 ± 1.055 μM）。对 Compound B 的研究提供了一个概念验证，以便下一代抑制剂可以开发出来，重点是使用前药基团代替碘乙酰胺部分，这代表了未来治疗恰加斯病的一个有吸引力的起点。
