Universidade Federal do Amazonas, UFAM, campus Humaitá, Amazonas, Brazil; Laboratório de Biotecnologia de Proteínas e Compostos Bioativos da Amazônia Ocidental, LABIOPROT, Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio, Fundação Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia e Universidade Federal de Rondônia, UNIR, Porto Velho, Rondônia, Brazil.
Centro de Pesquisa em Biologia Molecular e Funcional, PUCRS, Porto Alegre, Rio Grande do Sul, Brazil.
Int J Biol Macromol. 2020 Dec 15;165(Pt B):1832-1841. doi: 10.1016/j.ijbiomac.2020.10.062. Epub 2020 Oct 16.
Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: K 17 μM, k 1.2 s, V 2.2 U/mg and k/K 7 × 10; for MESG they were: K 131 μM, k 2.4 s, V 4.4 U/mg and k/K 1.8 × 10. The thermodynamic parameters for the substrate Phosphate were: Δ - 5.8 cal mol, Δ - 6.5 cal mol and Δ - 2.25 cal mol/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.
研究表明，抑制恶性疟原虫嘌呤核苷磷酸化酶（PfPNP）可阻断嘌呤补救途径在体外和体内。在这项研究中，PfPNP 被评估为一种模型，用于使用表面等离子体共振（SPR）寻找新的抑制剂。获得了 PfPNP 的表达、纯化、寡聚态、动力学常数、量热参数和动力学机制。将 PfPNP 固定在 CM5 传感器芯片上，并通过将酶与底物 MESG 结合以及天然提取物的 10 个馏分中包含的分子之间的相互作用产生传感器图谱。寡聚态表明重组 PfPNP 是六聚体。底物肌苷的真实稳态动力学参数为：K 17 μM，k 1.2 s，V 2.2 U/mg 和 k/K 7 × 10；MESG 的参数为：K 131 μM，k 2.4 s，V 4.4 U/mg 和 k/K 1.8 × 10。底物磷酸盐的热力学参数为：Δ-5.8 cal/mol，Δ-6.5 cal/mol 和 Δ-2.25 cal/mol/度。ITC 结果表明，磷酸盐与游离 PfPNP 的结合导致热和结合常数以及热力学参数发生显著变化。提出了一个顺序有序的机制作为动力学机制。三种植物提取物含有能够与 PfPNP 相互作用的分子，表现出不同的亲和力水平。使用 SPR 鉴定与重组 PfPNP 相互作用的植物提取物部分验证了该靶标作为寻找新抑制剂的高通量筛选模型。在这项研究中，我们首次展示了 PfPNP 催化反应的真实稳态动力学参数和使用 PfPNP 作为新抑制剂的高通量筛选目标的模型。这些知识将为寻找新的抗疟疾药物开发更有效的研究方法提供依据。
