Plasmodium falciparum purine nucleoside phosphorylase as a model in the search for new inhibitors by high throughput screening.

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: KM 17 µM, kcat 1.2 s-1, VMax 2.2 U/mg and kcat/KM 7 × 10-4; for MESG they were: KM 131 µM, kcat 2.4 s-1, VMax 4.4 U/mg and kcat/KM 1.8 × 10-4. The thermodynamic parameters for the substrate Phosphate were: ΔG - 5.8 cal mol-1, ΔH - 6.5 cal mol-1 and ΔS - 2.25 cal mol-1/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.

The effect of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene lupane compound isolated from Combretum leprosum Mart. on peripheral blood mononuclear cells.

BACKGROUND: The Combretum leprosum Mart. plant, popularly known as mofumbo, is used in folk medicine for inflammation, pain and treatment of wounds. From this species, it is possible to isolate three triterpenes: (3ß, 6ß, 16ß-trihydroxylup-20(29)-ene) called lupane, arjunolic acid and molic acid. In this study, through preclinical tests, the effect of lupane was evaluated on the cytotoxicity and on the ability to activate cellular function by the production of TNF-α, an inflammatory cytokine, and IL-10, an immuno regulatory cytokine was assessed. The effect of lupane on the enzymes topoisomerase I and II was also evaluated. METHODS: For this reason, peripheral blood mononuclear cells (PBMCs) were obtained and cytotoxicity was assessed by the MTT method at three different times (1, 15 and 24 h), and different concentrations of lupane (0.3, 0.7, 1.5, 6, 3 and 12 µg/mL). The cell function was assessed by the production of TNF-α and IL-10 by PBMCs quantified by specific enzyme immunoassay (ELISA). The activity of topoisomerases was assayed by in vitro biological assays and in silico molecular docking. RESULTS: The results obtained showed that lupane at concentrations below 1.5 µg/mL was not toxic to the cells. Moreover, lupane was not able to activate cellular functions and did not alter the production of IL-10 and TNF-α. Furthermore, the data showed that lupane has neither interfered in the action of topoisomerase I nor in the action of topoisomerase II. CONCLUSION: Based on preclinical results obtained in this study, we highlight that the compound studied (lupane) has moderate cytotoxicity, does not induce the production of TNF-α and IL-10, and does not act on human topoisomerases. Based on the results of this study and taking into consideration the reports about the anti-inflammatory and leishmanicidal activity of 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene, we suggest that this compound may serve as a biotechnological tool for the treatment of leishmaniasis in the future.