Classical and quantum mechanical methods to find specific allosteric inhibitors for papain-like cysteine proteases of Plasmodium falciparum and Plasmodium vivax

Worldwide, two are the most important species of the parasite causing malaria that infect humans: the Plasmodium falciparum, widely explored in the literature, and the Plasmodium vivax, more relevant to the Americas and less studied. The investigation of new inhibitory targets, involved in essential steps for the survival of such protozoa, is needed due to complications and the appearance of resistance to current drugs. Inhibition of the papain-like cysteine proteases, falcipains (FPs) and vivapains (VPs) of P. falciparum and P. vivax, respectively, is an interesting area of study because they are involved in the hydrolysis of haemoglobin, which releases substrates for the parasites to maintain their life cycle and proliferate. The main goal of this study is to design inhibitors specific for allosteric cavities of such enzymes through computational methods. To this, Molecular Dynamics (MD) simulations were performed for FPs 2 and 3 and VPs 2, 3 and 4 in replicates up to 2,5 μs in total using NAMD3.0 software. Clustering of conformations was carried out using the RMSD as the metric and a structure representative of each of the three main conformational clusters was chosen. Next, the CavityPlus server was used to identify cavities and their allosteric potential for each cluster, finding important mechanical allosteric sites as candidates. Two of them have presented the highest allosteric Z-scores of 1.8 (cavity A) and 2.57 (cavity B) among the conformational clusters. They involve residues such as K137 and Y225, and K13 and R48, respectively Additionally, quantum mechanics (QM) calculations were done using MOPAC2016 and were analysed using PRIMoRDiA to assess enzyme reactivity sites and the location of frontier orbitals, revealing amino acid residues in allosteric cavities that may be more focused in inhibitor design through electronic perturbation allostery. APBS software was used to analyse the electrostatics surface of each cluster, allowing the understanding of the changes in the potentials depending on the conformation analysed. Analysis with the Bio3D package of R revealed changes in the network of concerted motion among residues depending on the conformational cluster of the protein. Next, Virtual Screening of compounds from ZINC20 is going to be done, after filtering molecules with the knowledge obtained from studying pharmacophore groups in each cavity.