Abstract 1223 Structural modeling of P1 nuclease enzyme from promastigote of Leishmania infantum: Implications for design of natural prophylactic molecules for leishmaniasis

Background: Leishmaniasis is a vector borne neglected tropical disease infection caused by the protozoa Leishmania infantum. One million people are affected annually and one billion people in endemic areas are at risk. High fatality rate, risk of relapse, toxic chemotherapy and lack of effective vaccines has necessitated prophylactic drug development to prevent the infection. P1 nuclease enzyme in promastigote form is involved in purine salvage pathway in which nucleotides are synthesized from human host material for its own survival, growth and multiplication, and therefore makes it an attractive drug target. Methods: Protein sequence of P1 nuclease from Leishmania infantum (A4I5I0) was taken from uniport for sequence analysis. Homology modeling of the P1 nuclease was done based on the crystal structure of nuclease from E. coli (PDB ID: 1AKO) using SWISS MODEL. Conformational correctness and reliability were carried out using PROCHECK. Zinc ions were fitted into the best validated model and this complex was used as a receptor for structure based In-silico studies. Binding pocket prediction was done using program Sitemap in Maestro suit and resultant pockets with the best Dscore>0.9. Aurintricarboxylic acid (ATA), a general inhibitor of nuclease, was docked to the enzyme at the binding pocket using Glide, followed by Molecular Dynamics simulations for 100ns using Desmond simulation program. A library of molecules similar to scaffold of ATA was made from COCONUT, a natural product database using substructure search based on Ullmann algorithm. Screening was performed in virtual high throughput, standard precision, and extra precision docking modes using Glide. Complexes of molecules compliant for Lipinski rule were subjected to Molecular Dynamics simulations, and their binding parameters were delineated. Results: Structure of P1 nuclease shows a 268 residue globular protein with all its residues in core and allowed regions of Ramachandran plot. Fold comprises of secondary structural elements composed of nine helices, three short helices, and a single β-hairpin. Substrate binding cleft is nestled deep within the protein and comprises of an active site comprising of five histidine residues, three aspartate residues, one tryptophan and one bridging water molecule that together coordinate three zinc ions, a feature that is essential for structural stability. Met 92, Leu 86, and Ilu 195 provide hydrophobic moieties for bases. 2-[(4-hydroxyphenyl)-(4-methoxyphenyl) methyl]benzoic acid (244MBA) is the only screened natural molecule that fulfills all the applied screening criteria. Both ATA and 244MBA induces subtle conformational changes on binding to the enzyme. They make an array of hydrogen bonded and hydrophobic interactions with residues, and coordinate bonds with zinc ions at active site. 244MBA has an association constant of 0.13μM, and total binding energy of -85X103 kcal/mol. Conclusions: Structural modeling of P1 nuclease from Leishmania infantum reveals the three dimensional fold of enzyme. Analysis of enzyme molecule complexes establishes the binding modalities of respective molecules to P1 enzyme. 244MBA with a better potency than ATA has the potential to be developed as a prophylactic agent against leishmaniasis. Funding from Indian Council of Medical Research, New Delhi, India (ICMR/2020-4975) is acknowledged