Novel inhibitors designing against the potential drug target of Plasmodium falciparum M17 Leucyl Aminopeptidase – PfM17LAP

Malaria is one of the major disease of concern worldwide especially in the African regions. According to the recent WHO reports, African regions share 95% of the total deaths worldwide that occurs due to malaria. Plasmodium falciparum M17 Leucyl Aminopeptidase (PfM17LAP) plays an important role in the regulation of amino acids release and for the survival of the parasite. We performed molecular docking and simulation studies to find the potential inhibitors against PfM17LAP using ChEMBL antimalarial library. Molecular docking studies and post-docking analysis revealed that molecules CHEMBL369831 and CHEMBL176888 showed better binding than the reference molecule BESTATIN. LibDock and X-SCORES of molecules BES, CHEMBL369831 and CHEMBL176888 are 130.071, 230.38, 223.56 and -8.75 Kcal/mol, -10.90 Kcal/mol, -11.05 Kcal/mol respectively. ADMET profiling of the top ten ranked molecules was done by using the Discovery Studio. Molecular dynamic studies revealed that the complex PfM17LAP-CHEMBL369831 is stable throughout the simulation. Finally, we have reported novel inhibitors which possess more binding affinity towards PfM17LAP.


Introduction:
Malaria is one of the most lethal diseases transmitted by the Plasmodium spp through bite of female Anopheles mosquito. Plasmodium falciparum is one of the most lethal apicomplexan parasites among the Plasmodium species. Deaths due to malaria are more in countries with poor sanitization and in African countries. Children under age of 5 years are more prone to the malarial infection in African regions, along with that African regions shares 95% of the malarial cases and 96% of the overall malarial deaths throughout worldwide.
According to the WHO statistics, there are 229 million malaria cases and 409000 deaths reported worldwide in 2019. There are 241 million malaria cases and 627000 deaths reported worldwide in 2020. Deaths due to malaria increased in 69000 number as compared to the last year 2019. In October 2021, WHO approved the use of the malarial vaccine RTS,S/AS01 for children in nations with high transmission rate of P. falciparum. To achieve the success in the malarial treatment Artemisinin is being used either as a single drug or in combination with other drugs (ACT), use of Artemisinin in combination with other drugs helps to overcome the resistance against the other drugs. Other advantages of ACT are high efficacy, fast action and likelihood of resistance development.
ACT therapies are showing better results in the terms of efficacy and its control over the malaria is good. But in the recent years ACT therapy is also showing resistance in some areas of the Western Cambodia (Leang et al. 2015) and failure to the ACT therapies are rapidly increasing (Mok et al. 2011). Artemisinin resistance is emerging and spreading independently in the mainland Southeast Asia, Pursat, Western boarder of Thailand, Southern Myanmar and Vietnam (White et al. 2014) (Kyaw et al. 2013). A single nucleotide polymorphism in Kelch 13 gene of the Plasmodium parasite is reported as the causes of resistance related to Artemisinin (Noreen et al. 2021).
Malaria parasite utilizes proteases in the many stages of its life cycle. Aminopeptidase enzymes remove N-terminal amino acids from short peptides with high specificity. Two Aminopeptidases namely P. falciparum M1 Aminopeptidases (PfM1AAP) and P. falciparum M17 Leucyl Aminopeptidase (PfM17LAP) have important role in the growth and development of parasites inside the red blood cell by regulating release of amino acids that are required for the growth of parasite (Ruggeri et al. 2015). These two enzymes are present in the cytosol of parasite and are responsible for the survival of parasite. They also provide nutrients required for the growth and development of parasite. Gene targeting and in vivo experimental results have shown that, inactivation of PfM17LAP causes inhibition of growth in some parasites and in some parasites death also (Stack et al. 2007)(McGowan et al. 2009).
Hence we used Insilico tools to develop novel inhibitors of PfM17LAP with the aim to stop growth of parasite. PfM17LAP is 528 amino acid structure with two metals in the active site. The metal of the first site Mg 2+ is readily exchangeable with other metal ions Zn 2+ , Co 2+ and Mn +2 and acts as a regulatory site. But the second site with Zn 2+ is tightly bound to the active site and known as catalytic site. Metal replacement studies found that PfM17LAP retains activity when the tight binding catalytic site is occupied with the metal ion, but removal of the both metal ions leads to the irreversible inactivity (McGowan et al. 2010).
The structure of the PfM17LAP shows that only catalytic site 2 occupied by the Zn 2+ and no electron density was visible for the site occupied by Mg 2+ . Zn +2 coordinated by the Lys374, Asp379, Asp399 and Glu461. Along with the metal ions it also have inhibitors BESTATIN (BES) and Co4. BES is natural inhibitor of the leucine aminopeptidases. It is obtained from the culture filtrates of the Streptomyces olivoreticuli (Wilkes and Prescott 1985).
It has antibiotic and antimalarial activities as well (Wilkes and Prescott 1985). Inhibitory activity (Ki) for the BES and Co4 are 25 nM and 13 nM respectively (McGowan et al. 2010).
Plasmodium parasite uses Plasmepsins for intraerythrocytic hemoglobin degradation of human host and uses it as nutrient source (Kumar and Ghosh 2007). There are 10 different types of the Plasmepsins in the Plasmodium species expressed at different stages of the life cycle. Among the 10 types, PfPM I-IV are active in digestive vacuole and shares 50-70% identity; PfPM-V active in effector export and it shares 19-23% identity with other PfPM; PfPM-VI, VII & VIII are active during transmission stage and they shares 31-36% identity among each other; PfPM-IX and X shares 37% identity between each other (Nasamu et al. 2020). PfPM III is also known as Histidine Aspartic Protease (HAP), due to presence of Histidine in the active site. Expression data of the PfPM I-IV are mentioned in table 1. Studies have shown that removal of the any one of the PfPM from PfPM I-IV does not affect the parasitic activity but removal of multiple PfPMs show effective antiparasitic activity (Liu et al. 2005). Hence considering the importance of PfPM I-IV in parasitic activity, we selected PfPM To design novel inhibitors against PfM17LAP we performed molecular docking studies. We selected CHEMBL antimalarial library for screening, which have bioactivity information for every molecule. We selected top ten molecules with high docking score for post docking analysis and for Molecular Dynamic (MD) simulations.
For the selected the targets PfPM I-IV, to design the potential antimalarial drugs. We performed molecular docking and post-docking analysis of the top ten hits obtained from the high throughput screening of the ChEMBL database against the PfM17LAP, to propose the dual inhibitory activity of the top ten hits.

Material and Methods:
Protein preparation and chemical library preparation: PfM17LAP crystal structure is taken from the Protein Data Bank (PDB ID -3KR4).
Resolution of PDB structure is 2A 0 . Active site residues are defined based on the reference ligand BES. PDBSum analysis showed that residues ASP379, LYS386, ASP399, ASP459, Compounds are prepared by using Discovery studio. A maximum of 10 tautomers were generated for each compound. Isomers were generated and bad valancies were fixed during the library preparation. 3D co-ordinates were generated for each compound. A total of 281896 compounds generated after library preparation.

Plasmepsins
Expression data from TDR targets (

Post-docking analysis:
Top ten LibDock scored compounds were selected for the post-docking analysis.
Protein-ligand interactions were calculated and visualized by using Ligplot+ (Laskowski and Swindells 2011). Ligplot generates interaction diagrams of hydrogen and non-bonded interactions between the protein-ligand complexes along with labelling of distance between the bonds. Binding energies between receptor and ligand were calculated by using the X-SCORE (Wang, Lai, and Wang 2002) (Equation 1). X-score calculates hydrogen bonding, van der Waals, hydrophobic interactions and deformation penalties between the protein and ligand (Obiol-Pardo and Rubio-Martinez 2007).

Molecular Dynamic Simulations:
MD simulation were performed by using GROMACS version 2020.4 (Abraham et al.

2015)
. Gromos53a5 forcefield (Oostenbrink et al. 2004) was used for the simulation. Both protein and protein-ligand complex simulations were performed. Protein was placed in the center of the cubic box with distance of 10A 0 radius around the protein and SPCE water model was used for the solvation. Ligand topologies were generated by ATB server (Malde et al. 2011). NVT and NPT ensemble were ran for 500 Pico seconds before the minimization step at 300k temperature and 1 bar pressure. 50000 Minimization steps were done before the final production run. Particle Mesh Ewald method (Cerutti et al. 2009) was used for the long-range electrostatic interactions and Lenard Jones potential and coulombs charge was used for the short-range interactions with distance of 1.2 nm. Berendsen thermostat was used for the temperature coupling (Bussi, Donadio, and Parrinello 2008). LINCS algorithm was employed to constrain the bonds with hydrogen atoms. After equilibration, each system setup for the 50ns long production run was performed at 2 femto seconds (fs) time step, for every 10ps trajectory (pdb coordinates are saved) was saved. Results were analyzed by using the inbuilt gromacs modules and results were analyzed and visualized by using XMGRACE tool.

ADME and Toxicity predictions:
Pharmacokinetic (PK) properties namely absorption, distribution, metabolism and excretion (ADME) are the important properties to measure the movement of drug in the body with respect to time. In the present study ADME properties were predicted computationally with the help of ADME descriptor algorithm protocol in BIOVIA Discovery studio 2020 ChEMBL library have more affinity than reference ligand BES.

Molecular docking of Plasmepsins I-IV:
Experimental data from the ChEMBL database showed that top ten hits have already been reported to possess inhibitory activity (Ki) against PfPM I, PfPM II and Cathepsin D.
Among the top ten hits, 8 compounds have reported high Ki values against the target Plasmepsin I. Therefore, these top hits seem to be acting as probable dual inhibitors. To decipher this, we performed molecular docking analysis of the top ten hits against the four types of plasmepsins found in digestive vacuole. These plasmepsins are known to play an important role in the intraerythrocytic hemoglobin degradation (Nasamu et al. 2020  LibDock Score X-Score (K cal/mol)

ADMET and Mutagenesis analysis of top ten scored compounds:
ADMET analysis done with the help ADME predictor in Discovery studio. Toxicity predictions are done with of TOPKAT module from Discovery studio. ADME analysis showed that except BES, every ligand shows poor absorption. Solubility level is good for top eight ligands and extremely good for CHEMBL1086195 and CHEMBL5801112. Blood Brain Barrier penetration value is unknown for the top scored ligands including reference ligand BES.
As per prediction top ten scored ligands are non-toxic to humans except CHEMBL191130.
TOPKAT predictions showed that top scored ligands are non-mutagens.

Molecular Dynamic simulation analysis:
Through MD simulations we can observe the changes in the protein according to time. Hydrogen bonds analysis shows that Protein in complex with BES shows a greater number of hydrogen bonds (6) than the other system. Figure 7 shows that protein in complex with the CHEMBL369831 shows 2 consistent hydrogen bonds throughout the simulation.

Conclusion:
As resistance to first line antimalarial drugs is increasing rapidly, there is a rising need to find novel antimalarial molecules with grater potency. The aim of our study was to find novel molecules with greater affinity towards the selected target PfM17LAP. It is one of the most crucial enzymes required for the survival of the P. falciparum. In the present study, antimalarial database from ChEMBL was taken for the screening purpose whose results showed that CHEMBL369831 and CHEMBL176888 showed greater affinity towards the protein with highest LibDock and X-SCOREs. Further, stability of the protein in complex with CHEMBL39831 was studied by using molecular dynamic simulations. Overall MD simulation results showed that CHEMBL369831 is stable in the active site. Bioactivity data from the CHEMBL database shows that molecule CHEMBL369831 has minimum reported inhibitory constant (Ki) of 0.5 nM. Overall results showed that CHEMBL369831 may be the potential inhibitor for PfM17LAP. Further, clinical studies are required to use this molecule as an antimalarial drug. The experimental data from ChEMBL database and inferences drawn from our present investigation, together suggest that the top ten hits seem to act as dual inhibitors of Plasmepsins I-IV and PfM17LAP.