L-asparaginase as a new molecular target against leishmaniasis: insights

about mechanism of action and structure-based inhibitor design

Jasdeep Singha§, Ankit Srivastavaa§, Praveen Jhab, Kislay Kumar Sinhab, Bishwajit Kundua*

SUPPLEMENTARY DATA

Supplementary Table 1: The comparative genomics search of L-asparaginase gene sequences in human protozoan pathogens.

Organism Gene ID Uniprot ID Size Location ReferenceLeishmania Donovani 13392921(Cytoplasmic)

13388465(Putative)E9BC85 398 147557..148753 PMID:220

38251Plasmodium spp. - - - - -

- -Cryptosporidium spp. - - - - -Toxoplasma - - - - -Acanthamoeba castellanii

14923603 L8HE50 378 5799..7834

Balantidium coli - - - -Salmonella enteritidis Un-annotated X4ZXT1 338 - -Blastocystis - - - -Shewanella baltica Un-annotated E6SZ34 337 - -Cryptosporidium - - - -Dientamoeba fragilis - - - - -Entamoeba histolytica 3410905 C4LTU4 378 - -Giardia lamblia 5697871 (putative) A8BSW8 380 12719..13861 PMID:179

01334Isospora belli - - - - -Toxoplasma gondii Un-annotated S8EZS8 673 - -Trichomonas vaginalis - - - -Trypanosoma brucei - - - - -Trypanosoma cruzi 3541834 Q4D990 498 96383..105589

Electronic Supplementary Material (ESI) for Molecular BioSystems.This journal is © The Royal Society of Chemistry 2015

Supplementary Methods

ADMET parameters

Blood-Brain Barrier (BBB) penetration is represented as BBB(-). The values between

0.1-0.3 indicate high penetration, 0.4-0.6 indicates moderate penetration and 0.7-1.0

indicates minimal penetration.

Intestinal absorption (+) of a compound refers to sum total of bioavailability and

absorption evaluated from ratio of excretion or cumulative excretion in urine, bile and

feces. The values between 0.1-0.3 indicate poor absorption, 0.4-0.6 indicates moderate

absorption and 0.7-1.0 indicates high absorption.

Caco-2 cell (-) are derived from human colon adrenocarcinoma possessing multiple drug

transport pathways. The values between 0.1-0.3 indicate high permeability, 0.4-0.6

indicates moderate permeability and 0.7-1.0 indicates lowest permeability.

P-gp (P-glycoprotein) pumps are responsible for efflux of drugs leading to lower

available bioavailability. For a better bioavailability, a drug should ideally be moderate

inhibitors of P-gp to ensure adequate bioavailability in body. For P-gp (Non-substrate),

values between 0.1-0.3 indicate low probability of drug to be substrate of P-gp, 0.4-0.6

indicates moderate non-inhibition and 0.7-1.0 indicates high probability of the drug to be

substrate of P-gp.

CYP2D6 enzyme system is responsible for metabolism of plethora of drugs through

conjugation pathways. CYP2D6 induction may lead to formation of higher active

metabolites or higher excretion rates due to formation of polar metabolites. For CYP2D6

non-inhibition, a value between 0.1-0.3 indicates low probability of drugs to induce

CYP2D6, 0.4-0.6 indicates moderate induction and 0.7-1.0 indicates high probability of

induction of CYP2D6 by the drug.

Supplementary Figure 1: a. The chromosomal position of putative L-asparaginase in L.donovani; b.

Ramachandran plot of the LDA1 model; c. Prosa based residue-wise energy estimation of LdAI model.

Supplementary Figure 2: a. All-atom RMSD of Apo and Holo forms of LdAI; b. The total energy of Apo and Holo-complexes of LdAI for 10 ns MD simulations.

Supplementary Figure 3: a. Superposition of LdAI and EcAI monomers; b. The protruding C-terminal loop of LdAI shown in ribbon representation with superposed EcAI monomers shown as surface representation; c. The comparative depiction of solvent accessible surface area of active site residues in LdAI, EcAI and EcAII .

Supplementary Figure 4: Comparative free energy estimation of screened ligands (L1, L2, L5, L7, L12) binding with active site of LdAI and HLA.

Supplementary Figure 5: The surface representation of docked complexes in the LdAI active site pocket; a. L1, b. L2, c. L5, d. L7, e. L12.

Supplementary Figure 6: The total energy of ligand-complexes with LdAI for 10 ns MD simulations.