Repurposing DrugBank compounds as potential Plasmodium falciparum class 1a aminoacyl tRNA synthetase multi-stage pan-inhibitors with a specific focus on mitomycin

Abstract

Plasmodium falciparum aminoacyl tRNA synthetases (PfaaRSs) are potent antimalarial targets essential for proteome fidelity and overall parasite survival in every stage of the parasite's life cycle. So far, some of these proteins have been singly targeted yielding inhibitor compounds that have been limited by incidences of resistance which can be overcome via pan-inhibition strategies. Hence, herein, for the first time, we report the identification and in vitro antiplasmodial validation of Mitomycin (MMC) as a probable pan-inhibitor of class 1a (arginyl(A)-, cysteinyl(C), isoleucyl(I)-, leucyl(L), methionyl(M), and valyl(V)-) PfaaRSs which hypothetically may underlie its previously reported activity on the ribosomal RNA to inhibit protein translation and biosynthesis. We combined multiple in silico structure-based discovery strategies that first helped identify functional and druggable sites that were preferentially targeted by the compound in each of the plasmodial proteins: Ins1-Ins2 domain in Pf-ARS; anticodon binding domain in Pf-CRS; CP1-editing domain in Pf-IRS and Pf-MRS; C-terminal domain in Pf-LRS; and CP-core region in Pf-VRS. Molecular dynamics studies further revealed that MMC allosterically induced changes in the global structures of each protein. Likewise, prominent structural perturbations were caused by the compound across the functional domains of the proteins. More so, MMC induced systematic alterations in the binding of the catalytic nucleotide and amino acid substrates which culminated in the loss of key interactions with key active site residues and ultimate reduction in the nucleotide-binding affinities across all proteins, as deduced from the binding energy calculations. These altogether confirmed that MMC uniformly disrupted the structure of the target proteins and essential substrates. Further, MMC demonstrated IC₅₀ < 5 μM against the Dd2 and 3D7 strains of parasite making it a good starting point for malarial drug development. We believe that findings from our study will be important in the current search for highly effective multi-stage antimalarial drugs.

Keywords: Malaria; Mitomycin; Pan-inhibition; Plasmodium falciparum; Structure-based drug discovery; tRNA synthetases.

Graphical abstract

Fig. 1

A. The three-dimensional structural models of class 1a Plasmodium falciparum amino acyl tRNA synthetases (PfaaRSs) with color indications of their respective domains. 1. Pf-ARS 2. Pf-CRS 3. Pf-IRS 4. Pf-LRS 5. Pf-MRS 6. Pf-VRS. Substrate molecules for each protein are shown in surface representation and are colored pink (AMP) and light blue (amino acids; Arg, Cys, Ile, Leu, Met and Val) B. Schematic representation of the domain architectures of each of the class 1a PfaaRSs as previously described (Chung et al., 2020; Fukai et al., 2003; Hauenstein et al., 2004; Kim et al., 2014, 2021; Koh et al., 2012; Liu et al., 2020; Nakanishi et al., 2005; Silvian et al., 1999).

Fig. 2

ROC and Enrichment Curves for class 1a tRNA synthetases showing the AUC and BEDROC scores to validate the performance of the docking procedure for A. Pf-ARS B. Pf-IRS C. Pf-LRS D. Pf-MRS.

Fig. 3

Surface representation of the target class 1aPf aaRSs showing the allosteric sites predicted in each of them where the 7 hit compounds are binding. A. Pf-ARS B. Pf-CRS C. Pf-IRS D. Pf-LRS E. Pf-MRS F. Pf-VRS. The respective pockets are numbered based on the ranking from SiteMap as detailed in line with residue information presented in Supplementary Tables S4–S9. Corresponding information on the specific ligand binding at each of these sites is represented in Supplementary Fig. S3.

Fig. 4

Concentration-response curves of mitomycin (Catalog C474) on multi-resistant (PfDd2) and sensitive (Pf3D7) strains of P. falciparum. Data were normalized to percentage. Control activity and median inhibitory concentrations (IC_50s) were calculated using GraphPad Prism 8.0 software. Data was fitted by nonlinear regression to the variable slope sigmoidal dose-response formula y = 100/[1 + 10^{(logIC50− x)H}], where H is the Hill coefficient or slope factor.

Fig. 5

Comparative structural and sequence analyses of MMC target sites across plasmodial and human class 1a aaRSs. Each of the plasmodial protein is shown in A. ARS B. CRS C. IRS D. LRS, E. MRS and F. VRS and the associated numbering 1. Shows the structural superposition of MMC sites among the Pf- and Hs-aaRSs 2. Shows surface representation of the MMC binding pockets of PfaaRSs. Representation is colored by atom types: C (gray), N (blue), O (red) and S (orange) atoms 3. Shows target site sequence comparison between the Pf- and Hs-aaRSs indicating the conservation degree of constituent residues. PfaaRS sequences are outlined in gray in all cases and corresponding human aaRS sequences are highlighted in orange (HsARS), yellow (HsCRS), pink (HsIRS), blue (HsLRS), purple (HsMRS) and teal (HsVRS). These are highlighted in the pairwise sequence alignment in Supplementary Data S7–S12.

Fig. 6

Comparative structural and dynamic analyses of unbound (Holo) and MMC-bound forms of the plasmodial aaRSs over a uniformly defined trajectory from the last 20ns. A1-6 Last 20 ns RMSD line plots for each protein are shown in with Holo runs 1 and 2 respectively colored black and gray while MMC-bound systems are colored pink B. Violin plots of the respective RMSD results (last 20ns) for each protein systems as distinctly colored. C. Violin plot representations of the Rg calculations from the last 20ns for each plasmodial protein as distinctly colored.

Fig. 7

Comparative structural domain analyses of the plasmodial proteins in their Holo (h) and MMC-bound states. Calculations here were done using trajectories from the stable (last 20ns) time-frame A. Pf-ARS domain 1. RMSD and 2. RMSF plots B. Pf-CRS domain 1. RMSD and 2. RMSF plots C. Pf-IRS domain 1. RMSD and 2. RMSF plots D. Pf-LRS domain 1. RMSD and 2. RMSF plots E. Pf-MRS domain 1. RMSD and 2. RMSF plots F. Pf-VRS domain 1. RMSD and 2. RMSF plots. Abbreviations:ABD – anticodon binding domain; CATp – catalytic pocket; Ins 1 & 2 – Insertion domains 1 & 2; NTD – N-terminal domain; CP- connective peptide editing domain; CP1c – CP1 core region; CTD – C-terminal domain; HB – Helical bundle domain; AC stem – anticodon stem binding domain; CCD – C-terminal coiled coil domain. Color scheme presented accordingly from white through yellow, orange, red and black for low-to-high RMSF values.

Fig. 8

3D comparison of structural perturbations at key catalytic and allosteric domains of Holo (green) and MMC-bound (cyan) A. Pf-ARS B. Pf-CRS C. Pf-IRS D. Pf-LRS E. Pf-MRS and F. Pf-VRS. Some important residues with high fluctuations (from RMSF plots in Fig. 7) at each of the respective domains are also highlighted accordingly.

Fig. 9

Analyses of MMC's interaction dynamics, affinity, and mechanisms in the target plasmodial proteins. Calculations here were done using trajectories from the stable (last 20ns) time-frame A. RMSD violin plot of MMC across the six proteins B. 2D interaction profile of the ligand at the respective target sites of 1. Pf-ARS 2. Pf-CRS 3. Pf-IRS 4. Pf-LRS 5. Pf-MRS and 6. Pf-VRS C. Per-residue energy decomposition plots showing individual energy contributions of residues to the binding of MMC at the respective target sites of 1. Pf-ARS 2. Pf-CRS 3. Pf-IRS 4. Pf-LRS 5. Pf-MRS and 6. Pf-VRS.

Fig. 10

AMP interaction dynamics, mechanisms, and affinities at the respective active sites of the target plasmodial proteins. Calculations here were done using trajectories from the stable (last 20ns) time-frame A. Per-residue energy decomposition plots showing changes in individual energy contributions of catalytic site residues to the binding of AMP in 1. Pf-ARS 2. Pf-CRS 3. Pf-IRS 4. Pf-LRS 5. Pf-MRS and 6. Pf-VRS B. 3D structural superposition of AMP at the catalytic sites of Holo (green) and MMC-bound (cyan) 1. Pf-ARS 2. Pf-CRS 3. Pf-IRS 4. Pf-LRS 5. Pf-MRS and 6. Pf-VRS C. Comparative ΔG_bind plot showing differences in the binding affinities of AMP across the Holo (red) and MMC-bound (green) plasmodial proteins.