A Selective Inhibitor of Heme Biosynthesis in Endosymbiotic Bacteria Elicits Antifilarial Activity In Vitro

Abstract

Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms in vitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.

Overcoming the Challenges of Drug Discovery for Neglected Tropical Diseases: The A·WOL Experience

“…42 Hits from focused library screening have yielded several lead compounds/drugs, which are progressing through the screening funnel. Target-based screening of validated targets, including 5′-aminolevulinic acid dehydratase [43][44][45] and pyruvate phosphate dikinase, 46 has failed to deliver hits with sufficient potency or tractability compared with the wholeorganism cell-based screen. The A·WOL approach to macrofilaricidal drug discovery by using a phenotypic Wolbachia assay has generated hundreds of hit compounds, which reproducibly translate with good efficacy throughout the screening pipeline ( Table 1).…”

Cloning, Expression and Characterization of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Wolbachia Endosymbiont of Human Lymphatic Filarial Parasite Brugia malayi

“…Specific inhibitors of few Wolbachia enzymes have been investigated recently. The benzimidazoles have been shown to inhibit heme biosynthesis pathway [14], acyldepsipeptides inhibit Clp peptidase [15] and heteroaryl compounds target rsmD-like rRNA methyltransferase [16]. These inhibitors also exhibited antifilarial activity reassuring Wolbachia as a promising antifilarial drug target.…”

In Vitro Activity of wALADin Benzimidazoles against Different Life Cycle Stages of Plasmodium Parasites

“…ALADin benzimidazoles are a recently described class of species-specific inhibitors of the heme biosynthesis enzyme ␦-aminolevulinic acid dehydratase (ALAD; porphobilinogen synthase) of Wolbachia endobacteria of pathogenic filarial worms (1). The compounds killed filarial nematodes ex vivo in a Wolbachiadependent manner with no cytotoxicity below 500 M, thus representing drug lead candidates (1).…”

“…The compounds killed filarial nematodes ex vivo in a Wolbachiadependent manner with no cytotoxicity below 500 M, thus representing drug lead candidates (1).…”

“…1H and To elucidate whether the antiplasmodial effect was due to inhibition of PfALAD, we tested selected derivatives against recombinant PfALAD protein (5) (expression plasmid kindly provided by V. Arun Nagaraj). Enzymatic assays were performed as described previously for orthologs using 250 nM PfALAD in 100 mM Tris-HCl (pH 8.0), 1 mM MgCl 2 , 200 M 5-ALA for 60 min (1). wALADin1 and compound 9 showed marginal inhibitory activity at the highest concentration tested (533 M), whereas neither compound 4 nor 7 was inhibitory (Table 1).…”