| Abstrakt: |
Background: Filarial nematodes cause severe illnesses in humans and canines including limb deformities and disfigurement, heart failure, blindness, and death, among others. There are no vaccines, and current drugs against filarial nematodes infections have only modest effects and are prone to complications. Methodology/principal findings: We identified a gene (herein called DiMT) encoding an S-adenosyl-L-methionine (SAM)-dependent methyltransferase with orthologs in parasite filarial worms but not in mammals. By in silico analysis, DiMT possesses catalytic sites for binding SAM and catecholamines with high affinity. We expressed and purified recombinant DiMT protein and used it as an enzyme in a series of SAM-dependent methylation assays. DiMT acted specifically as a catechol-O-methyltransferase (COMT), catalyzing catabolic methylation of dopamine, and depicted Michaelis Menten kinetics on substrate and co-substrate. Among a set of SAM-dependent methyltransferase inhibitors, we identified compounds that bound with high affinity to DiMT's catalytic sites and inhibited its enzymatic activity. By testing the efficacy of DiMT inhibitors against microfilariae of Dirofilaria immitis in culture, we identified three inhibitors with concentration- and time-dependent effect of killing D. immitis microfilariae. Importantly, RNAi silencing of a DiMT ortholog in Caenorhabditis elegans has been shown to be lethal, likely as a result of excessive accumulation of active catecholamines that inhibit worm locomotion, pharyngeal pumping and fecundity. Conclusions/significance: Together, we have unveiled DiMT as an essential COMT that is conserved in parasitic filarial nematodes, but is significantly different from mammalian COMTs and, therefore, is a viable target for development of novel drugs against filarial nematode infections. Author summary: Parasitic filarial nematodes cause serious illnesses in humans and canines including limb deformities and disfigurement, blindness, heart failure, and death, among others. There are no vaccines, and current drugs in use against filarial nematodes infections have only modest effects and are prone to complications. In this study, we explored a unique enzyme (DiMT), conserved in all parasitic filarial nematodes but not in mammalian species. Using computation, biochemistry and molecular biology approaches, we validated DiMT as a catechol-O-methyltransferase that functions to inactivate catecholamines to prevent their accumulation and hence block their deleterious effects in filarial nematodes, including loss of worm viability, reproduction and survival. Subsequently, we identified DiMT's chemical inhibitors and demonstrated that they possess anti-filarial nematode efficacy through blocking the inactivation of catecholamines. Together, we have unveiled DiMT as a viable molecular target for the development of new drugs for treating filarial nematode infections. [ABSTRACT FROM AUTHOR] |
