Abstract
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.
Highlights
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease
Our results indicated that only dequalinium chloride possessed nanomolar potency and that 43% of the currently recommended drugs by the Centers for Disease Control and Prevention (CDC) for treating Balamuthia infections appeared to be only moderately to slightly efficacious, with IC50 values ranging from 18.35 μM to > 163.25 μM
We identified other ribosomal protein inhibitors for 50S, 23S of the 50S [mirincamycin], and the 16S ribosomal subunits suggesting protein synthesis would be a useful target for drug therapy against Balamuthia infections
Summary
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections. Though draft genomes have been published, no structural and functional annotation is currently available[18,19] This information is essential for the design of new drugs by SGDD/SBDD, as that methodology requires information about the molecular structure of the target protein. The clones were further validated by direct sequencing, providing the first step for recombinant expression and crystallization by the Seattle Structural Genomics Center for Infectious Disease (SSGCID) high-throughput gene-to-structure p ipeline[20]
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