Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Abstract

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri

“…The 50% inhibitory concentration (IC 50 ) of each drug was determined individually with three biological replicates for all 11 clinical isolates ( Table 1 ; also, see Table S1 in the supplemental material). We used isolate Nf69 as the reference for comparisons, because it has been used extensively in recent drug discovery research ( 17 , 23 , 32 , 35 – 38 ). As shown in Fig.…”

Differential growth rates and in vitro drug susceptibility to currently used drugs for multiple isolates of Naegleria fowleri

“…The IC50 for each drug was determined individually with three biological replicates for all 11 clinical isolates (Table 1, Table S1). We used the Nf69 isolate as the reference for comparisons because it has been used extensively in recent drug discovery research (17,23,31,(51)(52)(53)(54). As shown in Figure 1A, there was no statistically significant difference in the susceptibility of the isolates for amphotericin B when compared to the reference isolate as determined by one-way ANOVA (F(10,22)=1.711, p=0.1411).…”

The therapeutic potential of novel isobenzofuranones against Naegleria fowleri

“…With more than 430 cases officially reported ( Bellini et al, 2018 ; Maciver et al, 2020 ; Piñero et al, 2019 ), the United States of America (USA) and Pakistan are the most affected countries ( Debnath et al, 2020 ; Johnson et al, 2016 ; Yoder et al, 2010 ). Recently other countries including Brazil, Costa Rica, Philippines or India have reported new cases of PAM ( Ali et al, 2020 ; Graciaa et al, 2018a ; Henker et al, 2019 ; Milanez et al, 2017 ; Mittal et al, 2019 ), and at the present time it has been classified as a B category priority pathogen by the National Institute of Allergy and Infectious Diseases (NIAID) ( Tillery et al, 2021 ).…”