Structural and functional studies of histidine biosynthesis in Acanthamoeba spp. demonstrates a novel molecular arrangement and target for antimicrobials

Rice, Christopher A.; Campbell, Samantha A; Bisson, C.; Owen, H.J.; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W.; Henriquez, Fiona L.; Roberts, Craig W.

doi:10.1371/journal.pone.0198827

Cited by 12 publications

(8 citation statements)

References 37 publications

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“…More recently the MMV Pathogen Box was screened against non-pathogenic Acanthamoeba castellanii Neff [45]. These studies extrapolated translational activity of compounds from non-pathogenic to pathogenic amoebae despite several reports of drug variation responses from different species and pathogenicity within the Acanthamoeba genus [46][47][48]. For example, we previously identified 39 agents active against pathogenic N. fowleri from screening the U.S. FDA approved drug library, Medicines for Malaria Venture (MMV) Pathogen Box and MMV Malaria Box [49].…”

Section: Discussionmentioning

confidence: 99%

“…Structure-based drug design as a strategy is limited, since only a handful of proteins have been crystallized and structurally investigated as potential therapeutic targets. These include genes involved in shikimate, histidine, and steroid biosynthesis, and more recently, glucose metabolism [48,[54][55][56][57][58]. As a eukaryotic organism, these amoebae share common functional pathways with humans in which unwanted side effects can result due to the required effective concentrations needed from the current treatments used (e.g.…”

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 99%

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Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

et al. 2020

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Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC 50 <1 μM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new antiamoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.

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Section: Discussionmentioning

confidence: 99%

Section: Plos Neglected Tropical Diseasesmentioning

confidence: 99%

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

et al. 2020

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“…Structure-based drug design as a strategy is limited, since only a handful of proteins have been crystallized and structurally 378 investigated as potential therapeutic targets. These include genes involved in shikimate, histidine, and steroid 379 biosynthesis, and more recently, glucose metabolism 48,[54][55][56][57][58] . As a eukaryotic organism, these amoebae share common 380 functional pathways with humans in which unwanted side effects can result due to the required effective concentrations 381 needed from the current treatments used (e.g.…”

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confidence: 99%

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

Rice¹,

Colon

Chen

et al. 2020

Preprint

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33Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria 34 fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic 35 encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates 36 due to lack of effective therapeutics. In pursuit of repurposing drugs for chemotherapies, we conducted a high throughput 37 phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors 38 (IC 50 <1 M) against N. fowleri (n=19), A. castellanii (n=12), and B. mandrillaris (n=27) plus an additional 90 micromolar 39 inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or 40 Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit 41 N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, 42 solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, 43 acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the 44 drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for 45 discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many 46 of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for 47 structure-based drug design. 48 49 Author Summary 50 Free-living amoebae (FLA) are ubiquitous in soil and freshwater and most are non-pathogenic to people; 51 however, three different pathogenic FLA have been found to cause severe, most often fatal diseases in humans. Due to 52 poor detection and inadequate treatment options available for pathogenic FLA, the fatality rates are still > 90% for the 53 diseases caused by Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba spp. With hundreds of cases in the 54 United States and many more cases reported worldwide, there is still an urgent clinical need for effective diagnosis and 55 specific treatments discovered against these opportunistic parasites. Drug repurposing is a powerful approach for drug-56 discovery because it significantly improves the discovery time, reduces the amount of resources, and decreases costs 57 required to advance lead candidate drugs of interest into the clinic. This is extremely helpful for neglected diseases 58 including pathogenic FLA where there is a need for new active therapies with limited budgets. This report addresses the 59 discovery of new active drugs with potential for repurposing, multiple new drug classes that inhibit pathogenic FLA, and 60 numerous putative drug targets that can be used as tools for further investigation and structure-based drug design. 61 7 . Naegleria, being ther...

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“…All proteins discussed were PCR-amplified using cDNA as a template. RNA template of Naegleria fowleri ATCC30215 was provided by Dr. Christopher Rice (University of Georgia, Athens) through RNA extraction and cDNA synthesis using previously published methodology in Acanthamoeba (47). PCR, cloning, screening, sequencing, expression screening, large-scale expression and purification of proteins were performed as described in previous SSGCID publications (17,48).…”

Section: High-throughput Protein Expression and Purificationmentioning

confidence: 99%

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

Barrett

Tillery

Goldstein

et al. 2020

Preprint

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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. N. fowleri 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.

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Structural and functional studies of histidine biosynthesis in Acanthamoeba spp. demonstrates a novel molecular arrangement and target for antimicrobials

Cited by 12 publications

References 37 publications

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

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

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