Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis

Colon, Beatrice L.; Rice, Christopher A.; Guy, R. Kiplin; Kyle, Dennis E.

doi:10.1093/infdis/jiy622

Cited by 37 publications

(51 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, lipids are not only the preferred food source for N. gruberi , but oxidation of fatty acids also seems to be essential for growth. The current treatment of Miltefosine and Amphotericin B was also shown to be effective in inhibiting growth, which is in agreement with previous reports (17-19) and validates our high through-put assay to detect compounds that inhibit growth of N. gruberi .…”

Section: Discussionsupporting

confidence: 92%

Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs

Sarink

Verbon

Tielens

et al. 2019

Preprint

View full text Add to dashboard Cite

14Primary Amoebic Meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-15 living amoeba Naegleria fowleri. The disease mostly affects healthy children and young 16 adults after contaminated water enters the nose, generally during recreational water activities. 17 The amoeba migrate along the olfactory nerve to the brain, resulting in seizures, coma and 18 eventually death. Previous research has shown that Naegleria gruberi, a close relative of N. 19 fowleri, prefers lipids over glucose as an energy source. Therefore, we tested several 20 inhibitors of fatty acid oxidation alongside the currently used drugs amphotericin B and 21 miltefosine. Our data demonstrate that etomoxir, orlistat, perhexiline, thioridazine and 22 valproic acid inhibited growth of N. gruberi. Furthermore, additive effects were seen when 23 drugs were combined. Both thioridazine and valproic acid inhibit in vitro growth of N. 24 gruberi in concentrations that can be obtained at the site of infection, which is doubtful with 25 the currently used drugs amphotericin B and miltefosine. Both thioridazine and valproic acid 26 have already been used for other diseases. As the development of new drugs and randomized 27 controlled trials for this rare disease is nearly impossible, repurposing drugs is the most 28 promising way to obtain additional drugs to combat PAM. Thioridazine and valproic acid are 29 available drugs without major side-effects and can, therefore, be used as new complementary 30 options in PAM therapy. 31 49 the pathogen to exert their killing effect, such as the antimalarials atovaquone and proguanil, 50 and the broad-spectrum antihelminthic and antiprotozoal nitazoxanide (12, 13). 51Previous research by our group showed that N. gruberi, a close relative to N. fowleri, 52 prefers fatty acids as a food source (14). This led us to the hypothesis that inhibiting fatty acid 53 oxidation (FAO) could inhibit growth or even kill the amoeba. We identified several drugs 54 that inhibit fatty acid metabolism in different parts of this pathway. As the metabolic 55 machinery of N. gruberi is highly similar to that of N. fowleri (14-16), we used N. gruberi as a 56 4 model organism to determine the effects of these compounds. We then compared the effects 57 of these inhibitors to the currently used treatment (AMB and MIL) and determined additive 58 effects of the compounds when they were combined. 59 60 Materials and Methods 61 Chemicals and amoeba culture. N. gruberi strain NEG-M (ATCC® 30224) was grown 62 axenically at 25 ˚C in modified PYNFH medium (ATCC medium 1034), as described before 63 (14). Modified PYNFH is composed of peptone, yeast extract, yeast nucleic acid, folic acid, 64 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin and 65 40 µg/ml gentamicin. All experiments were performed using trophozoites harvested during 66 the logarithmic phase of growth. Amphotericin B (AMB), etomoxir (ETO), miltefosine 67 (MIL), thioridazine (TDZ), orlistat (ORL), perhexiline (PHX...

show abstract

Section: Discussionsupporting

confidence: 92%

Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs

Sarink

Verbon

Tielens

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Comparison of all hits shared between to have any potency against FLA. Furthermore, this is the first description of a high-throughput (384 well plates) identified the same 8 agents from the ReFRAME library as from our previous screening of the FDA approved drug library butenafine and pitavastatin) 49 . This exemplifies the robustness of our high-throughput screening assays as well as true hit inhibitors of N. fowleri (IC 50 = 20 nM), it has a lag phase of ~30 hours before inhibiting growth.…”

mentioning

confidence: 77%

“…In vitro drug susceptibility assays. The phenotypic drug susceptibility assays were performed as previously described using CellTiter-Glo v2.0 (CTG) (Promega, Madison, WI) 44,49,63 . The ReFrame collection of 12,000 compounds concentrations of 5µM (30nL of 10mM stock concentration/well in dimethylsulfoxide (DMSO)) using the Labcyte Echo growth control (0.5% DMSO) and inhibitor controls that produced ~100% inhibition of amoebae cell populations.…”

mentioning

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

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…The phenotypic drug susceptibility assays were performed as previously described using Cell-Titer-Glo v2.0 (CTG) (Promega, Madison, WI) 44,49,63 . The ReFrame collection of 12,000 compounds assembled at Calibr-Scripps were prepared in 384-well (Corning, White Flat bottom 3570) plate format at screening concentrations of 5μM (30nL of 10mM stock concentration/ well in dimethylsulfoxide (DMSO)) using the Labcyte Echo 555 for acoustic compound dispensing.…”

Section: In Vitro Drug Susceptibility Assaysmentioning

confidence: 99%

“…RealTime-Glo MT Cell Viability Assay (RTG; Promega, Madison, WI) previously described by Colon et al, was used to assess the speed of inhibition of active compounds against N. fowleri [49]. Compounds were plated at 1x and 5x their respective IC 50 's with 3 replicates per plate.…”

Section: Rate Of Action Assaymentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Phenotypic Screens Reveal Posaconazole as a Rapidly Acting Amebicidal Combination Partner for Treatment of Primary Amoebic Meningoencephalitis

Cited by 37 publications

References 33 publications

Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs

Inhibition of fatty acid oxidation as a new target to treat Primary Amoebic Meningoencephalitis by repurposing two well-known drugs

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

Contact Info

Product

Resources

About