Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase 1

Flaherty, Daniel P.; Harris, Michael T.; Schroeder, Chad E.; Khan, Haaris; Kahney, Elizabeth W.; Hackler, Amber L.; Patrick, Stephen L.; Weiner, Warren S.; Aubé, Jeffrey; Sharlow, Elizabeth R.; Morris, James C.; Golden, Jennifer E.

doi:10.1002/cmdc.201700592

Cited by 15 publications

(13 citation statements)

References 26 publications

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“…Of the two agents, only ebselen showed inhibition of NfGlck, with an IC 50 of 0.88 M, a notably higher concentration than that required to inhibit the other parasite HKs ( Table 2). The second scaffold, a benzamidobenzoic acid-based inhibitor of TbHK1 (14), was developed through structure-activity relationship (SAR) optimization against the trypanosome to yield ML205, a potent inhibitor of TbHK1 (17). While this particular compound was not active against the Naegleria enzyme (IC 50 , Ͼ10 M), evaluation of related analogs revealed some promising activity.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

Milanes

Suryadi

Abendroth

et al. 2019

Antimicrob Agents Chemother

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Infection with the free-living amoeba Naegleria fowleri leads to lifethreatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (ϳ98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasitespecific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent K m values of 42.5 Ϯ 7.3 M and 141.6 Ϯ 9.9 M for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucosephosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of Ͻ1 M that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.

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Section: Resultsmentioning

confidence: 99%

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

Milanes

Suryadi

Abendroth

et al. 2019

Antimicrob Agents Chemother

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“…Of the two agents, only ebselen showed inhibition of NfGlck with an IC 50 of 0.88 μM, a notably higher concentration than that required to inhibit the other parasite HKs (Table 2). The second scaffold, a benzamidobenzoic acid-based inhibitor of TbHK1 (20), was developed through structure-activity relationship (SAR) optimization against the trypanosome to yield ML205, a potent inhibitor of TbHK1 (23). While this particular compound was not active against the Naegleria enzyme (IC 50 > 10 μM), evaluation of related analogs revealed some promising activity.…”

Section: Resultsmentioning

confidence: 99%

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

Milanes

Suryadi

Abendroth

et al. 2018

Preprint

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37Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary 38 amoebic meningoencephalitis. Efficacious treatment options for these infections are limited 39 and the mortality rate is very high (~98%). Parasite metabolism may provide suitable 40 targets for therapeutic design. Like most other organisms, glucose metabolism is critical for 41 parasite viability, being required for growth in culture. The genome of the parasite encodes 42 a single glucose phosphorylating enzyme, a glucokinase (Glck). The products of this 43 enzyme are required for both glycolysis and the pentose phosphate pathway. The N. 44fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host 45 enzyme (HsGlck), suggesting that parasite-specific inhibitors with anti-amoeba activity 46 could be generated. Following heterologous expression, NfGlck was found to have a 47 limited hexose substrate range, with greatest activity observed with glucose. The enzyme 48 had apparent Km values of 42.5 ± 7.3 µM and 141.6 ± 9.9 µM for glucose and ATP, 49 respectively. The NfGlck structure was determined and refined to 2.2 Å resolution, 50 revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi 51Glck. These similarities include binding modes and binding environments for substrates. 52To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose 53 phosphorylating enzymes and identified several small molecules with IC50 values < 1 µM 54 that may prove useful as hit chemotypes for further lead and therapeutic development 55 against N. fowleri. 56 57

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“… Recombinant TbHK1 activity was monitored in vitro in the presence of increasing amounts of FLII12Pglu-700μδ6 in order to achieve TbHK1 to sensor molar ratios of 1 to 0.5, 1, 2, 4, 8, and 10, respectively. K2354, a known benzamidobenzoic acid inhibitor of TbHK1 [ 31 ], was used at 10 μM as a control. …”

Section: Supporting Informationmentioning

confidence: 99%

A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites

Voyton¹,

Qiu²,

Morris³

et al. 2018

PLoS Negl Trop Dis

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The bloodstream lifecycle stage of the kinetoplastid parasite Trypanosoma brucei relies solely on glucose metabolism for ATP production, which occurs in peroxisome-like organelles (glycosomes). Many studies have been conducted on glucose uptake and metabolism, but none thus far have been able to monitor changes in cellular and organellar glucose concentration in live parasites. We have developed a non-destructive technique for monitoring changes in cytosolic and glycosomal glucose levels in T. brucei using a fluorescent protein biosensor (FLII12Pglu-700μδ6) in combination with flow cytometry. T. brucei parasites harboring the biosensor allowed for observation of cytosolic glucose levels. Appending a type 1 peroxisomal targeting sequence caused biosensors to localize to glycosomes, which enabled observation of glycosomal glucose levels. Using this approach, we investigated cytosolic and glycosomal glucose levels in response to changes in external glucose or 2-deoxyglucose concentration. These data show that procyclic form and bloodstream form parasites maintain different glucose concentrations in their cytosol and glycosomes. In procyclic form parasites, the cytosol and glycosomes maintain indistinguishable glucose levels (3.4 ± 0.4mM and 3.4 ± 0.5mM glucose respectively) at a 6.25mM external glucose concentration. In contrast, bloodstream form parasites maintain glycosomal glucose levels that are ~1.8-fold higher than the surrounding cytosol, equating to 1.9 ± 0.6mM in cytosol and 3.5 ± 0.5mM in glycosomes. While the mechanisms of glucose transport operating in the glycosomes of bloodstream form T. brucei remain unresolved, the methods described here will provide a means to begin to dissect the cellular machinery required for subcellular distribution of this critical hexose.

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Optimization and Evaluation of Antiparasitic Benzamidobenzoic Acids as Inhibitors of Kinetoplastid Hexokinase 1

Cited by 15 publications

References 26 publications

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase

Enzymatic and structural characterization of the Naegleria fowleri glucokinase

A FRET flow cytometry method for monitoring cytosolic and glycosomal glucose in living kinetoplastid parasites

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