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.
e Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the ␥-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening smallmolecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ؎ 1.2, a coefficient of variation of 6.8% ؎ 2.9%, and a Z=-factor of 0.75 ؎ 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ϳ1 M, and several were found to have lowmicromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.
Human African trypanosomiasis is a disease of sub-Saharan Africa, where millions are at risk for the illness. The disease, commonly referred to as African sleeping sickness, is caused by an infection by the eukaryotic pathogen, Trypanosoma brucei. Previously, a target-based high throughput screen revealed ebselen (EbSe), and its sulfur analog, EbS, to be potent in vitro inhibitors of the T. brucei hexokinase 1 (TbHK1). These molecules also exhibited potent trypanocidal activity in vivo. In this manuscript, we synthesized a series of sixteen EbSe and EbS derivatives bearing electron-withdrawing carboxylic acid and methyl ester functional groups, and evaluated the influence of these substituents on the biological efficacy of the parent scaffold. With the exception of one methyl ester derivative, these modifications ablated or blunted the potent TbHK1 inhibition of the parent scaffold. Nonetheless, a few of the methyl ester derivatives still exhibited trypanocidal effects with single-digit micromolar or high nanomolar EC50 values.
Kinetoplastid-based infections are neglected diseases that represent a significant human health issue. Chemotherapeutic options are limited due to toxicity, parasite susceptibility, and poor patient compliance. In response, we studied a molecular target-directed approach involving intervention of hexokinase activity – a pivotal enzyme in parasite metabolism. A benzamidobenzoic acid hit with modest biochemical inhibition of T. brucei hexokinase 1 (TbHK1, IC50 = 9.1 μM), low mammalian cytotoxicity (IMR-90, EC50 > 25 μM), and no appreciable activity on whole BSF parasites was optimized to afford probe 4f with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50 = 0.28 μM, BSF LD50 = 1.9 μM). Compound 4f and analogs also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency compared to TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple, disease-causing trypanosomatid protozoa.
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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