Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 µM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 µM) while the reported CC50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

Characterization of Glucokinases from Pathogenic Free-Living Amoebae.

Infection with pathogenic free-living amoebae, including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (NfGlck) as a potential target for therapeutic intervention, as glucose metabolism is critical for in vitro viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (AcGlck) and B. mandrillaris (BmGlck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, AcGlck and BmGlck have apparent Km values of 472.5 and 41.0 µM, while Homo sapiens Glck (HsGlck) has a value of 310 µM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent Km values of 45.9 µM (AcGlck) and 124 µM (BmGlck) compared to ~8 mM for HsGlck. Additionally, AcGlck and BmGlck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.

A Microfluidic-Based Microscopy Platform for Continuous Interrogation of Trypanosoma brucei during Environmental Perturbation.

The African trypanosome, Trypanosoma brucei, is the causative agent of human African trypanosomiasis (HAT). African trypanosomes are extracellular parasites that possess a single flagellum that imparts a high degree of motility to the microorganisms. In addition, African trypanosomes show significant metabolic and structural adaptation to environmental conditions. Analysis of the ways that environmental cues affect these organisms generally requires rapid perfusion experiments in combination with single-cell imaging, which are difficult to apply under conditions of rapid motion. Microfluidic devices have been used previously as a strategy for trapping small motile cells in a variety of organisms, including trypanosomes; however, in the past, such devices required individual fabrication in a cleanroom, limiting their application. Here we demonstrate that a commercial microfluidic device, typically used for bacterial trapping, can trap bloodstream and procyclic form trypanosomes, allowing for rapid buffer exchange via perfusion. As a result, time-lapse single-cell microscopy images of these highly motile parasites were acquired during environmental variations. Using these devices, we have been able to perform and analyze perfusion-based single-cell tracking experiments of the responses of the parasite to changes in glucose availability, which is a major step in resolving the mechanisms of adaptation of kinetoplasts to their individual biological niches; we demonstrate utility of this tool for making measurements of procyclic form trypanosome intracellular glucose levels as a function of changes in extracellular glucose concentrations. These experiments demonstrate that cytosolic glucose equilibrates with external conditions as fast as, or faster than, the rate of solution exchange in the instrument.

Enzymatic and Structural Characterization of the Naegleria fowleri Glucokinase.

Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). 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 parasite-specific 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 Km values of 42.5 ± 7.3 µM and 141.6 ± 9.9 µM 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 glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 µM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.

pH regulation in glycosomes of procyclic form Trypanosoma brucei.

Here we report the use of a fluorescein-tagged peroxisomal targeting sequence peptide (F-PTS1, acetyl-C{K(FITC)}GGAKL) for investigating pH regulation of glycosomes in live procyclic form Trypanosoma brucei When added to cells, this fluorescent peptide is internalized within vesicular structures, including glycosomes, and can be visualized after 30-60 min. Using F-PTS1 we are able to observe the pH conditions inside glycosomes in response to starvation conditions. Previous studies have shown that in the absence of glucose, the glycosome exhibits mild acidification from pH 7.4 ± 0.2 to 6.8 ± 0.2. Our results suggest that this response occurs under proline starvation as well. This pH regulation is found to be independent from cytosolic pH and requires a source of Na+ ions. Glycosomes were also observed to be more resistant to external pH changes than the cytosol; placement of cells in acidic buffers (pH 5) reduced the pH of the cytosol by 0.8 ± 0.1 pH units, whereas glycosomal pH decreases by 0.5 ± 0.1 pH units. This observation suggests that regulation of glycosomal pH is different and independent from cytosolic pH regulation. Furthermore, pH regulation is likely to work by an active process, because cells depleted of ATP with 2-deoxyglucose and sodium azide were unable to properly regulate pH. Finally, inhibitor studies with bafilomycin and EIPA suggest that both V-ATPases and Na+/H+ exchangers are required for glycosomal pH regulation.

Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids.

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC50=0.010µM) while several compounds showed ∼10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC50>25µM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.

Evaluation of substituted ebselen derivatives as potential trypanocidal agents.

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.

Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

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 γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule 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 ∼1 µM, and several were found to have low-micromolar 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.

Environmentally regulated glycosome protein composition in the African trypanosome.

Trypanosomes compartmentalize many metabolic enzymes in glycosomes, peroxisome-related microbodies that are essential to parasite survival. While it is understood that these dynamic organelles undergo profound changes in protein composition throughout life cycle differentiation, the adaptations that occur in response to changes in environmental conditions are less appreciated. We have adopted a fluorescent-organelle reporter system in procyclic Trypanosoma brucei by expressing a fluorescent protein (FP) fused to a glycosomal targeting sequence (peroxisome-targeting sequence 2 [PTS2]). In these cell lines, PTS2-FP is localized within import-competent glycosomes, and organelle composition can be analyzed by microscopy and flow cytometry. Using this reporter system, we have characterized parasite populations that differ in their glycosome composition. In glucose-rich medium, two parasite populations are observed; one population harbors glycosomes bearing the full repertoire of glycosome proteins, while the other parasite population contains glycosomes that lack the usual glycosome-resident proteins but do contain the glycosome membrane protein TbPEX11. Interestingly, these cells lack TbPEX13, a protein essential for the import of proteins into the glycosome. This bimodal distribution is lost in low-glucose medium. Furthermore, we have demonstrated that changes in environmental conditions trigger changes in glycosome protein composition. These findings demonstrate a level of procyclic glycosome diversity heretofore unappreciated and offer a system by which glycosome dynamics can be studied in live cells. This work adds to our growing understanding of how the regulation of glycosome composition relates to environmental sensing.

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications. This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics in the live cell setting.

Glucose metabolism in the pathogenic free-living amoebae: Tempting targets for treatment development.

Pathogenic free-living amoebae (pFLA) are single-celled eukaryotes responsible for causing intractable infections with high morbidity and mortality in humans and animals. Current therapeutic approaches include cocktails of antibiotic, antifungal, and antimicrobial compounds. Unfortunately, the efficacy of these can be limited, driving the need for the discovery of new treatments. Pan anti-amebic agents would be ideal; however, identifying these agents has been a challenge, likely due to the limited evolutionary relatedness of the different pFLA. Here, we discuss the potential of targeting amoebae glucose metabolic pathways as the differences between pFLA and humans suggest specific inhibitors could be developed as leads for new therapeutics.

Synthesis and Evaluation of Benzylamine Inhibitors of Neuropathogenic Naegleria fowleri "Brain-Eating" Amoeba.

Current therapy for primary amoebic meningoencephalitis (PAM), a highly lethal brain infection in humans caused by Naegleria fowleri amoeba, is restricted to repurposed drugs with limited efficacy and success. Discovery of an antiamoebic benzylamine scaffold 2 precipitated a medicinal chemistry effort to improve potency, cytotoxicity profile, and drug-like properties. Thirty-four compounds were prepared, leading to compound 28 with significant gains in potency (EC50 = 0.92 µM), solubility, and microsomal stability and a demonstrated absence of cytotoxicity in SH-SY5Y human neuroblastoma cells (CC50 > 20 µM). The compounds demonstrated excellent blood-brain barrier permeability in an in vitro assay, thereby providing a new structural scaffold that inhibits N. fowleri viability and permits the investigation of therapeutic interventions in an understudied neglected disease.

Pharmacophore Establishment and Optimization of Saturated 1,6-Naphthyridine-Fused Quinazolinones that Inhibit Meningoencephalitis-Causing Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) is a human brain infection caused by Naegleria fowleri with a 97% mortality rate. Quinazolinones resulting from a Mannich-coupled domino rearrangement were recently identified as inhibitors of the amoeba. Herein, we resolved the effective concentrations for 25 pilot compounds and then, using the Mannich protocol and a key late-stage, N-demethylation/functionalization, we synthesized 53 additional analogs to improve potency, solubility and microsomal stability. We established an antiamoebic quinazolinone pharmacophore, culminating in (±)-trans-57b which featured the best combination of potency, selectivity index, solubility, and microsomal stability. Enantiomeric separation afforded (4aS,13bR)-57b (BDGR-20237) with a 41-fold potency advantage over its enantiomer. ADME and mouse pharmacokinetic profiling for BDGR-20237 revealed high brain penetrance but a limited half-life which did not statistically enhance the mouse survival in a pilot efficacy study. The pharmacophoric model, supported by 88 quinazolinones, several of which exhibit subnanomolar potency, will guide further scaffold optimization.

A multiplexed high throughput screening assay using flow cytometry identifies glycolytic molecular probes in bloodstream form Trypanosoma brucei.

Kinetoplastid organisms, including Trypanosoma brucei, are a significant health burden in many tropical and semitropical countries. Much of their metabolism is poorly understood. To better study kinetoplastid metabolism, chemical probes that inhibit kinetoplastid enzymes are needed. To discover chemical probes, we have developed a high-throughput flow cytometry screening assay that simultaneously measures multiple glycolysis-relevant metabolites in live T. brucei bloodstream form parasites. We transfected parasites with biosensors that measure glucose, ATP, or glycosomal pH. The glucose and ATP sensors were FRET biosensors, while the pH sensor was a GFP-based biosensor. The pH sensor exhibited a different fluorescent profile from the FRET sensors, allowing us to simultaneously measure pH and either glucose or ATP. Cell viability was measured in tandem with the biosensors using thiazole red. We pooled sensor cell lines, loaded them onto plates containing a compound library, and then analyzed them by flow cytometry. The library was analyzed twice, once with the pooled pH and glucose sensor cell lines and once with the pH and ATP sensor cell lines. Multiplexing sensors provided some internal validation of active compounds and gave potential clues for each compound's target(s). We demonstrated this using the glycolytic inhibitor 2-deoxyglucose and the alternative oxidase inhibitor salicylhydroxamic acid. Individual biosensor-based assays exhibited a Z'-factor value acceptable for high-throughput screening, including when multiplexed. We tested assay performance in a pilot screen of 14,976 compounds from the Life Chemicals Compound Library. We obtained hit rates from 0.2 to 0.4% depending on the biosensor, with many compounds impacting multiple sensors. We rescreened 44 hits, and 28 (64%) showed repeatable activity for one or more sensors. One compound exhibited EC50 values in the low micromolar range against two sensors. We expect this method will enable the discovery of glycolytic chemical probes to improve metabolic studies in kinetoplastid parasites.

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC 50 value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC 50 value of 0.21 ± 0.02 µM) while the reported CC 50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

Peptide-targeted delivery of a pH sensor for quantitative measurements of intraglycosomal pH in live Trypanosoma brucei.

Studies of dynamic changes in organelles of protozoan parasite Trypanosoma brucei have been limited, in part because of the difficulty of targeting analytical probes to specific subcellular compartments. Here we demonstrate application of a ratiometric probe for pH quantification in T. brucei glycosomes. The probe consists of a peptide encoding the peroxisomal targeting sequence (F-PTS1, acetyl-CKGGAKL) coupled to fluorescein, which responds to pH. When incubated with living parasites, the probe is internalized within vesicular structures that colocalize with a glycosomal marker. Inhibition of uptake of F-PTS1 at 4 °C and pulse-chase colocalization with fluorescent dextran suggested that the probe is initially taken up by non-receptor-mediated endocytosis but is subsequently transported separately from dextran and localized within glycosomes, prior to the final fusion of labeled glycosomes and lysosomes as part of glycosomal turnover. Intraorganellar measurements and pH calibration with F-PTS1 in T. brucei glycosomes indicate that the resting glycosomal pH under physiological conditions is 7.4 ± 0.2. However, incubation in glucose-depleted buffer triggered mild acidification of the glycosome over a period of 20 min, with a final observed pH of 6.8 ± 0.3. This glycosomal acidification was reversed by reintroduction of glucose. Coupling of ratiometric fluorescent sensors and reporters to PTS peptides offers an invaluable tool for monitoring in situ glycosomal response(s) to changing environmental conditions and could be applied to additional kinetoplastid parasites.

Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase.

Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 µM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 µM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Enolase Inhibitors as Early Lead Therapeutics against Trypanosoma brucei.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of -6.8 and -7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of -4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted.

Glycerol 3-phosphate alters Trypanosoma brucei hexokinase activity in response to environmental change.

The African trypanosome, Trypanosoma brucei, compartmentalizes some metabolic enzymes within peroxisome-like organelles called glycosomes. The amounts, activities, and types of glycosomal enzymes are modulated coincident with developmental and environmental changes. Pexophagy (fusion of glycosomes with acidic lysosomes) has been proposed to facilitate this glycosome remodeling. Here, we report that, although glycosome-resident enzyme T. brucei hexokinase 1 (TbHK1) protein levels are maintained during pexophagy, acidification inactivates the activity. Glycerol 3-phosphate, which is produced in vivo by a glycosome-resident glycerol kinase, mitigated acid inactivation of lysate-derived TbHK activity. Using recombinant TbHK1, we found that glycerol 3-P influenced enzyme activity at pH 6.5 by preventing substrate and product inhibition by ATP and ADP, respectively. Additionally, TbHK1 inhibition by the flavonol quercetin (QCN) was partially reversed by glycerol 3-P at pH 7.4, whereas at pH 6.5, enzyme activity in the presence of QCN was completely maintained by glycerol 3-P. However, glycerol 3-P did not alter the interaction of QCN with TbHK1, as the lone Trp residue (Trp-177) was quenched under all conditions tested. These findings suggest potential novel mechanisms for the regulation of TbHK1, particularly given the acidification of glycosomes that can be induced under a variety of parasite growth conditions.

Quercetin, a fluorescent bioflavanoid, inhibits Trypanosoma brucei hexokinase 1.

Hexokinases from the African trypanosome, Trypanosoma brucei, are attractive targets for the development of anti-parasitic drugs, in part because the parasite utilizes glycolysis exclusively for ATP production during the mammalian infection. Here, we have demonstrated that the bioflavanoid quercetin (QCN), a known trypanocide, is a mixed inhibitor of Trypanosoma brucei hexokinase 1 (TbHK1) (IC(50) = 4.1 ± 0.8µM). Spectroscopic analysis of QCN binding to TbHK1, taking advantage of the intrinsically fluorescent single tryptophan (Trp177) in TbHK1, revealed that QCN quenches emission of Trp177, which is located near the hinge region of the enzyme. ATP similarly quenched Trp177 emission, while glucose had no impact on fluorescence. Supporting the possibility that QCN toxicity is a consequence of inhibition of the essential hexokinase, in live parasites QCN fluorescence localizes to glycosomes, the subcellular home of TbHK1. Additionally, RNAi-mediated silencing of TbHK1 expression expedited QCN induced death, while over-expressing TbHK1 protected trypanosomes from the compound. In summary, these observations support the suggestion that QCN toxicity is in part attributable to inhibition of the essential TbHK1.