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.

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.

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.

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.

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.

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.

The transcriptome of Balamuthia mandrillaris trophozoites for structure-guided drug design.

Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a 'brain-eating' disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.

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

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. The 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.

Dual-Stage Picolinic Acid-Derived Inhibitors of Toxoplasma gondii.

Toxoplasma gondii causes a prevalent human infection for which only the acute stage has an FDA-approved therapy. To find inhibitors of both the acute stage parasites and the persistent cyst stage that causes a chronic infection, we repurposed a compound library containing known inhibitors of parasitic hexokinase, the first step in the glycolysis pathway, along with a larger collection of new structural derivatives. The focused screen of 22 compounds showed a 77% hit rate (>50% multistage inhibition) and revealed a series of aminobenzamide-linked picolinic acids with submicromolar potency against both T. gondii parasite forms. Picolinic acid 23, designed from an antiparasitic benzamidobenzoic acid class with challenging ADME properties, showed 60-fold-enhanced solubility, a moderate LogD7.4, and a 30% improvement in microsomal stability. Furthermore, isotopically labeled glucose tracing revealed that picolinic acid 23 does not function by hexokinase inhibition. Thus, we report a new probe scaffold to interrogate dual-stage inhibition of T. gondii.

Plasmodium vivax and human hexokinases share similar active sites but display distinct quaternary architectures.

Malaria is a devastating disease caused by a protozoan parasite. It affects over 300 million individuals and results in over 400 000 deaths annually, most of whom are young children under the age of five. Hexokinase, the first enzyme in glucose metabolism, plays an important role in the infection process and represents a promising target for therapeutic intervention. Here, cryo-EM structures of two conformational states of Plasmodium vivax hexokinase (PvHK) are reported at resolutions of ∼3 Å. It is shown that unlike other known hexokinase structures, PvHK displays a unique tetrameric organization (∼220 kDa) that can exist in either open or closed quaternary conformational states. Despite the resemblance of the active site of PvHK to its mammalian counterparts, this tetrameric organization is distinct from that of human hexokinases, providing a foundation for the structure-guided design of parasite-selective antimalarial drugs.

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.

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.

Glucose Signaling Is Important for Nutrient Adaptation during Differentiation of Pleomorphic African Trypanosomes.

The African trypanosome has evolved mechanisms to adapt to changes in nutrient availability that occur during its life cycle. During transition from mammalian blood to insect vector gut, parasites experience a rapid reduction in environmental glucose. Here we describe how pleomorphic parasites respond to glucose depletion with a focus on parasite changes in energy metabolism and growth. Long slender bloodstream form parasites were rapidly killed as glucose concentrations fell, while short stumpy bloodstream form parasites persisted to differentiate into the insect-stage procyclic form parasite. The rate of differentiation was lower than that triggered by other cues but reached physiological rates when combined with cold shock. Both differentiation and growth of resulting procyclic form parasites were inhibited by glucose and nonmetabolizable glucose analogs, and these parasites were found to have upregulated amino acid metabolic pathway component gene expression. In summary, glucose transitions from the primary metabolite of the blood-stage infection to a negative regulator of cell development and growth in the insect vector, suggesting that the hexose is not only a key metabolic agent but also an important signaling molecule.IMPORTANCE As the African trypanosome Trypanosoma brucei completes its life cycle, it encounters many different environments. Adaptation to these environments includes modulation of metabolic pathways to parallel the availability of nutrients. Here, we describe how the blood-dwelling life cycle stages of the African trypanosome, which consume glucose to meet their nutritional needs, respond differently to culture in the near absence of glucose. The proliferative long slender parasites rapidly die, while the nondividing short stumpy parasite remains viable and undergoes differentiation to the next life cycle stage, the procyclic form parasite. Interestingly, a sugar analog that cannot be used as an energy source inhibited the process. Furthermore, the growth of procyclic form parasite that resulted from the event was inhibited by glucose, a behavior that is similar to that of parasites isolated from tsetse flies. Our findings suggest that glucose sensing serves as an important modulator of nutrient adaptation in the parasite.

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

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-700µÎ´6) 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.

FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei.

Trypanosoma brucei, which causes human African typanosomiasis (HAT), derives cellular ATP from glucose metabolism while in the mammalian host. Targeting glucose uptake or regulation in the parasite has been proposed as a potential therapeutic strategy. However, few methods have been described to identify and characterize potential inhibitors of glucose uptake and regulation. Here, we report development of a screening assay that identifies small molecule disrupters of glucose levels in the cytosol and glycosomes. Using an endogenously expressed fluorescent protein glucose sensor expressed in cytosol or glycosomes, we monitored intracellular glucose depletion in the different cellular compartments. Two glucose level disrupters were identified, one of which only exhibited inhibition of glycosomal glucose and did not affect cytosolic levels. In addition to inhibiting glucose uptake with relatively high potency (EC50 = 700 nM), the compound also showed modest bloodstream form parasite killing activity. Expanding this assay will allow for identification of candidate compounds that disrupt parasite glucose metabolism.

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

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 Trypanosoma brucei hexokinase 1 (TbHK1, IC50 =9.1 µm), low mammalian cytotoxicity (IMR90 cells, EC50 >25 µm), and no appreciable activity on whole bloodstream-form (BSF) parasites was optimized to afford a probe with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50 =0.28 µm; BSF, ED50 =1.9 µm). Compounds in this series also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency than toward TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple disease-causing trypanosomatid protozoa.

A targeted delivery strategy for the development of potent trypanocides.

A new drug delivery strategy was investigated for the development of potent anti-parasitic compounds against Trypanosoma brucei, the causative agent of African sleeping sickness. Thus, potent in vitro hexokinase inhibitors were rendered cytotoxic by appending a tripeptide peroxosomal targeting sequence that facilitated delivery of the molecular cargo to the appropriate organelle in the parasite.

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.