Evaluating the Leishmania tarentolae response to inorganic strontium-based oxyfluorides.

The increasing global prevalence of the parasitic vector-borne disease leishmaniasis combined with rising resistance to current therapeutics necessitates the search for novel approaches to combat leishmania. This study evaluates the effects of novel strontium-based oxyfluorides for potential therapeutic use by testing cultures of Leishmania tarentolae, a species of Leishmania found in reptiles, as a model species. Cells were cultured with a range of mixed metal strontium oxyfluoride compounds selected to systematically test the relationship between compound structure and cell viability and enzyme activity over time.

Finding the best location: Improving the anti-amyloid ability of ruthenium(III) complexes with pyridine ligands.

Alzheimer's disease (AD) is a devastating neurological disorder where one of the primary pathological hallmarks are aggregate deposits of the peptide amyloid-beta (Aß). Although the Food and Drug Administration (FDA) has recently approved therapeutics that specifically target Aß, resulting in the removal of these deposits, the associated costs of such treatments create a need for effective, yet cheaper, alternatives. Metal-based compounds are propitious therapeutic candidates as they exploit the metal-binding properties of Aß, forming stable interactions with the peptide, thereby limiting its aggregation and toxicity. Previously, ruthenium-based complexes have shown a strong ability to modulate the aggregation and cytotoxicity of Aß, where the incorporation of a primary amine on the coordinated heterocyclic ligand gave the greatest activity. To determine the importance of the location of the primary amine on the pyridine ligand, thereby establishing structure-activity relationships (SAR), four complexes (RuP1-4) were prepared and evaluated for their ability to coordinate and subsequently modulate the aggregation and cytotoxicity of Aß. Coordination to Aß was determined using three complementary spectroscopic methods: UV-Vis, 1H NMR, and circular dichroism (CD). Similarly, the impact of the complexes on Aß aggregation was evaluated using three sequential methods of turbidity, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Overall, the location of the primary amine on the pyridine ligand did affect the resultant anti-Aß performance, with the 2-aminopyridine complex (RuP2) being the most active. This SAR will provide another guiding principle in the design of future metal-based anti-Aß complexes.

A comprehensive review of 30 years of pediatric clinical trial radiotherapy dose constraints.

BACKGROUND: Radiation therapy normal tissue dose constraints are critical when treating pediatric patients. However, there is limited evidence supporting proposed constraints, which has led to variations in constraints over the years. In this study, we identify these variations in dose constraints within pediatric trials both in the United States and in Europe used in the past 30 years. PROCEDURE: All pediatric trials from the Children's Oncology Group website were queried from inception until January 2022 and a sampling of European studies was included. Dose constraints were identified and built into an organ-based interactive web application with filters to display data by organs at risk (OAR), protocol, start date, dose, volume, and fractionation scheme. Dose constraints were evaluated for consistency over time and compared between pediatric US and European trials RESULTS: One hundred five closed trials were included-93 US trials and 12 European trials. Thirty-eight separate OAR were found with high-dose constraint variability. Across all trials, nine organs had greater than 10 different constraints (median 16, range 11-26), including serial organs. When comparing US versus European dose tolerances, the United States constraints were higher for seven OAR, lower for one, and identical for five. No OAR had constraints change systematically over the last 30 years. CONCLUSION: Review of pediatric dose-volume constraints in clinical trials showed substantial variability for all OAR. Continued efforts focused on standardization of OAR dose constraints and risk profiles are essential to increase consistency of protocol outcomes and ultimately to reduce radiation toxicities in the pediatric population.

Potent Inhibitory Activity of Natural Product Anaephene B and Analogues against Leishmania tarentolae In Vitro.

In this study, a specific alkylphenol natural product, anaephene B, and its unique synthesized derivatives were tested for their inhibitory effect on the protozoan parasite Leishmania tarentolae. In a series of cell viability tests and enzyme assays, these test compounds have produced interesting results with regard to their antibiotic effect, showing similar potency against L. tarentolae as they do against drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). All compounds tested in this study have shown the ability to completely inhibit our model system, L. tarentolae, in vitro. This study helps increase our understanding of the structure-activity relationship (SAR) between anaephene B and its analogues for a new class of potential pharmaceuticals for the treatment of Leishmania infections.

Development of a C6 Glioma Cell Model System to Assess Effects of Cathodic Passively Balanced Electrical Stimulation on Responses to Neurotransmitters: Implications for Modulation of Intracellular Nitric Oxide, Chloride, and Calcium Ions.

This research focused on the development of an astrocyte cell model system (C6 glioma) for the assessment of molecular changes in response to cathodic passively balanced pulsed electrical stimulation at a rate of 50 Hz (60 µs duration, 0.15 mA intensity). Cells treated with selected neurotransmitters (glutamate, adenosine, D-serine, and γ-aminobutyric acid) were monitored (using specific fluorescent probes) for changes in levels of intracellular nitric oxide, calcium ions, and/or chloride. ES exerted an inhibitory effect on NO, increased calcium and had no effect on chloride. Using this model, cells can be assessed qualitatively and quantitatively for changes and these changes can be correlated with the putative molecular effects that electrical stimulation has on astrocytes and their role in glia-mediated diseases. This model system allows for faster and cheaper experiments than those involving animal models due to the potential to easily vary the conditions, reduce the number of variables (especially problematic in animal models), and closely monitor the cellular effects.

A Dual-Pronged Approach: A Ruthenium(III) Complex That Modulates Amyloid-ß Aggregation and Disrupts Its Formed Aggregates.

Alzheimer's disease (AD) is a devastating neurological disorder for which soluble oligomers of the peptide amyloid-ß (Aß) are now recognized as the neurotoxic species. Metal-based therapeutics are uniquely suited to target Aß, with ruthenium-based (Ru) complexes emerging as propitious candidates. Recently, azole-based Ru(III) complexes were observed to modulate the aggregation of Aß in solution, where the inclusion of a primary amine proximal to the ligand coordination site improved the activity of the complexes. To advance these structure-activity relationships, a series of oxazole-based Ru complexes were prepared and evaluated for their ability to modulate Aß aggregation. From these studies, a lead candidate, Oc, emerged that had superior activity relative to its azole predecessors in modulating the aggregation of soluble Aß and diminishing its cytotoxicity. Further evaluation of Oc demonstrated its ability to disrupt formed Aß aggregates, resulting in smaller amorphous species. Because altering both sides of the aggregation equilibrium for Aß has not been previously suggested for metal-based complexes for AD, this work represents an exciting new avenue for improved therapeutic success.

Leishmania tarentolae novel responses to Bi3+-doped strontium aluminum oxyfluorides.

Novel therapeutics for the treatment of leishmaniasis are of interest as the disease not only is becoming more prevalent, but drug resistance is increasing in certain regions of the world. Reported here is the use of Bi3+-doped strontium aluminum oxyfluoride phosphors and protease inhibitors to test in vitro inhibitory activity against cultured promastigote Leishmania tarentolae and effects on L. tarentolae secreted acid phosphatase (SAP) activity. Cell viability did not significantly decrease in the presence of 50 µM anti-perovskite compounds, implying limited cytotoxicity. Yet SAP activity did increase in the cell free preparations with time in the presence of strontium compounds. Of interest was the observation that cell free SAP activity did not increase in the presence of protease inhibitors with or without added strontium compounds. Since secreted proteases may play a role in the maturation of Leishmania SAP and thus be involved with parasite-host infection establishment, this is in further need of evaluation. Nitric oxide production on day 4 post-addition of the strontium compounds was evaluated and showed an approximately 50% decrease in NO production in the presence of two test compounds relative to DMSO control cells. This is the first report of anti-perovskite compound inhibition of NO production by Leishmania.

Importance of Hydrogen Bonding: Structure-Activity Relationships of Ruthenium(III) Complexes with Pyridine-Based Ligands for Alzheimer's Disease Therapy.

Alzheimer's disease (AD) is the most common form of dementia, where one of the pathological hallmarks of AD is extracellular protein deposits, the primary component of which is the peptide amyloid-ß (Aß). Recently, the soluble form of Aß has been recognized as the primary neurotoxic species, making it an important target for therapeutic development. Metal-based drugs are promising candidates to target Aß, as the interactions with the peptide can be tuned by ligand design. In the current study, 11 ruthenium complexes containing pyridine-based ligands were prepared, where the functional groups at the para position on the coordinated pyridine ligand were varied to determine structure-activity relationships. Overall, the complexes with terminal primary amines had the greatest impact on modulating the aggregation of Aß and diminishing its cytotoxicity. These results identify the importance of specific intermolecular interactions and are critical in the advancement of metal-based drugs for AD therapy.

2'-3'-Cyclic Nucleotide 3'-Phosphodiesterase Inhibition by Organometallic Vanadium Complexes: A Potential New Paradigm for Studying CNS Degeneration.

The enzyme, 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) has been known for over fifty years. Nevertheless, the roles this membrane-bound enzyme play have yet to be described completely. Recently, there has been renewed interest in the study of this enzyme due to studies that suggest that CNPase plays a role in the mediation of cellular inflammatory responses in renal and nervous system tissues. Also, this enzyme, found in oligodendrocytes of the nervous system, has been reported to participate in significant regulatory changes associated with age which may be involved in age-related CNS degeneration. Consequently, development of CNPase inhibitors is of interest and should aid in the study of this, as yet, poorly understood enzyme. In this work we utilized a spectrophotometric enzyme assay to determine the effect a panel of organo-vanadium complexes had on isolated hamster myelin CNPase activity. Our group has now identified several potent in vitro CNPase inhibitors that could prove useful in clarifying the important roles of this enzyme.

Ruthenium(III) complexes with imidazole ligands that modulate the aggregation of the amyloid-ß peptide via hydrophobic interactions.

Alzheimer's disease (AD) is the most common form of dementia, characterized by extracellular protein deposits, comprised primarily of the peptide amyloid-beta (Aß), are a pathological indicator of the disease. Commonly known as Aß plaques, these deposits contain a relatively high concentration of metals, making metallotherapeutics uniquely suited to target soluble Aß, thereby limiting its aggregation and cytotoxicity. Ruthenium-based complexes are promising candidates for advancement, as the complex PMRU20 (2-aminothiazolium [trans-RuCl4(2-aminothiazole)2]) and several thiazole-based derivatives were found to prevent the aggregation of Aß, with hydrogen-bonding functional groups improving their performance. Further investigation into the impact of the heteroatom in the azole ring on the activity of Ru complexes was achieved through the synthesis and evaluation of a small set of imidazole-based compounds. The ability of the complexes to prevent the aggregation of Aß was determined where the same sample was subjected to analysis by three complementary methods: ThT fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM). It was found that hydrophobic interactions, along with hydrogen-bonding via the imidazole nitrogen heteroatom, promoted interactions with the Aß peptide, thereby limiting its aggregation. Furthermore, it was found that having rapid and sequential exchange proved detrimental as it resulted in a decreased association with Aß. These results highlight important considerations between a balance of intermolecular interactions and ligand exchange kinetics in the design of further therapeutic candidates.

Synthesis of Polycyclic Ether-Benzopyrans and In Vitro Inhibitory Activity against Leishmania tarentolae.

Construction of a focused library of polycyclic ether-benzopyrans was undertaken in order to discover new therapeutic compounds that affect Leishmania growth and infectivity. This is especially of interest since there are few drug therapies for leishmaniasis that do not have serious drawbacks such high cost, side effects, and emerging drug resistance. The construction of these polycyclic ether-benzopyrans utilized an acetoxypyranone-alkene [5+2] cycloaddition and the Suzuki-Miyaura cross-coupling. The multi-gram quantity of the requisite aryl bromide was obtained followed by effective Pd-catalyzed coupling with boronic acid derivatives. Compounds were tested in vitro using the parasitic protozoan, Leishmania tarentolae. Effects of concentration, time, and exposure to light were evaluated. In addition, the effects on secreted acid phosphatase activity and nitric oxide production were investigated, since both have been implicated in parasite infectivity. The data presented herein are indicative of disruption of the Leishmania tarentolae and thus provide impetus for the development and testing of a more extensive library.

Ruthenium(iii) complexes containing thiazole-based ligands that modulate amyloid-ß aggregation.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder where one of the commonly observed pathological hallmarks is extracellular deposits of the peptide amyloid-ß (Aß). These deposits contain a high concentration of metals and initially presented a promising target for therapy; however it has become increasingly evident that the soluble form of the peptide is neurotoxic, not the amyloidogenic species. Metal-based therapeutics are uniquely suited to target soluble Aß and have shown considerable promise to prevent the aggregation and induced cytotoxicity of the peptide in vitro. Herein, we have prepared a small series of derivatives of two promising Ru(iii) complexes NAMI-A (imidazolium [trans-RuCl4(1H-imidazole)(dimethyl sulfoxide-S)]) and PMRU20 (2-aminothiazolium [trans-RuCl4(2-aminothiazole)2]), to determine structure-activity relationships (SAR) for Ru(iii) therapeutics for AD. Using the three complementary methods of Thioflavin T fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM), it was determined that the symmetry around the metal center did not significantly impact the activity of the complexes, but rather the attached thiazole ligand(s) mitigated Aß aggregation. Across both families of Ru(iii) complexes the determined SAR for the functional groups on the thiazole ligands to modulate Aß aggregation were NH2 > CH3 > H. These results highlight the importance of secondary interactions between the metallotherapeutic and the Aß peptide where hydrogen-bonding has the greatest impact on modulating Aß aggregation.

Development of a new and reliable assay for choline kinase using 31P NMR.

Choline kinase catalyzes the conversion of choline to phosphocholine (PC) by transferring a phosphate group from adenosine triphosphate (ATP) as the first step in the biosynthetic pathway for the membrane phospholipid phosphatidylcholine, an essential pathway in the Leishmania parasitic protozoan. Commonly used methods for kinetically quantifying the enzyme include a radioisotope assay utilizing labeled choline and a coupled spectrophotometric assay with multiple enzymes and substrates that indirectly measures choline kinase activity. When testing potential inhibitors with the coupled assay, results can cast doubt on whether choline kinase is being inhibited or one of the coupled enzymes. Therefore, 31P NMR spectroscopy was used to quantitatively measure the formation of the key product, phosphocholine, and to evaluate choline kinase activity. Interrogation of 31P NMR spectroscopy offers a number of benefits. Since this isotope is 100% abundant and has a relatively large gyromagnetic ratio, it is considered one of the more sensitive nuclides. As such, the need for costly isotopic enriched phosphorous is not required and detection of the 31P signal is possible even at relatively low concentrations. The enzymatic activity of Leishmania infantum choline kinase was able to be directly measured via integration of the 31P resonance associated with the phosphocholine product (δ = 3.94 ppm). These initial studies reveal that a 31P NMR spectroscopic-based assay could be used for testing substrate or transition state analogs as competitive inhibitors of Leishmania choline kinase that may prevent phosphatidylcholine synthesis in the parasite.

Electrical Stimulation of C6 Glia-Precursor Cells In Vitro Differentially Modulates Gene Expression Related to Chronic Pain Pathways.

Glial cells comprise the majority of cells in the central nervous system and exhibit diverse functions including the development of persistent neuropathic pain. While earlier theories have proposed that the applied electric field specifically affects neurons, it has been demonstrated that electrical stimulation (ES) of neural tissue modulates gene expression of the glial cells. This study examines the effect of ES on the expression of eight genes related to oxidative stress and neuroprotection in cultured rodent glioma cells. Concentric bipolar electrodes under seven different ES types were used to stimulate cells for 30 min in the presence and absence of extracellular glutamate. ES consisted of rectangular pulses at 50 Hz in varying proportions of anodic and cathodic phases. Real-time reverse-transcribed quantitative polymerase chain reaction was used to determine gene expression using the ∆∆Cq method. The results demonstrate that glutamate has a significant effect on gene expression in both stimulated and non-stimulated groups. Furthermore, stimulation parameters have differential effects on gene expression, both in the presence and absence of glutamate. ES has an effect on glial cell gene expression that is dependent on waveform composition. Optimization of ES therapy for chronic pain applications can be enhanced by this understanding.

The relationship between ventricular volume and whole-brain irradiation dose in central nervous system germ cell tumors.

BACKGROUND: Advanced irradiation techniques, including intensity-modulated radiation therapy (IMRT), aim to limit irradiation to adjoining tissues by conforming beams to a well-defined volume. In intracranial germinomas, whole-ventricular IMRT decreases the volume of irradiation to surrounding parenchyma. This study examined the relationship between ventricular volume and radiation dose to surrounding tissue. PROCEDURE: We retrospectively reviewed age, sex, ventricular and brain volume, ventricular dose, and volume of brain that received 12 Gy (V12) for patients diagnosed with germ cell tumors at our institution treated with whole-ventricular IMRT between 2002 and 2016. Variables were assessed for correlation and statistical significance. RESULTS: Forty-seven patients were analyzed. The median whole-ventricular irradiation dose was 24 Gy with a median boost dose of 30 Gy. The median ventricular volume was 234.3 cm3 , and median brain volume was 1408 cm3 . There was no significant difference between mean ventricular volume of suprasellar versus pineal tumors (P = .95). The median V12 of the brain, including the ventricles, was 58.9%. The strongest correlation was between ventricular volume and V12, with an r2 (coefficient of determination) of .47 (P < .001). Multiple regression analysis indicated that total boost dose and boost planning target volume significantly predicted V12 (P < .001). CONCLUSIONS: Although whole-ventricular IMRT limited irradiation to surrounding tissue in our cohort, a significant percentage of the brain received at least 12 Gy. This study suggests that there is a positive correlation between ventricular volume and the volume of brain parenchyma receiving at least 12 Gy with an important contribution from the boost phase of treatment.

Lens Dose-Response Prediction Modeling and Cataract Incidence in Patients With Retinoblastoma After Lens-Sparing or Whole-Eye Radiation Therapy.

PURPOSE: We retrospectively assessed the incidence of cataracts in patients with retinoblastoma (Rb) treated with either lens-sparing radiation therapy (LSRT) or whole-eye radiation therapy (WERT). A secondary aim of this study was to model the dose-response risk of cataract. METHODS AND MATERIALS: We reviewed 65 patients with Rb treated with radiation therapy (RT) at Children's Hospital, Los Angeles from 1997 to 2015. Eyes that were enucleated before RT or lacked follow-up eye examinations were excluded. All patients underwent computed tomography simulation, and mean lens dose data were collected. Follow-up ophthalmologic examinations and intraocular lens implant history were reviewed for cataracts. The primary event-free survival (EFS) outcome was cataract development. Eyes without cataracts were censored on the last date of eye examination or post-RT enucleation, if applicable. Kaplan-Meier estimates were used to compare EFS outcomes, and dose response was projected with Cox regression and logistic regression models. RESULTS: Sixty-one patients (94 eyes) were analyzed with a median follow-up of 51.8 months. For eyes treated with WERT, cataracts developed in 71.7% versus 35.3% for LSRT. Median EFS for WERT and LSRT were 20.8 and 67.9 months, respectively. Compared with WERT, a significant EFS benefit was demonstrated for LSRT (P < .001). Mean lens dose had a significant effect on cataracts in both Cox regression and logistic regression models (P < .01). The mean lens dose of 7 Gy was projected to have a 5-year cataract incidence of 20% and 25% with the logistic and Cox regression models, respectively. CONCLUSIONS: We report the first clinical data demonstrating significantly improved EFS in patients with Rb treated with LSRT. Through lens dose-response modeling, we validate a mean lens dose threshold of 7 Gy to keep cataract risk below 25%. Although RT is used less often for Rb owing to advances in chemotherapy delivery options, these findings are relevant for refining lens dose constraints, particularly in children who have received radiation dose near the orbit.

Effects of Specific Electric Field Stimulation on the Release and Activity of Secreted Acid Phosphatases from Leishmania tarentolae and Implications for Therapy.

Leishmaniasis is a neglected tropical disease with 1.6 million new cases reported each year. However, there are few safe, effective, and affordable treatments provided to those affected by this disease. Still under-appreciated as potential pharmaceutical targets, especially for cutaneous leishmaniasis infections, are the two isozymes of secreted acid phosphatase (SAP). These enzymes are involved in the survival of the parasite in the sand fly vector, and in infecting host macrophages. While the application of electric or electromagnetic fields as a medicinal therapeutic is not new, the utility of electric field application for the treatment of leishmaniasis is under studied. Studies involving the effects of electric fields on the cell secretion of SAP or the activity of SAP that has been secreted prior to electrical stimulation have not yet been reported. This work is the first report on the effect of specific electric fields on the activity of Leishmania tarentolae secreted acid phosphatases and the modulation of this secretion from the cells. In addition, the kinetic constants for the enzyme isoforms were determined as a function of days in culture and removal of carbohydrate from the glycosylated enzymes, while using a glycosidase, was shown to affect these kinetic constants.

Evidence That Speciation of Oxovanadium Complexes Does Not Solely Account for Inhibition of Leishmania Acid Phosphatases.

Leishmaniasis is an endemic disease affecting a diverse spectra of populations, with 1.6 million new cases reported each year. Current treatment options are costly and have harsh side effects. New therapeutic options that have been previously identified, but still underappreciated as potential pharmaceutical targets, are Leishmania secreted acid phosphatases (SAP). These acid phosphatases, which are reported to play a role in the survival of the parasite in the sand fly vector, and in homing to the host macrophage, are inhibited by orthovanadate and decavanadate. Here, we use L. tarentolae to further evaluate these inhibitors. Using enzyme assays, and UV-visible spectroscopy, we investigate which oxovanadium starting material (orthovanadate or decavanadate) is a better inhibitor of L. tarentolae secreted acid phosphatase activity in vitro at the same total moles of vanadium. Considering speciation and total vanadium concentration, decavanadate is a consistently better inhibitor of SAP in our conditions, especially at low substrate:inhibitor ratios.

In vivo studies of the effectiveness of novel N-halomethylated and non-halomethylated quaternary ammonium salts in the topical treatment of cutaneous leishmaniasis.

The physicochemical properties of four N-halomethylated and one non-halomethylated ammonium salts, with proven in vitro antileishmanial activity, were determined according to pharmaceutical standard procedures. The effectiveness and toxicity of these compounds were assessed in hamsters infected with Leishmania (Viannia) braziliensis and compared to that showed by meglumine antimoniate. Animals were followed during 90 days after the completion of treatment. Therapeutic response was determined according to the reduction of size of skin lesions. Toxicity was determined by the effect of compounds on body weight changes and serum levels of renal and hepatic metabolites. The effectiveness of compound 4 was similar to that showed by intralesional administration of meglumine antimoniate and better than that of the other ammonium salts. Levels of creatinine, alanine amino transferase, and blood urea nitrogen in serum were not significantly different between treatment groups, including healthy or untreated hamsters. Results imply that compound 4 has potential as a pharmaceutical active ingredient in the development of new and better formulations for the treatment of cutaneous leishmaniasis.

Insights into the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in Leishmania parasites and characterization of a choline kinase from Leishmania infantum.

The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a Vmax of 3.52µmol/min/mg and an apparent Km value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent Km for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the Vmax/Km decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited.