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1.  Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects 
Trends in Parasitology  2014;30(6):289-298.
Highlights
•Two nitro drugs are currently used in the treatment of trypanosomatid diseases.•Several new nitroaromatics are being developed against the trypanosomatid diseases.•Many nitro drugs and drug candidates act as prodrugs which require bioactivation.•Nitroaromatics can have disparate mechanisms of action in trypanosomatid parasites.
There is an urgent need for new, safer, and effective treatments for the diseases caused by the protozoan parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. In the search for more effective drugs to treat these ‘neglected diseases’ researchers have chosen to reassess the therapeutic value of nitroaromatic compounds. Previously avoided in drug discovery programs owing to potential toxicity issues, a nitro drug is now being used successfully as part of a combination therapy for human African trypanosomiasis. We describe here the rehabilitation of nitro drugs for the treatment of trypanosomatid diseases and discuss the future prospects for this compound class.
doi:10.1016/j.pt.2014.04.003
PMCID: PMC4045206  PMID: 24776300
trypanosomatids; nitroaromatics; nitroreductase; pro-drugs; bioactivation
2.  Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy 
Molecular microbiology  2013;90(2):10.1111/mmi.12376.
Summary
African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.
doi:10.1111/mmi.12376
PMCID: PMC3868941  PMID: 23980694
3.  Comparison of Stress-Hemoconcentration Correction Techniques for Stress-Induced Coagulation 
BioMed Research International  2013;2013:480648.
When examining stress effects on coagulation, arithmetic correction is typically used to adjust for concomitant hemoconcentration but may be inappropriate for coagulation activity assays. We examined a new physiologically relevant method of correcting for stress-hemoconcentration. Blood was drawn from healthy men (N = 40) during baseline, mental stress, and recovery, and factor VII activity (FVII:C), factor VIII activity (FVIII:C), activated partial thromboplastin time (APTT), prothrombin time (PT%), fibrinogen, D-dimer, and plasma volume were determined. Three hemoconcentration correction techniques were assessed: arithmetic correction and two reconstitution techniques using baseline plasma or physiological saline. Area-under-the-curve (AUC) was computed for each technique. For FVII:C, uncorrected AUC was significantly greater than AUC corrected arithmetically. For PT%, uncorrected AUC was significantly greater than AUC corrected with saline or arithmetically. For APTT, uncorrected AUC was significantly less than AUC corrected with saline and greater than AUC corrected arithmetically. For fibrinogen, uncorrected AUC was significantly greater than AUC corrected with saline or arithmetically. For D-dimer, uncorrected AUC was significantly greater than AUC corrected arithmetically. No differences in AUC were observed for FVIII:C. Saline reconstitution seems most appropriate when adjusting for hemoconcentration effects on clotting time and activity. Stress-hemoconcentration accounted for the majority of coagulation changes.
doi:10.1155/2013/480648
PMCID: PMC3814105  PMID: 24222908
4.  The R Enantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis 
Antimicrobial Agents and Chemotherapy  2013;57(10):4699-4706.
The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.
doi:10.1128/AAC.00722-13
PMCID: PMC3811480  PMID: 23856774
5.  Assessing the Essentiality of Leishmania donovani Nitroreductase and Its Role in Nitro Drug Activation 
The nitroimidazole fexinidazole has potential as a safe and effective oral drug therapy for the treatment of visceral leishmaniasis. To date, nitroheterocyclics have not been used in the treatment of leishmaniasis, and relatively little is known about their mechanism of action. In African trypanosomes, nitro drugs are reductively activated by a type I nitroreductase (NTR), absent in mammalian cells. Modulation of nitroreductase levels in Trypanosoma brucei directly affected sensitivity to nitro compounds, with reduced concentrations of the enzyme leading to moderate nitro drug resistance. In view of the progression of fexinidazole into clinical development for visceral leishmaniasis, here we assess the essentiality of the nitroreductase in Leishmania donovani and the effect of modulating nitroreductase levels on susceptibility to fexinidazole. The failure to directly replace both endogenous copies of the NTR gene, except in the presence of an ectopic copy of the gene, suggests that the NTR gene is essential for the growth and survival of L. donovani promastigotes. Loss of a single chromosomal copy of the L. donovani NTR gene resulted in parasites that were mildly resistant (<2-fold) to the predominant in vivo metabolite of fexinidazole, while parasites overexpressing NTR were 18-fold more susceptible. These data confirm that Leishmania NTR plays a pivotal role in fexinidazole activation. Reliance on a single enzyme for prodrug activation may leave fexinidazole vulnerable to the emergence of drug resistance. However, the essentiality of the NTR in L. donovani promastigotes, combined with the limited resistance shown by NTR single knockout cells, suggests that the potential for the spread of NTR-based resistance to fexinidazole may be limited.
doi:10.1128/AAC.01788-12
PMCID: PMC3553740  PMID: 23208716
6.  The anti-trypanosome drug fexinidazole shows potential for treating visceral leishmaniasis 
Science translational medicine  2012;4(119):119re1.
Safer and more effective oral drugs are required to treat visceral leishmaniasis, a parasitic disease that kills 50-60,000 people each year. Here we report that fexinidazole, a drug currently in phase I clinical trials for treating African trypanosomiasis, shows promise for treating visceral leishmaniasis. This 2-substituted 5-nitroimidazole drug is rapidly oxidized in vivo in mice, dogs and humans to sulfoxide and sulfone metabolites. Both metabolites of fexinidazole were active against Leishmania donovani amastigotes grown in macrophages, whereas the parent compound was inactive. Pharmacokinetic studies with fexinidazole (200 mg kg−1) showed that fexinidazole sulfone achieves blood concentrations in mice above the EC99 value for at least 24h following a single oral dose. A once daily regimen for 5 days at this dose resulted in a 98.4% suppression of infection in a mouse model of visceral leishmaniasis, equivalent to that seen with the drugs miltefosine and Pentostam, which are currently used clinically to treat visceral leishmaniasis. In African trypanosomes, the mode of action of nitro-drugs involves reductive activation via an NADH-dependent bacterial-like nitroreductase. Overexpression of the leishmanial homologue of this nitroreductase in L. donovani increased sensitivity to fexinidazole by 19-fold indicating that a similar mechanism is involved in both parasites. These findings illustrate the potential of fexinidazole as an oral drug therapy for treating visceral leishmaniasis.
doi:10.1126/scitranslmed.3003326
PMCID: PMC3457684  PMID: 22301556
7.  Quinol derivatives as potential trypanocidal agents 
Bioorganic & Medicinal Chemistry  2012;20(4):1607-1615.
Graphical abstract
Quinols have been developed as a class of potential anti-cancer compounds. They are thought to act as double Michael acceptors, forming two covalent bonds to their target protein(s). Quinols have also been shown to have activity against the parasite Trypanosoma brucei, the causative organism of human African trypanosomiasis, but they demonstrated little selectivity over mammalian MRC5 cells in a counter-screen. In this paper, we report screening of further examples of quinols against T. brucei. We were able to derive an SAR, but the compounds demonstrated little selectivity over MRC5 cells. In an approach to increase selectivity, we attached melamine and benzamidine motifs to the quinols, because these moieties are known to be selectively concentrated in the parasite by transporter proteins. In general these transporter motif-containing analogues showed increased selectivity; however they also showed reduced levels of potency against T. brucei.
doi:10.1016/j.bmc.2011.12.018
PMCID: PMC3281193  PMID: 22264753
Inhibitors; Medicinal chemistry; Trypanosoma brucei; P2 transporter; Quinols
8.  Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis▿ ‖ 
The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.
doi:10.1128/AAC.00332-10
PMCID: PMC2897277  PMID: 20439607
9.  A comparative study of methylglyoxal metabolism in trypanosomatids 
The Febs Journal  2009;276(2):376-386.
The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d-lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione-dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1-like protein on immunoblots of cell lysates, and lack of d-lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4-fold and 5.7-fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the ‘Tritryp’ parasites in metabolizing methylglyoxal, producing l-lactate rather than d-lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d-lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH-dependent methylglyoxal reductase and NAD+-dependent l-lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l-lactate.
doi:10.1111/j.1742-4658.2008.06788.x
PMCID: PMC2702497  PMID: 19076214
glyoxalase; lactate; methylglyoxal metabolism; Trypanosoma brucei; trypanothione
10.  The Effects of Cold Whirlpool on Power, Speed, Agility, and Range of Motion 
The purpose was to determine if cold whirlpool treatment decreases functional performance equally regardless of gender. A secondary aim was to determine if there is a gradual increase in functional performance across time. Twenty-one college-aged subjects volunteered to participate in this study and were required to perform four measures of functional performance including: counter movement vertical jump, T-test, 36.58-meter dash (40-yard), and active range of motion of the ankle. Participants were treated with a 20 minute, 10 degree Celsius cold whirlpool following the pre-test of a given functional performance measure. Participants demonstrated significant decreases in counter movement vertical jump, T-test, and 40-yard dash performance immediately following treatment. Vertical jump performance remained impaired for at least 32 minutes. While both the T-test and 40-yard dash were affected for 7 and 22 minutes post- treatment, respectively. Participants also demonstrated significant decreases in peak power and average power immediately after and for 32 minutes post-treatment. Dorsiflexion was significantly decreased 7 and 12 minutes following treatment. There were no differences for plantar flexion, inversion, or eversion. These data suggest functional performance was affected immediately following and for up to 32 minutes after cold whirlpool treatment. It was also evident that there is a gradual performance increase for each measure of functional performance across time. Therefore, the consequences should be carefully considered before returning athletes to activity following cold whirlpool treatment.
Key pointsCryotherapy is a common and highly effective modality in treating acute and chronic athletic injuries.The results indicated that cold whirlpool does have an immediate and subsequent effect on functional performance.Understanding how cold whirlpool adversely affects functional performance allows clinicians to continue using this modality before vigorous athletic activity.
PMCID: PMC3761888  PMID: 24149907
Functional performance; cryotherapy; therapeutic modalities
11.  Cuboid Syndrome: a Review of the Literature 
The purpose of this review was to inform all medical health care professionals about cuboid syndrome, which has been described as difficult to recognize and is commonly misdiagnosed, by explaining the etiology of this syndrome, its clinical diagnosis in relation to differential diagnoses, commonly administered treatment techniques, and patient outcomes. A comprehensive review of the relevant literature was conducted with MEDLINE, EBSCO, and PubMed (1960 - Present) using the key words cuboid, cuboid syndrome, foot anatomy, tarsal bones, manual therapy, and manipulation. Medical professionals must be aware that any lateral foot and ankle pain may be the result of cuboid syndrome. Once properly diagnosed, cuboid syndrome responds exceptionally well to conservative treatment involving specific cuboid manipulation techniques. Other methods of conservative treatment including therapeutic modalities, therapeutic exercises, padding, and low dye taping techniques are used as adjuncts in the treatment of this syndrome. Immediately after the manipulation is performed, the patient may note a decrease or a complete cessation of their symptoms. Occasionally, if the patient has had symptoms for a longer duration, several manipulations may be warranted throughout the course of time. Due to the fact radiographic imaging is of little value, the diagnosis is largely based on the patient’s history and a collection of signs and symptoms associated with the condition. Additionally, an understanding of the etiology behind this syndrome is essential, aiding the clinician in the diagnosis and treatment of this syndrome. After the correct diagnosis is made and a proper treatment regimen is utilized, the prognosis is excellent.
Key PointsDefine the poorly understood condition of cuboid syndrome.Provide an understanding of the anatomical structures involved.Provide an explanation as to the cause of this syndrome.Demonstrate ways to evaluate by making a differential diagnosis.To inform health care professionals about management and treatment of cuboid syndrome.
PMCID: PMC3861761  PMID: 24357955
Subluxation; manipulation; tarsal; syndrome; manual therapy
12.  Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy 
Molecular Microbiology  2013;90(2):443-455.
African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.
doi:10.1111/mmi.12376
PMCID: PMC3868941  PMID: 23980694
13.  Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods 
Molecular Microbiology  2009;74(3):529-540.
The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.
doi:10.1111/j.1365-2958.2009.06761.x
PMCID: PMC2784880  PMID: 19558432
14.  Synthesis and Evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) Analogues as Inhibitors of Trypanothione Reductase 
Chemmedchem  2009;4(8):1341-1353.
Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1-(1-benzo[b]thiophen-2-yl-cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1 μm) and biologically active against bloodstream T. brucei (EC50=10 μm), but with poor selectivity against mammalian MRC5 cells (EC50=29 μm). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.
doi:10.1002/cmdc.200900098
PMCID: PMC2929374  PMID: 19557802
BTCP; inhibitors; medicinal chemistry; trypanosoma brucei; trypanothione reductase
15.  Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography 
Journal of Medicinal Chemistry  2011;54(19):6514-6530.
Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR–ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.
doi:10.1021/jm200312v
PMCID: PMC3188286  PMID: 21851087

Results 1-15 (15)