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.
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.
The genome of Leishmania major, the causative agent of cutaneous Leishmaniasis, contains three nearly identical genes encoding putative glutathione peroxidases, which only differ at their N- and C-termini. Since the gene homologues are essential in trypanosomes, they may also represent potential drug targets in leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as electron donor indicating that it is not a bone fide glutathione peroxidase. In contrast, high peroxidase activity was obtained with tryparedoxin indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in formation of an intramolecular disulphide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than to t-butyl hydroperoxide whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 M−1s−1 whereas TryP1 exhibits higher rate constants ~106 M−1s−1. All three TDPX proteins together constitute about 0.05 % of the Leishmania major promastigote protein content whereas the TryPs are ~40 times more abundant. Possible specific functions of TDPXs are discussed.
Glutathione peroxidase; tryparedoxin peroxidase; peroxiredoxin; trypanothione; Leishmania
Visceral leishmaniasis is a neglected tropical disease with significant health impact. The current treatments are poor, and there is an urgent need to develop new drugs. Primary screening assays used for drug discovery campaigns have typically used free-living forms of the Leishmania parasite to allow for high-throughput screening. Such screens do not necessarily reflect the physiological situation, as the disease-causing stage of the parasite resides inside human host cells. Assessing the drug sensitivity of intracellular parasites on scale has recently become feasible with the advent of high-content screening methods. We describe here a 384-well microscopy-based intramacrophage Leishmania donovani assay and compare it to an axenic amastigote system. A panel of eight reference compounds was tested in both systems, as well as a human counterscreen cell line, and our findings show that for most clinically used compounds both axenic and intramacrophage assays report very similar results. A set of 15,659 diverse compounds was also screened using both systems. This resulted in the identification of seven new antileishmanial compounds and revealed a high false-positive rate for the axenic assay. We conclude that the intramacrophage assay is more suited as a primary hit-discovery platform than the current form of axenic assay, and we discuss how modifications to the axenic assay may render it more suitable for hit-discovery.
Background: Deoxyhypusine synthase (DHS) catalyzes the spermidine-dependent modification of translation factor eIF5A.
Results: Trypanosomatid DHS activity is increased 3000-fold by heterotetramer formation with a catalytically dead paralog, and both gene products are essential for parasite growth.
Conclusion: Trypanosomatid DHS is a complex between catalytically impaired and inactive DHS subunits.
Significance: This activation mechanism uniquely evolved for two independent enzymes within the trypanosomatid polyamine pathway.
Polyamine biosynthesis is a key drug target in African trypanosomes. The “resurrection drug” eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.
Parasite Metabolism; Polyamines; Protozoan; Trypanosoma brucei; Trypanosome; Deoxyhypusine; Deoxyhypusine Synthase; eIF5A; Spermidine
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.
Human African Trypanosomiasis is a vector-borne disease of sub-Saharan Africa that causes significant morbidity and mortality. Current therapies have many drawbacks, and there is an urgent need for new, better medicines. Ideally such new treatments should be fast-acting cidal agents that cure the disease in as few doses as possible. Screening assays used for hit-discovery campaigns often do not distinguish cytocidal from cytostatic compounds and further detailed follow-up experiments are required. Such studies usually do not have the throughput required to test the large numbers of hits produced in a primary high-throughput screen. Here, we present a 384-well assay that is compatible with high-throughput screening and provides an initial indication of the cidal nature of a compound. The assay produces growth curves at ten compound concentrations by assessing trypanosome counts at 4, 24 and 48 hours after compound addition. A reduction in trypanosome counts over time is used as a marker for cidal activity. The lowest concentration at which cell killing is seen is a quantitative measure for the cidal activity of the compound. We show that the assay can identify compounds that have trypanostatic activity rather than cidal activity, and importantly, that results from primary high-throughput assays can overestimate the potency of compounds significantly. This is due to biphasic growth inhibition, which remains hidden at low starting cell densities and is revealed in our static-cidal assay. The assay presented here provides an important tool to follow-up hits from high-throughput screening campaigns and avoid progression of compounds that have poor prospects due to lack of cidal activity or overestimated potency.
Trypanosoma brucei is a protozoan parasite causing African sleeping sickness. Current treatments for this disease have significant limitations, underlining the need for better and safer drugs. To identify new chemical starting points for drug development, large compound collections are screened against the parasite. Such screens typically do not distinguish between compounds that slow the growth of the parasite and compounds that actually kill the parasite (cidal compounds). Here, we present the development of an assay to identify such compounds. The main advantage of our assay is that it marries a relatively high-throughput to increased understanding of mode of action. Many active compounds (hits) are usually identified in T. brucei primary screening campaigns, making it difficult to select which compounds should undergo further development. Our assay allows testing of all of the hits for cidal activity so that only the most promising compounds are progressed. We show that the starting cell density used in the T. brucei growth assay can have a significant effect on the shape of dose response curves, and that important information regarding the mode of action of a compound can remain hidden at low starting densities as used commonly in T. brucei screening assays.
Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible.
enzyme-inhibitor complex; Trypanosoma cruzi; trypanothione reductase; quinacrine mustard; X-ray diffraction
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.
Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play significant in clinical resistance to antimonials.
antimonial resistance; Leishmania donovani; tryparedoxin peroxidase
A high-throughput screening campaign of a library of 100,000 lead-like compounds identified 2-iminobenzimidazoles as a novel class of trypanothione reductase inhibitors. These 2-iminobenzimidazoles display potent trypanocidal activity against Trypanosoma brucei rhodesiense, do not inhibit closely related human glutathione reductase and have low cytotoxicity against mammalian cells.
Tropical diseases; Trypanosomiasis therapeutics; Trypanothione reductase inhibitors; High-throughput screening; Medicinal chemistry; Imino benzimidazoles
Hexanic, methanolic, and hydroalcoholic extracts, and 34 isolated compounds from Vitex polygama Cham. (Lamiaceae, formely Verbenaceae) and Siphoneugena densiflora O. Berg (Myrtaceae) were screened for their trypanocidal effects on bloodstream forms of Trypanosoma cruzi and T. brucei, as well as for their enzymatic inhibitory activities on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) and trypanothione reductase (TR) enzymes from T. cruzi and adeninephosphoribosyl transferase (APRT) enzyme from Leishmania tarentolae. In general, polar extracts displayed strong effects and some of the tested compounds have shown good results in comparison to positive controls of the bioassays.
Myrtaceae; Trypanosoma; Leishmania
Trypanothione reductase (TR), an enzyme that buffers oxidative stress in trypanosomatid parasites, was screened against commercial libraries containing approximately 134,500 compounds. After secondary screening, four chemotypes were identified as screening positives with selectivity for TR over human glutathione reductase. Thirteen compounds from these four chemotypes were purchased, and their in vitro activity against TR and Trypanosoma brucei are described.
Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defences. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular mass of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine, and only used 1-chloro-2,4- dinitrobenzene from a range of glutathione S-transferases substrates. Peptide sequencing revealed that the three components were the alpha, beta and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight-binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4- dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bγ genes were cloned from C. fasciculata but recombinant C. fasciculata eEF1Bγ had no S-transferase activity, suggesting that eEF1Bγ is unstable in the absence of the other subunits.
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.
Inhibitors; Medicinal chemistry; Trypanosoma brucei; P2 transporter; Quinols
N-Myristoyltransferase (NMT) represents
drug target for human African trypanosomiasis (HAT), which is caused
by the parasitic protozoa Trypanosoma brucei. We
report the optimization of a high throughput screening hit (1) to give a lead molecule DDD85646 (63), which
has potent activity against the enzyme (IC50 = 2 nM) and T. brucei (EC50 = 2 nM) in culture. The compound
has good oral pharmacokinetics and cures rodent models of peripheral
HAT infection. This compound provides an excellent tool for validation
of T. brucei NMT as a drug target for HAT as well
as a valuable lead for further optimization.
Methylglyoxal is a toxic by-product of glycolysis and other metabolic pathways. In mammalian cells, the principal route for detoxification of this reactive metabolite is via the glutathione-dependent glyoxalase pathway forming d-lactate, involving lactoylglutathione lyase (GLO1; EC 126.96.36.199) and hydroxyacylglutathione hydrolase (GLO2; EC 188.8.131.52). In contrast, the equivalent enzymes in the trypanosomatid parasites Trypanosoma cruzi and Leishmania spp. show >200-fold selectivity for glutathionylspermidine and trypanothione over glutathione and are therefore sensu stricto lactoylglutathionylspermidine lyases (EC 4.4.1.-) and hydroxyacylglutathionylspermidine hydrolases (EC 3.2.1.-). The unique substrate specificity of the parasite glyoxalase enzymes can be directly attributed to their unusual active site architecture. The African trypanosome differs from these parasites in that it lacks GLO1 and converts methylglyoxal to l-lactate rather than d-lactate. Since Trypanosoma brucei is the most sensitive of the trypanosomatids to methylglyoxal toxicity, the absence of a complete and functional glyoxalase pathway in these parasites is perplexing. Alternative routes of methylglyoxal detoxification in T. brucei are discussed along with the potential of exploiting trypanosomatid glyoxalase enzymes as targets for anti-parasitic chemotherapy.
GLO1, glyoxalase I; GLO2, glyoxalase II; T[SH]2, trypanothione, N1,N8-bis(glutathionyl)spermidine; GSH, glutathione; LADH, lactaldehyde dehydrogenase; Trypanosoma; Leishmania; Methylglyoxal; Glyoxalase; Trypanothione; Drug discovery
Leishmania parasites are pteridine auxotrophs that use an NADPH-dependent pteridine reductase 1 (PTR1) and NADH-dependent quinonoid dihydropteridine reductase (QDPR) to salvage and maintain intracellular pools of tetrahydrobiopterin (H4B). However, the African trypanosome lacks a credible candidate QDPR in its genome despite maintaining apparent QDPR activity. Here we provide evidence that the NADH-dependent activity previously reported by others is an assay artifact. Using an HPLC-based enzyme assay, we demonstrate that there is an NADPH-dependent QDPR activity associated with both TbPTR1 and LmPTR1. The kinetic properties of recombinant PTR1s are reported at physiological pH and ionic strength and compared with LmQDPR. Specificity constants (kcat/Km) for LmPTR1 are similar with dihydrobiopterin (H2B) and quinonoid dihydrobiopterin (qH2B) as substrates and about 20-fold lower than LmQDPR with qH2B. In contrast, TbPTR1 shows a 10-fold higher kcat/Km for H2B over qH2B. Analysis of Trypanosoma brucei isolated from infected rats revealed that H4B (430 nm, 98% of total biopterin) was the predominant intracellular pterin, consistent with a dual role in the salvage and regeneration of H4B. Gene knock-out experiments confirmed this: PTR1-nulls could only be obtained from lines overexpressing LmQDPR with H4B as a medium supplement. These cells grew normally with H4B, which spontaneously oxidizes to qH2B, but were unable to survive in the absence of pterin or with either biopterin or H2B in the medium. These findings establish that PTR1 has an essential and dual role in pterin metabolism in African trypanosomes and underline its potential as a drug target.
Enzyme Kinetics; Gene Knockout; Parasite Metabolism; Pterin; Trypanosome; Biopterin; Leishmania; Pteridine Reductase; Quinonoid Pteridine Reductase; Substrate Inhibition
Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class (“quinols”) and identified several with potent trypanocidal activity (EC50 < 100 nm). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)2), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)2, TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and l-cysteine bound in a 2:1 ratio with PMX464 with Kd values of 6 and 27 μm, respectively, whereas T(SH)2 bound more tightly in a 1:1 ratio with a Kd value of 430 nm. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)2 is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.
Drug Action; Metabolism; Peroxidase; Thiol; Trypanosome; Quinol; Trypanothione; Tryparedoxin Peroxidase
Phenotypic screening of the LOPAC library identified several potent and selective inhibitors of African trypanosomes. The κ-opioid agonist (+)-U50,488 represents a novel lead for drug discovery against sleeping sickness.
A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 ‘library of pharmacologically active compounds’ against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC50 values <1 μM. Counter-screening vs. normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (<70 nM) and selective (>700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC50 value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (−)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several μ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.
Phenotypic screening; African trypanosomiasis; Target identification; Target validation; U50,488
Drug discovery is a high-risk, expensive and lengthy process taking at least 12 years and costing upwards of US$500 million per drug to reach the clinic. For neglected diseases, the drug discovery process is driven by medical need and guided by pre-defined target product profiles. Assessment and prioritisation of the most promising targets for entry into screening programmes is crucial for maximising chances of success. Here we describe criteria used in our drug discovery unit for target assessment and introduce the ‘traffic light’ system as a prioritisation and management tool. We hope this brief review will stimulate basic scientists to acquire additional information necessary for drug discovery.
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.
Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase–thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.
Drug discovery; Screening; Pteridine reductase; Dihydrofolate reductase
As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3 Å and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6 Å over 482 Cα atoms). Kinetically, the Km for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The Km for NADPH for the T. brucei enzyme was found to be 0.77 μM using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S = Km values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thioridazine and citalopram. The relative IC50 values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.
TryR, trypanothione reductase; T(S)2, trypanothione disulphide; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid); HAT, human African trypanosomiasis; Trypanothione metabolism; Trypanosome; Thiol; Enzymology; Drug discovery