RESUMEN
Trypanosoma cruzi (T. cruzi) is the causative agent of Chagas' disease, a parasitic infection responsible for significant morbidity and mortality in Latin America. The current treatments have many serious drawbacks and new drugs are urgently required. In the UK, T. cruzi is classified by the Advisory Committee on Dangerous Pathogens (ACDP) as a Hazard Group 3 organism and strict safety practices must be adhered to when handling this pathogen in the laboratory. Validated inactivation techniques are required for safe T. cruzi waste disposal and removal from Containment Level 3 (CL3) facilities for storage, transportation and experimental analysis. Here we assess three T. cruzi. inactivation methods. These include three freeze-thaw cycles, chemical inactivation with Virkon disinfectant, and air drying on Whatman FTA cards (A, B, C, Elute) and on a Mitra microsampling device. After each treatment parasite growth was monitored for 4-6 weeks by microscopic examination. Three freeze-thaw cycles were sufficient to inactivate all T. cruzi CLBrener Luc life cycle stages and Silvio x10/7 A1 large epimastigote cell pellets up to two grams wet weight. Virkon treatment for one hour inactivated T. cruzi Silvio x10/7 subclone A1 and CLBrener Luc both in whole blood and cell culture medium when incubated at a final concentration of 2.5% Virkon, or at ≥1% Virkon when in tenfold excess of sample volume. Air drying also inactivated T. cruzi CLBrener Luc spiked blood when dried on FTA A, B or Elute cards for ≥30 minutes and on a Mitra Microsampler for two hours. However, T. cruzi CLBrener Luc were not inactivated on FTA C cards when dried for up to two hours. These experimentally confirmed conditions provide three validated T. cruzi inactivation methods which can be applied to other related ACDP Hazard Group 2-3 kinetoplastid parasites.
Asunto(s)
Aminopiridinas , Enfermedad de Chagas , Ácidos Sulfúricos , Trypanosoma cruzi , Humanos , Enfermedad de Chagas/parasitología , PeróxidosRESUMEN
BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 µM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 µM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Humanos , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/farmacología , Leucil Aminopeptidasa/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Descubrimiento de Drogas , Antiparasitarios/uso terapéutico , Tripanocidas/uso terapéuticoRESUMEN
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions of people in the Americas and across the world, leading to considerable morbidity and mortality. Current treatment options, benznidazole (BNZ) and nifurtimox, offer limited efficacy and often lead to adverse side effects because of long treatment durations. Better treatment options are therefore urgently required. Here, we describe a pyrrolopyrimidine series, identified through phenotypic screening, that offers an opportunity to improve on current treatments. In vitro cell-based washout assays demonstrate that compounds in the series are incapable of killing all parasites; however, combining these pyrrolopyrimidines with a subefficacious dose of BNZ can clear all parasites in vitro after 5 days. These findings were replicated in a clinically predictive in vivo model of chronic Chagas disease, where 5 days of treatment with the combination was sufficient to prevent parasite relapse. Comprehensive mechanism of action studies, supported by ligand-structure modeling, show that compounds from this pyrrolopyrimidine series inhibit the Qi active site of T. cruzi cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Knowledge of the molecular target enabled a cascade of assays to be assembled to evaluate selectivity over the human cytochrome b homolog. As a result, a highly selective and efficacious lead compound was identified. The combination of our lead compound with BNZ rapidly clears T. cruzi parasites, both in vitro and in vivo, and shows great potential to overcome key issues associated with currently available treatments.
Asunto(s)
Enfermedad de Chagas , Parásitos , Tripanocidas , Trypanosoma cruzi , Animales , Humanos , Citocromos b , Tripanocidas/efectos adversos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/inducido químicamente , Enfermedad de Chagas/parasitologíaRESUMEN
There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.
Asunto(s)
Enfermedad de Chagas , Leishmaniasis Visceral , Tripanocidas , Trypanosoma cruzi , Ratones , Animales , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Complejo de la Endopetidasa Proteasomal , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Leishmaniasis Visceral/tratamiento farmacológico , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Tripanocidas/químicaRESUMEN
While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei, the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.
Asunto(s)
Citostáticos , Tripanocidas , Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Humanos , Tripanosomiasis Africana/tratamiento farmacológico , Tripanocidas/uso terapéutico , Tripanocidas/farmacocinética , Citostáticos/uso terapéutico , Barrera HematoencefálicaRESUMEN
Leishmaniasis (visceral and cutaneous), Chagas disease and human African trypanosomiasis cause substantial death and morbidity, particularly in low- and middle-income countries. Although the situation has improved for human African trypanosomiasis, there remains an urgent need for new medicines to treat leishmaniasis and Chagas disease; the clinical development pipeline is particularly sparse for Chagas disease. In this Review, we describe recent advances in our understanding of the biology of the causative pathogens, particularly from the drug discovery perspective, and we explore the progress that has been made in the development of new drug candidates and the identification of promising molecular targets. We also explore the challenges in developing new clinical candidates and discuss potential solutions to overcome such hurdles.
Asunto(s)
Enfermedad de Chagas , Leishmaniasis , Tripanosomiasis Africana , Animales , Humanos , Tripanosomiasis Africana/tratamiento farmacológico , Enfermedad de Chagas/tratamiento farmacológico , Leishmaniasis/tratamiento farmacológico , Descubrimiento de DrogasRESUMEN
Approximately 6-7 million people around the world are estimated to be infected with Trypanosoma cruzi, the causative agent of Chagas disease. The current treatments are inadequate and therefore new medical interventions are urgently needed. In this paper we describe the identification of a series of disubstituted piperazines which shows good potency against the target parasite but is hampered by poor metabolic stability. We outline the strategies used to mitigate this issue such as lowering logD, bioisosteric replacements of the metabolically labile piperazine ring and use of plate-based arrays for quick diversity scoping. We discuss the success of these strategies within the context of this series and highlight the challenges faced in phenotypic programs when attempting to improve the pharmacokinetic profile of compounds whilst maintaining potency against the desired target.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Humanos , Piperazinas/farmacologíaRESUMEN
Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi, is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo, that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T. cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy.
Asunto(s)
Automatización/métodos , Enfermedad de Chagas/parasitología , Evaluación Preclínica de Medicamentos/métodos , Microscopía Fluorescente/métodos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Automatización/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Humanos , Microscopía Fluorescente/instrumentación , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologíaRESUMEN
There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.
Asunto(s)
Diseño de Fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Proteínas Protozoarias/metabolismo , Animales , Antiprotozoarios/química , Antiprotozoarios/metabolismo , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Sitios de Unión , Línea Celular , Evaluación Preclínica de Medicamentos , Semivida , Humanos , Leishmania donovani/efectos de los fármacos , Leishmania donovani/metabolismo , Leishmaniasis Visceral/tratamiento farmacológico , Leishmaniasis Visceral/parasitología , Ratones , Simulación de Dinámica Molecular , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/metabolismo , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/química , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Solubilidad , Relación Estructura-ActividadRESUMEN
Visceral leishmaniasis (VL) is a parasitic infection that results in approximately 26â¯000-65â¯000 deaths annually. The available treatments are hampered by issues such as toxicity, variable efficacy, and unsuitable dosing options. The need for new treatments is urgent and led to a collaboration between the Drugs for Neglected Diseases initiative (DNDi), GlaxoSmithKline (GSK), and the University of Dundee. An 8-hydroxynaphthyridine was identified as a start point, and an early compound demonstrated weak efficacy in a mouse model of VL but was hampered by glucuronidation. Efforts to address this led to the development of compounds with improved in vitro profiles, but these were poorly tolerated in vivo. Investigation of the mode of action (MoA) demonstrated that activity was driven by sequestration of divalent metal cations, a mechanism which was likely to drive the poor tolerability. This highlights the importance of investigating MoA and pharmacokinetics at an early stage for phenotypically active series.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Diseño de Fármacos , Leishmania/efectos de los fármacos , Naftiridinas/química , Naftiridinas/farmacología , Animales , Concentración 50 Inhibidora , Ratones , Solubilidad , Relación Estructura-Actividad , Agua/químicaRESUMEN
Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in kinetoplastid parasites Trypanosoma brucei and Leishmania donovani. To date, all kinetoplastid MetRS inhibitors described bind in a similar way to an expanded methionine pocket and an adjacent, auxiliary pocket. In the current study, we have identified a structurally novel class of inhibitors containing a 4,6-diamino-substituted pyrazolopyrimidine core (the MetRS02 series). Crystallographic studies revealed that MetRS02 compounds bind to an allosteric pocket in L. major MetRS not previously described, and enzymatic studies demonstrated a noncompetitive mode of inhibition. Homology modeling of the Trypanosoma cruzi MetRS enzyme revealed key differences in the allosteric pocket between the T. cruzi and Leishmania enzymes. These provide a likely explanation for the lower MetRS02 potencies that we observed for the T. cruzi enzyme compared to the Leishmania enzyme. The identification of a new series of MetRS inhibitors and the discovery of a new binding site in kinetoplastid MetRS enzymes provide a novel strategy in the search for new therapeutics for kinetoplastid diseases.
Asunto(s)
Sitio Alostérico , Metionina-ARNt Ligasa/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/enzimología , MetioninaRESUMEN
Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
Asunto(s)
Antiparasitarios/farmacología , Citocromos b/antagonistas & inhibidores , Descubrimiento de Drogas , Leishmania donovani/efectos de los fármacos , Leishmania donovani/genética , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Antiparasitarios/química , Antiparasitarios/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Citocromos b/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Leishmaniasis Visceral/tratamiento farmacológicoRESUMEN
Visceral leishmaniasis (VL) affects millions of people across the world, largely in developing nations. It is fatal if left untreated and the current treatments are inadequate. As such, there is an urgent need for new, improved medicines. In this paper, we describe the identification of a 6-amino-N-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine scaffold and its optimization to give compounds which showed efficacy when orally dosed in a mouse model of VL.
RESUMEN
In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.
Asunto(s)
Control de Enfermedades Transmisibles , Enfermedades Transmisibles/tratamiento farmacológico , Congresos como Asunto , Terapia Combinada , Enfermedades Transmisibles/epidemiología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Infecciones por VIH/tratamiento farmacológico , Humanos , Pobreza , Reino UnidoRESUMEN
Chagas' disease, which is caused by the Trypanosoma cruzi parasite, has become a global health problem that is currently treated with poorly tolerated drugs that require prolonged dosing. Therefore, there is a clinical need for new therapeutic agents that can mitigate these issues. The phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GDP-MP) enzymes form part of the de novo biosynthetic pathway to the nucleotide sugar GDP-mannose. This nucleotide sugar is used either directly, or indirectly via the formation of dolichol-phosphomannose, for the assembly of all mannose-containing glycoconjugates. In T. cruzi, mannose-containing glycoconjugates include the cell-surface glycoinositol-phospholipids and the glycosylphosphatidylinositol-anchored mucin-like glycoproteins that dominate the cell surface architectures of all life cycle stages. This makes PMM and GDP-MP potentially attractive targets for a drug discovery program against Chagas' disease. To assess the ligandability of these enzymes in T. cruzi, we have screened 18,117 structurally diverse compounds exploring drug-like chemical space and 16,845 small polar fragment compounds using an assay interrogating the activities of both PMM and GDP-MP enzymes simultaneously. This resulted in 48 small fragment hits, and on retesting 20 were found to be active against the enzymes. Deconvolution revealed that these were all inhibitors of T. cruzi GDP-MP, with compounds 2 and 3 acting as uncompetitive and competitive inhibitors, respectively. Based on these findings, the T. cruzi PMM and GDP-MP enzymes were deemed not ligandable and poorly ligandable, respectively, using small molecules from conventional drug discovery chemical space. This presents a significant hurdle to exploiting these enzymes as therapeutic targets for Chagas' disease.
Asunto(s)
Antiprotozoarios/farmacología , Manosa/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfotransferasas (Fosfomutasas)/metabolismo , Trypanosoma cruzi/enzimología , Enfermedad de Chagas/parasitología , Descubrimiento de Drogas/métodos , Manosafosfatos/metabolismo , Nucleotidiltransferasas/genética , Fosfotransferasas (Fosfomutasas)/genéticaRESUMEN
Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening condition that has become a global issue. Current treatment is limited to two medicines that require prolonged dosing and are associated with multiple side effects, which often lead to treatment discontinuation and failure. One way to address these shortcomings is through target-based drug discovery on validated T. cruzi protein targets. One such target is the proteasome, which plays a crucial role in protein degradation and turnover through chymotrypsin-, trypsin-, and caspase-like catalytic activities. In order to initiate a proteasome drug discovery program, we isolated proteasomes from T. cruzi epimastigotes and characterized their activity using a commercially available glow-like luminescence-based assay. We developed a high-throughput biochemical assay for the chymotrypsin-like activity of the T. cruzi proteasome, which was found to be sensitive, specific, and robust but prone to luminescence technology interference. To mitigate this, we also developed a counterscreen assay that identifies potential interferers at the levels of both the luciferase enzyme reporter and the mechanism responsible for a glow-like response. Interestingly, we also found that the peptide substrate for chymotrypsin-like proteasome activity was not specific and was likely partially turned over by other catalytic sites of the protein. Finally, we utilized these biochemical tools to screen 18,098 compounds, exploring diverse drug-like chemical space, which allowed us to identify 39 hits that were active in the primary screening assay and inactive in the counterscreen assay.