RESUMEN
Identification and assessment of novel targets is essential to combat drug resistance in the treatment of HIV/AIDS. HIV Capsid (HIV-CA), the protein playing a major role in both the early and late stages of the viral life cycle, has emerged as an important target. We have applied an NMR fragment screening platform and identified molecules that bind to the N-terminal domain (NTD) of HIV-CA at a site close to the interface with the C-terminal domain (CTD). Using X-ray crystallography, we have been able to obtain crystal structures to identify the binding mode of these compounds. This allowed for rapid progression of the initial, weak binding, fragment starting points to compounds 37 and 38, which have 19F-pKi values of 5.3 and 5.4 respectively.
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Fármacos Anti-VIH , Cristalografía por Rayos X , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/síntesis química , Sitios de Unión , Descubrimiento de Drogas , VIH-1/efectos de los fármacos , Unión Proteica , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/antagonistas & inhibidores , Humanos , Estructura Molecular , Modelos Moleculares , Espectroscopía de Resonancia Magnética , Relación Estructura-ActividadRESUMEN
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
There is a need for novel chemical matter for phenotypic and target-based screens to find starting points for drug discovery programmes in neglected infectious diseases and non-hormonal contraceptives that disproportionately affect Low- and Middle-Income Countries (LMICs). In some disease areas multiple screens of corporate and other libraries have been carried out, giving rise to some valuable starting points and leading to preclinical candidates. Whilst in other disease areas, little screening has been carried out. Much screening against pathogens has been conducted phenotypically as there are few robustly validated protein targets. However, many of the active compound series identified share the same molecular targets. To address the need for new chemical material, in this article we describe the design of a new library, designed for screening in drug discovery programmes for neglected infectious diseases. The compounds have been selected from the Enamine REAL (REadily AccessibLe) library, a virtual library which contains approximately 4.5 billion molecules. The molecules theoretically can be synthesized quickly using commercially available intermediates and building blocks. The vast majority of these have not been prepared before, so this is a source of novel compounds. In this paper we describe the design of a diverse library of 30,000 compounds from this collection (graphical abstract). The new library will be made available to laboratories working in neglected infectious diseases, subject to a review process. The project has been supported by the Bill & Melinda Gates Foundation and the Wellcome Trust (Wellcome).
Asunto(s)
Enfermedades Transmisibles , Salud Global , Humanos , Bibliotecas de Moléculas Pequeñas/química , Descubrimiento de Drogas , Enfermedades Transmisibles/diagnósticoRESUMEN
There is an urgent need for the development of new therapeutics with novel modes of action to target Gram-negative bacterial infections, due to resistance to current drugs. Previously, FabA, an enzyme in the bacterial type II fatty acid biosynthesis pathway, was identified as a potential drug target in Pseudomonas aeruginosa, a Gram-negative bacteria of significant clinical concern. A chemical starting point was also identified. There is a cysteine, Cys15, in the active site of FabA, adjacent to where this compound binds. This paper describes the preparation of analogues containing an electrophilic warhead with the aim of covalent inhibition of the target. A wide variety of analogues were successfully prepared. Unfortunately, these analogues did not increase inhibition, which may be due to a loop within the enzyme partially occluding access to the cysteine.
RESUMEN
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.
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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
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.
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Antivirales/farmacología , Exorribonucleasas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Nitrocompuestos/farmacología , Caperuzas de ARN/antagonistas & inhibidores , ARN Viral/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Tiazoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antiparasitarios/química , Antiparasitarios/farmacología , Antivirales/química , COVID-19/virología , Clonación Molecular , Reposicionamiento de Medicamentos , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Cinética , Espectrometría de Masas/métodos , Metilación , Nitrocompuestos/química , Medicamentos bajo Prescripción/química , Medicamentos bajo Prescripción/farmacología , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/enzimología , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tiazoles/química , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacología , Ensayos Analíticos de Alto Rendimiento , Mycobacterium tuberculosis/genética , Relación Estructura-ActividadRESUMEN
Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Linfoma de Células B/tratamiento farmacológico , Ácido Mirístico/metabolismo , Adenina/análogos & derivados , Aminopiridinas/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dasatinib/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Ratones SCID , Modelos Biológicos , Piperidinas , Pirazoles/farmacología , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/metabolismoRESUMEN
Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 µM) and arphamenine A (IC50 = 15.75 µM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
Asunto(s)
Enfermedad de Chagas/diagnóstico , Ensayos Analíticos de Alto Rendimiento , Leucil Aminopeptidasa/aislamiento & purificación , Trypanosoma cruzi/aislamiento & purificación , Secuencia de Aminoácidos/genética , Animales , Enfermedad de Chagas/enzimología , Enfermedad de Chagas/parasitología , Humanos , Cinética , Leucil Aminopeptidasa/genética , Espectrometría de Masas , Especificidad por Sustrato , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/patogenicidadRESUMEN
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
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
BACKGROUND AND PURPOSE: Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. EXPERIMENTAL APPROACH: A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. KEY RESULTS: Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. CONCLUSION AND IMPLICATIONS: Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.
Asunto(s)
Inhibidores de Agregación Plaquetaria/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Tetrahidroisoquinolinas/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Voluntarios Sanos , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
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.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Sistema Libre de Células , Luminiscencia , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Reproducibilidad de los Resultados , Trypanosoma cruzi/químicaRESUMEN
Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N-(3-chloro-4-fluorophenyl)-2-methyl-2-{[4-methyl-3-(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low-nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter-screen up to 25â µm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for inâ vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification.
Asunto(s)
Acetamidas/farmacología , Antimaláricos/farmacología , Acetamidas/síntesis química , Acetamidas/farmacocinética , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacocinética , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium berghei/efectos de los fármacos , Plasmodium cynomolgi/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 µM and 102 µM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.
Asunto(s)
Péptidos/antagonistas & inhibidores , Fosfotransferasas/antagonistas & inhibidores , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/dietoterapia , Animales , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinetocoros/efectos de los fármacos , Cinetocoros/enzimología , Péptidos/química , Fosfotransferasas/química , Fosfotransferasas/genética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Estaurosporina/farmacología , Trypanosoma brucei brucei/enzimología , Tripanosomiasis Africana/parasitología , Zearalenona/análogos & derivados , Zearalenona/farmacologíaRESUMEN
Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.
Asunto(s)
Antiprotozoarios/administración & dosificación , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/diagnóstico por imagen , Inhibidores de Proteasoma/administración & dosificación , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antiprotozoarios/química , Sitios de Unión , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Leishmania donovani/química , Leishmania donovani/enzimología , Leishmania infantum/química , Leishmania infantum/enzimología , Leishmaniasis Visceral/parasitología , Masculino , Ratones , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.
Asunto(s)
Leishmaniasis Visceral/tratamiento farmacológico , Morfolinas/uso terapéutico , Pirazoles/uso terapéutico , Pirimidinas/uso terapéutico , Tripanocidas/uso terapéutico , Animales , Femenino , Células Hep G2 , Humanos , Leishmania donovani/efectos de los fármacos , Masculino , Ratones Endogámicos BALB C , Estructura Molecular , Morfolinas/síntesis química , Morfolinas/toxicidad , Pruebas de Sensibilidad Parasitaria , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/toxicidad , Pirazoles/síntesis química , Pirazoles/toxicidad , Pirimidinas/síntesis química , Pirimidinas/toxicidad , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/toxicidadRESUMEN
The structures of nifurtimox in Table 1 were incorrect and have been updated in the pdf and online. The authors apologize for any confusion caused.
RESUMEN
Chagas' disease is responsible for significant mortality and morbidity in Latin America. Current treatments display variable efficacy and have adverse side effects, hence more effective, better tolerated drugs are needed. However, recent efforts have proved unsuccessful with failure of the ergosterol biosynthesis inhibitor posaconazole in phase II clinical trials despite promising in vitro and in vivo studies. The lack of translation between laboratory experiments and clinical outcome is a major issue for further drug discovery efforts. Our goal was to identify cell-based assays that could differentiate current nitro-aromatic drugs nifurtimox and benznidazole from posaconazole. Using a panel of T. cruzi strains including the six major lineages (TcI-VI), we found that strain PAH179 (TcV) was markedly less susceptible to posaconazole in vitro. Determination of parasite doubling and cycling times as well as EdU labelling experiments all indicate that this lack of sensitivity is due to the slow doubling and cycling time of strain PAH179. This is in accordance with ergosterol biosynthesis inhibition by posaconazole leading to critically low ergosterol levels only after multiple rounds of division, and is further supported by the lack of effect of posaconazole on the non-replicative trypomastigote form. A washout experiment with prolonged posaconazole treatment showed that, even for more rapidly replicating strains, this compound cannot clear all parasites, indicative of a heterogeneous parasite population in vitro and potentially the presence of quiescent parasites. Benznidazole in contrast was able to kill all parasites. The work presented here shows clear differentiation between the nitro-aromatic drugs and posaconazole in several assays, and suggests that in vitro there may be clinically relevant heterogeneity in the parasite population that can be revealed in long-term washout experiments. Based on these findings we have adjusted our in vitro screening cascade so that only the most promising compounds are progressed to in vivo experiments.