RESUMO
Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.
Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Percepção de Quorum/fisiologia , Trypanosoma/metabolismo , Diferenciação Celular , Sequência Conservada/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Filogenia , Proteínas de Protozoários/metabolismo , Percepção de Quorum/genética , Transdução de Sinais , Trypanosoma/fisiologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Virulência/fisiologiaRESUMO
BACKGROUND: LCP1 encodes L-plastin, an actin-bundling protein primarily expressed in hematopoietic cells. In mouse and fish models, LCP1 deficiency has been shown to result in hematologic and immune defects. OBJECTIVE: This study aimed to determine the nature of a human inborn error of immunity resulting from a novel genetic variant of LCP1. METHODS: We performed genetic, protein, and cellular analysis of PBMCs from a kindred with apparent autosomal dominant immune deficiency. We identified a candidate causal mutation in LCP1, which we evaluated by engineering the orthologous mutation in mice and Jurkat cells. RESULTS: A splice-site variant in LCP1 segregated with lymphopenia, neutropenia, and thrombocytopenia. The splicing defect resulted in at least 2 aberrant transcripts, producing an in-frame deletion of 24 nucleotides, and a frameshift deletion of exon 8. Cellular analysis of the kindred revealed a proportionate reduction of T and B cells and a mild expansion of transitional B cells. Similarly, mice carrying the orthologous genetic variant exhibited the same in-frame aberrant transcript, reduced expression Lcp1 and gene dose-dependent leukopenia, mild thrombocytopenia, and lymphopenia, with a significant reduction of T-cell populations. Functional analysis revealed that LCP1c740-1G>A confers a defect in platelet development and function with aberrant spreading on collagen. Immunologic analysis revealed defective actin organization in T cells, reduced migration of PBMCs from patients, splenocytes from mutant mice, and a mutant Jurkat cell line in response to CXCL12; impaired germinal center B-cell expansion after immunization; and reduced cytokinesis during T cell proliferation. CONCLUSIONS: We describe a unique human hematopoietic defect affecting neutrophils, lymphocytes, and platelets arising from partial LCP1 deficiency.
RESUMO
The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 µM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 µM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.
Assuntos
Antiprotozoários , Leishmania donovani , Nitroimidazóis , Humanos , Camundongos , Animais , Antiprotozoários/farmacologia , Antiprotozoários/metabolismo , Nitroimidazóis/farmacologia , Nitroimidazóis/metabolismo , Macrófagos , Nitrorredutases/metabolismoRESUMO
Artemisinin-based combination therapies have been crucial in driving down the global burden of malaria, the world's largest parasitic killer. However, their efficacy is now threatened by the emergence of resistance in Southeast Asia and sub-Saharan Africa. Thus, there is a pressing need to develop new antimalarials with diverse mechanisms of action. One area of Plasmodium metabolism that has recently proven rich in exploitable antimalarial targets is protein synthesis, with a compound targeting elongation factor 2 now in clinical development and inhibitors of several aminoacyl-tRNA synthetases in lead optimization. Given the promise of these components of translation as viable drug targets, we rationalized that an assay containing all functional components of translation would be a valuable tool for antimalarial screening and drug discovery. Here, we report the development and validation of an assay platform that enables specific inhibitors of Plasmodium falciparum translation (PfIVT) to be identified. The primary assay in this platform monitors the translation of a luciferase reporter in a P. falciparum lysate-based expression system. Hits identified in this primary assay are assessed in a counterscreen assay that enables false positives that directly interfere with the luciferase to be triaged. The remaining hit compounds are then assessed in an equivalent human IVT assay. This platform of assays was used to screen MMV's Pandemic and Pathogen Box libraries, identifying several selective inhibitors of protein synthesis. We believe this new high-throughput screening platform has the potential to greatly expedite the discovery of antimalarials that act via this highly desirable mechanism of action.
Assuntos
Antimaláricos , Malária Falciparum , Malária , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Humanos , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/genéticaRESUMO
Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between ß4 and ß5 subunits that catalyze chymotrypsin-like activity. A mutation in the ß5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and in vitro-selected clones resistant to proteasome inhibitors known to bind at the ß4/ß5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Doença de Chagas/tratamento farmacológico , Descoberta de Drogas , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Trypanosoma cruzi/químicaRESUMO
New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the PfKRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing PfKRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with PfKRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed. IMPORTANCE: An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase (PfKRS). Screening of this high-coverage library with characterized PfKRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.
RESUMO
Here, we provide a protocol using chemical pulldown combined with mass spectrometry (LC-MS/MS) to identify drug targets in Plasmodium falciparum. This approach works upon the principle that a resin-bound inhibitor selectively binds its molecular target(s) in cell-free lysates. We describe the preparation of drug beads and P. falciparum lysate, followed by chemical pulldown, sample fractionation, and LC-MS/MS analysis. We then detail how to identify specifically bound proteins by comparing protein enrichment in DMSO-treated relative to drug-treated lysates via quantitative proteomics. For complete details on the use and execution of this protocol, please refer to Milne et al. (2022).1.
Assuntos
Antimaláricos , Cromatografia Líquida/métodos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Antimaláricos/metabolismo , Espectrometria de Massas em Tandem/métodos , Proteínas/metabolismo , Plasmodium falciparumRESUMO
Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.
Assuntos
Antimaláricos , Malária Falciparum , Humanos , Plasmodium falciparum/metabolismo , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Mutação , Ligases/metabolismoRESUMO
There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
Assuntos
Antimaláricos , Lisina-tRNA Ligase , Malária , Antimaláricos/química , Antimaláricos/farmacologia , Descoberta de Drogas , Humanos , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/genética , Lisina-tRNA Ligase/metabolismo , Plasmodium falciparum/metabolismoRESUMO
African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.
Assuntos
Trypanosoma congolense , Tripanossomíase Africana , Animais , Bovinos , Quinases Ciclina-Dependentes , Reposicionamento de Medicamentos , Trypanosoma vivax , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/veterináriaRESUMO
The synthesis and preliminary structure-activity relationships (SAR) of a novel class of vasopressin V(1B) receptor antagonists are described. Hit compound 5, identified via high throughput screening of the corporate collection, showed good activity in a V(1B) binding assay (K(i) 63 nM) but did not possess the lead-like physicochemical properties typically required in a hit compound. A 'deletion approach' on the HTS hit 5 was performed, with the focus on improvement of physicochemical properties, yielding the selective V(1B) antagonist 9f (K(i) 190 nM), with improved druglike characteristics.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
The previously described lead compound 5 is a potent and selective V(1A) antagonist with affinity at both the rat and human receptor, but displays poor oral bioavailability and moderate clearance. We report herein the successful optimisation of the pharmacokinetic (PK) properties to afford the potent, selective, orally bioavailable and CNS penetrant compound 15f. A custom optimisation approach was required which demonstrated the value of using early, rapid in vivo PK studies to show improvements in oral exposure. Such assays may be of particular value where low oral bioavailability is anticipated to be multifactorial (e.g., permeability, gut wall metabolism and/or transport) where satisfactory modelling of in vitro data is likely to be difficult within a drug discovery context.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Fenilalanina/análogos & derivados , Administração Oral , Animais , Disponibilidade Biológica , Humanos , Masculino , Peptídeos/química , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacocinética , Ligação Proteica , Ratos , Ratos Wistar , Receptores de Vasopressinas/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.
Assuntos
Acetamidas/síntese química , Antagonistas dos Receptores de Hormônios Antidiuréticos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Quinazolinonas/síntese química , Quinazolinonas/farmacologia , Acetamidas/química , Acetamidas/farmacologia , Animais , Células CACO-2 , Humanos , Concentração Inibidora 50 , Masculino , Estrutura Molecular , Quinazolinonas/química , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Antagonistas dos Receptores de Hormônios Antidiuréticos , Descoberta de Drogas , Amidas/química , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Células Cultivadas , Ciclização , Humanos , Fígado/metabolismo , Estrutura Molecular , Ligação Proteica/efeitos dos fármacosRESUMO
Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V(1b) (V(3)) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V(1b) receptor and good selectivity with respect to related receptors V(1a), V(2) and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Quinazolinas/síntese química , Quinazolinas/farmacologia , Animais , Humanos , Quinazolinas/química , Quinazolinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti- Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and -tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.
RESUMO
An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.
RESUMO
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.
Assuntos
Antiparasitários/farmacologia , Citocromos b/antagonistas & inibidores , Descoberta de Drogas , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/genética , Antiparasitários/química , Antiparasitários/isolamento & purificação , Doença de Chagas/tratamento farmacológico , Citocromos b/genética , Ensaios de Triagem em Larga Escala , Humanos , Leishmaniose Visceral/tratamento farmacológicoRESUMO
Twenty nine original 3-nitroimidazo[1,2-a]pyridine derivatives, bearing a phenylthio (or benzylthio) moiety at position 8 of the scaffold, were synthesized. In vitro evaluation highlighted compound 5 as an antiparasitic hit molecule displaying low cytotoxicity for the human HepG2 cell line (CC50 > 100 µM) alongside good antileishmanial activities (IC50 = 1-2.1 µM) against L. donovani, L. infantum, and L. major; and good antitrypanosomal activities (IC50 = 1.3-2.2 µM) against T. brucei brucei and T. cruzi, in comparison to several reference drugs such as miltefosine, fexinidazole, eflornithine, and benznidazole (IC50 = 0.6 to 13.3 µM). Molecule 5, presenting a low reduction potential (E° = -0.63 V), was shown to be selectively bioactivated by the L. donovani type 1 nitroreductase (NTR1). Importantly, molecule 5 was neither mutagenic (negative Ames test), nor genotoxic (negative comet assay), in contrast to many other nitroaromatics. Molecule 5 showed poor microsomal stability; however, its main metabolite (sulfoxide) remained both active and nonmutagenic, making 5 a good candidate for further in vivo studies.
RESUMO
White rot fungi (WRF) are applicable to biodegradation of recalcitrant pollutants. However, excessive biomass growth typical for WRF cultivation can hinder their large scale applications. Therefore, immobilization of Irpex lacteus to liquid-core alginate beads restricting excessive mycelium growth and simultaneously keeping high degradation rate of pollutants was tested. Effective diffusivities of dyes to the beads varied from (2.98 ± 0.69) × 10-10 to (10.27 ± 2.60) × 10-10 m2/s. Remazol Brilliant Blue R (RBBR), Reactive Orange 16 (RO16), and Naphthol Blue Black (NBB) were used as model dyes. The immobilized fungus decolorized model dyes when applied both in microwell plates and in fluidized bed reactors. Using the microwell plates, the apparent reaction rate constants ranged from (2.06 ± 0.11) × 10-2 to (11.06 ± 0.27) × 10-2 1/h, depending on the dye used and its initial concentration. High initial concentrations negatively affected the dye decolorization rate. No fungal growth outside the beads was observed in fluidized bed reactors and thus no operational problems linked to an excessive biomass growth occurred. When RBBR was decolorized in subsequent batches in the fluidized bed reactor, the apparent reaction rate constant increased from (11.63 ± 0.35) × 10-2 to (29.26 ± 7.19) × 10-2 1/h.