RESUMEN
The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Estadios del Ciclo de Vida , Proteínas Protozoarias , Criptosporidiosis/parasitología , Criptosporidiosis/tratamiento farmacológico , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Estadios del Ciclo de Vida/efectos de los fármacos , Cryptosporidium/efectos de los fármacos , Cryptosporidium/genética , Cryptosporidium/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Humanos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Asunto(s)
Criptosporidiosis , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Ratones SCID , Proteínas Protozoarias/metabolismoRESUMEN
Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.
Asunto(s)
Antiprotozoarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Imidazoles/farmacología , Metionina-ARNt Ligasa/antagonistas & inhibidores , Piridinas/farmacología , Animales , Cryptosporidium parvum/genética , Ciclooxigenasa 1/efectos de los fármacos , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Femenino , Células Hep G2 , Humanos , Imidazoles/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Piridinas/químicaRESUMEN
FIKKs are parasite-specific protein kinases with distinctive sequence motifs and their biological roles have not been completely elucidated. Here, we report the first potent Cryptosporidium FIKK (CpFIKK) inhibitor. We identified 4b as a potent (IC50=0.2nM) inhibitor of CpFIKK catalytic activity. In addition, we identified both CpCDPK1 selective as well as dually acting CpFIKK-CDPK1 inhibitors from the same structural class of compounds. We evaluated these CpFIKK inhibitors for inhibition of parasite growth in vitro. The observed effects on parasite growth did not correlate with CpFIKK inhibition, suggesting that CpFIKK may not be involved in parasite growth.
Asunto(s)
Cryptosporidium/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/química , Secuencia de Aminoácidos , Cryptosporidium/crecimiento & desarrollo , Descubrimiento de Drogas , Humanos , Homología de Secuencia de Aminoácido , Análisis Espectral/métodos , Relación Estructura-ActividadRESUMEN
We previously reported interferon gamma secretion by human CD4⺠and CD8⺠T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-ß unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.
Asunto(s)
Inmunidad Adaptativa , Vacuna BCG/efectos adversos , Proteínas Bacterianas/efectos adversos , Células Dendríticas/inmunología , Glicoproteínas/efectos adversos , Memoria Inmunológica , Mycobacterium tuberculosis/inmunología , Animales , Vacuna BCG/antagonistas & inhibidores , Vacuna BCG/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/uso terapéutico , Polaridad Celular , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Femenino , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicoproteínas/uso terapéutico , Glicosilación , Mediadores de Inflamación/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Ratones Endogámicos BALB C , Mycobacterium bovis/inmunología , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Bazo/inmunología , Bazo/metabolismo , Bazo/microbiología , Células TH1/inmunología , Células TH1/metabolismo , Células TH1/microbiología , Células Th17/inmunología , Células Th17/metabolismo , Células Th17/microbiología , Tuberculosis/inmunología , Tuberculosis/metabolismo , Tuberculosis/microbiología , Tuberculosis/prevención & controlRESUMEN
The apicomplexan parasites Cryptosporidium parvum and Cryptosporidium hominis are major etiologic agents of human cryptosporidiosis. The infection is typically self-limited in immunocompetent adults, but it can cause chronic fulminant diarrhea in immunocompromised patients and malnutrition and stunting in children. Nitazoxanide, the current standard of care for cryptosporidiosis, is only partially efficacious for children and is no more effective than a placebo for AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology impede the development of better drugs for treating cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture (MMV) Open Access Malaria Box for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited submicromolar potency against C. parvum. Potency was conserved in a subset of compounds from each scaffold with varied physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of the compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Cryptosporidium parvum/efectos de los fármacos , Reposicionamiento de Medicamentos/métodos , Humanos , Hidroxiquinolinas/química , Nitrocompuestos , Quinazolinas/química , Tiazoles/farmacología , Toxoplasma/efectos de los fármacosRESUMEN
Diseases caused by trypanosomatid parasites remain a significant unmet medical need for millions of people globally. Trypanosomatid parasites such as Trypanosoma cruzi and subspecies of Trypanosoma brucei cause Chagas disease and human African trypanosomiasis (HAT), respectively. Although efforts to find novel treatments have been successful for HAT, Chagas disease is still treated with decades-old therapies that suffer from long treatment durations and severe safety concerns. We recently described the identification and characterization of the cyanotriazole compound class that kills trypanosomes, in vitro and in vivo, by selective inhibition of the trypanosome nuclear topoisomerase II enzyme. To evaluate whether inhibition of the topoisomerase II enzyme led to parasite death due to lethal double-strand DNA breaks, we developed assays for detecting DNA damage in both intracellular amastigotes of T. cruzi and bloodstream-form T. brucei by using the canonical DNA damage marker γH2A. Herein, this article describes the protocols for detecting DNA damage using an immunofluorescence assessment of γH2A by microscopy in trypanosome parasites. Key features ⢠Immunofluorescence-based assay to detect the γH2A response in T. brucei and T. cruzi parasites. ⢠Robust DNA damage pathway-based cellular assays to evaluate topoisomerase II poisons' ability to cause DNA damage. ⢠A 384-well plate-based T. cruzi protocol allows high-resolution and high-throughput evaluation of compounds that cause DNA damage by measuring γH2A in intracellular parasites. ⢠This assay could be modifiable for evaluation of DNA damage responses in various intracellular and extracellular eukaryotic pathogens.
RESUMEN
Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.
Asunto(s)
Enfermedad de Chagas , Inhibidores de Topoisomerasa II , Triazoles , Trypanosoma , Tripanosomiasis Africana , Animales , Humanos , Ratones , Enfermedad de Chagas/tratamiento farmacológico , Microscopía por Crioelectrón , ADN-Topoisomerasas de Tipo II/metabolismo , Trypanosoma/efectos de los fármacos , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Triazoles/química , Triazoles/farmacología , Triazoles/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Evaluación Preclínica de MedicamentosRESUMEN
Cryptosporidiosis is a leading cause of moderate-to-severe diarrhea in low- and middle-income countries, responsible for high mortality in children younger than two years of age, and it is also strongly associated with childhood malnutrition and growth stunting. There is no vaccine for cryptosporidiosis and existing therapeutic options are suboptimal to prevent morbidity and mortality in young children. Recently, novel therapeutic agents have been discovered through high-throughput phenotypic and target-based screening strategies, repurposing malaria hits, etc., and these agents have a promising preclinical in vitro and in vivo anti-Cryptosporidium efficacy. One key step in bringing safe and effective new therapies to young vulnerable children is the establishment of some prospect of direct benefit before initiating pediatric clinical studies. A Cryptosporidium controlled human infection model (CHIM) in healthy adult volunteers can be a robust clinical proof of concept model for evaluating novel therapeutics. CHIM could potentially accelerate the development path to pediatric studies by establishing the safety of a proposed pediatric dosing regimen and documenting preliminary efficacy in adults. We present, here, perspectives regarding the opportunities and perceived challenges with the Cryptosporidium human challenge model.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Malaria , Adulto , Antiparasitarios/farmacología , Niño , Preescolar , Criptosporidiosis/tratamiento farmacológico , Diarrea/tratamiento farmacológico , HumanosRESUMEN
Diarrhea has long been recognized as an important cause of mortality during childhood. In parallel with ensuring access to proven care practices is the imperative to apply modern advances in medicine, science, and technology to accelerate progress against diarrheal disease, particularly in developing countries where the burden of avoidable harm is the greatest. In order to highlight achievements and identify outstanding areas of need, we reviewed the landscape of recent innovations that have significance for the study and clinical management of pediatric diarrhea in low resource settings.
Asunto(s)
Países en Desarrollo/estadística & datos numéricos , Diarrea/epidemiología , Diarrea/prevención & control , Recursos en Salud/provisión & distribución , Infecciones Bacterianas/prevención & control , Niño , Control de Enfermedades Transmisibles , Países en Desarrollo/economía , Diarrea/mortalidad , Recursos en Salud/estadística & datos numéricos , Humanos , Enfermedades Parasitarias/prevención & control , Salud Pública/métodos , Vacunas , Virosis/prevención & controlRESUMEN
Cryptosporidium is a protozoan parasite and a leading cause of diarrheal disease and mortality in young children. Currently, there are no fully effective treatments available to cure infection with this diarrheal pathogen. In this study, we report a broad drug repositioning effort that led to the identification of bicyclic azetidines as a new anticryptosporidial series. Members of this series blocked growth in in vitro culture of three Cryptosporidium parvum isolates with EC50 's in 1% serum of <0.4 to 96 nM, had comparable potencies against Cryptosporidium hominis and C. parvum, and was effective in three of four highly susceptible immunosuppressed mice with once-daily dosing administered for 4 days beginning 2 weeks after infection. Comprehensive genetic, biochemical, and chemical studies demonstrated inhibition of C. parvum phenylalanyl-tRNA synthetase (CpPheRS) as the mode of action of this new lead series. Introduction of mutations directly into the C. parvum pheRS gene by CRISPR-Cas9 genome editing resulted in parasites showing high degrees of compound resistance. In vitro, bicyclic azetidines potently inhibited the aminoacylation activity of recombinant ChPheRS. Medicinal chemistry optimization led to the identification of an optimal pharmacokinetic/pharmacodynamic profile for this series. Collectively, these data demonstrate that bicyclic azetidines are a promising series for anticryptosporidial drug development and establish a broad framework to enable target-based drug discovery for this infectious disease.
Asunto(s)
Azetidinas , Criptosporidiosis , Cryptosporidium , Parásitos , Fenilalanina-ARNt Ligasa , Animales , Azetidinas/farmacología , Criptosporidiosis/tratamiento farmacológico , Diarrea , RatonesRESUMEN
Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.
Asunto(s)
Antiparasitarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/efectos de los fármacos , Diarrea/tratamiento farmacológico , Inhibidores de Crecimiento/farmacología , Algoritmos , Animales , Antiparasitarios/uso terapéutico , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Análisis por Conglomerados , Criptosporidiosis/parasitología , Cryptosporidium/crecimiento & desarrollo , Diarrea/parasitología , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Inhibidores de Crecimiento/uso terapéutico , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , FenotipoRESUMEN
Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.
Asunto(s)
Amidas/administración & dosificación , Antiprotozoarios/administración & dosificación , Compuestos de Boro/administración & dosificación , Criptosporidiosis/tratamiento farmacológico , Isoxazoles/administración & dosificación , Amidas/efectos adversos , Amidas/química , Animales , Antiprotozoarios/efectos adversos , Antiprotozoarios/química , Compuestos de Boro/efectos adversos , Compuestos de Boro/química , Criptosporidiosis/parasitología , Cryptosporidium/efectos de los fármacos , Cryptosporidium/crecimiento & desarrollo , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Isoxazoles/efectos adversos , Isoxazoles/química , Masculino , Ratones , RatasRESUMEN
Cryptosporidiosis causes life-threatening diarrhea in infants, but the best available treatment is only modestly efficacious. Rodents infected with relevant Cryptosporidium species do not develop diarrhea, which complicates drug development. Cryptosporidium parvum infection of dairy calves, however, causes an illness like that seen in infants. Here, the clinical and microbiologic anti-Cryptosporidium efficacy of the piperazine-based compound MMV665917 was demonstrated in neonatal calves. Oral administration of MMV665917 (22 mg/kg once daily) was begun two days after the onset of severe diarrhea and continued for seven days. Treatment resulted in prompt resolution of diarrhea, and reduced total fecal oocyst shedding by ~94%. MMV665917 was useful for treatment, rather than just prophylaxis, since it was safe and effective when administered well after the onset of diarrhea. Furthermore, even though all animals received intensive supportive care, there was a strong trend towards improved secondary health outcomes, including general health, appetite, and dehydration measures amongst treated animals. These data establish MMV665917 as an outstanding lead compound for Cryptosporidium drug development.
Asunto(s)
Antiprotozoarios/administración & dosificación , Antiprotozoarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/efectos de los fármacos , Piperazinas/administración & dosificación , Piperazinas/farmacología , Administración Oral , Animales , Animales Recién Nacidos , Antinematodos , Bovinos , Criptosporidiosis/parasitología , Diarrea/tratamiento farmacológico , Diarrea/parasitología , Modelos Animales de Enfermedad , Heces/parasitología , Carga de Parásitos , PiperazinaRESUMEN
Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.
Asunto(s)
Antiprotozoarios/farmacología , Clofazimina/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Reposicionamiento de Medicamentos , Animales , Automatización de Laboratorios , Línea Celular , Criptosporidiosis/parasitología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células Epiteliales/parasitología , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Resultado del TratamientoRESUMEN
Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Inhibidores Enzimáticos/administración & dosificación , Infecciones por Protozoos/tratamiento farmacológico , Quinazolinonas/administración & dosificación , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Animales , Dominio Catalítico/efectos de los fármacos , Coccidiosis/tratamiento farmacológico , Criptosporidiosis/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Leishmaniasis/tratamiento farmacológico , Malaria/tratamiento farmacológico , Ratones , Modelos Moleculares , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Quinazolinonas/química , Quinazolinonas/farmacología , Relación Estructura-Actividad , Toxoplasmosis/tratamiento farmacológicoRESUMEN
We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+)and CD8(+)T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.