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
Our previous work identified compound 1 (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite Cryptosporidium (EC50 = 0.17 µM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (52, EC50 = 0.07 µΜ), which was found to be orally efficacious with an ED90 < 5 mg/kg BID in a Cryptosporidium-infection mouse model, superior to SLU-2633.
Asunto(s)
Criptosporidiosis , Cryptosporidium , Ratones , Animales , Criptosporidiosis/tratamiento farmacológico , Flúor , Relación Estructura-ActividadRESUMEN
Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.
Asunto(s)
Antiprotozoarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Línea Celular , Relación Dosis-Respuesta a Droga , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Relación Estructura-ActividadRESUMEN
The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last â¼10 years, with numerous lead compounds identified, including several tRNA synthetase inhibitors. Here, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (Cryptosporidium parvum MetRS [CpMetRS]) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day 5. Parasites shed by each recrudescent calf had different amino acid-altering mutations in the gene encoding CpMetRS (CpMetRS), yielding either an aspartate 243-to-glutamate (D243E) or a threonine 246-to-isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant-expressing parasites, respectively, had 2093 half-maximal effective concentrations (EC50s) that were 613- and 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.
Asunto(s)
Enfermedades de los Bovinos , Criptosporidiosis , Cryptosporidium parvum , Cryptosporidium , Animales , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Niño , Preescolar , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/genética , Cryptosporidium parvum/genética , Resistencia a Medicamentos/genética , Heces , HumanosRESUMEN
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
Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.
Asunto(s)
Anisomicina/farmacología , Antiparasitarios/farmacología , Reposicionamiento de Medicamentos , Parásitos/efectos de los fármacos , Prodigiosina/farmacología , Pirroles/farmacología , Animales , Anisomicina/efectos adversos , Anisomicina/farmacocinética , Antiparasitarios/efectos adversos , Antiparasitarios/farmacocinética , Línea Celular , Supervivencia Celular , Fibroblastos/efectos de los fármacos , Humanos , Indoles , Ratones , Pruebas de Sensibilidad Parasitaria , Prodigiosina/efectos adversos , Prodigiosina/farmacocinética , Pirroles/efectos adversos , Pirroles/farmacocinética , RatasRESUMEN
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 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
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
Cryptosporidium species cause significant morbidity in malnourished children. Nitazoxanide (NTZ) is the only approved treatment for cryptosporidiosis, but NTZ has diminished effectiveness during malnutrition. Here, we show that amixicile, a highly selective water-soluble derivative of NTZ diminishes Cryptosporidium infection severity in a malnourished mouse model despite a lack of direct anticryptosporidial activity. We suggest that amixicile, by tamping down anaerobes associated with intestinal inflammation, reverses weight loss and indirectly mitigates infection-associated pathology.
Asunto(s)
Benzamidas/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/efectos de los fármacos , Tiazoles/farmacología , Animales , Antiprotozoarios/farmacología , Criptosporidiosis/etiología , Cryptosporidium parvum/patogenicidad , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Nitrocompuestos , Piruvato-Sintasa/antagonistas & inhibidores , Piruvato-Sintasa/metabolismo , Pérdida de Peso/efectos de los fármacosRESUMEN
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.