RESUMEN
There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (ß1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos/métodos , Células Epiteliales/virología , Humanos , ARN Viral , SARS-CoV-2/aislamiento & purificación , Células VeroRESUMEN
The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos/métodos , Pandemias , SARS-CoV-2 , Animales , COVID-19/prevención & control , COVID-19/virología , Línea Celular , Citidina/administración & dosificación , Citidina/análogos & derivados , Citidina/farmacología , Bases de Datos Farmacéuticas , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Células HeLa , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Hidroxilaminas/administración & dosificación , Hidroxilaminas/farmacología , Mesocricetus , Nelfinavir/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Daño del ADN , Isoxazoles/farmacología , Pirazinas/farmacología , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacos , Células A549 , Animales , COVID-19/metabolismo , COVID-19/patología , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Células HEK293 , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Células VeroRESUMEN
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.
Asunto(s)
Antivirales/análisis , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , COVID-19 , Línea Celular , Inhibidores de Cisteína Proteinasa/análisis , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Hidrazonas , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Morfolinas/análisis , Morfolinas/farmacología , Pandemias , Pirimidinas , Reproducibilidad de los Resultados , SARS-CoV-2 , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Triazinas/análisis , Triazinas/farmacología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections.
Asunto(s)
Antivirales/farmacología , Arenaviridae/efectos de los fármacos , Bases de Datos Farmacéuticas , Evaluación Preclínica de Medicamentos/métodos , Reposicionamiento de Medicamentos/métodos , Células A549 , Animales , Apoptosis , Arenaviridae/fisiología , Infecciones por Arenaviridae/tratamiento farmacológico , Infecciones por Arenaviridae/inmunología , Infecciones por Arenaviridae/virología , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Complejo III de Transporte de Electrones/metabolismo , Células HEK293 , Humanos , Interferones/genética , Virus Junin/efectos de los fármacos , Virus Lassa/efectos de los fármacos , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Purinas/biosíntesis , Pirimidinas/biosíntesis , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (â¼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.
Asunto(s)
Antiprotozoarios/farmacología , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium/efectos de los fármacos , Bases de Datos Farmacéuticas , Descubrimiento de Drogas , Reposicionamiento de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Criptosporidiosis/parasitología , Evaluación Preclínica de Medicamentos/métodos , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The typeâ II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90â nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.
Asunto(s)
Antibacterianos/farmacología , Indazoles/farmacología , Mycobacterium tuberculosis/enzimología , NADH Deshidrogenasa/antagonistas & inhibidores , Quinazolinas/farmacología , Evaluación Preclínica de Medicamentos , Viabilidad Microbiana/efectos de los fármacosRESUMEN
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
A variable region fusion strategy was used to generate an immunosuppressive antibody based on a novel "stalk-knob" structural motif in the ultralong complementary-determining region (CDR) of a bovine antibody. The potent Kv1.3 channel inhibitory peptides Moka1-toxin and Vm24-toxin were grafted into different CDRs of the humanized antibodies BVK and Synagis (Syn) using both ß-sheet and coiled-coil linkers. Structure-activity relationship efforts led to generation of the fusion protein Syn-Vm24-CDR3L, which demonstrated excellent selectivity and potency against effector human memory T cells (subnanomolar to picomolar EC50 values). This fusion antibody also had significantly improved plasma half-life and serum stability in rodents compared with the parent Vm24 peptide. Finally, this fusion protein showed potent in vivo efficacy in the delayed type hypersensitivity in rats. These results illustrate the utility of antibody CDR fusions as a general and effective strategy to generate long-acting functional antibodies, and may lead to a selective immunosuppressive antibody for the treatment of autoimmune diseases.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Diseño de Fármacos , Inmunosupresores/farmacología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células CHO , Bovinos , Regiones Determinantes de Complementariedad/química , Cricetinae , Cricetulus , Células HEK293 , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Línea Celular , Enfermedad de Chagas/tratamiento farmacológico , Colorimetría/métodos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Enfermedades Desatendidas/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas , Tripanocidas/química , Xantina/química , Xantina/farmacologíaRESUMEN
Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.
Asunto(s)
Antimaláricos/farmacología , Imidazoles/farmacología , Malaria/tratamiento farmacológico , Plasmodium cynomolgi/efectos de los fármacos , Pirazinas/farmacología , Animales , Antimaláricos/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Femenino , Hepatocitos/parasitología , Imidazoles/uso terapéutico , Técnicas In Vitro , Hígado/parasitología , Macaca mulatta/parasitología , Malaria/parasitología , Malaria/prevención & control , Ratones , Ratones Endogámicos ICR , Pirazinas/uso terapéutico , Esporozoítos/efectos de los fármacosRESUMEN
New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.
Asunto(s)
Antituberculosos/farmacología , Indoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Administración Oral , Animales , Antituberculosos/administración & dosificación , Antituberculosos/farmacocinética , Antituberculosos/toxicidad , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Disponibilidad Biológica , Modelos Animales de Enfermedad , Perros , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Indoles/administración & dosificación , Indoles/farmacocinética , Indoles/toxicidad , Inyecciones Intravenosas , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Ratas , Ratas Wistar , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Malaria elimination has recently been reinstated as a global health priority but current therapies seem to be insufficient for the task. Elimination efforts require new drug classes that alleviate symptoms, prevent transmission and provide a radical cure. To develop these next-generation medicines, public-private partnerships are funding innovative approaches to identify compounds that target multiple parasite species at multiple stages of the parasite life cycle. In this Review, we discuss the cell-, chemistry- and target-based approaches used to discover new drug candidates that are currently in clinical trials or undergoing preclinical testing.
Asunto(s)
Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , HumanosRESUMEN
A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Efedrina/análisis , Alcaloides Indólicos/análisis , Malvaceae/química , Extractos Vegetales/química , Quinolinas/análisis , Efedrina/química , Alcaloides Indólicos/química , Análisis de los Mínimos Cuadrados , Quinolinas/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.
Asunto(s)
Antimaláricos/farmacología , Descubrimiento de Drogas , Imidazoles/farmacología , Hígado/parasitología , Malaria/tratamiento farmacológico , Piperazinas/farmacología , Plasmodium/efectos de los fármacos , Animales , Antimaláricos/química , Antimaláricos/farmacocinética , Antimaláricos/uso terapéutico , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , Eritrocitos/parasitología , Humanos , Imidazoles/química , Imidazoles/farmacocinética , Imidazoles/uso terapéutico , Malaria/parasitología , Malaria/prevención & control , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Plasmodium/citología , Plasmodium/crecimiento & desarrollo , Plasmodium/fisiología , Plasmodium berghei/citología , Plasmodium berghei/efectos de los fármacos , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/fisiología , Plasmodium falciparum/citología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/fisiología , Plasmodium yoelii/citología , Plasmodium yoelii/efectos de los fármacos , Plasmodium yoelii/crecimiento & desarrollo , Plasmodium yoelii/fisiología , Polimorfismo de Nucleótido Simple , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Distribución Aleatoria , Bibliotecas de Moléculas Pequeñas , Esporozoítos/efectos de los fármacos , Esporozoítos/crecimiento & desarrolloRESUMEN
The efficacy of most marketed antimalarial drugs has been compromised by evolution of parasite resistance, underscoring an urgent need to find new drugs with new mechanisms of action. We have taken a high-throughput approach toward identifying novel antimalarial chemical inhibitors of prioritized drug targets for Plasmodium falciparum, excluding targets which are inhibited by currently used drugs. A screen of commercially available libraries identified 5655 low molecular weight compounds that inhibit growth of P. falciparum cultures with EC(50) values below 1.25µM. These compounds were then tested in 384- or 1536-well biochemical assays for activity against nine Plasmodium enzymes: adenylosuccinate synthetase (AdSS), choline kinase (CK), deoxyuridine triphosphate nucleotidohydrolase (dUTPase), glutamate dehydrogenase (GDH), guanylate kinase (GK), N-myristoyltransferase (NMT), orotidine 5'-monophosphate decarboxylase (OMPDC), farnesyl pyrophosphate synthase (FPPS) and S-adenosylhomocysteine hydrolase (SAHH). These enzymes were selected using TDRtargets.org, and are believed to have excellent potential as drug targets based on criteria such as their likely essentiality, druggability, and amenability to high-throughput biochemical screening. Six of these targets were inhibited by one or more of the antimalarial scaffolds and may have potential use in drug development, further target validation studies and exploration of P. falciparum biochemistry and biology.
Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/aislamiento & purificación , Inhibidores Enzimáticos/aislamiento & purificación , Enzimas/metabolismo , Concentración 50 Inhibidora , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/antagonistas & inhibidoresRESUMEN
The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.
Asunto(s)
Antimaláricos/análisis , Antimaláricos/farmacología , Biología Computacional , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Análisis por Conglomerados , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos/efectos de los fármacos , Antagonistas del Ácido Fólico/análisis , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Malaria/tratamiento farmacológico , Modelos Moleculares , Parásitos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Reproducibilidad de los Resultados , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.