RESUMEN
Since the start of the COVID-19 outbreak, pharmaceutical companies and research groups have focused on the development of vaccines and antiviral drugs against SARS-CoV-2. Here, we apply a drug repurposing strategy to identify drug candidates that are able to block the entrance of the virus into human cells. By combining virtual screening with in vitro pseudovirus assays and antiviral assays in Human Lung Tissue (HLT) cells, we identify entrectinib as a potential antiviral drug.
Asunto(s)
Benzamidas/farmacología , Tratamiento Farmacológico de COVID-19 , Indazoles/farmacología , SARS-CoV-2/efectos de los fármacos , Animales , Antivirales/farmacología , Benzamidas/metabolismo , COVID-19/metabolismo , Línea Celular , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos/métodos , Humanos , Indazoles/metabolismo , Pulmón/patología , Pulmón/virología , Simulación del Acoplamiento Molecular , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Células Vero , Acoplamiento Viral/efectos de los fármacosRESUMEN
Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker γ-H2AX after DHODH inhibition is preventable by cotreatment with the pan-caspase inhibitor Z-VAD-FMK. Additional solubility and in vitro metabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Indazoles/química , Indazoles/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Dihidroorotato Deshidrogenasa , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indazoles/farmacología , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismoRESUMEN
In this study, we report the identification of potent pyrimidoindazoles as phosphodiesterase10A (PDE10A) inhibitors by using the method of fragment-based drug discovery (FBDD). The pyrazolopyridine derivative 2 was found to be a fragment hit compound which could occupy a part of the binding site of PDE10A enzyme by using the method of the X-ray co-crystal structure analysis. On the basis of the crystal structure of compound 2 and PDE10A protein, a number of compounds were synthesized and evaluated, by means of structure-activity relationship (SAR) studies, which culminated in the discovery of a novel pyrimidoindazole derivative 13 having good physicochemical properties.
Asunto(s)
Indazoles/química , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Indazoles/metabolismo , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Inhibidores de Fosfodiesterasa/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Relación Estructura-ActividadRESUMEN
Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.
Asunto(s)
Antineoplásicos/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Inhibidores Enzimáticos/metabolismo , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Fosfatidilinositol 3-Quinasa Clase I , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Medición de Intercambio de Deuterio , Evaluación Preclínica de Medicamentos/métodos , Transporte de Electrón , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Indazoles/química , Indazoles/metabolismo , Indazoles/farmacología , Peso Molecular , Oligonucleótidos/antagonistas & inhibidores , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Conformación Proteica , Purinas/química , Purinas/metabolismo , Purinas/farmacología , Piridazinas , Quinazolinonas/química , Quinazolinonas/metabolismo , Quinazolinonas/farmacología , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Procesamiento de Señales Asistido por Computador , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Espectrometría de Masas en Tándem , Triazinas/química , Triazinas/metabolismo , Triazinas/farmacologíaRESUMEN
NEW FINDINGS: What is the central question of this study? Our previous study demonstrates that elevated orexin 2 receptor (OX2R) activity within the rostral ventrolateral medulla (RVLM) contributes to hypertension in spontaneously hypertensive rats (SHRs), and a lower OX2R protein level was detected in their RVLM. The present study aims to explore the mechanisms underlying elevated orexinergic activity in the RVLM of SHRs, compared with their normotensive counterparts, Wistar-Kyoto rats. What is the main finding and its importance? Increased orexinergic input into the RVLM and enhanced OX2R responsiveness in the RVLM, which was mainly mediated by augmented OX2R-neuronal nitric oxide synthase signalling, may underlie the elevated OX2R activity within the RVLM of SHRs. Our previous study showed that elevated orexin 2 receptor (OX2R) activity within the rostral ventrolateral medulla (RVLM) contributes to hypertension in spontaneously hypertensive rats (SHRs). Herein, we investigated the mechanism(s) underlying the elevated OX2R activity. The following results were found. (i) More hypothalamic orexin A-immunoreactive (OXA-IR) cells existed in SHRs than in Wistar-Kyoto (WKY) rats at either 4 (2217 ± 43 versus 1809 ± 69) or 16 weeks of age (1829 ± 59 versus 1230 ± 84). The number of OXA-IR cells that project to the RVLM was higher in 16-week-old SHRs than in WKY rats (91 ± 11 versus 52 ± 11). (ii) Higher numbers of OXA-IR and RVLM-projecting OXA-IR cells were found in the dorsomedial and perifornical hypothalamus of 16-week-old SHRs. (iii) Spontaneously hypertensive rats had higher levels of orexin A and B in the hypothalamus and higher levels of orexin A in the RVLM than did WKY rats. (iv) Unilateral intra-RVLM application of OX2R agonist, orexin A or [Ala(11), d-Leu(15)]-orexin B (50 pmol) induced a larger pressor response in SHRs than in WKY rats. (v) Intra-RVLM pretreatment with a neuronal nitric oxide synthase (NOS) inhibitor, 7-nitro-indazole (2.5 pmol), or a soluble guanylate cyclase inhibitor, methylene blue (250 pmol), reduced the intra-RVLM [Ala(11), d-Leu(15) ]-orexin B-induced pressor response in both WKY rats and SHRs. In contrast, an inducible NOS inhibitor, aminoguanidine (100 pmol), was ineffective. (vi) Neuronal NOS was co-expressed with OX2R in RVLM neurons. In conclusion, increased orexinergic input and enhanced OX2R-neuronal NOS signalling may underlie elevated OX2R activity in the RVLM and contribute to the pathophysiology of hypertension in SHRs.
Asunto(s)
Hipertensión/metabolismo , Bulbo Raquídeo/metabolismo , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Animales , Presión Sanguínea/fisiología , Frecuencia Cardíaca/fisiología , Hipotálamo/metabolismo , Indazoles/metabolismo , Masculino , Neuropéptidos/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
A series of tetrahydroindazole derivatives were synthesized and evaluated for their affinities for both sigma-1 and sigma-2 receptors. These compounds are hybrid structures of a tetrahydroindazole substituted benzamide and a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety or a 9-azabicyclo[3.3.1]nonan-3-yl-amine moiety. These newly synthesized hybrid analogs showed various affinities for sigma-2 receptor without any significant affinities for sigma-1 receptor. In particular, compounds 12, 15b, 15c, and 15d, demonstrated moderate affinity and excellent selectivity for sigma-2 receptor. It is interesting to note that 3-5 carbon units between the tetrahydroindazole substituted benzamide and the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety are appropriate for sigma-2 receptor binding. No substitution on the 9-aza nitrogen is proper for sigma-2 affinity in the 2-(9-azabicyclo[3.3.1]nonan-3-yl-amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide analogs.
Asunto(s)
Indazoles/síntesis química , Indazoles/metabolismo , Receptores sigma/metabolismo , Animales , Encéfalo/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Cobayas , Ligandos , Hígado/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).
Asunto(s)
Sistema Nervioso Central/metabolismo , Descubrimiento de Drogas , Indazoles/metabolismo , Indazoles/farmacología , Piperidinas/metabolismo , Piperidinas/farmacología , Receptor Muscarínico M5/química , Receptor Muscarínico M5/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Indazoles/química , Indazoles/farmacocinética , Masculino , Piperidinas/química , Piperidinas/farmacocinética , Ratas , Especificidad por Sustrato , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
OBJECTIVE: Chemokines play a fundamental role in trafficking and activation of leukocytes in colonic inflammation. We investigated the ability of bindarit, an inhibitor of monocyte chemoattractant protein-1 (MCP-1/CCL2) synthesis, to inhibit chemokine production by human intestinal epithelial cells (HT-29) and its effect in trinitro-benzene sulfonic acid (TNBS)-induced colitis in mice. MATERIALS AND METHODS: HT-29 cells were incubated with bindarit in the presence of TNF-alpha/IFN-gamma and 24 h later supernatants were collected for MCP-1, IL-8 and RANTES measurement. A 1 mg enema of TNBS was given to BALB/c mice, and bindarit (100 mg/kg) was orally administered twice daily starting from two days before colitis induction. Weight loss, histology, and MCP-1 level and myeloperoxidase (MPO) activity in colon extracts were assessed. RESULTS: In HT-29 cells, bindarit concentration-dependently and selectively inhibited MCP-1 secretion (as well as mRNA expression) primed by TNF-alpha/IFN-gamma. Moreover treatment with bindarit reduced clinical and histopathological severity of TNBS-induced colitis. These effects were associated with significant inhibition of MCP-1 and MPO in colon extracts. CONCLUSIONS: Bindarit exhibits a potent bioactivity in reducing leukocyte infiltration, down-regulating MCP-1 synthesis, and preventing the development of severe colitis in a mice model of TNBS-induced colitis. These observations suggest a potential use of MCP-1 synthesis blockers in intestinal inflammation in humans.
Asunto(s)
Quimiocina CCL2/antagonistas & inhibidores , Colitis/inducido químicamente , Colitis/prevención & control , Indazoles , Propionatos , Ácido Trinitrobencenosulfónico/farmacología , Animales , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Colitis/tratamiento farmacológico , Colitis/patología , Progresión de la Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Indazoles/metabolismo , Indazoles/uso terapéutico , Interferón gamma/inmunología , Ratones , Ratones Endogámicos BALB C , Propionatos/metabolismo , Propionatos/uso terapéutico , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
A novel class of potent and selective inhibitors of KDR incorporating an indazole moiety 1 is reported. The discovery, synthesis, and structure-activity relationships of this series of inhibitors have been investigated. The most promising compounds were also profiled to determine their pharmacokinetic properties and evaluated in a VEGF-induced vascular permeability assay.
Asunto(s)
Indazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Indazoles/síntesis química , Indazoles/química , Indazoles/metabolismo , Masculino , Ratones , Ratones Desnudos , Unión Proteica/fisiología , Inhibidores de Proteínas Quinasas/clasificación , Ratas , Ratas Sprague-Dawley , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Prolactin (PRL) and vasoactive intestinal polypeptide (VIP) mRNA levels were elevated in the brainstem of neuronal nitric oxide synthase (nNOS) gene knockout (KO) mice compared to the levels in nNOS control mice. In addition, PRL mRNA levels increased in the hypothalamus and the brainstem of nNOS control mice after administration of 7-nitro-indazole (7-NI), a relatively selective nNOS inhibitor. The results suggest that NO inhibits PRL. No differences in the genes measured were observed in inducible NOS KO mice.