RESUMEN
Dual-targeting anticancer agents 4-29 are designed by combining the structural features of purine-type microtubule-disrupting compounds and HDAC inhibitors. A library of the conjugate compounds connected by appropriate linkers was synthesized and found to possess HDACs inhibitory activity and render microtubule fragmentation by activating katanin, a microtubule-severing protein. Among various zinc-binding groups, hydroxamic acid shows the highest inhibitory activity of Class I HDACs, which was also reconfirmed by three-dimensional quantitative structure-activity relationship (3D-QSAR) pharmacophore prediction. The purine-hydroxamate conjugates exhibit enhanced cytotoxicity against MDA-MB231 breast cancer cells, H1975 lung cancer cells, and various clinical isolated non-small-cell lung cancer cells with different epidermal growth factor receptor (EGFR) status. Pyridyl substituents could be used to replace the C2 and N9 phenyl moieties in the purine-type scaffold, which can help to improve the solubility under physiological conditions, thus increasing cytotoxicity. In mice treated with the purine-hydroxamate conjugates, the tumor growth rate was significantly reduced without causing toxic effects. Our study demonstrates the potential of the dual-targeting purine-hydroxamate compounds for cancer monotherapy.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Ratones , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Línea Celular Tumoral , Histona Desacetilasas/metabolismo , Antineoplásicos/química , Inhibidores de Histona Desacetilasas/química , Microtúbulos/metabolismo , Purinas/farmacología , Ácidos Hidroxámicos/química , Relación Estructura-Actividad , Proliferación CelularRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43. METHODS: By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43. RESULTS: Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system. CONCLUSIONS: Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Enfermedades Neurodegenerativas/genética , Proteolisis , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , Animales Modificados GenéticamenteRESUMEN
Peramivir is an efficacious neuraminidase (NA) inhibitor for treatment of influenza by intravenous administration. However, the efficacy of peramivir toward the H275Y mutant is appreciably reduced. To address this drawback, conjugation of peramivir with caffeic acid is devised in this study to enhance the binding affinity with neuraminidases. The C2-OH group of peramivir is elaborated to link with caffeate derivatives, giving the desired conjugates 8 and 9 that possess potent NA inhibitory activity against both wild-type and H275Y viruses with the IC50 values in nanomolar range. The molecular modeling reveals that the caffeate moiety of conjugate 9 prefers to reside in the 295-cavity of H275Y neuraminidase, thus providing additional hydrogen bonds and hydrophobic interactions to compensate the reduced binding affinity of the peramivir moiety due to Glu-276 dislocation in H275Y mutant. In comparison with peramivir, the lipophilicity of conjugates 8 and 9 also increases by incorporation of the caffeate moiety. Thus, conjugates 8 and 9 offer better effect to protect MDCK cells from infection of H275Y virus with low EC50 value (â¼17â¯nM). Administration of conjugates 8 or 9 by oral gavage is effective in treatment of mice that are infected by lethal dose of wild-type or H275Y influenza viruses. Considering drug metabolism, since the ester linkage in conjugate 8 is susceptible to hydrolysis in plasma, conjugate 9 with robust amide linkage may be a better candidate for development into orally available anti-influenza drug that is also active to mutant viruses.
Asunto(s)
Antivirales/farmacología , Ciclopentanos/farmacología , Guanidinas/farmacología , Virus de la Influenza A/efectos de los fármacos , Ácidos Carbocíclicos , Administración Oral , Animales , Antivirales/administración & dosificación , Antivirales/química , Ciclopentanos/administración & dosificación , Ciclopentanos/química , Perros , Relación Dosis-Respuesta a Droga , Guanidinas/administración & dosificación , Guanidinas/química , Células HEK293 , Humanos , Virus de la Influenza A/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mutación , Conejos , Relación Estructura-ActividadRESUMEN
Peramivir is a potent neuraminidase (NA) inhibitor for treatment of influenza infection by intravenous administration. By replacing the carboxylate group in peramivir with a phosphonate group, phosphono-peramivir (6a), the dehydration and deoxy derivatives (7a and 8a) as well as their corresponding monoalkyl esters are prepared from a pivotal intermediate epoxide 12. Among these phosphonate compounds, the dehydration derivative 7a that has a relatively rigid cyclopentene core structure exhibits the strongest inhibitory activity (IC50 = 0.3-4.1 nM) against several NAs of wild-type human and avian influenza viruses (H1N1, H3N2, H5N1, and H7N9), although the phosphonate congener 6a is unexpectedly less active than peramivir. The inferior binding affinity of 6a is attributable to the deviated orientations of its phosphonic acid and 3-pentyl groups in the NA active site as inferred from the NMR, X-ray diffraction, and molecular modeling analyses. Compound 7a is active to the oseltamivir-resistant H275Y strains of H1N1 and H5N1 viruses (IC50 = 73-86 nM). The phosphonate monoalkyl esters (6b, 6c, 7b, 7c, 8b, and 8c) are better anti-influenza agents (EC50 = 19-89 nM) than their corresponding phosphonic acids (EC50 = 50-343 nM) in protection of cells from the viral infection. The phosphonate monoalkyl esters are stable in buffer solutions (pH 2.0-7.4) and rabbit serum; furthermore, the alkyl group is possibly tuned to attain the desired pharmacokinetic properties.