RESUMEN
As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure-activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand-protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharmacology. Further characterization of 19f in vivo demonstrated an antidepressant-like signature in the mouse forced-swim test (mFST) assay when brain levels of this compound exceeded the cellular mGlu2 IC50 value.
Asunto(s)
Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Descubrimiento de Drogas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Antidepresivos/síntesis química , Antidepresivos/química , Encéfalo/efectos de los fármacos , Ciclohexanos/síntesis química , Ciclohexanos/química , Ciclohexanos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Ratones Endogámicos , Modelos Moleculares , Estructura Molecular , Actividad Motora/efectos de los fármacos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/aislamiento & purificación , Relación Estructura-Actividad , NataciónRESUMEN
The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/química , Pirimidinas/farmacología , Proteínas ras/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral/efectos de los fármacos , Técnicas de Química Sintética , Perros , Femenino , Semivida , Humanos , Masculino , Ratones Desnudos , Terapia Molecular Dirigida , Mutación , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacocinética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas Sprague-Dawley , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genéticaRESUMEN
Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.