RESUMO
To improve pironetin's metabolic stability we prepared four analogs by replacing its C12-14 segment with an aryl group. The antiproliferative activity of phenyl analog 4 was reduced two-fold and dihydroxy-4-fluorophenyl analog 5 was slightly more effective against OVCAR5 and A2780 ovarian cancer cell lines compared with the parent compound pironetin (1). The activity of 4-fluorophenyl analog 6 was reduced 3-fold in both cell lines. The activity of 7-O-methyl analog 7 was reduced 36-fold in OVCAR5 cells and 47-fold and A2780 cells, compared with pironetin. Phenylpironetin (4) was rapidly metabolized by mouse and human liver microsomes. We identified 17 human metabolites for phenyl analog 4 and 14 human metabolites for pironetin. Metabolism occurred at the C12-13 moiety, the α,ß-unsaturated lactone and the side chains of the molecules (C6-C11 segments). The significant extent of oxidative metabolism suggests that it may not be possible to attain a metabolically stable pironetin analog by structural modifications of the parent compound.
Assuntos
Neoplasias Ovarianas , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Lactonas , Camundongos , Pironas/químicaRESUMO
Pironetin is an α-tubulin-binding natural product with potent antiproliferative activity against several cancer cell lines that inhibits cell division by forming a covalent adduct with α-tubulin via a Michael addition into the natural product's α,ß-unsaturated lactone. We designed and prepared analogs carrying electron-withdrawing groups at the α-position (C2) of the α,ß-unsaturated lactone with the goal to generate potent and selective binding analogs. We prepared derivatives containing halogens, a phenyl, and a methyl group at the C2 position to evaluate the structure-activity relationship at this position. Testing of the analogs in ovarian cancer cell lines demonstrated 100-1000-fold decreased antiproliferative activity.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Pironas/farmacologia , Tubulina (Proteína)/química , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/química , Sítios de Ligação , Produtos Biológicos/síntese química , Produtos Biológicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Pironas/síntese química , Pironas/química , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismoRESUMO
The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal (Cryptococcus neoformans, Candida albicans) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.
Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Fungos/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirazóis/farmacologia , Animais , Antifúngicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Ligação Proteica , Pirazóis/química , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
The molecular chaperone Hsp90, essential in all eukaryotes, plays a multifaceted role in promoting survival, virulence, and drug resistance across diverse pathogenic fungal species. The chaperone is also critically important, however, to the pathogen's human host, preventing the use of known clinical Hsp90 inhibitors in antifungal applications due to concomitant host toxicity issues. With the goal of developing Hsp90 inhibitors with acceptable therapeutic indices for the treatment of invasive fungal infections, we initiated a program to design and synthesize potent inhibitors with selective activity against fungal Hsp90 isoforms over their human counterparts. Building on our previously reported derivatization of resorcylate natural products to produce fungal-selective compounds, we have developed a series of synthetic aminopyrazole-substituted resorcylate amides with broad, potent, and fungal-selective Hsp90 inhibitory activity. Herein we describe the synthesis of this series, as well as biochemical structure-activity relationships driving selectivity for the Hsp90 isoforms expressed by Cryptococcus neoformans and Candida albicans, two pathogenic fungi of major clinical importance.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Pirazóis/farmacologia , Aminação , Animais , Antifúngicos/síntese química , Antifúngicos/química , Candida albicans/metabolismo , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/metabolismo , Desenho de Fármacos , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Pironetin, the only crystallographically confirmed natural product to target α-tubulin, displays potent cytotoxic activity against sensitive and resistant A2780 ovarian cancer cell lines but is only marginally active in vivo. We now report that pironetin has a short half-life (<7 min) in human liver microsomes, suggesting that its limited in vivo efficacy is due to rapid metabolism. Further, we describe the discovery of epoxypironetin as pironetin's major metabolite in human liver microsomes.
Assuntos
Produtos Biológicos/metabolismo , Pironas/metabolismo , Moduladores de Tubulina/metabolismo , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/farmacocinética , Linhagem Celular Tumoral , Meia-Vida , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Pironas/síntese química , Pironas/farmacocinética , Ratos , Streptomyces/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/farmacocinéticaRESUMO
New strategies are needed to counter the escalating threat posed by drug-resistant fungi. The molecular chaperone Hsp90 affords a promising target because it supports survival, virulence and drug-resistance across diverse pathogens. Inhibitors of human Hsp90 under development as anticancer therapeutics, however, exert host toxicities that preclude their use as antifungals. Seeking a route to species-selectivity, we investigate the nucleotide-binding domain (NBD) of Hsp90 from the most common human fungal pathogen, Candida albicans. Here we report structures for this NBD alone, in complex with ADP or in complex with known Hsp90 inhibitors. Encouraged by the conformational flexibility revealed by these structures, we synthesize an inhibitor with >25-fold binding-selectivity for fungal Hsp90 NBD. Comparing co-crystals occupied by this probe vs. anticancer Hsp90 inhibitors revealed major, previously unreported conformational rearrangements. These insights and our probe's species-selectivity in culture support the feasibility of targeting Hsp90 as a promising antifungal strategy.
Assuntos
Antifúngicos/farmacologia , Candida albicans/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Proteínas Fúngicas/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Linhagem Celular , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP90/genética , Compostos Heterocíclicos de 4 ou mais Anéis/antagonistas & inibidores , Humanos , Isoxazóis/antagonistas & inibidores , Camundongos , Modelos Moleculares , Chaperonas Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes , Resorcinóis/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Triazóis/antagonistas & inibidores , Virulência/efeitos dos fármacosRESUMO
Pironetin is a natural product with potent antiproliferative activity that forms a covalent adduct with α-tubulin via conjugate addition into the natural product's α,ß-unsaturated lactone. Although pironetin's α,ß-unsaturated lactone is involved in its binding to tubulin, the structure-activity relationship at different positions of the lactone have not been thoroughly evaluated. For a systematic evaluation of the structure-activity relationships at the C4 and C5 positions of the α,ß-unsaturated lactone of pironetin, twelve analogues of the natural product were prepared by total synthesis. Modifying the stereochemistry at the C4 and/or C5 positions of the α,ß-unsaturated lactone of pironetin resulted in loss of antiproliferative activity in OVCAR5 ovarian cancer cells. While changing the C4 ethyl substituent with groups such as methyl, propyl, cyclopropyl, and isobutyl were tolerated, groups with larger steric properties such as an isopropyl and benzyl groups were not.
Assuntos
Antineoplásicos/química , Antineoplásicos/síntese química , Lactonas/química , Pironas/química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ligação Proteica , Pironas/síntese química , Pironas/metabolismo , Pironas/toxicidade , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/toxicidadeAssuntos
Acetais/química , Etilenos/química , Cetonas/química , Paládio/química , Catálise , Complexos de Coordenação/química , ÉsteresRESUMO
Aging alters numerous aspects of circadian biology, including the amplitude of rhythms generated by the suprachiasmatic nuclei (SCN) of the hypothalamus, the site of the central circadian pacemaker in mammals, and the response of the pacemaker to environmental stimuli such as light. Although previous studies have described molecular correlates of these behavioral changes, to date only 1 study in rats has attempted to determine if there are age-related changes in the expression of genes that comprise the circadian clock itself. We used in situ hybridization to examine the effects of age on the circadian pattern of expression of a subset of the genes that comprise the molecular machinery of the circadian clock in golden hamsters. Here we report that age alters the 24-h expression profile of Clock and its binding partner Bmal1 in the hamster SCN. There is no effect of age on the 24-h profile of either Per1 or Per2 when hamsters are housed in constant darkness. We also found that light pulses, which induce smaller phase shifts in old animals than in young, lead to decreased induction of Per1, but not of Per2, in the SCN of old hamsters.