RESUMEN
With structural guidance, tropane-derived HTS hits were modified to optimize for HSP90 inhibition and a desirable in vivo profile. Through an iterative SAR development process 12i (XL888) was discovered and shown to reduce HSP90 client protein content in PD studies. Furthermore, efficacy experiments performed in a NCI-N87 mouse xenograft model demonstrated tumor regression in some dosing regimens.
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Antineoplásicos/química , Antineoplásicos/uso terapéutico , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Ácidos Ftálicos/química , Ácidos Ftálicos/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Compuestos de Azabiciclo/farmacocinética , Compuestos de Azabiciclo/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Descubrimiento de Drogas , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patología , Ácidos Ftálicos/farmacocinética , Ácidos Ftálicos/farmacologíaRESUMEN
Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of betaAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin. Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of betaAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, like nicastrin, are required for the activity and accumulation of gamma-secretase.
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Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Caenorhabditis elegans/aislamiento & purificación , Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Proteínas de Homeodominio/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Clonación Molecular , Proteínas de Drosophila , Drosophila melanogaster , Elementos de Facilitación Genéticos/genética , Glucagón/metabolismo , Péptido 1 Similar al Glucagón , Proteínas del Helminto/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación/genética , Fragmentos de Péptidos/metabolismo , Presenilina-1 , Precursores de Proteínas/metabolismo , Receptores Notch , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.
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Compuestos de Azabiciclo/farmacología , Ciclo Celular , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Ácidos Ftálicos/farmacología , Benzoquinonas/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Citometría de Flujo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Lactamas Macrocíclicas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Tiempo , Quinasa Tipo Polo 1RESUMEN
Asymmetric cell division depends on coordinating the position of the mitotic spindle with the axis of cellular polarity. We provide evidence that LET-99 is a link between polarity cues and the downstream machinery that determines spindle positioning in C. elegans embryos. In let-99 one-cell embryos, the nuclear-centrosome complex exhibits a hyperactive oscillation that is dynein dependent, instead of the normal anteriorly directed migration and rotation of the nuclear-centrosome complex. Furthermore, at anaphase in let-99 embryos the spindle poles do not show the characteristic asymmetric movements typical of wild type animals. LET-99 is a DEP domain protein that is asymmetrically enriched in a band that encircles P lineage cells. The LET-99 localization pattern is dependent on PAR polarity cues and correlates with nuclear rotation and anaphase spindle pole movements in wild-type embryos, as well as with changes in these movements in par mutant embryos. In particular, LET-99 is uniformly localized in one-cell par-3 embryos at the time of nuclear rotation. Rotation fails in spherical par-3 embryos in which the eggshell has been removed, but rotation occurs normally in spherical wild-type embryos. The latter results indicate that nuclear rotation in intact par-3 embryos is dictated by the geometry of the oblong egg and are consistent with the model that the LET-99 band is important for rotation in wild-type embryos. Together, the data indicate that LET-99 acts downstream of PAR-3 and PAR-2 to determine spindle positioning, potentially through the asymmetric regulation of forces on the spindle.