RESUMEN
Histone deacetylases (HDACs) play diverse roles in many diseases including cancer, sarcopenia, and Alzheimer's. Different isoforms of HDACs appear to play disparate roles in the cell and are associated with specific diseases; as such, a substantial effort has been made to develop isoform-selective HDAC inhibitors. Our group focused on developing HDAC1/HDAC2-specific inhibitors as a cancer therapeutic. In the course of characterizing the mechanism of inhibition of a novel HDAC1/2-selective inhibitor, it was determined that it did not exhibit classical Michaelis-Menten kinetic behavior; this result is in contrast to the seminal HDAC inhibitor SAHA. Enzymatic assays, along with a newly developed binding assay, were used to determine the rates of binding and the affinities of both the HDAC1/2-selective inhibitor and SAHA. The mechanism of action studies identified a potential conformational change required for optimal binding by the selective inhibitor. A model of this putative conformational change is proposed.
Asunto(s)
Antineoplásicos/química , Benzoatos/química , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/química , Ácidos Hidroxámicos/química , Xantenos/química , Animales , Antineoplásicos/farmacología , Benzoatos/farmacología , Línea Celular Tumoral , Femenino , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Xenoinjertos , Histona Desacetilasa 1/química , Histona Desacetilasa 2/química , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ácidos Hidroxámicos/farmacología , Cinética , Ratones , Ratones Desnudos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Vorinostat , Xantenos/farmacologíaRESUMEN
We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.
Asunto(s)
Antineoplásicos/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Inhibidores de Histona Desacetilasas , Organofosfonatos/farmacocinética , Proteínas Represoras/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/metabolismo , Ratones , Ratones Desnudos , Organofosfonatos/síntesis química , Organofosfonatos/química , Proteínas Represoras/metabolismo , Trasplante HeterólogoRESUMEN
The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An initial lead from a small parallel library was found to be potent and selective in biochemical assays. Advanced compounds were the culmination of iterative library design and possess excellent biochemical and cellular potency, as well as acceptable PK and efficacy in animal models.
Asunto(s)
Inhibidores de Histona Desacetilasas , Animales , Técnicas Químicas Combinatorias , Perros , Diseño de Fármacos , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Humanos , Estructura Molecular , Ratas , Proteínas Represoras/antagonistas & inhibidores , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-life in dogs.
Asunto(s)
Sitio Alostérico , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/química , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Perros , Humanos , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Neoplasias de la Próstata/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células Tumorales CultivadasRESUMEN
A class of biaryl benzamides was identified and optimized as selective HDAC1&2 inhibitors (SHI-1:2). These agents exhibit selectivity over class II HDACs 4-7, as well as class I HDACs 3 and 8; providing examples of selective HDAC inhibitors for the HDAC isoforms most closely associated with cancer. The hypothesis for the increased selectivity is the binding of a pendant aromatic group in the internal cavity of the HDAC1&2 enzymes. SAR development based on an initial lead led to a series of potent and selective inhibitors with reduced off-target activity and tumor growth inhibition activity in a HCT-116 xenograft model.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Proteínas Represoras/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Ratones , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Ongoing clinical studies indicate that inhibitors of Class I and Class II histone deacetylase (HDAC) enzymes show great promise for the treatment of cancer. Zolinza (SAHA, Zolinza) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma. As a part of an ongoing effort to identify novel small molecules to target these important enzymes, we have prepared several classes of amino acid-derived HDAC1 inhibitors. The design rationale and in vitro activity against the HDAC1 enzyme and HCT116 cell line are described in this letter.
Asunto(s)
Aminoácidos/química , Aminoácidos/farmacología , Inhibidores de Histona Desacetilasas , Aminoácidos/síntesis química , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Histona Desacetilasa 1 , Humanos , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacología , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.
Asunto(s)
Sitio Alostérico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/química , Pirimidinas/química , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Perros , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica , Estructura Molecular , Neoplasias Ováricas/metabolismo , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Células Tumorales CultivadasRESUMEN
This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1 and Akt2. This series also shows a high level of selectivity over other closely related kinases and significantly improved caspase-3 activity with the more optimized compounds.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacología , Aminas/síntesis química , Aminas/química , Aminas/farmacología , Caspasa 3/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Relación Estructura-Actividad , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Canales de Potasio Éter-A-Go-Go/metabolismo , Inhibidores de Histona Desacetilasas , Niacinamida/síntesis química , Niacinamida/farmacología , Compuestos de Espiro/síntesis química , Compuestos de Espiro/farmacología , Animales , Antineoplásicos/química , Benzamidas/síntesis química , Benzamidas/química , Benzamidas/farmacología , Técnicas Químicas Combinatorias , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Células HCT116 , Histona Desacetilasas , Histonas/análisis , Humanos , Ratones , Ratones Desnudos , Estructura Molecular , Niacinamida/química , Isoformas de Proteínas , Compuestos de Espiro/química , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Inhibidores de Histona Desacetilasas , Fenilalanina/química , Acetilación , Amidas , Animales , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Perros , Canal de Potasio ERG1 , Inhibidores Enzimáticos/farmacocinética , Canales de Potasio Éter-A-Go-Go/metabolismo , Glicina/química , Histona Desacetilasa 1 , Humanos , Macaca mulatta , Ratones , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.
Asunto(s)
Derivados del Benceno/síntesis química , Derivados del Benceno/farmacología , Inhibidores de Histona Desacetilasas , Modelos Moleculares , Derivados del Benceno/química , Sitios de Unión/efectos de los fármacos , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Humanos , Estructura Molecular , Isoformas de Proteínas , Proteínas Represoras , Relación Estructura-ActividadRESUMEN
We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Péptidos/química , Péptidos/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Homología de Secuencia de Aminoácido , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Adenosina Trifosfato/metabolismo , Proteínas Reguladoras de la Apoptosis , Bencilaminas/farmacología , Unión Competitiva , Proteínas Sanguíneas/inmunología , Carcinoma/química , Carcinoma/metabolismo , Carcinoma/patología , Caspasas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Masculino , Glicoproteínas de Membrana/farmacología , Estructura Molecular , Péptidos/inmunología , Péptidos/metabolismo , Fosfoproteínas/inmunología , Fosforilación/efectos de los fármacos , Neoplasias de la Próstata/química , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Quinoxalinas/farmacología , Transducción de Señal/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacología , Neoplasias del Cuello Uterino/química , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.
RESUMEN
Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Acetilación/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/administración & dosificación , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/farmacología , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/mortalidad , Linfoma/patología , Ratones , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Histona Desacetilasas/metabolismo , Histonas/química , Espectrometría de Masas/métodos , Proteómica/métodos , Antineoplásicos/farmacología , Línea Celular Tumoral , Cromatografía Liquida/métodos , Reacciones Falso Positivas , Histonas/metabolismo , Humanos , Péptidos/química , Isoformas de Proteínas , Proteoma , Curva ROC , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas , Diseño de Fármacos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.