RESUMEN
The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.
Asunto(s)
Proteínas Bacterianas/química , Liasas de Carbono-Carbono/química , Variación Genética , Estructura Cuaternaria de Proteína , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Liasas de Carbono-Carbono/clasificación , Liasas de Carbono-Carbono/genética , Dominio Catalítico , Cristalografía por Rayos X , Deinococcus/enzimología , Deinococcus/genética , Enterococcus faecalis/enzimología , Enterococcus faecalis/genética , Evolución Molecular , Mutación INDEL , Listeria/enzimología , Listeria/genética , Modelos Moleculares , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
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
Thermobifida fusca o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily that catalyzes a step in menaquinone biosynthesis, has an amino acid sequence that is 22 and 28% identical with those of two previously characterized OSBS enzymes from Escherichia coli and Amycolatopsis sp. T-1-60, respectively. These values are considerably lower than typical levels of sequence identity among homologous proteins that have the same function. To determine how such divergent enzymes catalyze the same reaction, we determined the structure of T. fusca OSBS and identified amino acids that are important for ligand binding. We discovered significant differences in structure and conformational flexibility between T. fusca OSBS and other members of the enolase superfamily. In particular, the 20s loop, a flexible loop in the active site that permits ligand binding and release in most enolase superfamily proteins, has a four-amino acid deletion and is well-ordered in T. fusca OSBS. Instead, the flexibility of a different region allows the substrate to enter from the other side of the active site. T. fusca OSBS was more tolerant of mutations at residues that were critical for activity in E. coli OSBS. Also, replacing active site amino acids found in one protein with the amino acids that occur at the same place in the other protein reduces the catalytic efficiency. Thus, the extraordinary divergence between these proteins does not appear to reflect a higher tolerance of mutations. Instead, large deletions outside the active site were accompanied by alteration of active site size and electrostatic interactions, resulting in small but significant differences in ligand binding.
Asunto(s)
Actinomycetales/enzimología , Evolución Biológica , Liasas de Carbono-Carbono/metabolismo , Escherichia coli/enzimología , Magnesio/metabolismo , Sitios de Unión , Liasas de Carbono-Carbono/química , Liasas de Carbono-Carbono/genética , Catálisis , Dominio Catalítico , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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
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
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
Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.
Asunto(s)
Adenosina Trifosfato/metabolismo , Boro/química , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Adenosina Trifosfato/análogos & derivados , Secuencia de Bases , Sitios de Unión , Terapia por Captura de Neutrón de Boro , Evolución Molecular Dirigida , Datos de Secuencia Molecular , Neoplasias/radioterapia , Conformación de Ácido Nucleico , Alineación de SecuenciaRESUMEN
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
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.
Asunto(s)
6-Aminonicotinamida/análogos & derivados , 6-Aminonicotinamida/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , 6-Aminonicotinamida/síntesis química , Animales , Área Bajo la Curva , Benzamidas/química , Disponibilidad Biológica , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Perros , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacocinética , Semivida , Humanos , Isoenzimas/antagonistas & inhibidores , Modelos Moleculares , Trasplante de Neoplasias , Unión Proteica , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A new mode of allosteric regulation of nucleic acid enzymes is described and shown to operate effectively with hammerhead ribozymes. In the "TRAP" design (for targeted ribozyme-attenuated probe), a 3' terminal "attenuator" anneals to conserved bases in the catalytic core to form the "off" state of the ribozyme. Binding of RNA or DNA to an antisense sequence linking the ribozyme and attenuator frees the core to fold into an active conformation, even though the antisense sequence itself does not interfere with the ribozyme. TRAP hammerheads based on the previously characterized HH8 ribozyme were shown to be activated more than 250-fold upon addition of the sense strand. RNA oligonucleotides were more effective activators than DNA oligos, consistent with the known relative helix stabilities (RNA-RNA > RNA-DNA). Oligonucleotides that directly paired with the attenuator gave up to 1760-fold activation. The magnitude of the activation was greater when the oligo was added prior to folding than if it was added during the cleavage reaction. The TRAP design requires no prior knowledge of (deoxy)ribozyme structure beyond identification of the essential core. Thus, this approach should be readily generalizable to other systems for biomedicine, sensor technology, and additional applications.