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1.
Proc Natl Acad Sci U S A ; 119(27): e2200260119, 2022 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-35771941

RESUMEN

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.


Asunto(s)
Antirretrovirales , Descubrimiento de Drogas , Retrovirus Endógenos , ADN Polimerasa Dirigida por ARN , Inhibidores de la Transcriptasa Inversa , Antirretrovirales/química , Antirretrovirales/farmacología , Retrovirus Endógenos/enzimología , Retrovirus Endógenos/genética , Genes Virales , Transcriptasa Inversa del VIH/química , Humanos , Multimerización de Proteína , ADN Polimerasa Dirigida por ARN/química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología
2.
Bioorg Med Chem Lett ; 29(1): 83-88, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30463802

RESUMEN

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ∼150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC50 levels for ∼8 h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.


Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Ratones , Estructura Molecular , Relación Estructura-Actividad
3.
PLoS One ; 17(4): e0266812, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35395060

RESUMEN

Huntington's disease (HD) is caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin gene that results in expression of a mutant huntingtin protein (mHTT) containing an expanded polyglutamine tract in the amino terminus. A number of therapeutic approaches that aim to reduce mHTT expression either locally in the CNS or systemically are in clinical development. We have previously described sensitive and selective assays that measure human HTT proteins either in a polyglutamine-independent (detecting both mutant expanded and non-expanded proteins) or in a polyglutamine length-dependent manner (detecting the disease-causing polyglutamine repeats) on the electrochemiluminescence Meso Scale Discovery detection platform. These original assays relied upon polyclonal antibodies. To ensure an accessible and sustainable resource for the HD field, we developed similar assays employing monoclonal antibodies. We demonstrate that these assays have equivalent sensitivity compared to our previous assays through the evaluation of cellular and animal model systems, as well as HD patient biosamples. We also demonstrate cross-site validation of these assays, allowing direct comparison of studies performed in geographically distinct laboratories.


Asunto(s)
Enfermedad de Huntington , Animales , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Péptidos/genética , Péptidos/metabolismo , Expansión de Repetición de Trinucleótido
4.
Bioorg Med Chem Lett ; 21(18): 5244-7, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21820899

RESUMEN

Caspase-6 is a cysteine protease implicated in neuronal survival and apoptosis. Deregulation of caspase-6 activity was linked to several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Several recent studies on the structure of caspase-6 feature the caspase-6 zymogen, mature apo-caspase-6 as well as the Ac-VEID-CHO peptide complex. All structures share the same typical dimeric caspase conformation. However, mature apo-caspase-6 crystallized at low pH revealed a novel, non-canonical inactive caspase conformation speculated to represent a latent state of the enzyme suitable for the design of allosteric inhibitors. In this treatise we present the structure of caspase-6 in the non-canonical inactive enzyme conformation bound to the irreversible inhibitor Z-VAD-FMK. The complex features a unique peptide binding mode not observed previously.


Asunto(s)
Clorometilcetonas de Aminoácidos/farmacología , Inhibidores de Caspasas , Inhibidores de Cisteína Proteinasa/farmacología , Clorometilcetonas de Aminoácidos/química , Sitios de Unión/efectos de los fármacos , Caspasa 6/química , Caspasa 6/metabolismo , Inhibidores de Cisteína Proteinasa/química , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad
5.
J Med Chem ; 64(8): 5018-5036, 2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33783225

RESUMEN

Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have potential as novel therapeutics for the treatment of Huntington's disease (HD). However, the previously described pyranone-thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-thioxanthenes.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Piridonas/química , Pirimidinonas/química , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Encéfalo/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Semivida , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Morfolinos/química , Piridonas/metabolismo , Piridonas/uso terapéutico , Pirimidinonas/metabolismo , Pirimidinonas/uso terapéutico , Relación Estructura-Actividad
6.
J Med Chem ; 63(21): 12887-12910, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33105987

RESUMEN

We describe the hit-to-lead exploration of a [1,2,4]triazolo[1,5-a]pyrimidine phosphodiesterase 2A (PDE2A) inhibitor arising from high-throughput screening. X-ray crystallography enabled structure-guided design, leading to the identification of preferred substructural components. Further rounds of optimization used relative binding free-energy calculations to prioritize different substituents from the large accessible chemical space. The free-energy perturbation (FEP) calculations were performed for 265 putative PDE2A inhibitors, and 100 compounds were synthesized representing a relatively large prospective application providing unexpectedly active molecules with IC50's from 2340 to 0.89 nM. Lead compound 46 originating from the FEP calculations showed PDE2A inhibition IC50 of 1.3 ± 0.39 nM, ∼100-fold selectivity versus other PDE enzymes, clean cytochrome P450 profile, in vivo target occupancy, and promise for further lead optimization.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/química , Pirimidinas/química , Triazoles/química , Animales , Sitios de Unión , Encéfalo/metabolismo , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/metabolismo , Diseño de Fármacos , Semivida , Humanos , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Inhibidores de Fosfodiesterasa/metabolismo , Inhibidores de Fosfodiesterasa/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratas , Ratas Wistar , Estereoisomerismo , Relación Estructura-Actividad , Termodinámica , Triazoles/metabolismo , Triazoles/farmacocinética
7.
J Med Chem ; 62(6): 2988-3008, 2019 03 28.
Artículo en Inglés | MEDLINE | ID: mdl-30840447

RESUMEN

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Modelos Animales de Enfermedad , Perros , Humanos , Células de Riñón Canino Madin Darby , Ratones , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacocinética , Prueba de Estudio Conceptual
8.
J Med Chem ; 60(9): 3580-3590, 2017 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-28414242

RESUMEN

Autotaxin is a circulating enzyme with a major role in the production of lysophosphatic acid (LPA) species in blood. A role for the autotaxin/LPA axis has been suggested in many disease areas including pulmonary fibrosis. Structural modifications of the known autotaxin inhibitor lead compound 1, to attenuate hERG inhibition, remove CYP3A4 time-dependent inhibition, and improve pharmacokinetic properties, led to the identification of clinical candidate GLPG1690 (11). Compound 11 was able to cause a sustained reduction of LPA levels in plasma in vivo and was shown to be efficacious in a bleomycin-induced pulmonary fibrosis model in mice and in reducing extracellular matrix deposition in the lung while also reducing LPA 18:2 content in bronchoalveolar lavage fluid. Compound 11 is currently being evaluated in an exploratory phase 2a study in idiopathic pulmonary fibrosis patients.


Asunto(s)
Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Imidazoles/uso terapéutico , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Pirimidinas/uso terapéutico , Animales , Humanos , Imidazoles/farmacología , Ratones , Ratones Noqueados , Hidrolasas Diéster Fosfóricas/genética , Pirimidinas/farmacología , Ratas
9.
ACS Med Chem Lett ; 7(1): 34-9, 2016 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-26819662

RESUMEN

Potent and selective class IIa HDAC tetrasubstituted cyclopropane hydroxamic acid inhibitors were identified with high oral bioavailability that exhibited good brain and muscle exposure. Compound 14 displayed suitable properties for assessment of the impact of class IIa HDAC catalytic site inhibition in preclinical disease models.

10.
J Med Chem ; 59(11): 5221-37, 2016 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-27167172

RESUMEN

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.


Asunto(s)
4-Aminopiridina/análogos & derivados , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , 4-Aminopiridina/síntesis química , 4-Aminopiridina/química , 4-Aminopiridina/farmacología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirazinas/síntesis química , Pirazinas/química , Relación Estructura-Actividad
11.
J Med Chem ; 58(7): 2967-87, 2015 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-25760409

RESUMEN

Through medicinal chemistry lead optimization studies focused on calculated properties and guided by X-ray crystallography and computational modeling, potent pan-JNK inhibitors were identified that showed submicromolar activity in a cellular assay. Using in vitro ADME profiling data, 9t was identified as possessing favorable permeability and a low potential for efflux, but it was rapidly cleared in liver microsomal incubations. In a mouse pharmacokinetics study, compound 9t was brain-penetrant after oral dosing, but exposure was limited by high plasma clearance. Brain exposure at a level expected to support modulation of a pharmacodynamic marker in mouse was achieved when the compound was coadministered with the pan-cytochrome P450 inhibitor 1-aminobenzotriazole.


Asunto(s)
Proteína Quinasa 10 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Técnicas de Química Sintética , Cristalografía por Rayos X , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Modelos Animales de Enfermedad , Perros , Evaluación Preclínica de Medicamentos/métodos , Semivida , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/metabolismo , Concentración 50 Inhibidora , Células de Riñón Canino Madin Darby/efectos de los fármacos , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Proteína Quinasa 10 Activada por Mitógenos/química , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Pirazoles/química , Pirimidinas/química , Relación Estructura-Actividad
12.
PLoS One ; 9(5): e96854, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24816435

RESUMEN

The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD in vitro cellular and in vivo animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.


Asunto(s)
Bioensayo/métodos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Encéfalo/metabolismo , Línea Celular , Femenino , Humanos , Proteína Huntingtina , Mediciones Luminiscentes , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares/química , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Ratas , Solubilidad
13.
J Med Chem ; 56(24): 9934-54, 2013 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-24261862

RESUMEN

Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.


Asunto(s)
Diseño de Fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacocinética , Histona Desacetilasas/clasificación , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad
14.
J Mol Biol ; 410(2): 307-15, 2011 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-21621544

RESUMEN

Caspase-6 has been identified as a key component in the pathway of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. It has been the focus of drug development for some time, but only recently have structural data become available. The first study identified a novel noncanonical conformation of apo-caspase-6 contrasting with the typical caspase conformation. Then, the structures of both caspase-6 zymogen and the Ac-VEID-CHO peptide inhibitor complex described caspase-6 in the canonical conformation, raising the question of why the intermediate between these two structures (mature apo-caspase-6) would adopt the noncanonical conformation. In this study, we present a new crystal form of the apoenzyme in the canonical conformation by identifying the previous apostructure as a pH-inactivated form of caspase-6. Our new apostructure is further compared to the Ac-VEID-CHO caspase-6 inhibitor complex. The structural comparison allows us to visualize the organization of loops L2, L3, and L4 upon ligand binding and how the catalytic groove forms to accommodate the inhibitor.


Asunto(s)
Caspasa 6/química , Apoenzimas/química , Apoenzimas/metabolismo , Caspasa 6/metabolismo , Inhibidores de Caspasas , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Ligandos , Unión Proteica , Conformación Proteica
15.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 3): 451-5, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11856830

RESUMEN

Cathepsin S (EC 3.4.22.27), a cysteine proteinase of the papain superfamily, plays a critical role in the generation of a major histocompatibility complex (MHC) class II restricted T-cell response by antigen-presenting cells. Therefore, selective inhibition of this enzyme may be useful in modulating class II restricted T-cell responses in immune-related disorders such as rheumatoid arthritis, multiple sclerosis and extrinsic asthma. The three-dimensional structure at 2.2 A resolution of the active-site Cys25-->Ser mutant presented here in an unliganded state provides further insight useful for the design of selective enzyme inhibitors.


Asunto(s)
Catepsinas/química , Sustitución de Aminoácidos , Catepsinas/genética , Cristalización , Cristalografía por Rayos X , Cisteína/genética , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Proteínas Recombinantes/química , Serina/genética
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