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

RESUMO

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.


Assuntos
Antirretrovirais , Descoberta de Drogas , Retrovirus Endógenos , DNA Polimerase Dirigida por RNA , Inibidores da Transcriptase Reversa , Antirretrovirais/química , Antirretrovirais/farmacologia , Retrovirus Endógenos/enzimologia , Retrovirus Endógenos/genética , Genes Virais , Transcriptase Reversa do HIV/química , Humanos , Multimerização Proteica , DNA Polimerase Dirigida por RNA/química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia
2.
PLoS One ; 17(4): e0266812, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35395060

RESUMO

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.


Assuntos
Doença de Huntington , Animais , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Expansão das Repetições de Trinucleotídeos
3.
J Med Chem ; 64(8): 5018-5036, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33783225

RESUMO

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.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Piridonas/química , Pirimidinonas/química , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Meia-Vida , Humanos , Doença de Huntington/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Morfolinos/química , Piridonas/metabolismo , Piridonas/uso terapêutico , Pirimidinonas/metabolismo , Pirimidinonas/uso terapêutico , Relação Estrutura-Atividade
4.
J Med Chem ; 63(21): 12887-12910, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33105987

RESUMO

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.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Inibidores de Fosfodiesterase/química , Pirimidinas/química , Triazóis/química , Animais , Sítios de Ligação , Encéfalo/metabolismo , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Termodinâmica , Triazóis/metabolismo , Triazóis/farmacocinética
5.
J Med Chem ; 62(6): 2988-3008, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30840447

RESUMO

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.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Doença de Huntington/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Modelos Animais de Doenças , Cães , Humanos , Células Madin Darby de Rim Canino , Camundongos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacocinética , Estudo de Prova de Conceito
6.
Bioorg Med Chem Lett ; 29(1): 83-88, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463802

RESUMO

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.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade
7.
J Med Chem ; 60(9): 3580-3590, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28414242

RESUMO

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.


Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Imidazóis/uso terapêutico , Diester Fosfórico Hidrolases/efeitos dos fármacos , Pirimidinas/uso terapêutico , Animais , Humanos , Imidazóis/farmacologia , Camundongos , Camundongos Knockout , Diester Fosfórico Hidrolases/genética , Pirimidinas/farmacologia , Ratos
8.
J Med Chem ; 59(11): 5221-37, 2016 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-27167172

RESUMO

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.


Assuntos
4-Aminopiridina/análogos & derivados , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , 4-Aminopiridina/síntese química , 4-Aminopiridina/química , 4-Aminopiridina/farmacologia , Quinase 1 do Ponto de Checagem/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazinas/síntese química , Pirazinas/química , Relação Estrutura-Atividade
9.
ACS Med Chem Lett ; 7(1): 34-9, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26819662

RESUMO

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 ; 58(7): 2967-87, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25760409

RESUMO

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.


Assuntos
Proteína Quinase 10 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Modelos Animais de Doenças , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Meia-Vida , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Concentração Inibidora 50 , Células Madin Darby de Rim Canino/efeitos dos fármacos , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Proteína Quinase 10 Ativada por Mitógeno/química , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Pirazóis/química , Pirimidinas/química , Relação Estrutura-Atividade
11.
PLoS One ; 9(5): e96854, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24816435

RESUMO

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.


Assuntos
Bioensaio/métodos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Encéfalo/metabolismo , Linhagem Celular , Feminino , Humanos , Proteína Huntingtina , Medições Luminescentes , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Ratos , Solubilidade
12.
J Med Chem ; 56(24): 9934-54, 2013 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-24261862

RESUMO

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.


Assuntos
Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Doença de Huntington/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/classificação , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
13.
Bioorg Med Chem Lett ; 21(18): 5244-7, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21820899

RESUMO

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.


Assuntos
Clorometilcetonas de Aminoácidos/farmacologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Clorometilcetonas de Aminoácidos/química , Sítios de Ligação/efeitos dos fármacos , Caspase 6/química , Caspase 6/metabolismo , Inibidores de Cisteína Proteinase/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
14.
J Mol Biol ; 410(2): 307-15, 2011 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-21621544

RESUMO

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.


Assuntos
Caspase 6/química , Apoenzimas/química , Apoenzimas/metabolismo , Caspase 6/metabolismo , Inibidores de Caspase , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Ativação Enzimática , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Ligantes , Ligação Proteica , Conformação Proteica
15.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 3): 451-5, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11856830

RESUMO

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.


Assuntos
Catepsinas/química , Substituição de Aminoácidos , Catepsinas/genética , Cristalização , Cristalografia por Raios X , Cisteína/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Serina/genética
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