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1.
Cell Mol Neurobiol ; 41(4): 669-685, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32424773

RESUMO

Tau-tubulin kinase 1 (TTBK1) is a CNS-specific, kinase that has been implicated in the pathological phosphorylation of tau in Alzheimer's Disease (AD) and Frontotemporal Dementia (FTD). TTBK1 is a challenging therapeutic target because it shares a highly conserved catalytic domain with its homolog, TTBK2, a ubiquitously expressed kinase genetically linked to the disease spinocerebellar ataxia type 11. The present study attempts to elucidate the functional distinctions between the TTBK isoforms and increase our understanding of them as distinct targets for the treatment of neurodegenerative disease. We demonstrate that in cortical neurons, TTBK1, not TTBK2, is the isoform responsible for tau phosphorylation at epitopes enriched in tauopathies such as Serine 422. In addition, although our elucidation of the crystal structure of the TTBK2 kinase domain indicates almost identical structural similarity with TTBK1, biochemical and cellular assays demonstrate that the enzymatic activity of these two proteins is regulated by a combination of unique extra-catalytic sequences and autophosphorylation events. Finally, we have identified an unbiased list of neuronal interactors and phosphorylation substrates for TTBK1 and TTBK2 that highlight the unique cellular pathways and functional networks that each isoform is involved in. This data address an important gap in knowledge regarding the implications of targeting TTBK kinases and may prove valuable in the development of potential therapies for disease.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Córtex Cerebral/patologia , Epitopos/metabolismo , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos Transgênicos , Neurônios/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteômica , Serina/metabolismo , Homologia Estrutural de Proteína , Proteínas tau/metabolismo
2.
BMC Struct Biol ; 16(1): 7, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27246200

RESUMO

BACKGROUND: The nuclear hormone receptor RORγ regulates transcriptional genes involved in the production of the pro-inflammatory interleukin IL-17 which has been linked to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. This transcriptional activity of RORγ is modulated through a protein-protein interaction involving the activation function 2 (AF2) helix on the ligand binding domain of RORγ and a conserved LXXLL helix motif on coactivator proteins. Our goal was to develop a RORγ specific inverse agonist that would help down regulate pro-inflammatory gene transcription by disrupting the protein protein interaction with coactivator proteins as a therapeutic agent. RESULTS: We identified a novel series of synthetic benzoxazinone ligands having an agonist (BIO592) and inverse agonist (BIO399) mode of action in a FRET based assay. We show that the AF2 helix of RORγ is proteolytically sensitive when inverse agonist BIO399 binds. Using x-ray crystallography we show how small modifications on the benzoxazinone agonist BIO592 trigger inverse agonism of RORγ. Using an in vivo reporter assay, we show that the inverse agonist BIO399 displayed specificity for RORγ over ROR sub-family members α and ß. CONCLUSION: The synthetic benzoxazinone ligands identified in our FRET assay have an agonist (BIO592) or inverse agonist (BIO399) effect by stabilizing or destabilizing the agonist conformation of RORγ. The proteolytic sensitivity of the AF2 helix of RORγ demonstrates that it destabilizes upon BIO399 inverse agonist binding perturbing the coactivator protein binding site. Our structural investigation of the BIO592 agonist and BIO399 inverse agonist structures identified residue Met358 on RORγ as the trigger for RORγ specific inverse agonism.


Assuntos
Benzoxazinas/química , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Benzoxazinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
3.
ChemMedChem ; 19(11): e202400093, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38482564

RESUMO

Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair enzyme, has proven to be a successful strategy for the treatment of various cancers. With the appropriate selection conditions and protein design, DNA-encoded library (DEL) technology provides a powerful avenue to identify small molecules with the desired mechanism of action towards a target of interest. However, DNA-binding proteins, such as PARP1, can be challenging targets for DEL screening due to non-specific protein-DNA interactions. To overcome this, we designed and screened a PARP1 catalytic domain construct without the autoinhibitory helical domain. This allowed us to interrogate an active, functionally-relevant form of the protein resulting in the discovery of novel isoindolinone PARP1 inhibitors with single-digit nanomolar potency. These inhibitors also demonstrated little to no PARP1-DNA trapping, a property that could be advantageous in the clinic.


Assuntos
DNA , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , DNA/química , DNA/metabolismo , Relação Estrutura-Atividade , Descoberta de Drogas , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Relação Dose-Resposta a Droga , Isoindóis/química , Isoindóis/farmacologia , Isoindóis/síntese química , Domínio Catalítico
4.
Bioorg Med Chem Lett ; 22(12): 4033-7, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22607669

RESUMO

This Letter reports the optimization of a pyrrolopyrimidine series as dual inhibitors of Aurora A/B kinases. This series derived from a pyrazolopyrimidine series previously reported as inhibitors of aurora kinases and CDKs. In an effort to improve the selectivity of this chemotype, we switched to the pyrrolopyrimidine core which allowed functionalization on C-2. In addition, the modeling rationale was based on superimposing the structures of Aurora-A kinase and CDK2 which revealed enough differences leading to a path for selectivity improvement. The synthesis of the new series of pyrrolopyrimidine analogs relied on the development of a different route for the two key intermediates 7 and 19 which led to analogs with both tunable activity against CDK1 and maintained cell potency.


Assuntos
Antineoplásicos/síntese química , Proteína Quinase CDC2/química , Quinase 2 Dependente de Ciclina/química , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/síntese química , Pirróis/síntese química , Antineoplásicos/farmacologia , Aurora Quinases , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Desenho de Fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Pirimidinas/farmacologia , Pirróis/farmacologia , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
5.
J Med Chem ; 64(20): 15402-15419, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34653340

RESUMO

Apoptosis signal-regulating kinase 1 (ASK1) is one of the key mediators of the cellular stress response that regulates inflammation and apoptosis. To probe the therapeutic value of modulating this pathway in preclinical models of neurological disease, we further optimized the profile of our previously reported inhibitor 3. This effort led to the discovery of 32, a potent (cell IC50 = 25 nM) and selective ASK1 inhibitor with suitable pharmacokinetic and brain penetration (rat Cl/Clu = 1.6/56 L/h/kg and Kp,uu = 0.46) for proof-of-pharmacology studies. Specifically, the ability of 32 to inhibit ASK1 in the central nervous system (CNS) was evaluated in a human tau transgenic (Tg4510) mouse model exhibiting elevated brain inflammation. In this study, transgenic animals treated with 32 (at 3, 10, and 30 mg/kg, BID/PO for 4 days) showed a robust reduction of inflammatory markers (e.g., IL-1ß) in the cortex, thus confirming inhibition of ASK1 in the CNS.


Assuntos
Encéfalo/efeitos dos fármacos , Descoberta de Drogas , Inflamação/tratamento farmacológico , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Animais , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamação/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Ratos , Relação Estrutura-Atividade
6.
J Med Chem ; 64(9): 6358-6380, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33944571

RESUMO

Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer's disease and other tauopathies.


Assuntos
Encéfalo/metabolismo , Desenho de Fármacos , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Humanos , Indazóis/química , Indazóis/metabolismo , Indazóis/farmacologia , Camundongos , Terapia de Alvo Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Ratos
7.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 3): 103-108, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32133995

RESUMO

Tau proteins play an important role in the proper assembly and function of neurons. Hyperphosphorylation of tau by kinases such as tau tubulin kinase (TTBK) has been hypothesized to cause the aggregation of tau and the formation of neurofibrillary tangles (NFTs) that lead to the destabilization of microtubules, thereby contributing to neurodegenerative diseases such as Alzheimer's disease (AD). There are two TTBK isoforms with highly homologous catalytic sites but with distinct tissue distributions, tau phosphorylation patterns and loss-of-function effects. Inhibition of TTBK1 reduces the levels of NFT formation involved in neurodegenerative diseases such as AD, whereas inhibition of TTBK2 may lead to the movement disorder spinocerebellar ataxia type 11 (SCA11). Hence, it is critical to obtain isoform-selective inhibitors. Structure-based drug design (SBDD) has been used to design highly potent and exquisitely selective inhibitors. While structures of TTBK1 have been reported in the literature, TTBK2 has evaded structural characterization. Here, the first crystal structure of the TTBK2 kinase domain is described. Furthermore, the crystal structure of human TTBK2 in complex with a small-molecule inhibitor has successfully been determined to elucidate the structural differences in protein conformations between the two TTBK isoforms that could aid in SBDD for the design of inhibitors that selectively target TTBK1 over TTBK2.


Assuntos
Domínio Catalítico/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Difração de Raios X/métodos , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , Humanos , Proteínas Serina-Treonina Quinases/metabolismo
8.
ACS Med Chem Lett ; 11(4): 485-490, 2020 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-32292554

RESUMO

Apoptosis signal-regulating kinase 1 (ASK1) is a key mediator in the apoptotic and inflammatory cellular stress response. To investigate the therapeutic value of modulating this pathway in neurological disease, we have completed medicinal chemistry studies to identify novel CNS-penetrant ASK1 inhibitors starting from peripherally restricted compounds reported in the literature. This effort led to the discovery of 21, a novel ASK1 inhibitor with good potency (cell IC50 = 138 nM), low clearance (rat Cl/Clu = 0.36/6.7 L h-1 kg-1) and good CNS penetration (rat K p,uu = 0.38).

9.
J Med Chem ; 62(23): 10740-10756, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31710475

RESUMO

Structural analysis of a known apoptosis signal-regulating kinase 1 (ASK1) inhibitor bound to its kinase domain led to the design and synthesis of the novel macrocyclic inhibitor 8 (cell IC50 = 1.2 µM). The profile of this compound was optimized for CNS penetration following two independent strategies: a rational design approach leading to 19 and a parallel synthesis approach leading to 26. Both analogs are potent ASK1 inhibitors in biochemical and cellular assays (19, cell IC50 = 95 nM; 26, cell IC50 = 123 nM) and have moderate to low efflux ratio (ER) in an MDR1-MDCK assay (19, ER = 5.2; 26, ER = 1.5). In vivo PK studies revealed that inhibitor 19 had moderate CNS penetration (Kpuu = 0.17) and analog 26 had high CNS penetration (Kpuu = 1.0).


Assuntos
MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Animais , Encéfalo/metabolismo , Desenho de Fármacos , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Compostos Macrocíclicos/química , Estrutura Molecular , Ratos
10.
Protein Sci ; 27(3): 672-680, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29280296

RESUMO

The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1-GluA2 protein-protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1-GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure-aided drug design but were unsuccessful in producing a co-crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co-crystal structure of the PDZ domain of PICK1 bound to BIO124.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Cristalografia , Desenho de Fármacos , Humanos , Modelos Moleculares , Conformação Molecular , Domínios PDZ , Ligação Proteica/efeitos dos fármacos , Receptores de AMPA/metabolismo , Relação Estrutura-Atividade
11.
Sci Rep ; 8(1): 13438, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194389

RESUMO

Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aß-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Espinhas Dendríticas/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Sinapses/metabolismo , Animais , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Espinhas Dendríticas/patologia , Desenho de Fármacos , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Proteínas Nucleares/metabolismo , Domínios PDZ , Receptores de AMPA/metabolismo , Sinapses/patologia
12.
Protein Sci ; 19(3): 429-39, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20052711

RESUMO

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 A resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 A resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.


Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Cristalografia por Raios X , Dasatinibe , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Tiazóis/química
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