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1.
iScience ; 27(2): 108839, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38303712

RESUMO

ERBB receptor tyrosine kinases are involved in development and diseases like cancer, cardiovascular, neurodevelopmental, and mental disorders. Although existing drugs target ERBB receptors, the next generation of drugs requires enhanced selectivity and understanding of physiological pathway responses to improve efficiency and reduce side effects. To address this, we developed a multilevel barcoded reporter profiling assay, termed 'ERBBprofiler', in living cells to monitor the activity of all ERBB targets and key physiological pathways simultaneously. This assay helps differentiate on-target therapeutic effects from off-target and off-pathway side effects of ERBB antagonists. To challenge the assay, eight established ERBB antagonists were profiled. Known effects were confirmed, and previously uncharacterized properties were discovered, such as pyrotinib's preference for ERBB4 over EGFR. Additionally, two lead compounds selectively targeting ERBB4 were profiled, showing promise for clinical trials. Taken together, this multiparametric profiling approach can guide early-stage drug development and lead to improved future therapeutic interventions.

2.
Eur J Med Chem ; 265: 116053, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38141285

RESUMO

The colony-stimulating factor 1 receptor (CSF1R) is an attractive target for inflammation disorders and cancers. Based on a series of pyrrolo[2,3-d]pyrimidine containing two carbo-aromatic rings, we have searched for new CSF1R inhibitors having a higher fraction of sp3-atoms. The phenyl unit in the 4-amino group could efficiently be replaced by tetrahydropyran (THP) retaining inhibitor potency. Exchanging the 6-aryl group with cyclohex-2-ene units also resulted in highly potent compounds, while fully saturated ring systems at C-6 led to a loss of activity. The structure-activity relationship study evaluating THP containing pyrrolo[2,3-d]pyrimidine derivates identified several highly active inhibitors by enzymatic studies. A comparison of 11 pairs of THP and aromatic compounds showed that inhibitors containing THP had clear benefits in terms of enzymatic potency, solubility, and cell toxicity. Guided by cellular experiments in Ba/F3 cells, five CSF1R inhibitors were further profiled in ADME assays, indicating the para-aniline derivative 16t as the most attractive compound for further development.


Assuntos
Pirimidinas , Receptores Proteína Tirosina Quinases , Pirimidinas/farmacologia , Pirróis/farmacologia , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologia
3.
Virus Res ; 335: 199200, 2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37591314

RESUMO

Human cytomegalovirus (HCMV) infection is shaped by a tightly regulated interplay between viral and cellular proteins. Distinct kinase activities, such as the viral cyclin-dependent kinase ortholog (vCDK) pUL97 and cellular CDK7 are both crucial for efficient viral replication. Previously, we reported that both kinases, vCDK/pUL97 and CDK7, interact with cyclin H, thereby achieving an enhanced level of kinase activity and overall functionality in viral replication. Here we provide a variety of novel results, as generated on a methodologically extended basis, and present a concept for the codetermination of viral replication efficiency through these kinase activities: (i) cyclin H expression, in various human cell types, is substantially upregulated by strains of HCMV including the clinically relevant HCMV Merlin; (ii) vCDK/pUL97 interacts with human cyclin H in both HCMV-infected and plasmid-transfected cell systems; (iii) a doxycycline-inducible shRNA-dependent knock-down (KD) of cyclin H significantly reduces pUL97 activity (qSox in vitro kinase assay); (iv) accordingly, pUL97 in vitro kinase activity is seen significantly increased upon addition of recombinant cyclin H; (v) as a point of specific importance, human CDK7 activity shows an increase by vCDK/pUL97-mediated trans-stimulation (whereas pUL97 is not stimulated by CDK7); (vi) phosphosite-specific antibodies indicate an upregulated CDK7 phosphorylation upon HCMV infection, as mediated through a pUL97-specific modulatory effect (i.e. shown by pUL97 inhibitor treatment or pUL97-deficient viral mutant); (vii) finally, an efficient KD of cyclin H in primary fibroblasts generally results in an impaired HCMV replication efficiency as measured on protein and genomic levels. These results show evidence for the codetermination of viral replication by vCDK/pUL97, cyclin H and CDK7, thus supporting the specific importance of cyclin H as a central regulatory factor, and suggesting novel targeting options for antiviral drugs.


Assuntos
Quinases Ciclina-Dependentes , Citomegalovirus , Humanos , Antivirais , Ciclina H , Quinases Ciclina-Dependentes/genética , Citomegalovirus/genética , Fosforilação
4.
ChemMedChem ; 18(11): e202200631, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36883965

RESUMO

Due to worldwide increasing resistances, there is a considerable need for antibacterial compounds with modes of action not yet realized in commercial antibiotics. One such promising structure is the acetyl-CoA carboxylase (ACC) inhibitor moiramide B which shows strong antibacterial activity against gram-positive bacteria such as Bacillus subtilis and weaker activities against gram-negative bacteria. However, the narrow structure-activity relationship of the pseudopeptide unit of moiramide B represents a formidable challenge for any optimization strategy. In contrast, the lipophilic fatty acid tail is considered an unspecific vehicle responsible only for the transport of moiramide into the bacterial cell. Here we show that the sorbic acid unit, in fact, is highly relevant for ACC inhibition. A hitherto undescribed sub-pocket at the end of the sorbic acid channel binds strongly aromatic rings and allows the development of moiramide derivatives with altered antibacterial profiles including anti-tubercular activity.


Assuntos
Antibacterianos , Ácido Sórbico , Antibacterianos/farmacologia , Antibacterianos/química , Amidas/farmacologia , Succinimidas/farmacologia , Testes de Sensibilidade Microbiana
5.
Commun Biol ; 5(1): 1206, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352263

RESUMO

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.


Assuntos
Receptores Acoplados a Proteínas G , Fosforilação , Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Imunoensaio
6.
ChemMedChem ; 17(22): e202200392, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-35979853

RESUMO

Ras proteins are implicated in some of the most common life-threatening cancers. Despite intense research during the past three decades, progress towards small-molecule inhibitors of mutant Ras proteins still has been limited. Only recently has significant progress been made, in particular with ligands for binding sites located in the switch II and between the switch I and switch II region of K-Ras4B. However, the structural diversity of inhibitors identified for those sites to date is narrow. Herein, we show that hydrazones and oxime ethers of specific bis(het)aryl ketones represent structurally variable chemotypes for new GDP/GTP-exchange inhibitors with significant cellular activity.


Assuntos
Éteres , Proteínas ras , Sítios de Ligação , Proteínas Fúngicas , Hidrazonas/farmacologia
7.
ChemMedChem ; 16(16): 2504-2514, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-33899342

RESUMO

Oncogenic Ras proteins are implicated in the most common life-threatening cancers. Despite intense research over the past two decades, the progress towards small-molecule inhibitors has been limited. One reason for this failure is that Ras proteins interact with their effectors only via protein-protein interactions, which are notoriously difficult to address with small organic molecules. Herein we describe an alternative strategy, which prevents farnesylation and subsequent membrane insertion, a prerequisite for the activation of Ras proteins. Our approach is based on sequence-selective supramolecular receptors which bind to the C-terminal farnesyl transferase recognition unit of Ras and Rheb proteins and covalently modify the essential cysteine in the so-called CaaX-box.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/química , Proteínas Quinases Ativadas por Mitógeno/química , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositol 3-Quinases/química , Ligação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Transdução de Sinais
8.
Antimicrob Agents Chemother ; 59(4): 2062-71, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25624324

RESUMO

Protein kinases represent central and multifunctional regulators of a balanced virus-host interaction. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. Previously, we developed a CDK7 inhibitor, LDC4297, that inhibits CDK7 in vitro in the nano-picomolar range. Novel data from a kinome-wide evaluation (>330 kinases profiled in vitro) demonstrate a kinase selectivity. Importantly, we provide first evidence for the antiviral potential of the CDK7 inhibitor LDC4297, i.e., in exerting a block of the replication of human cytomegalovirus (HCMV) in primary human fibroblasts at nanomolar concentrations (50% effective concentration, 24.5 ± 1.3 nM). As a unique feature compared to approved antiherpesviral drugs, inhibition occurred already at the immediate-early level of HCMV gene expression. The mode of antiviral action was considered multifaceted since CDK7-regulated cellular factors that are supportive of HCMV replication were substantially affected by the inhibitors. An effect of LDC4297 was identified in the interference with HCMV-driven inactivation of retinoblastoma protein (Rb), a regulatory step generally considered a hallmark of herpesviral replication. In line with this finding, a broad inhibitory activity of the drug could be demonstrated against a selection of human and animal herpesviruses and adenoviruses, whereas other viruses only showed intermediate drug sensitivity. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.


Assuntos
Antivirais/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Pirazóis/farmacologia , Triazinas/farmacologia , Adenoviridae/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Fibroblastos/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesviridae/efeitos dos fármacos , Humanos , Camundongos , Fosforilação , Replicação Viral/efeitos dos fármacos
9.
Chem Biol ; 11(2): 211-23, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15123283

RESUMO

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.


Assuntos
Adenina/análogos & derivados , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Adenina/química , Adenina/metabolismo , Animais , Sítios de Ligação , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Inibidores Enzimáticos/química , Ligantes , Análise Serial de Proteínas , Proteoma/genética , Saccharomyces/genética
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