RESUMO
The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.
Assuntos
Administração Intravenosa , Animais , Administração Oral , Camundongos , Relação Estrutura-Atividade , Humanos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Estrutura MolecularRESUMO
As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ MÏ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ MÏ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ MÏ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.
Assuntos
Diferenciação Celular/fisiologia , Inflamação/metabolismo , Interferon gama/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Psoríase/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fenótipo , Proteômica/métodos , Pele/metabolismoRESUMO
The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Biomarcadores , Células Cultivadas , Citocinas/metabolismo , Humanos , Imunofenotipagem , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/efeitos adversos , Macrófagos/efeitos dos fármacos , Células THP-1RESUMO
A novel series of indazole non-steroidal glucocorticoid receptor agonist has been discovered. This series features a sulfonamide central core and meta amides which interact with the extended ligand binding domain. This series has produced some of the most potent and least lipophilic agonists of which we are aware such as 20a (NFκB pIC(50) 8.3 (100%), clogP 1.9). Certain analogues in this series also display evidence for modulated pharmacology.
Assuntos
Indazóis/química , Receptores de Glucocorticoides/agonistas , Sulfonamidas/síntese química , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indazóis/síntese química , Indazóis/farmacologia , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.