RESUMO
Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Peptídeos/farmacologia , beta Catenina/antagonistas & inibidores , Compostos Bicíclicos Heterocíclicos com Pontes/química , Modelos Moleculares , Peptídeos/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.
Assuntos
Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Medição da Troca de Deutério , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Espectrometria de Massas , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.
Assuntos
Neoplasias da Mama , Receptores de Estrogênio , Camundongos , Humanos , Animais , Feminino , Receptores de Estrogênio/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Antagonistas de Estrogênios/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptor alfa de Estrogênio/metabolismo , Linhagem CelularRESUMO
ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ligação a DNA/antagonistas & inibidores , Linhagem Celular Tumoral , Cristalografia por Raios X , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Especificidade por Substrato , Ensaio Tumoral de Célula-TroncoRESUMO
Herein we describe our efforts using a late stage functionalization together with more traditional synthetic approaches to generate fluorinated analogues of the clinical candidate AZD9833. The effects of the addition of fluorine on the lipophilicity, permeability, and metabolism are discussed. Many of these changes were tolerated in terms of pharmacology and resulted in high quality molecules which reached advanced stages of profiling in the testing cascade.
RESUMO
Herein we report the optimization of a series of tricyclic indazoles as selective estrogen receptor degraders (SERD) and antagonists for the treatment of ER+ breast cancer. Structure based design together with systematic investigation of each region of the molecular architecture led to the identification of N-[1-(3-fluoropropyl)azetidin-3-yl]-6-[(6S,8R)-8-methyl-7-(2,2,2-trifluoroethyl)-6,7,8,9-tetrahydro-3H-pyrazolo[4,3-f]isoquinolin-6-yl]pyridin-3-amine (28). This compound was demonstrated to be a highly potent SERD that showed a pharmacological profile comparable to fulvestrant in its ability to degrade ERα in both MCF-7 and CAMA-1 cell lines. A stringent control of lipophilicity ensured that 28 had favorable physicochemical and preclinical pharmacokinetic properties for oral administration. This, combined with demonstration of potent in vivo activity in mouse xenograft models, resulted in progression of this compound, also known as AZD9833, into clinical trials.
Assuntos
Antineoplásicos/administração & dosagem , Moduladores Seletivos de Receptor Estrogênico/administração & dosagem , Administração Oral , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ciclização , Descoberta de Drogas , Feminino , Humanos , Lipídeos/química , Estrutura Molecular , Moduladores Seletivos de Receptor Estrogênico/química , Moduladores Seletivos de Receptor Estrogênico/farmacocinética , Relação Estrutura-AtividadeRESUMO
Herein we report the use of metathesis to construct a novel tetracyclic core in a series of estrogen receptor degraders. This improved the chemical stability, as assessed using an NMR-MS based assay, and gave a molecule with excellent physicochemical properties and pharmacokinetics in rat. X-ray crystallography established minimal perturbation of the bridged compounds relative to the unbridged analogues in the receptor binding pocket. Unfortunately, despite retaining excellent binding to ERα, this adversely affected the ability of the compounds to degrade the receptor.
RESUMO
Herein, we report the identification and synthesis of a series of tricyclic indazoles as a novel class of selective estrogen receptor degrader antagonists. Replacement of a phenol, present in our previously reported tetrahydroisoquinoline scaffold, with an indazole group led to the removal of a reactive metabolite signal in an in vitro glutathione trapping assay. Further optimization, guided by X-ray crystal structures and NMR conformational work, varied the alkyl side chain and pendant aryl group and resulted in compounds with low turnover in human hepatocytes and enhanced chemical stability. Compound 9 was profiled as a representative of the series in terms of pharmacology and demonstrated the desired estrogen receptor α degrader-antagonist profile and demonstrated activity in a xenograft model of breast cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Antagonistas do Receptor de Estrogênio/uso terapêutico , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Indazóis/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Antagonistas do Receptor de Estrogênio/síntese química , Antagonistas do Receptor de Estrogênio/farmacocinética , Receptor alfa de Estrogênio/metabolismo , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Humanos , Indazóis/síntese química , Indazóis/farmacocinética , Células MCF-7 , Masculino , Camundongos SCID , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.