RESUMO
Aim: To quantify the free form of a protein as a target-engagement biomarker in nonhuman primate serum, a Meso Scale Discovery ligand-binding assay was developed and qualified. Results: The initial assay produced an unexpected artifact when used to measure the free target in study samples dosed with drug. By using incurred study samples dosed with high drug levels to test assay performance, we developed an alternative assay that does not suffer from drug interference. Conclusion: Our work demonstrated that an assay designed to measure free target may not necessarily deliver reliable quantitation. In our case, incurred study samples dosed with drug proved to be useful in developing an alternative free assay that does not suffer from drug interference.
Assuntos
Desenvolvimento de Medicamentos , Preparações Farmacêuticas/sangue , Animais , Biomarcadores/sangue , LigantesRESUMO
Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [KD]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (kon) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 106 and 1.83 × 105, respectively. The dissociation constant (koff) values were 4.29 × 10â»4 and 2.14 × 10â»4, respectively. Calculated KD values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the kon values for human IL-5 for reslizumab and mepolizumab were 3.17 × 106 and 1.32 × 105, respectively. The koff values were 1.36 × 10â»5 and 1.48 × 10â»5, respectively. Measured KD values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC50) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC50 values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.
RESUMO
TL1A is an attractive therapeutic target for the treatment of mucosal inflammation associated with inflammatory bowel disease (IBD) and asthma. Blockade of the TL1A pathway has been shown to reduce inflammatory responses while leaving baseline immunity intact, and to be beneficial in animal models of colitis and asthma. Given the therapeutic potential of blocking this pathway in IBD and asthma, we developed C03V, a human antibody that binds with high affinity to soluble and membrane-bound TL1A. In an assay measuring apoptosis induced by exogenous TL1A, C03V was 43-fold more potent than the next most potent anti-TL1A antibody analyzed. C03V also potently inhibited endogenous TL1A activity in a primary cell-based assay. This potency was linked to the C03V-binding epitope on TL1A, encompassing the residue R32. This residue is critical for the binding of TL1A to its signaling receptor DR3 but not to its decoy receptor DcR3, and explains why C03V inhibited TL1A-DR3 binding to a much greater extent than TL1A-DcR3 binding. This characteristic may be advantageous to preserve some of the homeostatic functions of DcR3, such as TL1A antagonism. In colitis models, C03V significantly ameliorated microscopic, macroscopic and clinical aspects of disease pathology, and in an asthma model it significantly reduced airways inflammation. Notable in both types of disease model was the reduction in fibrosis observed after C03V treatment. C03V has the potential to address unmet medical needs in asthma and IBD.
Assuntos
Anticorpos Monoclonais/farmacologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Anticorpos Monoclonais/química , Asma/imunologia , Humanos , Doenças Inflamatórias Intestinais/imunologiaRESUMO
During SAR development of previously reported pyrrolocarbazole 1, a potent PARP-1 inhibitor, compound 14, was discovered serendipitously to be a prodrug of compound 1.
Assuntos
Carbazóis/química , Inibidores Enzimáticos/química , Ftalimidas/química , Inibidores de Poli(ADP-Ribose) Polimerases , Pró-Fármacos/química , Animais , Carbazóis/metabolismo , Carbazóis/farmacocinética , Galinhas , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Meia-Vida , Ftalimidas/metabolismo , Ftalimidas/farmacocinética , Poli(ADP-Ribose) Polimerases/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
An understanding of the dynamics of drug-target interactions is important in the drug discovery process. Information related to the binding kinetics of a drug toward its target or off-target aids in determining the efficacy or toxicity of a drug. Biophysical techniques such as surface plasmon resonance (SPR) have been available for over 20 years, but have been predominantly utilized to characterize protein-protein interactions. With improvements in instrument sensitivity and data analysis software, interactions between proteins (such as kinases) and small molecules have been successfully evaluated. More recently, the LanthaScreen Eu kinase binding assay for characterizing kinase inhibitors has been described. This assay monitors displacement of an Alexa Fluor 647-labeled tracer from the ATP-binding site of an epitope-tagged kinase by a test compound. Such behavior results in a decrease in time-resolved fluorescence energy transfer signal. In this report, a side-by-side comparison of the LanthaScreen Eu kinase binding assay and the SPR method was performed using inhibitors of focal adhesion kinase. The two methods yielded comparable results and identified compounds with time-dependent inhibition and relatively slow dissociation.
Assuntos
Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/farmacocinética , Estaurosporina/metabolismo , Estaurosporina/farmacocinéticaRESUMO
Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de ProteassomaRESUMO
Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.
Assuntos
Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/análise , Anticorpos/análise , Anticorpos/imunologia , Anticorpos Anti-Idiotípicos/análise , Anticorpos Anti-Idiotípicos/imunologia , Linhagem Celular , Corantes , DNA Complementar/genética , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceína , Proteína-Tirosina Quinases de Adesão Focal/análise , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Humanos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , c-Mer Tirosina QuinaseRESUMO
Proteasome, a large multicatalytic proteinase complex that plays an important role in processing of proteins, has been shown to possess multiple catalytic activities. Among its various activities, the 'chymotrypsin-like' activity of proteasome has emerged as the focus of drug discovery efforts in cancer therapy. Herein we report chiral boronate derived novel, potent, selective and cell-permeable peptidomimetic inhibitors 6 and 7 that displayed activity against various rodent and human tumor cell lines (in vitro).
Assuntos
Ácidos Borônicos/química , Nitrocompostos/química , Inibidores de Proteases/química , Inibidores de Proteassoma , Animais , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Fígado/enzimologia , Camundongos , Neoplasias/tratamento farmacológico , Nitrocompostos/farmacologia , Nitrocompostos/uso terapêutico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Synthesis and activity of a series of 3-aroyl-derived analogs of novel pyrrolocarbazole 1 as poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors are disclosed.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Antineoplásicos/farmacologia , Carbazóis/química , Química Farmacêutica/métodos , Dano ao DNA , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , NAD/química , Neoplasias/tratamento farmacológico , Células PC12 , Poli(ADP-Ribose) Polimerase-1 , RatosRESUMO
The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/química , Janus Quinase 2/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Progressão da Doença , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Mutação , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/síntese química , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Fatores de TempoRESUMO
The hepatocyte growth factor (HGF)-c-Met signaling axis is involved in the mediation of many biological activities, including angiogenesis, proliferation, cell survival, cell motility, and morphogenesis. Dysregulation of c-Met signaling (e.g., overexpression or increased activation) is associated with the proliferation and metastasis of a wide range of tumor types, including breast, liver, lung, colorectal, gastric, bladder, and prostate, among others. Inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many cancers. Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which pharmaceutical properties were modulated by substituents appended on the C2-benzazepinone ring. In particular, certain-3-amidobenzazepin-2-one analogs had improved oral bioavailability and were evaluated in PK/PD and efficacy models. Lead compounds demonstrated tumor stasis with partial regressions when evaluated in a GTL-16 tumor xenograft mouse model.
Assuntos
Benzazepinas/química , Benzazepinas/farmacocinética , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacocinética , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Benzazepinas/síntese química , Disponibilidade Biológica , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/enzimologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirimidinas/síntese química , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
The HGF-c-Met signaling axis is an important paracrine mediator of epithelial-mesenchymal cell interactions involving the regulation of multiple cellular activities including cell motility, mitogenesis, morphogenesis, and angiogenesis. Dysregulation of c-Met signaling (e.g., overexpression or increased activation) is associated with the development of a wide range of tumor types; thus, inhibiting the HGF-c-Met pathway is predicted to lead to anti-tumor effects in many cancers. Elaboration of a 2-arylaminopyrimidine scaffold led to a series of potent c-Met inhibitors bearing a C4-2-amino-N-methylbenzamide group. Specifically, a series of C2-benzazepinone analogs demonstrated potent inhibition of c-Met in enzymatic and cellular assays. Kinase selectivity could be tuned by varying the nature of the alkyl group on the benzazepinone nitrogen.
Assuntos
Compostos Bicíclicos com Pontes/química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirimidinas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Relação Estrutura-AtividadeRESUMO
Elaboration of the SAR around a series of 2,4-diaminopyrimidines led to a number of c-Met inhibitors in which kinase selectivity was modulated by substituents appended on the C4-aminobenzamide ring and the nature of the C2-aminoaryl ring. Further lead optimization of the C2-aminoaryl group led to benzoxazepine analogs whose pharmaceutical properties were modulated by the nature of the substituent on the benzoxazepine nitrogen. Tumor stasis (with partial regressions) were observed when an orally bioavailable analog was evaluated in a GTL-16 tumor xenograft mouse model. Subsequent PK/PD studies suggested that a metabolite contributed to the overall in vivo response.
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/uso terapêutico , Administração Oral , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Humanos , Camundongos , Camundongos Nus , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K(m), V(max), Z' factor analysis, and IC(50) value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 microM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 microM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC(50) values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this cost-effective method.
Assuntos
Bioensaio/métodos , Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Moduladores de Transporte de Membrana/análise , Moduladores de Transporte de Membrana/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Glicina/metabolismo , Humanos , Cinética , Padrões de Referência , Trítio/metabolismoRESUMO
The optimization of the dihydronaphthyl[3,4-a]pyrrolo[3,4-c]carbazole-5-one R(2) and R(12) positions led to the identification of the first MLK1 and MLK3 subtype-selective inhibitors within the MLK family. Compounds 14 (CEP-5104) and 16 (CEP-6331) displayed good potency for MLK1 and MLK3 inhibition with a greater than 30- to 100-fold selectivity for related family members MLK2 and DLK. Compounds 14 and 16 were orally active in vivo in a mouse MPTP biochemical efficacy model that was comparable to the first-generation pan-MLK inhibitor 1 (CEP-1347). The MLK1 structure-activity relationships were supported by the first-reported X-ray crystal structure of MLK1 bound with 16.
Assuntos
Carbazóis/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Pirrolidinonas/farmacologia , Administração Oral , Animais , Carbazóis/administração & dosagem , Carbazóis/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Pirrolidinonas/administração & dosagem , Pirrolidinonas/química , Ratos , MAP Quinase Quinase Quinase 11 Ativada por MitógenoRESUMO
The effect of the potent and selective poly(ADP-ribose) (PAR) polymerase-1 [and PAR polymerase-2] inhibitor CEP-8983 on the ability to sensitize chemoresistant glioblastoma (RG2), rhabdomyosarcoma (RH18), neuroblastoma (NB1691), and colon carcinoma (HT29) tumor cells to temozolomide- and camptothecin-induced cytotoxicity, DNA damage, and G(2)-M arrest and on the potentiation of chemotherapy-induced myelotoxicity was evaluated using in vitro assays. In addition, the effect of the prodrug CEP-9722 in combination with temozolomide and/or irinotecan on PAR accumulation and tumor growth was also determined using glioblastoma and/or colon carcinoma xenografts relative to chemotherapy alone. CEP-8983 sensitized carcinoma cells to the growth-inhibitory effects of temozolomide and/or SN38 increased the fraction of and/or lengthened duration of time tumor cells accumulated in chemotherapy-induced G(2)-M arrest and sensitized tumor cells to chemotherapy-induced DNA damage and apoptosis. A granulocyte-macrophage colony-forming unit colony formation assay showed that coincubation of CEP-8983 with temozolomide or topotecan did not potentiate chemotherapy-associated myelotoxicity. CEP-9722 (136 mg/kg) administered with temozolomide (68 mg/kg for 5 days) or irinotecan (10 mg/kg for 5 days) inhibited significantly the growth of RG2 tumors (60%) or HT29 tumors (80%) compared with temozolomide or irinotecan monotherapy, respectively. In addition, CEP-9722 showed "stand alone" antitumor efficacy in these preclinical xenografts. In vivo biochemical efficacy studies showed that CEP-9722 attenuated PAR accumulation in glioma xenografts in a dose- and time-related manner. These data indicate that CEP-8983 and its prodrug are effective chemosensitizing agents when administered in combination with select chemotherapeutic agents against chemoresistant tumors.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Carbazóis/farmacologia , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ftalimidas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Camptotecina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Dacarbazina/farmacologia , Sinergismo Farmacológico , Fase G2/efeitos dos fármacos , Humanos , Irinotecano , Camundongos , Poli Adenosina Difosfato Ribose/metabolismo , Ratos , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Synthesis and activity of a series of 4-thiazol-yl substituted analogs of novel pyrrolocarbazole 1 as poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been disclosed.
Assuntos
Carbazóis/síntese química , Carbazóis/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Tiazóis/síntese química , Tiazóis/farmacologia , Animais , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Células PC12 , Poli(ADP-Ribose) Polimerase-1 , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
A series of novel pyrrolocarbazoles was synthesized as potential PARP-1 inhibitors. Pyrrolocarbazole 1 was identified as a potent PARP-1 inhibitor (IC50 = 36 nM) from our internal database. Synthesis of analogs around this template with the aid of modeling studies led to the identification of the truncated imide 14. Compound 14 (IC50 = 40 nM), with deleted B-ring, was found to be an equipotent PARP-1 inhibitor.
Assuntos
Carbazóis/síntese química , Carbazóis/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Carbazóis/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Poli(ADP-Ribose) Polimerase-1 , Relação Estrutura-AtividadeRESUMO
A series of novel pyrrolocarbazole lactams was identified as potent PARP-1 inhibitors in vitro and in a PC12 cellular NAD(+) depletion assay. The SAR trends of substituents at the 3-position, as well as the effect of blocking the indole or lactam NH-groups of the template by methylation or formylation, are discussed in relation to molecular modeling studies.