RESUMO
Thermal shift assays (TSAs) are among the most commonly used biophysical approaches in drug discovery in both academic and industrial settings. However, the most common interpretation of the data generated by a TSA is purely qualitative, using only the change in melting temperature (ΔTm) as a metric. This has left many questions surrounding the interpretation of the data as well as whether the TSA truly correlates with other assays. TSAs also lack theoretical descriptions of the melt behavior of proteins in the presence of multiple ligands. Here we describe a novel simplified analytical framework based on "pseudoisothermal" models as well as exact thermodynamic descriptions of protein-ligand melt behavior rooted in changes in the entropy of melting. We show how the models are broad and independently applicable, in that they can describe the behavior of any macromolecule such as a protein or DNA and demonstrate good correlations with other techniques. These models are shown to give good descriptions of assay systems containing single or multiple ligands and can determine the mechanism of interaction. The models are derived from first principles, and the theoretical justification is discussed.
Assuntos
Anidrase Carbônica II/química , DNA/química , Entropia , Glutationa Transferase/química , Modelos Teóricos , Termodinâmica , Anidrase Carbônica II/metabolismo , DNA/metabolismo , Glutationa Transferase/metabolismo , Humanos , Cinética , LigantesRESUMO
We report the further optimization of our series 1H-pyrazolo[3,4-g]hexahydro-isoquinoline sulfonamides as GR antagonists. By incorporating a heteroaryl ketone group at the ring junction, we have obtained compounds with excellent functional GR antagonism. Optimization of the sulfonamide substituent has provided compounds with a very desirable overall profile, including minimal hERG activity, good bioavailability and in vivo efficacy.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Isoquinolinas/química , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Células Hep G2 , Humanos , Isoquinolinas/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Pirazóis/química , Ratos , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.
Assuntos
Inibidores Enzimáticos/farmacologia , Hidrocarbonetos Aromáticos/farmacologia , Ácidos Hidroxâmicos/farmacologia , Peroxidase/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Humanos , Hidrocarbonetos Aromáticos/síntese química , Hidrocarbonetos Aromáticos/química , Ácidos Hidroxâmicos/química , Cinética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Neutrófilos/enzimologia , Peroxidase/antagonistas & inibidores , Peroxidase/metabolismo , Ligação ProteicaRESUMO
G-protein-coupled receptors (GPCRs) are the largest and most versatile cell surface receptor family with a broad repertoire of ligands and functions. We've learned an enormous amount about discovering drugs of this receptor class since the first GPCR was cloned and expressed in 1986, such that it's now well-recognized that GPCRs are the most successful target class for approved drugs. Here we take the reader through a GPCR drug discovery journey from target to the clinic, highlighting the key learnings, best practices, challenges, trends and insights on discovering drugs that ultimately modulate GPCR function therapeutically in patients. The future of GPCR drug discovery is inspiring, with more desirable drug mechanisms and new technologies enabling the delivery of better and more successful drugs.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Herein, we report the discovery of a first-in-class chemotype 2-(alkylsulfonamido)thiazol-4-yl)acetamides that act as pan-selective inhibitors of cytidine 5'-triphosphate synthetase (CTPS1/2), critical enzymes in the de novo pyrimidine synthesis pathway. Weak inhibitors identified from a high-throughput screening of 240K compounds have been optimized to a potent, orally active agent, compound 27, which has shown significant pharmacological responses at 10 mg/kg dose BID in a well-established animal model of inflammation.
Assuntos
Carbono-Nitrogênio Ligases , Inibidores Enzimáticos , Animais , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Carbono-Nitrogênio Ligases/metabolismo , Proliferação de Células , Ensaios de Triagem em Larga EscalaRESUMO
To try and generate broad spectrum human rhinovirus VP1 inhibitors with more attractive physicochemical, DMPK and safety profiles, we explored the current SAR of known VP1 compounds. This lead to the identification of specific structural regions where reduction in polarity can be achieved, so guiding chemistry to analogues with significantly superior profiles to previously reported inhibitors.
Assuntos
Antivirais/química , Antivirais/farmacologia , Proteínas do Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Infecções por Picornaviridae/tratamento farmacológico , Rhinovirus/efeitos dos fármacos , Animais , Antivirais/metabolismo , Humanos , Microssomos/metabolismo , Ligação Proteica , Piridazinas/química , Piridazinas/metabolismo , Piridazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The identification of two novel series of formyl peptide receptor 1 (FPR1) antagonists are reported, represented by methionine benzimidazole 6 and diamide 7. Both series specifically inhibited the binding of labelled fMLF to hrFPR1 and selectively antagonized FPR1 function in human neutrophils, making them useful in vitro validation tools for the target.
Assuntos
Descoberta de Drogas , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/química , Humanos , Estrutura MolecularRESUMO
A series of pyrazole inhibitors of the human FPR1 receptor have been identified from high throughput screening. The compounds demonstrate potent inhibition in human neutrophils and attractive physicochemical and in vitro DMPK profiles to be of further interest.
Assuntos
Pirazóis/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Descoberta de Drogas , Humanos , Neutrófilos/efeitos dos fármacos , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
A novel series of small molecule C5a antagonists is reported. In particular, in vitro metabolic studies and solution based combinatorial synthesis are demonstrated as useful tools for the rapid identification of antagonists with low in vitro clearance. Members of this series specifically inhibited the binding of (125)I-labeled C5a to human recombinant C5a receptor (C5aR). In functional cell assays these compounds displayed surmountable antagonism against C5a and did not demonstrate any detectable agonist activity.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Derivados de Benzeno/síntese química , Derivados de Benzeno/farmacologia , Furanos/síntese química , Furanos/farmacologia , Receptores de Complemento/antagonistas & inibidores , Amidas/química , Animais , Derivados de Benzeno/química , Técnicas de Química Combinatória , Cães , Furanos/química , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The nonselective glucocorticoid receptor (GR) antagonist mifepristone has been approved in the U.S. for the treatment of selected patients with Cushing's syndrome. While this drug is highly effective, lack of selectivity for GR leads to unwanted side effects in some patients. Optimization of the previously described fused azadecalin series of selective GR antagonists led to the identification of CORT125134, which is currently being evaluated in a phase 2 clinical study in patients with Cushing's syndrome.
Assuntos
Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Cromatografia Líquida , Células Hep G2 , Humanos , Espectrometria de MassasRESUMO
N-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 1 was identified as a hit in a CCR4 receptor antagonist high-throughput screen (HTS) of a subset of the AstraZeneca compound bank. As a hit with a lead-like profile, it was an excellent starting point for a CCR4 receptor antagonist program and enabled the rapid progression through the Lead Identification and Lead Optimization phases resulting in the discovery of two bioavailable CCR4 receptor antagonist candidate drugs.
RESUMO
CONTEXT: Endogenous Cushing's syndrome is caused by chronically elevated levels of cortisol. Mifepristone, a glucocorticoid receptor (GR) antagonist, is approved for the treatment of Cushing's syndrome. Currently there is an unmet clinical need for a direct biochemical method for monitoring the immediate effectiveness of mifepristone in patients with Cushing's syndrome. The glucocorticoid induction of FK506-binding protein 5 (FKBP5) expression is rapid and has been shown to be attenuated by GR antagonists in a range of in vitro and in vivo models. OBJECTIVE: The objective of the study was to develop a quantitative PCR assay for FKBP5 mRNA expression in blood and apply it to measure the inhibition of glucocorticoid-induced FKBP5 expression by GR antagonists in healthy human subjects. METHODS: Briefly, blood samples were acquired from a phase I study in which healthy human subjects were administered either a single dose of the GR agonist prednisone with and without coadministration of a single oral dose of mifepristone or glucocorticoid receptor antagonist (CORT125134) or multiple daily doses of CORT125134 over 14 days with coadministration of prednisone with the final dose. FKBP5 mRNA levels were analyzed by quantitative PCR in blood samples collected at selected time points. SETTING: The study was conducted at Quotient Clinical (Nottingham, United Kingdom). RESULTS: Oral administration of the glucocorticoid prednisone to healthy human subjects resulted in a time-dependent increase of FKBP5 mRNA to peak levels of approximately 12-fold compared with unstimulated levels within 4 hours of steroid administration, followed by a reduction to baseline levels within 24 hours. Furthermore, oral administration of mifepristone or the selective GR antagonist CORT125134 had the desired effect of inhibiting prednisone-mediated activation of GR as seen by a reduction of FKBP5 mRNA levels. CONCLUSIONS: The inhibition of FKBP5 mRNA expression by a selective GR antagonist is a potential clinical biomarker of GR antagonism.
Assuntos
Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Prednisona/farmacologia , RNA Mensageiro/sangue , Receptores de Glucocorticoides/antagonistas & inibidores , Proteínas de Ligação a Tacrolimo/sangue , Adulto , Animais , Bioensaio , Biomarcadores/sangue , Glucocorticoides/administração & dosagem , Antagonistas de Hormônios/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Mifepristona/administração & dosagem , Reação em Cadeia da Polimerase , Prednisona/administração & dosagem , RNA Mensageiro/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas de Ligação a Tacrolimo/efeitos dos fármacos , Adulto JovemRESUMO
Functional activity of N- and C-terminal fluorescent fusion proteins between STAT6 and EGFP was demonstrated through IL-4-dependent transcriptional activation and nuclear translocation. The N-terminal (EGFP-STAT6) fusion protein appeared to be more active than the C-terminal fusion. In HEK-293 cells both fusion proteins formed fluorescent nuclear foci following IL-4 stimulation, but in HeLa cells nuclear accumulation was homogeneous. Stimulation of the NF-kappaB pathway through TNFalpha treatment, or expression of p65-EGFP fusion protein, repressed both basal STAT6-dependent transcriptional activity and the extent of activation in response to IL-4. This indicates a novel mechanism of inhibition of STAT6 signalling by NF-kappaB activation.
Assuntos
Transdução de Sinais , Transativadores/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Interleucina-4/farmacologia , Cinética , Proteínas Luminescentes/genética , Microscopia Confocal , Microscopia de Fluorescência , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT6 , Transativadores/genética , Transativadores/fisiologia , Fator de Transcrição RelA , Ativação TranscricionalRESUMO
The T cell mitogen and plant glycoprotein, phytohaemagglutinin (PHA), is commonly used to stimulate peripheral blood mononuclear cell (PBMC) preparations to produce IL-2, IL-5, GM-CSF and IFN-γ and so provide an assay to detect immunosuppressants like FK506 and anti-inflammatories such as PDE IV inhibitors. During the early discovery of novel TLR agonists for the treatment of asthma we initially showed that PHA-L is a specific human TLR4 agonist, devoid of effects on equivalent TLR4 null cells. This TLR4 agonism was not due to LPS contamination of PHA-L, as polymyxin B was ineffective and unlike PHA-L, LPS did not stimulate TLR5 or TLR2/6. Also this specific PHA-L agonism of TLR4 was shown for different PHA forms, for example, PHA-P. This TLR lectin pharmacology finding was further explored by testing a broader panel of plant lectin representatives for agonism against a suite of hrTLR cell reporter assays (2/6, 3, 4, 5, 7, 8 and 9). Soybean agglutinin (SBA), concanavalin A (ConA) and PHA lectin family members only stimulated extracellular TLRs (2/6, 4 and 5) probably due to lack of intracellular access, whilst other lectins were either pan-active (WGA) or inactive (AIL). Interestingly SBA only stimulated TLR4, ConA, TLR2/6 and PHA-L, TLR2/6, 4 and 5. As each lectin family exhibits different sugar ligand specificity for interaction, these results suggest that the pharmacology of this TLR agonism is encoded by the lectin's carbohydrate recognition motifs and the appropriate surface presentation of these motifs on different TLRs.
Assuntos
Lectinas/farmacologia , Plantas/química , Receptores Toll-Like/agonistas , Linhagem Celular , Humanos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fito-Hemaglutininas/farmacologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Asthma and chronic obstructive pulmonary disease exacerbations are associated with human rhinovirus (HRV) lung infections for which there are no current effective antiviral therapies. To date, HRV infectivity of cells in vitro has been measured by a variety of biochemical and immunological methods. This paper describes the development of a high-throughput HRV infectivity assay using HeLa OHIO cells and a chemiluminescent-based ATP cell viability system, CellTiter-Glo from Promega, to measure HRV-induced cytopathic effect (CPE). This CellTiter-Glo assay was validated with standard antiviral agents and employed to screen AstraZeneca compounds for potential antiviral activity. Compound potency values in this assay correlated well with the quantitative RT-PCR assay measuring HRV infectivity and replication in human primary airway epithelial cells. In order to improve pan-HRV screening capability, compound potency was also measured in the CellTiter-Glo assay with a combination of 3 different HRV serotypes. This HRV serotype combination assay could be used to identify quickly compounds with desirable broad spectrum antiviral activity.
Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Rhinovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células HeLa , Humanos , Medições Luminescentes/métodos , Testes de Sensibilidade Microbiana/métodos , Rhinovirus/crescimento & desenvolvimentoRESUMO
Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.
Assuntos
Aminocumarinas/metabolismo , Bioensaio , Catepsina C/análise , Dipeptídeos/metabolismo , Corantes Fluorescentes/metabolismo , Lisossomos/enzimologia , Animais , Linhagem Celular Tumoral , Fluorescência , Humanos , Espaço Intracelular/enzimologia , Especificidade por SubstratoRESUMO
The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-kappaB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-kappaB-dependent gene expression, which supports the idea that oscillation frequency has a functional role.
Assuntos
Expressão Gênica , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Retroalimentação Fisiológica , Humanos , Proteínas I-kappa B/metabolismo , Camundongos , Modelos Biológicos , Modelos Estatísticos , Inibidor de NF-kappaB alfa , Fosforilação , Proteínas Recombinantes de Fusão/metabolismo , Processos Estocásticos , TransfecçãoRESUMO
A Hit-to-Lead optimisation programme was carried out on a high throughput screening hit, the thiazolopyrimidine 1, resulting in the discovery of the potent, orally bioavailable CXCR2 antagonist 29.
Assuntos
Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Receptores de Interleucina-8B/antagonistas & inibidores , Tiazóis/administração & dosagem , Tiazóis/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Estrutura Molecular , Pirimidinas/síntese química , Ratos , Relação Estrutura-Atividade , Tiazóis/síntese químicaRESUMO
A Hit-to-Lead optimisation programme was carried out on the high throughput screening hit, the triazolethiol 1, resulting in the discovery of the potent, orally bioavailable triazolethiol CXCR2 receptor antagonist 45.
Assuntos
Receptores de Interleucina-8B/antagonistas & inibidores , Triazóis/farmacologia , Administração Oral , Animais , Quimiocinas/antagonistas & inibidores , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Técnicas de Química Combinatória , Bases de Dados Factuais , Humanos , Estrutura Molecular , Ratos , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/metabolismoRESUMO
We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.