RESUMO
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
Assuntos
Aldeído Oxidase/metabolismo , Inibidores de Janus Quinases/farmacocinética , Pulmão/metabolismo , Administração Intranasal , Administração Intravenosa , Animais , Sítios de Ligação , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Inibidores de Janus Quinases/administração & dosagem , Inibidores de Janus Quinases/síntese química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Simulação de Acoplamento Molecular , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.
Assuntos
Furanos/farmacologia , Proteínas/antagonistas & inibidores , Pirazóis/farmacologia , Relação Dose-Resposta a Droga , Furanos/química , Humanos , Estrutura Molecular , Proteínas/metabolismo , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Domain-specific BET bromodomain ligands represent an attractive target for drug discovery with the potential to unlock the therapeutic benefits of antagonizing these proteins without eliciting the toxicological aspects seen with pan-BET inhibitors. While we have reported several distinct classes of BD2 selective compounds, namely, GSK620, GSK549, and GSK046, only GSK046 shows high aqueous solubility. Herein, we describe the lead optimization of a further class of highly soluble compounds based upon a picolinamide chemotype. Focusing on achieving >1000-fold selectivity for BD2 over BD1 ,while retaining favorable physical chemical properties, compound 36 was identified as being 2000-fold selective for BD2 over BD1 (Brd4 data) with >1 mg/mL solubility in FaSSIF media. 36 represents a valuable new in vivo ready molecule for the exploration of the BD2 phenotype.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Piridinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
A number of reports have recently been published describing the discovery and optimization of bromo and extraterminal inhibitors which are selective for the second bromodomain (BD2); these include our own work toward GSK046 (3) and GSK620 (5). This paper describes our approach to mitigating the genotoxicity risk of GSK046 by replacement of the acetamide functionality with a heterocyclic ring. This was followed by a template-hopping and hybridization approach, guided by structure-based drug design, to incorporate learnings from other BD2-selective series, optimize the vector for the amide region, and explore the ZA cleft, leading to the identification of potent, selective, and bioavailable compounds 28 (GSK452), 39 (GSK737), and 36 (GSK217).
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Humanos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.
Assuntos
Anti-Inflamatórios/química , Proteínas de Ciclo Celular/antagonistas & inibidores , Desenho de Fármacos , Fatores de Transcrição/antagonistas & inibidores , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Sítios de Ligação , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Meia-Vida , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Simulação de Dinâmica Molecular , Filogenia , Domínios Proteicos , Quinolonas/química , Quinolonas/metabolismo , Quinolonas/farmacologia , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismoRESUMO
Pan-BET inhibitors have shown profound efficacy in a number of in vivo preclinical models and have entered the clinic in oncology trials where adverse events have been reported. These inhibitors interact equipotently with the eight bromodomains of the BET family of proteins. To better understand the contribution of each domain to their efficacy and to improve from their safety profile, selective inhibitors are required. This Letter discloses the profile of GSK973, a highly selective inhibitor of the second bromodomains of the BET proteins that has undergone extensive preclinical in vitro and in vivo characterization.
RESUMO
The profound efficacy, yet associated toxicity of pan-BET inhibitors is well documented. The possibility of an ameliorated safety profile driven by significantly selective (>100-fold) inhibition of a subset of the eight bromodomains is enticing, but challenging given the close homology. Herein, we describe the X-ray crystal structure-directed optimization of a novel weak fragment ligand with a pan-second bromodomain (BD2) bias, to potent and highly BD2 selective inhibitors. A template hopping approach, enabled by our parallel research into an orthogonal template (15, GSK046), was the basis for the high selectivity observed. This culminated in two tool molecules, 20 (GSK620) and 56 (GSK549), which showed an anti-inflammatory phenotype in human whole blood, confirming their cellular target engagement. Excellent broad selectivity, developability, and in vivo oral pharmacokinetics characterize these tools, which we hope will be of broad utility to the field of epigenetics research.
Assuntos
Anti-Inflamatórios/química , Ligantes , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Amidas/química , Amidas/metabolismo , Amidas/farmacocinética , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Cães , Meia-Vida , Humanos , Ligação de Hidrogênio , Masculino , Simulação de Dinâmica Molecular , Domínios Proteicos , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Over the last decade, a number of computational methods have been developed, which attempt to evaluate the thermodynamic properties of individual water molecules at the solute-solvent interface, in order to assess contributions to protein-ligand binding. In some cases, these tools tell us what we already know, e.g. that hydrophobic pockets prefer lipophilic substituents, and in other cases the methods only seem to add clarity when retrospectively applied. Hence we have grappled with how to utilize such approaches to understand non-intuitive results and to generate chemistry ideas that otherwise would not have been developed. Here we provide our perspective on these methods and describe how results have been interpreted and applied. We include examples from GSK and elsewhere that highlight how water methods have been (1) utilized retrospectively to explain non-intuitive structure- activity relationships and (2) applied prospectively for chemistry design. Finally, we discuss where this field of study could lead to maximal impact in drug discovery research.
Assuntos
Desenho de Fármacos , Água/química , Ligantes , Estrutura Molecular , Proteínas/química , TermodinâmicaRESUMO
The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.
Assuntos
Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Indazóis/farmacologia , Receptores de Glucocorticoides/metabolismo , Androstadienos/química , Androstadienos/farmacologia , Anti-Inflamatórios/química , Benzamidas/química , Benzoquinonas/farmacologia , Dexametasona/química , Dexametasona/farmacologia , Fluticasona , Células HeLa , Humanos , Doenças do Sistema Imunitário , Indazóis/química , Lactamas Macrocíclicas/farmacologia , Ligantes , Terapia de Alvo Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico , Receptores de Glucocorticoides/agonistas , Ativação Transcricional/efeitos dos fármacosRESUMO
Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays.
RESUMO
Bromodomain-containing proteins are key epigenetic regulators of gene transcription and readers of the histone code. However, the therapeutic benefits of modulating this target class are largely unexplored due to the lack of suitable chemical probes. This article describes the generation of lead molecules for the BET bromodomains through screening a fragment set chosen using structural insights and computational approaches. Analysis of 40 BRD2/fragment X-ray complexes highlights both shared and disparate interaction features that may be exploited for affinity and selectivity. Six representative crystal structures are then exemplified in detail. Two of the fragments are completely new bromodomain chemotypes, and three have never before been crystallized in a bromodomain, so our results significantly extend the limited public knowledge-base of crystallographic small molecule/bromodomain interactions. Certain fragments (including paracetamol) bind in a consistent mode to different bromodomains such as CREBBP, suggesting their potential to act as generic bromodomain templates. An important implication is that the bromodomains are not only a phylogenetic family but also a system in which chemical and structural knowledge of one bromodomain gives insights transferrable to others.
Assuntos
Modelos Moleculares , Peptidomiméticos/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Acetaminofen/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Indolizinas/química , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Piridinas/química , Pirrolidinonas/química , Relação Quantitativa Estrutura-Atividade , Quinazolinonas/química , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/químicaRESUMO
Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC(50)=0.12 µM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.
Assuntos
Amidinas/síntese química , Amidinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Heme/antagonistas & inibidores , Compostos Heterocíclicos/síntese química , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Prolina/análogos & derivados , Amidinas/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacologia , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Prolina/síntese química , Prolina/química , Prolina/farmacologiaRESUMO
Aryl aminopyrazole amides capped with N-alkylbenzamides 13-16 are selective glucocorticoid receptor agonists. 2,6-Disubstituted benzamides have prednisolone-like potency or better in vitro. Good oral exposure was demonstrated in the rat, with compounds with lower lipophilicity, for example N-hydroxyethyl benzamides (e.g., 16e).
Assuntos
Benzamidas/síntese química , Pirazóis/síntese química , Receptores de Glucocorticoides/agonistas , Administração Oral , Animais , Benzamidas/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Prednisolona , Pirazóis/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Docking is a computational technique that samples conformations of small molecules in protein binding sites; scoring functions are used to assess which of these conformations best complements the protein binding site. An evaluation of 10 docking programs and 37 scoring functions was conducted against eight proteins of seven protein types for three tasks: binding mode prediction, virtual screening for lead identification, and rank-ordering by affinity for lead optimization. All of the docking programs were able to generate ligand conformations similar to crystallographically determined protein/ligand complex structures for at least one of the targets. However, scoring functions were less successful at distinguishing the crystallographic conformation from the set of docked poses. Docking programs identified active compounds from a pharmaceutically relevant pool of decoy compounds; however, no single program performed well for all of the targets. For prediction of compound affinity, none of the docking programs or scoring functions made a useful prediction of ligand binding affinity.
Assuntos
Ligantes , Proteínas/química , Relação Quantitativa Estrutura-Atividade , Algoritmos , Sítios de Ligação , Desenho de Fármacos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , SoftwareRESUMO
A series of chiral, (S)-proline-alpha-methylpyrrolidine-5,5-trans-lactam serine protease inhibitors has been developed as antivirals of human cytomegalovirus (HCMV). The SAR of the functionality on the proline nitrogen has shown that derivatives of para-substituted phenyl ureas > para-substituted phenyl sulfonamides > para-substituted phenyl carboxamide for activity against HCMV deltaAla protease, producing para-substituted phenyl ureas with single figure nM potency (K(i)) against the viral enzyme. The SAR of the functionality on the lactam nitrogen has defined the steric and electronic requirements for high human plasma stability while retaining good activity against HCMV protease. The combination of high potency against HCMV deltaAla protease and high human plasma stability has produced compounds with significant in vitro antiviral activity against human cytomegalovirus with the 6-hydroxymethyl benzothiazole derivative 72 being equivalent in potency to ganciclovir. The parent benzothiazole 56 had good pharmacokinetics in dogs with 29% bioavailability and good brain and ocular penetration in guinea pigs.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/enzimologia , Lactamas/síntese química , Lactamas/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Serina Endopeptidases/metabolismo , Animais , Antivirais/sangue , Disponibilidade Biológica , Encéfalo/metabolismo , Células Cultivadas , Cães , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Olho/metabolismo , Ganciclovir/farmacologia , Cobaias , Meia-Vida , Humanos , Indicadores e Reagentes , Cinética , Espectrometria de Massas , Modelos Moleculares , Inibidores de Proteases/sangue , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The stereospecific synthesis of a series of alpha-methylpyrrolidine-5,5-trans-lactam inhibitors of human cytomegalovirus (HCMV) protease is described. Examination of the SAR in this series has defined the size and chirality of the alpha-substituent, optimized the acyl substituent on the lactam nitrogen, and defined the steric constraint of this functionality. The SAR of the functionality on the pyrrolidine nitrogen of the trans-lactam has been investigated, and this has led to the discovery of potent serine protease inhibitors that are highly selective for the viral enzyme over the mammalian enzymes elastase, thrombin, and acetylcholine esterase. The mechanism of action of our lead compounds has been established by mass spectrometry, and enzymatic degradation of HCMV deltaAla protease acylated with these inhibitors showed that Ser 132 is the active site nucleophile. The crystal structure of HCMV protease was obtained and used to model the conformationally restricted, chiral (S)-proline-alpha-methyl-5,5-trans-lactams into the active site groove of the enzyme, enabling us to direct and rationalize the SAR in this series. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the dansyl-(S)-proline alpha-methyl-5,5-trans-lactam template, which have low nanomolar activity against the viral enzyme.