RESUMO
Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.
RESUMO
The p38α mitogen-activated protein kinase regulates the synthesis of pro-inflammatory cytokines in response to stimulation by a diverse set of stress signals. Various different chemotypes and clinical candidates that inhibit p38α function have been reported over the years. In this publication, the novel structure of p38α cocrystallized with the clinical candidate TAK-715 is reported. Owing to the impact of crystallization conditions on the conformation of protein kinases (and in particular p38α), the structures of complexes of p38α with SB-203580, SCIO-469 and VX-745 have also been determined to enable in-depth comparison of ligand-induced protein conformations. The impact of experimental conditions on p38α-inhibitor complex structures, most importantly soaking versus cocrystallization, is discussed. Analysis of the structures and quantification of the protein-ligand interactions couples ligand-induced protein conformations to the number of interactions and to inhibitor selectivity against the human kinome. This shows that for the design of novel kinase inhibitors, selectivity is best obtained through maximization of the number of interactions throughout the ATP pocket and the exploitation of specific features in the active site.
Assuntos
Benzamidas/farmacologia , Tiazóis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/química , Trifosfato de Adenosina/química , Domínio Catalítico , Clonagem Molecular , Cristalização , Cristalografia por Raios X/métodos , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Ligantes , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Factor XI (FXI) is a key enzyme in the coagulation pathway and an attractive target for the development of anticoagulant drugs. A small number of high-resolution crystal structures of FXIa in complex with small synthetic inhibitors have been published to date. All of these ligands have a basic P1 group and bind exclusively in the nonprime side of the active site of FXIa. Here, two structures of FXIa in complex with nonbasic inhibitors that occupy both the prime and nonprime sides of the active site are presented. These new structures could be valuable in the design and optimization of new FXIa synthethic inhibitors.
Assuntos
Inibidores Enzimáticos/química , Fator XIa/química , Domínios e Motivos de Interação entre Proteínas , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Fator XIa/antagonistas & inibidores , Fator XIa/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Homologia Estrutural de ProteínaRESUMO
We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11ß substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.
Assuntos
Estrenos/química , Mifepristona/química , Oximas/química , Receptores de Progesterona/agonistas , Receptores de Progesterona/química , Cristalografia por Raios X , Humanos , Ligantes , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Optimization of our previously described pyrrolopiperidone series led to the identification of a new benzamide sub-series, which exhibits consistently high potency in biochemical and cell-based assays throughout the series. Strong inhibition of LPS-induced production of the cytokine TNFα is coupled to the regulation of HSP27 phosphorylation, indicating that the observed cellular effects result from the inhibition of MK2. X-ray crystallographic and computational analyses provide a rationale for the high potency of the series.
Assuntos
Benzamidas/farmacologia , Química Farmacêutica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Piperidonas/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Simulação por Computador , Cristalografia por Raios X/métodos , Citocinas/metabolismo , Desenho de Fármacos , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Modelos Químicos , Chaperonas Moleculares , Fosforilação , Pirróis/químicaRESUMO
MK2 kinase is a promising drug discovery target for the treatment of inflammatory diseases. Here, we describe the discovery of novel MK2 inhibitors using X-ray crystallography and structure-based drug design. The lead has in vivo efficacy in a short-term preclinical model.
Assuntos
Trifosfato de Adenosina , Desenho de Fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/química , Animais , Ligação Competitiva , Células CACO-2 , Cristalografia por Raios X , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Ratos , Relação Estrutura-AtividadeRESUMO
The identification of a potent, selective, and orally available MK2 inhibitor series is described. The initial absence of oral bioavailability was successfully tackled by moving the basic nitrogen of the spiro-4-piperidyl moiety towards the electron-deficient pyrrolepyridinedione core, thereby reducing the pK(a) and improving Caco-2 permeability. The resulting racemic spiro-3-piperidyl analogues were separated by chiral preparative HPLC, and the activity towards MK2 inhibition was shown to reside mostly in the first eluting stereoisomer. This led to the identification of new MK2 inhibitors, such as (S)-23, with low nanomolar biochemical inhibition (EC(50) 7.4 nM) and submicromolar cellular target engagement activity (EC(50) 0.5 µM).
Assuntos
Descoberta de Drogas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Piperidinas/síntese química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Compostos de Espiro/síntese química , Administração Oral , Animais , Ligação Competitiva , Disponibilidade Biológica , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/química , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.
Assuntos
Desenho de Fármacos , Indanos/química , Indanos/farmacologia , Receptores de AMPA/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação Alostérica , Animais , Linhagem Celular , Cristalografia por Raios X , Humanos , Indanos/metabolismo , Indanos/farmacocinética , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirazóis/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinéticaRESUMO
Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor.
Assuntos
Indazóis/química , Receptores de AMPA/química , Tiofenos/química , Regulação Alostérica , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Indazóis/síntese química , Indazóis/farmacologia , Microssomos/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/metabolismo , Tiofenos/síntese química , Tiofenos/farmacologiaRESUMO
Starting from lead compound 1, we demonstrate how X-ray structural data can be used to understand SAR and expediently optimize bioavailability in a novel series of AMPA receptor modulators, furnishing 5 with improved bioavailability and robust in vivo activity.