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1.
J Med Chem ; 64(17): 12978-13003, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34432979

RESUMO

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.


Assuntos
Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Animais , Desenho de Fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Meia-Vida , Humanos , Masculino , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Células THP-1
2.
ACS Chem Biol ; 15(9): 2316-2323, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32697072

RESUMO

The Bcl-2 family of proteins, such as Bcl-xL and Bcl-2, play key roles in cancer cell survival. Structural studies of Bcl-xL formed the foundation for the development of the first Bcl-2 family inhibitors and FDA approved drugs. Recently, Proteolysis Targeting Chimeras (PROTACs) that degrade Bcl-xL have been proposed as a therapeutic modality with the potential to enhance potency and reduce toxicity versus antagonists. However, no ternary complex structures of Bcl-xL with a PROTAC and an E3 ligase have been successfully determined to guide this approach. Herein, we report the design, characterization, and X-ray structure of a VHL E3 ligase-recruiting Bcl-xL PROTAC degrader. The 1.9 Å heterotetrameric structure, composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xL, reveals an extensive network of neo-interactions, between the E3 ligase and the target protein, and between noncognate parts of the PROTAC and partner proteins. This work illustrates the challenges associated with the rational design of bifunctional molecules where interactions involve composite interfaces.


Assuntos
Benzotiazóis/metabolismo , Isoquinolinas/metabolismo , Oligopeptídeos/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína bcl-X/antagonistas & inibidores , Benzotiazóis/química , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Proteína bcl-X/química , Proteína bcl-X/metabolismo
3.
Commun Biol ; 3(1): 140, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198438

RESUMO

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.


Assuntos
Anti-Inflamatórios/farmacologia , Desenho de Fármacos , Inflamação/prevenção & controle , Leucócitos Mononucleares/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/enzimologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/enzimologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Feminino , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Injeções Intravenosas , Leucócitos Mononucleares/enzimologia , Masculino , Proteólise , Ratos Sprague-Dawley , Ratos Wistar , Células THP-1 , Técnicas de Cultura de Tecidos , Ubiquitinação
4.
Pharmacology ; 97(3-4): 114-25, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26734728

RESUMO

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvß6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvß6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (K(D): 0.22 nmol/l) and selectivity (at least 85-fold) for αvß6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvß6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvß6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvß6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvß6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvß6 following RGD ligand engagement.


Assuntos
Antígenos de Neoplasias/metabolismo , Vírus da Febre Aftosa , Integrinas/metabolismo , Peptídeos/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Cinética , Ligantes , Ligação Proteica , Ensaio Radioligante
5.
Int J Clin Pharmacol Ther ; 52(4): 267-76, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24472402

RESUMO

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1)-expressing sensory C-fibers may play a role in the development of nasal hyper-responsiveness and symptoms of non-allergic rhinitis (NAR). OBJECTIVE: To evaluate the effects of a TRPV1-antagonist, SB-705498, on cold dry air (CDA)-induced symptoms in patients with NAR. METHODS: This randomized, double-blind, placebo-controlled, crossover study evaluated 14 days of once daily, topical intranasal SB-705498 12 mg in 40 patients with NAR using a CDA challenge experimental model in an environmental exposure chamber (EEC, Cetero Research, Mississauga, Ontario). The primary endpoint was total symptom score (TSS), expressed as weighted mean over 60 minutes (WM0-60) or maximum TSS at 1 hour and 24 hours postdosing. RESULTS: Treatment with SB-705498, relative to placebo, did not improve WM0-60 or maximum TSS at 1 hour and 24 hours post-dosing on days 1 or 14. Mean (95% CI) treatment differences (SB-705498 - placebo) on day 14 were, for WM0-60 at 1 hour: -0.12 (-0.60, 0.36); for maximum TSS at 1 hour: -0.03 (-0.58, 0.51). SB-705498 had no impact on any other efficacy parameters. SB-705498 was well tolerated and pharmacokinetics analysis supported the dosing regimen. CONCLUSION: SB-705498 12 mg for 14 days did not alleviate the CDA-induced symptoms of NAR. Despite engagement of the TRPV1 receptor, there was no translation to clinical efficacy, suggesting redundancy in symptom pathways.


Assuntos
Pirrolidinas/uso terapêutico , Rinite/prevenção & controle , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Adulto , Temperatura Baixa , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirrolidinas/efeitos adversos , Pirrolidinas/farmacocinética , Ureia/efeitos adversos , Ureia/farmacocinética , Ureia/uso terapêutico
6.
Br J Clin Pharmacol ; 77(5): 777-88, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23909699

RESUMO

AIMS: To assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of intranasal SB-705498, a selective TRPV1 antagonist. METHODS: Two randomized, double-blind, placebo-controlled, clinical studies were performed: (i) an intranasal SB-705498 first time in human study to examine the safety and PK of five single escalating doses from 0.5 to 12 mg and of repeat dosing with 6 mg and 12 mg twice daily for 14 days and (ii) a PD efficacy study in subjects with non-allergic rhinitis (NAR) to evaluate the effect of 12 mg intranasal SB-705498 against nasal capsaicin challenge. RESULTS: Single and repeat dosing with intranasal SB-705498 was safe and well tolerated. The overall frequency of adverse events was similar for SB-705498 and placebo and no dose-dependent increase was observed. Administration of SB-705498 resulted in less than dose proportional AUC(0,12 h) and Cmax , while repeat dosing from day 1 to day 14 led to its accumulation. SB-705498 receptor occupancy in nasal tissue was estimated to be high (>80%). Administration of 12 mg SB-705498 to patients with NAR induced a marked reduction in total symptom scores triggered by nasal capsaicin challenge. Inhibition of rhinorrhoea, nasal congestion and burning sensation was associated with 2- to 4-fold shift in capsaicin potency. CONCLUSIONS: Intranasal SB-705498 has an appropriate safety and PK profile for development in humans and achieves clinically relevant attenuation of capsaicin-provoked rhinitis symptoms in patients with NAR. The potential impact intranasal SB-705498 may have in rhinitis treatment deserves further evaluation.


Assuntos
Capsaicina/antagonistas & inibidores , Pirrolidinas/farmacologia , Rinite/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Adolescente , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirrolidinas/efeitos adversos , Pirrolidinas/farmacocinética , Ureia/efeitos adversos , Ureia/farmacocinética , Ureia/farmacologia , Escala Visual Analógica
7.
Int J Clin Pharmacol Ther ; 51(7): 576-84, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23735181

RESUMO

BACKGROUND: Current pharmacotherapy for allergic rhinitis (AR) does not totally ameliorate all symptoms for all patients. Residual symptoms could be due to nasal neuronal hyperresponsiveness caused by stimulation of the ion channel transient receptor potential vanilloid 1 (TRPV1). SB-705498 is a TRPV1 antagonist that has been developed in an intranasal formulation for treatment of AR. METHODS: This randomized, double-blind, 3-way incomplete block crossover study evaluated the effects of 8 days treatment with SB-705498 12 mg alone, SB-705498 12 mg plus fluticasone propionate 200 µg (FP), FP 200 µg alone or placebo on allergen-induced symptoms in 70 patients with AR. The primary endpoint was total nasal symptom score (TNSS), expressed as mean over 4 hours or maximum TNSS during allergen challenge in the Vienna Challenge Chamber on 8th day of treatment. RESULTS: At the end of treatment, there were no differences in allergen-induced mean TNSS between SB-705498 alone and placebo or between SB-705498 plus FP and FP alone. Treatment with FP and SB-705498 plus FP resulted in a significant decrease in TNSS vs. placebo. Mean (90% CI) treatment differences in TNSS over 0 - 4 hours were: SB-705498 - placebo: -0.2 (-0.9, 0.4); SB-705498 plus FP - FP: 0.7 (0.2, 1.2); FP - placebo: -2.9 (-3.4, -2.5); SB-705498 plus FP - placebo: -2.3 (-2.8, -1.8). SB-705498 had no impact on diary card symptoms, nasal airflow or Rhinoconjunctivitis Quality of Life Questionnaire scores. SB-705498 was well tolerated and pharmacokinetics exposure results supported the dosing regimen. CONCLUSION: SB-705498 12 mg for 8 days did not alleviate the allergen-induced symptoms of AR, or provide additional relief of symptoms when in combination with FP. Despite engagement of the TRPV1 receptor there was no translation to clinical efficacy, suggesting redundancy in symptom pathways.


Assuntos
Antialérgicos/uso terapêutico , Pirrolidinas/uso terapêutico , Rinite Alérgica Sazonal/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Administração Intranasal , Adulto , Androstadienos/uso terapêutico , Antialérgicos/administração & dosagem , Antialérgicos/efeitos adversos , Antialérgicos/sangue , Antialérgicos/farmacocinética , Áustria , Estudos Cross-Over , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Fluticasona , Humanos , Masculino , Pirrolidinas/administração & dosagem , Pirrolidinas/efeitos adversos , Pirrolidinas/sangue , Pirrolidinas/farmacocinética , Rinite Alérgica Sazonal/diagnóstico , Rinite Alérgica Sazonal/metabolismo , Canais de Cátion TRPV/metabolismo , Resultado do Tratamento , Ureia/administração & dosagem , Ureia/efeitos adversos , Ureia/sangue , Ureia/farmacocinética , Ureia/uso terapêutico
8.
Br J Pharmacol ; 169(3): 580-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23441756

RESUMO

BACKGROUND AND PURPOSE: Nasal sensory nerves play an important role in symptoms associated with rhinitis triggered by environmental stimuli. Here, we propose that TRPV1 is pivotal in nasal sensory nerve activation and assess the potential of SB-705498 as an intranasal therapy for rhinitis. EXPERIMENTAL APPROACH: The inhibitory effect of SB-705498 on capsaicin-induced currents in guinea pig trigeminal ganglion cells innervating nasal mucosa was investigated using patch clamp electrophysiology. A guinea pig model of rhinitis was developed using intranasal challenge of capsaicin and hypertonic saline to elicit nasal secretory parasympathetic reflex responses, quantified using MRI. The inhibitory effect of SB-705498, duration of action and potency comparing oral versus intranasal route of administration were examined. KEY RESULTS: SB-705498 concentration-dependently inhibited capsaicin-induced currents in isolated trigeminal ganglion cells (pIC50 7.2). In vivo, capsaicin ipsilateral nasal challenge (0.03-1 mM) elicited concentration-dependent increases in contralateral intranasal fluid secretion. Ten per cent hypertonic saline initiated a similar response. Atropine inhibited responses to either challenge. SB-705498 inhibited capsaicin-induced responses by ∼50% at 10 mg·kg⁻¹ (oral), non-micronized 10 mg·mL⁻¹ or 1 mg·mL⁻¹ micronized SB-705498 (intranasal) suspension. Ten milligram per millilitre intranasal SB-705498, dosed 24 h prior to capsaicin challenge produced a 52% reduction in secretory response. SB-705498 (10 mg·mL⁻¹, intranasal) inhibited 10% hypertonic saline responses by 70%. CONCLUSIONS AND IMPLICATIONS: The paper reports the development of a guinea pig model of rhinitis. SB-705498 inhibits capsaicin-induced trigeminal currents and capsaicin-induced contralateral nasal secretions via oral and intranasal routes; efficacy was optimized using particle-reduced SB-705498. We propose that TRPV1 is pivotal in initiating symptoms of rhinitis.


Assuntos
Modelos Animais de Doenças , Mucosa Nasal/efeitos dos fármacos , Sistema Nervoso Parassimpático/efeitos dos fármacos , Parassimpatolíticos/uso terapêutico , Pirrolidinas/uso terapêutico , Rinite Alérgica Perene/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Administração Intranasal , Administração Oral , Animais , Antialérgicos/administração & dosagem , Antialérgicos/química , Antialérgicos/farmacologia , Antialérgicos/uso terapêutico , Capsaicina/administração & dosagem , Capsaicina/antagonistas & inibidores , Capsaicina/toxicidade , Células Cultivadas , Relação Dose-Resposta a Droga , Composição de Medicamentos , Feminino , Cobaias , Masculino , Mucosa Nasal/inervação , Mucosa Nasal/metabolismo , Sistema Nervoso Parassimpático/metabolismo , Sistema Nervoso Parassimpático/patologia , Parassimpatolíticos/administração & dosagem , Parassimpatolíticos/química , Parassimpatolíticos/farmacologia , Tamanho da Partícula , Pirrolidinas/administração & dosagem , Pirrolidinas/química , Pirrolidinas/farmacologia , Rinite Alérgica , Rinite Alérgica Perene/metabolismo , Rinite Alérgica Perene/patologia , Via Secretória/efeitos dos fármacos , Fármacos do Sistema Sensorial/administração & dosagem , Fármacos do Sistema Sensorial/antagonistas & inibidores , Fármacos do Sistema Sensorial/toxicidade , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/efeitos dos fármacos , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/patologia , Ureia/administração & dosagem , Ureia/química , Ureia/farmacologia , Ureia/uso terapêutico
9.
Drug News Perspect ; 22(3): 146-50, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19440557

RESUMO

Recent advances in the understanding of allergic mechanisms have highlighted the role of immunoglobulin E (IgE) signaling in the mast cell. One of the most important kinases in the IgE signaling pathway is spleen tyrosine kinase (Syk) which has a critical function early in the signaling cascade following binding of allergen to receptor bound IgE on the mast cell. A number of Syk inhibitors have now been developed which have been shown to effectively inhibit IgE-driven mast cell degranulation and release of inflammatory cytokines in vitro and inhibit allergic responses in a variety of in vivo models. In humans, allergen-driven symptoms were reduced in allergic rhinitic patients exposed to tree pollen in an outdoor environment following intranasal dosing of the Syk inhibitor R-112. Syk therefore represents a promising target for therapeutic intervention in allergic diseases.


Assuntos
Hipersensibilidade/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/fisiologia , Animais , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Mastócitos/fisiologia , Modelos Biológicos , Estrutura Molecular , Quinase Syk
10.
Anal Biochem ; 384(1): 56-67, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18762159

RESUMO

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Inibidores de Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/metabolismo , Quinase Syk
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