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-1RESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].
RESUMO
Receptor Interacting Protein 2 (RIP2) kinase inhibitors have been reported for therapeutic opportunities in inflammatory bowel diseases such as Ulcerative Colitis and Crohn's disease. During lead optimization, team identified 4-aminoquinoline series and several compounds from this series were investigated in rat and dog pharmacokinetic studies. While compounds such as GSKA and GSKB demonstrated acceptable pharmacokinetics in rat and dog, further progression of these compounds was halted due to adverse findings in advanced safety studies. Structurally similar analogues incorporating polarity at C-7 position of 4-aminoquinoline resulted in identification of GSKC - GSKF. Interestingly, following oral administration to rat at similar low dose, GSKC - GSKF demonstrated significantly low systemic drug exposure compared to GSKA and GSKB (3-17-fold difference). However, in dog, dose normalized oral systemic exposure for GSKC - GSKF was comparable to GSKA and GSKB (within 2-fold). A series of studies were conducted to understand the disconnect which highlighted that an intrinsic reduction in permeability and high P-glycoprotein (P-gp) efflux ratio for C-7 substituted analogues were driving pharmacokinetic disconnect between rat and dog. Oral absorption was minimally impacted in dog by P-gp mediated efflux compared to rat because the leakier gastrointestinal tract in dog likely overcomes this effect.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Aminoquinolinas/farmacocinética , Administração Oral , Animais , Transporte Biológico , Cães , Permeabilidade , RatosRESUMO
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 , UbiquitinaçãoRESUMO
Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.
RESUMO
RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.
Assuntos
Benzotiazóis/farmacologia , Fosfatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Benzotiazóis/química , Benzotiazóis/farmacocinética , Benzotiazóis/uso terapêutico , Colite/tratamento farmacológico , Cães , Descoberta de Drogas , Humanos , Masculino , Camundongos , Simulação de Acoplamento Molecular , Fosfatos/química , Fosfatos/farmacocinética , Fosfatos/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/química , Quinazolinas/farmacocinética , Quinazolinas/uso terapêutico , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Suínos , Porco MiniaturaRESUMO
RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 µM).
RESUMO
RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.
Assuntos
Aminoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Sulfonas/farmacologia , Aminoquinolinas/sangue , Aminoquinolinas/química , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Relação Estrutura-Atividade , Sulfonas/sangue , Sulfonas/químicaRESUMO
Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.
Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Animais , Caspase 8/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Técnicas de Introdução de Genes , Células HT29 , Humanos , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Necrose/enzimologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidoresRESUMO
NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.
Assuntos
Benzotiazóis/farmacologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Xantinas/farmacologia , Animais , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosforilação , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.
Assuntos
Amidas/química , Benzimidazóis/química , Benzimidazóis/farmacologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Citocinas/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Relação Estrutura-Atividade , Receptor 2 Toll-Like/metabolismoRESUMO
A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.
Assuntos
Receptores CCR2/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Administração Oral , Animais , Técnicas de Introdução de Genes , Guanosina 5'-O-(3-Tiotrifosfato)/sangue , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/efeitos dos fármacos , Ratos , Receptores CCR2/genética , Receptores CCR2/metabolismo , Albumina Sérica/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/farmacocinéticaRESUMO
(-)-Isopulegol derivatives undergo a ring contraction under silylene-mediated conditions to provide cyclopentane products. Silylene transfer to other homoallylic ethers did not provide the ring contraction products. Allylic silane products were elaborated to determine the stereochemical course of the ring contraction reaction. A mechanism for the transformation is proposed.
Assuntos
Compostos Alílicos/síntese química , Éteres/química , Metano/análogos & derivados , Silanos/síntese química , Silício/química , Compostos Alílicos/química , Catálise , Monoterpenos Cicloexânicos , Ciclopentanos/síntese química , Ciclopentanos/química , Espectroscopia de Ressonância Magnética , Metano/química , Modelos Químicos , Silanos/química , Terpenos/químicaRESUMO
Brevetoxin A is a decacyclic ladder toxin that possesses 5-, 6-, 7-, 8-, and 9-membered oxacycles, as well as 22 tetrahedral stereocenters. Herein, we describe a unified approach to the B, E, G, and J rings based upon a ring-closing metathesis strategy from the corresponding dienes. The enolate technologies developed in our laboratory allowed access to the precursor acyclic dienes for the B, E, and G medium-ring ethers. The strategies developed for the syntheses of these four monocycles ultimately provided multigram quantities of each of the rings, supporting our efforts toward the completion of a convergent synthesis of brevetoxin A.
Assuntos
Éteres/síntese química , Toxinas Marinhas/síntese química , Oxocinas/síntese química , Alquilação , Ciclização , Éteres/química , Toxinas Marinhas/química , Estrutura Molecular , Oxocinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Allylic ethers undergo insertions of silylenes into C-O bonds to form allylic silanes. Silylene insertion into C-O acetal bonds was also observed. Formation of silylene ylide intermediates led to [1,2]-Stevens rearrangement products as well as [2,3]-sigmatropic products depending upon the steric environment of the starting allylic ether.
RESUMO
A total synthesis of brevetoxin A is reported. Two tetracyclic coupling partners, prepared from previously reported advanced fragments, were effectively united via a Horner-Wittig olefination. The resulting octacycle was progressed to substrates that were explored for reductive etherification, the success of which led to a penultimate tetraol intermediate. The tetraol was converted to the natural product through an expeditious selective oxidative process followed by methylenation.
Assuntos
Toxinas Marinhas/síntese química , Oxocinas/síntese química , Aldeídos/química , Alcenos/química , Toxinas Marinhas/química , Oxirredução , Oxocinas/química , Especificidade por SubstratoRESUMO
Molecular clusters of BBr3 were subjected to electron ionization and mass analysis in a reflectron time-of-flight mass spectrometer. Five series of cluster ions were observed, with formulas corresponding to each of the possible fragment ions of BBr3 being solvated by neutral BBr3 molecules. Geometry optimizations on the observed cluster ions using density functional theory (B3LYP/6-31G*) predict that fragment ions smaller than BBr3+ undergo reactions with neutral BBr3 molecules to form covalently bound adduct species that function as core ions within the clusters. Once all boron atoms are saturated, the reactions cease, and larger cluster ions consist of BBr3 molecules loosely bound to the core ions. Divalent bromine atoms are present in at least three of the cluster ions, and most of the intermolecular contact within the clusters is between Br atoms. Enthalpies of formation, addition reactions, and BBr3 elimination from the cluster ions were derived from B3LYP and MP2 calculations at the B3LYP/6-31G* geometries using both the 6-31G* and the 6-311++G(2df,2p) basis sets. The results are compared to limiting expectations based on known bulk thermochemistry.