RESUMO
IRAK4 is a key mediator of innate immunity. There is a high interest in identifying novel IRAK4 inhibitors for the treatment of inflammatory autoimmune diseases. We describe here a highly potent and selective IRAK4 inhibitor (HS271) that exhibited superior enzymatic and cellular activities, as well as excellent pharmacokinetic properties. HS271 displayed robust in vivo anti-inflammatory efficacy as evaluated in rat models of LPS induced TNFα production and collagen-induced arthritis.
Assuntos
Aminas/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Descoberta de Drogas , Indazóis/farmacologia , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Aminas/síntese química , Aminas/química , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Relação Dose-Resposta a Droga , Haplorrinos , Humanos , Indazóis/síntese química , Indazóis/química , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Our recently discovered, selective, on-resin route to N(τ)-alkylated imidazolium-containing histidine residues affords new strategies for peptide mimetic design. In this, we demonstrate the use of this chemistry to prepare a series of macrocyclic phosphopeptides, in which imidazolium groups serve as ring-forming junctions. Interestingly, these cationic moieties subsequently serve to charge-mask the phosphoamino acid group that directed their formation. Neighbor-directed histidine N(τ)-alkylation opens the door to new families of phosphopeptidomimetics for use in a range of chemical biology contexts.
Assuntos
Histidina/química , Imidazóis/química , Compostos Macrocíclicos/química , Fosfopeptídeos/química , Alquilação , Cristalografia por Raios XRESUMO
There are few methodologies that yield peptides containing His residues with selective N(τ), N(π)-bis-alkylated imidazole rings. We have found that, under certain conditions, on-resin Mitsunobu coupling of alcohols with peptides having a N(π)-alkylated His residue results in selective and high-yield alkylation of the imidazole N(τ) nitrogen. The reaction requires the presence of a proximal phosphoric, carboxylic or sulfonic acid, and proceeds through an apparent intramolecular mechanism involving Mitsunobu intermediates. These transformations have particular application to phosphopeptides, where "charge masking" of one phosphoryl anionic charge by the cationic histidine imidazolium ion is now possible. This chemistry opens selective access to peptides containing differentially functionalized imidazolium heterocycles, which provide access to new classes of peptides and peptide mimetics.
Assuntos
Histidina/química , Imidazóis/química , Peptidomiméticos/química , Alquilação , Esterificação , Estrutura MolecularRESUMO
Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Histidina/metabolismo , Fosfopeptídeos/síntese química , Fosfopeptídeos/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Alquilação , Ânions , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Cristalização , Estabilidade Enzimática/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Esterases/metabolismo , Polarização de Fluorescência , Células HeLa , Histidina/química , Humanos , Fosfopeptídeos/química , Pró-Fármacos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem , Quinase 1 Polo-LikeRESUMO
The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.
Assuntos
Aminoácidos/química , Fluorenos/química , Peptídeos/química , Fosfotreonina/química , Pró-Fármacos/química , Aminoácidos/síntese química , Desenho de Fármacos , Fluorenos/síntese química , Peptídeos/síntese química , Fosfotreonina/síntese química , Pró-Fármacos/síntese químicaRESUMO
We obtained unanticipated synthetic byproducts from alkylation of the δ(1) nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest-affinity byproduct, bearing a C(6)H(5)(CH(2))(8)- group, a Plk1 PBD cocrystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically stable phosphothreonyl residue (pT) bound the Plk1 PBD with affinity equal to that of the non-PEGylated parent but showed markedly less interaction with the PBDs of the two closely related proteins Plk2 and Plk3. Treatment of cultured cells with this PEGylated peptide resulted in delocalization of Plk1 from centrosomes and kinetochores and in chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides insights that might advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer agents.
Assuntos
Antineoplásicos/química , Proteínas de Ciclo Celular/química , Histidina/química , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Alquilação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Centrossomo/efeitos dos fármacos , Células HeLa , Humanos , Cinetocoros/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Fosfotreonina/química , Polietilenoglicóis/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas Supressoras de Tumor , Quinase 1 Polo-LikeRESUMO
Reported herein are the synthesis and solid-phase peptide incorporation of N-Fmoc-(2S,3R)-2-amino-3-methyl-4-phosphonobutyric acid bis-pivaloyloxymethyl phosphoryl ester [Fmoc-Pmab(POM)2-OH, 2] as a phosphatase-stable phosphothreonine (pThr) mimetic bearing orthogonal protection suitable for the synthesis of Pmab-containing peptides having bio-reversible protection of the phosphonic acid moiety. This represents the first report of a bio-reversibly protected pThr mimetic in a form suitable for facile solid-phase peptide synthesis.
Assuntos
Aminobutiratos/química , Peptídeos/síntese química , Fosfotreonina/química , Pró-Fármacos/síntese química , Estrutura Molecular , Peptídeos/química , Pró-Fármacos/química , Técnicas de Síntese em Fase SólidaRESUMO
Protein-protein interactions (PPIs) mediated by the polo-box domain (PBD) of polo-like kinase 1 (Plk1) serve important roles in cell proliferation. Critical elements in the high affinity recognition of peptides and proteins by PBD are derived from pThr/pSer-residues in the binding ligands. However, there has been little examination of pThr/pSer mimetics within a PBD context. Our current paper compares the abilities of a variety of amino acid residues and derivatives to serve as pThr/pSer replacements by exploring the role of methyl functionality at the pThr ß-position and by replacing the phosphoryl group by phosphonic acid, sulfonic acid and carboxylic acids. This work sheds new light on structure activity relationships for PBD recognition of phosphoamino acid mimetics.
Assuntos
Proteínas de Ciclo Celular/química , Modelos Moleculares , Peptídeos/química , Fosfoaminoácidos/química , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Proteínas de Ciclo Celular/metabolismo , Desenho de Fármacos , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Fosfoaminoácidos/síntese química , Fosfoaminoácidos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-Atividade , Quinase 1 Polo-LikeRESUMO
We replaced the amino terminal Pro residue of the Plk1 polo-box-domain-binding pentapeptide (PLHSpT) with a library of N-alkyl-Gly "peptoids", and identified long-chain tethered phenyl moieties giving greater than two-orders-of-magnitude affinity enhancement. Further simplification by replacing the peptoid residue with appropriate amides gave low-nanomolar affinity N-acylated tetrapeptides. Binding of the N-terminal long-chain phenyl extension was demonstrated by X-ray co-crystal data.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peptoides/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Ligantes , Modelos Moleculares , Glicinas N-Substituídas/química , Glicinas N-Substituídas/metabolismo , Peptoides/química , Estrutura Terciária de Proteína , Quinase 1 Polo-LikeRESUMO
We report herein that incorporating long-chain alkylphenyl-containing non-proteinogenic amino acids in place of His at the pT-2 position of the parent polo-like kinase 1 (Plk1) polo box domain (PBD)-binding pentapeptide, PLHSpT (1a) increases affinity. For certain analogs, approximately two orders-of-magnitude improvement in affinity was observed. Although, none of the new analogs was as potent as our previously described peptide 1b, in which the pT-2 histidine imidazole ring is alkylated at its π nitrogen (N3), our current finding that the isomeric His(N1)-analog (1c) binds with approximately 50-fold less affinity than 1b, indicates the positional importance of attachment to the His imidazole ring. Our demonstration that a range of modified residues at the pT-2 position can enhance binding affinity, should facilitate the development of minimally-sized Plk1 PBD-binding antagonists.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Modelos Moleculares , Quinase 1 Polo-LikeRESUMO
Due to the high homology of the ATP sites of the JAK family, the development of selective inhibitors for a certain JAK isoform is extremely challenging. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Based on the clinical compound BMS-986165, through structure-activity relationship studies, a class of acyl compounds with excellent TYK2 inhibitory activity and selectivity to other subtypes of the JAK family was discovered.
Assuntos
Janus Quinases , Piridinas/síntese química , TYK2 Quinase , Janus Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Relação Estrutura-Atividade , TYK2 Quinase/antagonistas & inibidoresRESUMO
We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo-box domain of polo-like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies.
Assuntos
Proteínas de Ciclo Celular/síntese química , Histidina/química , Histidina/síntese química , Imidazóis/química , Peptídeos/química , Peptídeos/síntese química , Fosfotreonina/química , Fosfotreonina/síntese química , Proteínas Serina-Treonina Quinases/síntese química , Proteínas Proto-Oncogênicas/síntese química , Alquilação , Proteínas de Ciclo Celular/química , Elétrons , Histidina/análogos & derivados , Estrutura Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Quinase 1 Polo-LikeRESUMO
Efficient methodologies for constructing several multiring frameworks have been developed utilizing the cascade reactions of air-stable isochromenylium tetrafluoroborates with olefins. Successive additions of two equivalents of styrene-type olefins to isochromenylium tetrafluoroborate (ICTB) 1 have been observed and their mechanisms were proposed and discussed. Intramolecular capture of the early stage cationic intermediates by predevised heteroatoms or Friedel-Crafts donors provided two different types of bridged-ring systems.
Assuntos
Alcenos/química , Boratos/química , Estirenos/química , Estrutura MolecularRESUMO
Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Peptídeos/química , Peptídeos/farmacologia , Fosfotreonina/química , Pró-Fármacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/química , Proteínas de Ciclo Celular/metabolismo , Estabilidade de Medicamentos , Ésteres , Células HeLa , Humanos , Modelos Moleculares , Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Especificidade por Substrato , Quinase 1 Polo-LikeRESUMO
In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Oximas/química , Peptídeos/química , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Técnicas de Síntese em Fase Sólida , Proteínas de Ciclo Celular/química , Linhagem Celular , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Quinase 1 Polo-LikeRESUMO
Molecular processes depending on proteinprotein interactions can use consensus recognition sequences that possess defined secondary structures. Left-handed polyproline II (PPII) helices are a class of secondary structure commonly involved with cellular signal transduction. However, unlike -helices, for which a substantial body of work exists regarding applications of ring-closing metathesis (RCM), there are few reports on the stabilization of PPII helices by RCM methodologies. The current study examined the effects of RCM macrocyclization on left-handed PPII helices involved with the SH3 domain-mediated binding of Sos1Grb2. Starting with the Sos1-derived peptide "Ac-V1-P2-P3-P4-V5-P6-P7-R8-R9-R10-amide," RCM macrocyclizations were conducted using alkenyl chains of varying lengths originating from the pyrrolidine rings of the Pro4 and Pro7 residues. The resulting macrocyclic peptides showed increased helicity as indicated by circular dichroism and enhanced abilities to block Grb2Sos1 interactions in cell lysate pull-down assays. The synthetic approach may be useful in RCM macrocyclizations, where maintenance of proline integrity at both ring junctures is desired.
Assuntos
Proteína Adaptadora GRB2/química , Dicroísmo Circular , Ciclização , Modelos Moleculares , Estrutura Secundária de Proteína , Domínios de Homologia de srcRESUMO
Transformation of reactive isochromenylium intermediates to the corresponding storable and stable reagents has been achieved, and a number of isochromenylium tetrafluoroborates (ICTBs, 1) have been conveniently prepared and characterized. Direct metal-free treatment of isochromenylium tetrafluoroborate 1a with olefins afforded a variety of polycyclic frameworks 4 via mild cascade reactions. Starting from the prefunctionalized o-alkynylbenzaldehydes, a one-pot metal-free procedure of intramolecular cascade annulation to 2,3-dihydrophenanthren-4(1H)-one derivatives was also developed.