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1.
Nat Commun ; 15(1): 52, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168038

RESUMO

The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.


Assuntos
Dinaminas , GTP Fosfo-Hidrolases , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Hidrólise , Fusão de Membrana , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34261790

RESUMO

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.


Assuntos
Cardiolipinas/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Dinâmica Mitocondrial/fisiologia , Motivos de Aminoácidos , Sítios de Ligação , Dinaminas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância Magnética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Mitofagia , Mutação , Ligação Proteica , Conformação Proteica
5.
J Pharm Sci ; 109(10): 3160-3171, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32565354

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 , Ratos
6.
J Am Chem Soc ; 142(4): 1882-1894, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31880439

RESUMO

Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.


Assuntos
Evolução Molecular Direcionada , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Peptídeos/química , Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular
7.
J Med Chem ; 62(14): 6482-6494, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31265286

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 Miniatura
8.
ACS Med Chem Lett ; 10(6): 857-862, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31223438

RESUMO

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

9.
Chem Commun (Camb) ; 55(41): 5777-5780, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31041432

RESUMO

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Mapas de Interação de Proteínas , Animais , Sistema Enzimático do Citocromo P-450/química , Humanos , Modelos Moleculares , NADPH-Ferri-Hemoproteína Redutase/química , Ressonância Magnética Nuclear Biomolecular/métodos , Oxirredução , Mapeamento de Interação de Proteínas/métodos , Coelhos
10.
J Med Chem ; 62(10): 5096-5110, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31013427

RESUMO

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Animais , Disponibilidade Biológica , Linhagem Celular , Doença Crônica , Desenho de Fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Esclerose Múltipla/tratamento farmacológico , Pirazóis/farmacocinética , Ratos , Retinose Pigmentar/tratamento farmacológico , Relação Estrutura-Atividade
11.
Cancer Cell ; 34(5): 757-774.e7, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30423296

RESUMO

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.


Assuntos
Carcinoma Ductal Pancreático/imunologia , Tolerância Imunológica/imunologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica/genética , Células L , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Transcrição STAT1/metabolismo , Células Th1/citologia , Células Th17/citologia
12.
ACS Med Chem Lett ; 9(10): 1039-1044, 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30344914

RESUMO

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).

13.
Angew Chem Int Ed Engl ; 57(28): 8458-8462, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29722926

RESUMO

Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome-b5 (cyt-b5 ), Arg 125 on the C-helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein-protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Mononucleotídeo de Flavina/metabolismo , Nanoestruturas/química , Peptídeos/química , Sistema Enzimático do Citocromo P-450/química , Mononucleotídeo de Flavina/química , Modelos Moleculares , Oxirredução
14.
Biochim Biophys Acta Biomembr ; 1860(2): 407-415, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28988778

RESUMO

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Fusão de Membrana , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Fosforilcolina/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Eletricidade Estática
15.
Chem Commun (Camb) ; 53(95): 12798-12801, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29143058

RESUMO

Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b5. These results highlight the importance of lipid membrane for the function of P450.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Sistema Enzimático do Citocromo P-450/química , Citocromos b5/química , Modelos Moleculares
16.
Sci Rep ; 7(1): 7793, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798301

RESUMO

Cytochrome b 5 (cytb 5) is a membrane protein vital for the regulation of cytochrome P450 (cytP450) metabolism and is capable of electron transfer to many redox partners. Here, using cyt c as a surrogate for cytP450, we report the effect of membrane on the interaction between full-length cytb 5 and cyt c for the first time. As shown through stopped-flow kinetic experiments, electron transfer capable cytb 5 - cyt c complexes were formed in the presence of bicelles and nanodiscs. Experimentally measured NMR parameters were used to map the cytb 5-cyt c binding interface. Our experimental results identify differences in the binding epitope of cytb 5 in the presence and absence of membrane. Notably, in the presence of membrane, cytb 5 only engaged cyt c at its lower and upper clefts while the membrane-free cytb 5 also uses a distal region. Using restraints generated from both cytb 5 and cyt c, a complex structure was generated and a potential electron transfer pathway was identified. These results demonstrate the importance of studying protein-protein complex formation in membrane mimetic systems. Our results also demonstrate the successful preparation of novel peptide-based lipid nanodiscs, which are detergent-free and possesses size flexibility, and their use for NMR structural studies of membrane proteins.


Assuntos
Citocromos b5/química , Citocromos c/química , Elétrons , Bicamadas Lipídicas/química , Animais , Simulação de Dinâmica Molecular , Ligação Proteica , Coelhos
17.
Sci Rep ; 7: 39925, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28051162

RESUMO

To become clinically effective, antimicrobial peptides (AMPs) should be non-cytotoxic to host cells. Piscidins are a group of fish-derived AMPs with potent antimicrobial and antiendotoxin activities but limited by extreme cytotoxicity. We conjectured that introduction of cationic residue(s) at the interface of polar and non-polar faces of piscidins may control their insertion into hydrophobic mammalian cell membrane and thereby reducing cytotoxicity. We have designed several novel analogs of piscidin-1 by substituting threonine residue(s) with L and D-lysine residue(s). L/D-lysine-substituted analogs showed significantly reduced cytotoxicity but exhibited either higher or comparable antibacterial activity akin to piscidin-1. Piscidin-1-analogs demonstrated higher efficacy than piscidin-1 in inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory responses in THP-1 cells. T15,21K-piscidin-1 (0.5 mg/Kg) and T15,21dK-piscidin-1 (1.0 mg/Kg) demonstrated 100% survival of LPS (12.0 mg/Kg)-administered mice. High resolution NMR studies revealed that both piscidin-1 and T15,21K-piscidin-1 adopted helical structures, with latter showing a shorter helix, higher amphipathicity and cationic residues placed at optimal distances to form ionic/hydrogen bond with lipid A of LPS. Remarkably, T15,21dK-piscidin-1 showed a helix-loop-helix structure in LPS and its interactions with LPS could be sustained by the distance of separation of side chains of R7 and D-Lys-15 which is close to the inter-phosphate distance of lipid A.


Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Peixes/administração & dosagem , Proteínas de Peixes/química , Lipopolissacarídeos/antagonistas & inibidores , Lisina/química , Animais , Antibacterianos/administração & dosagem , Linhagem Celular , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/administração & dosagem , Lisina/administração & dosagem , Lisina/análogos & derivados , Camundongos
18.
Chem Sci ; 8(1): 808, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30123471

RESUMO

[This corrects the article DOI: 10.1039/C5SC04108B.].

19.
J Med Chem ; 59(10): 4867-80, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27109867

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/química
20.
Chem Sci ; 7(4): 2563-2571, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-28660027

RESUMO

Designed peptides demonstrating well-defined structures and functioning in membrane environment are of significant interest in developing novel proteins for membrane active biological processes including enzymes, electron transfer, ion channels and energy conversion. Heme proteins' ability to carry out multiple functions in nature has inspired the design of several helical heme binding peptides and proteins soluble in water and also recently in membrane. Naturally occurring ß-sheet proteins are both water and membrane soluble, and are known to bind heme, however, designed heme binding ß-sheet proteins are yet to be reported, plausibly because of the complex folding and difficulty in introducing heme binding sites in the ß-sheet structures. Here, we describe the design, NMR structures and biochemical functional characterization of four stranded and six stranded membrane soluble ß-sheet peptides that bind heme and di-heme, respectively. The designed peptides contain either DP-G or DP-DA residues for the nucleation of ß-turns intended to stabilize multi-stranded ß-sheet topologies and ligate heme with bis-His coordination between adjacent antiparallel ß-strands. Furthermore, we have optimized a high affinity heme binding pocket, Kd ∼ nM range, in the adjacent ß-strands by utilizing a series of four stranded ß-sheet peptides employing ß- and ω-amino acids. We find that there is a progressive increase in cofactor binding affinity in the designed peptides with the alkyl chain length of ω-amino acids. Notably, the six stranded ß-sheet peptide binds two molecules of heme in a cooperative fashion. The designed peptides perform peroxidase activity with varying ability and efficiently carried out electron transfer with membrane associated protein cytochrome c. The current study demonstrates the designing of functional ß-sheet proteins in a membrane environment and expands the repertoire of heme protein design.

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