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1.
J Med Chem ; 63(23): 15050-15071, 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33261314

RESUMO

Scaffold hopping and structure-based drug design were employed to identify substituted 4-aminoquinolines and 4-aminonaphthyridines as potent, small molecule inhibitors of tumor necrosis factor alpha (TNFα). Structure-activity relationships in both the quinoline and naphthyridine series leading to the identification of compound 42 with excellent potency and pharmacokinetic profile are discussed. X-ray co-crystal structure analysis and ultracentrifugation experiments clearly demonstrate that these inhibitors distort the TNFα trimer upon binding, leading to aberrant signaling when the trimer binds to TNF receptor 1 (TNFR1). Pharmacokinetic-pharmacodynamic activity of compound 42 in a TNF-induced IL-6 mouse model and in vivo activity in a collagen antibody-induced arthritis model, where it showed biologic-like in vivo efficacy, will be discussed.

2.
ACS Med Chem Lett ; 11(3): 266-271, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32184955

RESUMO

Necroptosis has been implicated in a variety of disease states, and RIPK3 is one of the kinases identified to play a critical role in this signaling pathway. In an effort to identify RIPK3 kinase inhibitors with a novel profile, mechanistic studies were incorporated at the hit triage stage. Utilization of these assays enabled identification of a Type II DFG-out inhibitor for RIPK3, which was confirmed by protein crystallography. Structure-based drug design on the inhibitors targeting this previously unreported conformation enabled an enhancement in selectivity against key off-target kinases.

3.
J Biomol NMR ; 68(4): 237-247, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28711957

RESUMO

An improved expression protocol is proposed for amino acid type-specific [13C], [15N]-isotope labeling of proteins in baculovirus-infected (BV) insect cell cultures. This new protocol modifies the methods published by Gossert et al. (J Biomol NMR 51(4):449-456, 2011) and provides efficient incorporation of isotopically labeled amino acids, with similar yields per L versus unlabeled expression in rich media. Gossert et al. identified the presence of unlabeled amino acids in the yeastolate of the growth medium as a major limitation in isotope labeling using BV-infected insect cells. By reducing the amount of yeastolate in the growth medium ten-fold, a significant improvement in labeling efficiency was demonstrated, while maintaining good protein expression yield. We report an alternate approach to improve isotope labeling efficiency using BV-infected insect cells namely by replacing the yeast extracts in the medium with dialyzed yeast extracts to reduce the amount of low molecular weight peptides and amino acids. We report the residual levels of amino acids in various media formulations and the amino acid consumption during fermentation, as determined by NMR. While direct replacement of yeastolate with dialyzed yeastolate delivered moderately lower isotope labeling efficiencies compared to the use of ten-fold diluted undialized yeastolate, we show that the use of dialyzed yeastolate combined with a ten-fold dilution delivered enhanced isotope labeling efficiency and at least a comparable level of protein expression yield, all at a scale which economizes use of these costly reagents.


Assuntos
Marcação por Isótopo/métodos , Aminoácidos/análise , Aminoácidos/química , Animais , Baculoviridae , Antígenos CD4/biossíntese , Antígenos CD4/química , Antígenos CD4/isolamento & purificação , Isótopos de Carbono , Meios de Cultura/análise , Meios de Cultura/química , Quinase 1 de Adesão Focal/biossíntese , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/isolamento & purificação , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Proteínas , Células Sf9 , Spodoptera
4.
Bioconjug Chem ; 27(5): 1276-84, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27098672

RESUMO

A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.


Assuntos
Biotecnologia , Dissulfetos/química , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Modelos Moleculares , Peptídeos/genética , Multimerização Proteica , Estrutura Quaternária de Proteína
5.
Assay Drug Dev Technol ; 12(1): 80-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24547742

RESUMO

Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Interpretação de Imagem Assistida por Computador/métodos , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Bioensaio/métodos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Neoplasias Pulmonares/patologia , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Transdução de Sinais/efeitos dos fármacos
6.
Anal Biochem ; 314(2): 243-52, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12654311

RESUMO

Assays for two enzymes from Escherichia coli were developed and validated as antibacterial inhibitor screens. The MraY and MurG enzymes were overexpressed and purified as the membrane fraction or to homogeneity, respectively. The MurG enzyme was expressed with a six-histidine tag using an optimized minimal-medium protocol for subsequent purification. Although traditional assays were established, the enzymes were also assayed via a 96-well membrane plate assay and a 384-well scintillation proximity-based assay developed herein. These assays afford a more economical and high-throughput evaluation of inhibitors. A mureidomycin inhibitor mix was used as a control for the assay development and screen validation. Several inhibitors resulting from a high-throughput screen were found and evaluated for potential therapeutic use.


Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Transferases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Parede Celular/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Concentração Inibidora 50 , Estrutura Molecular , Peso Molecular , N-Acetilglucosaminiltransferases/antagonistas & inibidores , Nucleosídeos/farmacologia , Fatores de Tempo , Transferases/antagonistas & inibidores
7.
Arch Biochem Biophys ; 410(2): 307-16, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12573291

RESUMO

Amyloid precursor protein (APP) cleaving enzyme (BACE) is the enzyme responsible for beta-site cleavage of APP, leading to the formation of the amyloid-beta peptide that is thought to be pathogenic in Alzheimer's disease (AD). Hence, BACE is an attractive pharmacological target, and numerous research groups have begun searching for potent and selective inhibitors of this enzyme as a potential mechanism for therapeutic intervention in AD. The mature enzyme is composed of a globular catalytic domain that is N-linked glycosylated in mammalian cells, a single transmembrane helix that anchors the enzyme to an intracellular membrane, and a short C-terminal domain that extends outside the phospholipid bilayer of the membrane. Here we have compared the substrate and active site-directed inhibitor binding properties of several recombinant constructs of human BACE. The constructs studied here address the importance of catalytic domain glycosylation state, inclusion of domains other than the catalytic domain, and incorporation into a membrane bilayer on the interactions of the enzyme active site with peptidic ligands. We find no significant differences in ligand binding properties among these various constructs. These data demonstrate that the nonglycosylated, soluble catalytic domain of BACE faithfully reflects the ligand binding properties of the full-length mature enzyme in its natural membrane environment. Thus, the use of the nonglycosylated, soluble catalytic domain of BACE is appropriate for studies aimed at understanding the determinants of ligand recognition by the enzyme active site.


Assuntos
Ácido Aspártico Endopeptidases/química , Proteínas Recombinantes/química , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Células CHO , Catálise , Domínio Catalítico , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Cricetinae , Relação Dose-Resposta a Droga , Drosophila , Endopeptidases , Escherichia coli/metabolismo , Glicosilação , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Luz , Bicamadas Lipídicas/metabolismo , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Espalhamento de Radiação , Fatores de Tempo
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