Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Med ; 3(12): 860-882.e15, 2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36257298

RESUMO

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.


Assuntos
Barreira Hematoencefálica , Transcitose , Barreira Hematoencefálica/metabolismo , Transcitose/fisiologia , Receptores da Transferrina , Transporte Biológico/fisiologia , Anticorpos
2.
Bioorg Med Chem Lett ; 27(24): 5490-5495, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29126850

RESUMO

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity.


Assuntos
Anticorpos Biespecíficos/química , Antígenos de Superfície/imunologia , Glutamato Carboxipeptidase II/imunologia , Fragmentos Fab das Imunoglobulinas/química , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Complexo CD3/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Química Click , Dissulfetos/química , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/farmacologia , Leucócitos Mononucleares/citologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
3.
Bioorg Med Chem Lett ; 27(16): 3647-3652, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28720505

RESUMO

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an αHER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency.


Assuntos
Anticorpos Biespecíficos/química , Dissulfetos/química , Imunoglobulina G/imunologia , Anticorpos Biespecíficos/imunologia , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Química Click , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Células MCF-7 , Microscopia de Fluorescência , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo
4.
Bioconjug Chem ; 27(10): 2271-2275, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27666414

RESUMO

Bioorthogonal labeling of antibodies enables the conjugation of compounds, such as small molecules or peptides, which expand targeting capacity or enhance cytotoxicity. Taking advantage of a cyclohexene sulfonamide compound that site-selectively labels Lys64 in human serum albumin (HSA), we demonstrate that domain I of HSA can be used as a fusion protein for the preparation of antibody conjugates. Trastuzumab fusions were expressed at the N-terminus of the light chain or the C-terminus of the heavy chain enabling conjugation to small molecules. Moreover, these conjugates retained HER2 binding and proved to be highly stable in human plasma. Antibody conjugation via HSA domain I fusion should therefore have broad utility for making serum-stable antibody conjugates, particularly for antibody-drug conjugates.


Assuntos
Imunoconjugados/química , Proteínas Recombinantes de Fusão/química , Albumina Sérica/química , Anticorpos/química , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imunoconjugados/sangue , Imunoconjugados/metabolismo , Lisina/química , Domínios Proteicos , Engenharia de Proteínas/métodos , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química , Trastuzumab/química
5.
Bioconjug Chem ; 26(11): 2243-8, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26161903

RESUMO

Site-specific conjugation technologies enable the production of homogeneous antibody-drug conjugates (ADCs) with improved therapeutic indices compared to conventional ADCs. However, current site-specific conjugation methods can only attach one type of drug to a single antibody. Given the emergence of acquired resistance to current ADCs, arming single antibodies with different drugs may provide an attractive option in the development of next-generation ADCs. Here, we describe a site-specific dual conjugation strategy as a platform for dual warhead ADCs.


Assuntos
Cisteína/química , Imunoconjugados/química , Selenocisteína/química , Trastuzumab/química , Linhagem Celular Tumoral , Humanos
6.
Angew Chem Int Ed Engl ; 53(44): 11783-6, 2014 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-25196737

RESUMO

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo half-life of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK-242, a small molecule inhibitor of Toll-like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK-242-based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site-specific manner. Additionally, HSA-cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.


Assuntos
Lisina/química , Albumina Sérica/síntese química , Humanos
7.
Bioconjug Chem ; 25(8): 1402-7, 2014 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-25099687

RESUMO

Current routes for synthesizing antibody-drug conjugates commonly rely on maleimide linkers to react with cysteine thiols. However, thioether exchange with metabolites and serum proteins can compromise conjugate stability and diminish in vivo efficacy. We report the application of a phenyloxadiazole sulfone linker for the preparation of trastuzumab conjugates. This sulfone linker site-specifically labeled engineered cysteine residues in THIOMABs and improved antibody conjugate stability in human plasma at sites previously shown to be labile for maleimide conjugates. Similarly, sulfone conjugation with selenocysteine in an anti-ROR1 scFv-Fc improved human plasma stability relative to maleimide conjugation. Kinetically controlled labeling of a THIOMAB containing two cysteine substitutions was also achieved, offering a strategy for producing antibody conjugates with expanded valency.


Assuntos
Imunoconjugados/química , Sulfonas/química , Anticorpos Monoclonais Humanizados/química , Sítios de Ligação , Linhagem Celular , Humanos , Imunoconjugados/sangue , Fragmentos Fc das Imunoglobulinas/química , Modelos Moleculares , Oxidiazóis/química , Conformação Proteica , Estabilidade Proteica , Anticorpos de Cadeia Única/química , Especificidade por Substrato , Trastuzumab
8.
PLoS One ; 9(1): e85755, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465685

RESUMO

Transcription activator-like (TAL) effector nucleases (TALENs) have enabled the introduction of targeted genetic alterations into a broad range of cell lines and organisms. These customizable nucleases are comprised of programmable sequence-specific DNA-binding modules derived from TAL effector proteins fused to the non-specific FokI cleavage domain. Delivery of these nucleases into cells has proven challenging as the large size and highly repetitive nature of the TAL effector DNA-binding domain precludes their incorporation into many types of viral vectors. Furthermore, viral and non-viral gene delivery methods carry the risk of insertional mutagenesis and have been shown to increase the off-target activity of site-specific nucleases. We previously demonstrated that direct delivery of zinc-finger nuclease proteins enables highly efficient gene knockout in a variety of mammalian cell types with reduced off-target effects. Here we show that conjugation of cell-penetrating poly-Arg peptides to a surface-exposed Cys residue present on each TAL effector repeat imparted cell-penetrating activity to purified TALEN proteins. These modifications are reversible under reducing conditions and enabled TALEN-mediated gene knockout of the human CCR5 and BMPR1A genes at rates comparable to those achieved with transient transfection of TALEN expression vectors. These findings demonstrate that direct protein delivery, facilitated by conjugation of chemical functionalities onto the TALEN protein surface, is a promising alternative to current non-viral and viral-based methods for TALEN delivery into mammalian cells.


Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Endonucleases/administração & dosagem , Marcação de Genes/métodos , Engenharia Genética/métodos , Proliferação de Células , Peptídeos Penetradores de Células/genética , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Receptores CCR5/genética , Receptores CCR5/metabolismo
9.
Mol Metab ; 2(2): 86-91, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24199154

RESUMO

Structure-function studies have analyzed substitutions within the glucagon-like peptide-1 (GLP-1) sequence that increase resistance to proteolysis, however, the investigation into how such substitutions alter interactions at the GLP-1 receptor (GLP-1R) has captured less attention. This work describes our efforts at identifying relevant interactions between peptide ligands and the GLP-1R extracellular domain that contribute to the positioning of the peptide N-terminus for receptor activation. Alanine substitutions at hydrophilic (Glu127⁎ and Glu128⁎) and hydrophobic (Leu32⁎) GLP-1R residues were previously shown to differentially interact with GLP-1 and exendin-4. We examined if these receptor residues influence the activity of GLP-1- and exendin-4-based peptides containing either alanine or glycine at position 2. Additionally, a series of glucagon-based peptides were studied to determine how the central to C-terminal region affects activity. Our results suggest that peptide binding to the GLP-1R is largely driven by hydrophobic interactions with the extracellular domain that orient the N-terminus for activation.

10.
Am J Physiol Endocrinol Metab ; 305(2): E161-70, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23592482

RESUMO

Glucagon-like peptide-1 receptor (GLP-1R) plays a major role in promoting glucose-stimulated insulin secretion in pancreatic ß-cells. In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.


Assuntos
AMP Cíclico/biossíntese , Endossomos/metabolismo , Glucose/farmacologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de Glucagon/fisiologia , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , Exocitose/fisiologia , Imunofluorescência , Genes Reporter , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Luciferases/genética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Receptores de Glucagon/genética , Sacarose/farmacologia
11.
J Pept Sci ; 17(10): 659-66, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21661079

RESUMO

GLP-1 is an incretin peptide involved in the regulation of glucose metabolism and the glucose-dependent stimulation of insulin secretion. Ex-4 is a paralog of GLP-1 that has comparable GLP-1R potency but extended physiological action. GLP-1 and Ex-4 are helical peptides that share ∼50% sequence homology but differ at several residues, notably the second amino acid which controls susceptibility to DPP-IV cleavage. This single amino acid difference yields divergent receptor potency when studied in the context of the two hormone sequences. Ex-4 uniquely tolerates Gly2 through select amino acid differences in the middle region of the peptide that are absent in GLP-1. We report that substitution of Ex-4 amino acids Glu16, Leu21, and Glu24 to the GLP-1 sequence enabled Gly2 tolerance. The coordination of the N-terminus with these central residues shows an interaction of substantial importance not only to DPP-IV stability but also to receptor activation. Extension of this observation to glucagon-based co-agonist peptides showed different structural requirements for effective communication between the N-terminus and the mid-section of these peptides in achieving high potency agonism at the GLP-1 and GCGRs.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/química , Peptídeos/química , Peçonhas/química , Sequência de Aminoácidos , Animais , Dipeptidil Peptidase 4/metabolismo , Exenatida , Peptídeos Semelhantes ao Glucagon/química , Humanos , Receptores de Glucagon/química , Homologia de Sequência de Aminoácidos
12.
J Pept Sci ; 17(3): 218-25, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21308878

RESUMO

Glucagon and glucagon-like peptide-1 (GLP-1)are two structurally related hormones that acutely regulate glucose control in opposite directions through homologous receptors. The molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance. The application of co-agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported. Substitution of amino acids 16, 18, and 20 in glucagon with those found in GLP-1 and exendin-4 were identified as partial contributors to balanced, high potency receptor action. The amidation of the C-terminus was an additional glucagon-based structural change observed to be of seminal importance to discriminate recognition by both receptors. In this work, the molecular basis for receptor selectivity associated with differences in C-terminal peptide sequence has been determined. A single charge inversion in glucagon and GLP-1 receptor sequence at position 68* was determined to significantly alter hormone action. Changing E68* in GLP-1R to the corresponding Lys of GCGR reduced receptor activity for natural GLP-1 hormones by eightfold. The enhanced C-terminal positive charges in GLP-1 peptides favor the native receptor's negative charge at position 68*, while the unfavorable interaction with the C-terminal acid of native glucagon is minimized by amidation. The extension of these observations to other glucagon-related hormones such as oxyntomodulin and exendin, as well as other related receptors such as GIPR, should assist in the assembly of additional hormones with broadened pharmacology.


Assuntos
Glucagon/metabolismo , Receptores de Glucagon/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Glucagon/química , Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptores de Glucagon/química , Receptores de Glucagon/genética
13.
ACS Chem Biol ; 6(2): 135-45, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-20939591

RESUMO

Ex-4 (9-39)a is a well characterized GLP-1 receptor antagonist that suffers from two notable limitations, its nonhuman amino acid sequence and its relatively short in vivo duration of action. Comparable N-terminal shortening of human GLP-1 lessens agonism but does not provide a high potency antagonist. Through a series of GLP-1/Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1-based antagonist were identified as Glu16, Val19, and Arg20, yielding an antagonist of approximately 3-fold greater in vitro potency compared with Ex-4 (9-39)a. The structural basis of antagonism appears to result from stabilization of the α helix combined with enhanced electrostatic and hydrophobic interactions with the extracellular domain of the receptor. Site-specific acylation of the human-based antagonist yielded a peptide of increased potency as a GLP-1 receptor antagonist and 10-fold greater selectivity relative to the GIP receptor. The acylated antagonist demonstrated sufficient duration of action to maintain inhibitory activity when administered as a daily subcutaneous injection. The sustained pharmacokinetics and enhanced human sequence combine to form an antagonist optimized for clinical study. Daily administration of this antagonist by subcutaneous injection to diet-induced obese mice for 1 week caused a significant increase in food intake, body weight, and glucose intolerance, demonstrating endogenous GLP-1 as a relevant hormone in mammalian energy balance in the obese state.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptores de Glucagon/antagonistas & inibidores , Acilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Peso Corporal/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/farmacocinética , Humanos , Camundongos , Camundongos Obesos , Dados de Sequência Molecular , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacocinética , Receptores de Glucagon/agonistas , Receptores de Glucagon/metabolismo
14.
PLoS One ; 5(8): e12368, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20811490

RESUMO

BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.


Assuntos
Bacteriófago T4/enzimologia , DNA Ligases/metabolismo , Nucleotídeos/metabolismo , Fosfotransferases/metabolismo , RNA Catalítico/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Bases , Biocatálise , Fosfatos de Dinucleosídeos/metabolismo , Etilenoglicóis/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleotídeos/química , Nucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Fosforilação , Ribonuclease T1/metabolismo , Especificidade por Substrato
15.
Nat Neurosci ; 13(7): 877-82, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20526334

RESUMO

Cholesterol circulates in the blood in association with triglycerides and other lipids, and elevated blood low-density lipoprotein cholesterol carries a risk for metabolic and cardiovascular disorders, whereas high-density lipoprotein (HDL) cholesterol in the blood is thought to be beneficial. Circulating cholesterol is the balance among dietary cholesterol absorption, hepatic synthesis and secretion, and the metabolism of lipoproteins by various tissues. We found that the CNS is also an important regulator of cholesterol in rodents. Inhibiting the brain's melanocortin system by pharmacological, genetic or endocrine mechanisms increased circulating HDL cholesterol by reducing its uptake by the liver independent of food intake or body weight. Our data suggest that a neural circuit in the brain is directly involved in the control of cholesterol metabolism by the liver.


Assuntos
HDL-Colesterol/sangue , Grelina/fisiologia , Hipotálamo/metabolismo , Fígado/metabolismo , Melanocortinas/metabolismo , Animais , Peso Corporal , Antígenos CD36/metabolismo , Ingestão de Alimentos , Grelina/genética , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Homeostase/fisiologia , Camundongos , Camundongos Knockout , Sistemas Neurossecretores/metabolismo , Ratos , Ratos Wistar , Receptores de Melanocortina/genética , Receptores de Melanocortina/fisiologia , Receptores Depuradores Classe B/metabolismo
16.
Nat Chem Biol ; 5(10): 749-57, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19597507

RESUMO

We report the efficacy of a new peptide with agonism at the glucagon and GLP-1 receptors that has potent, sustained satiation-inducing and lipolytic effects. Selective chemical modification to glucagon resulted in a loss of specificity, with minimal change to inherent activity. The structural basis for the co-agonism appears to be a combination of local positional interactions and a change in secondary structure. Two co-agonist peptides differing from each other only in their level of glucagon receptor agonism were studied in rodent obesity models. Administration of PEGylated peptides once per week normalized adiposity and glucose tolerance in diet-induced obese mice. Reduction of body weight was achieved by a loss of body fat resulting from decreased food intake and increased energy expenditure. These preclinical studies indicate that when full GLP-1 agonism is augmented with an appropriate degree of glucagon receptor activation, body fat reduction can be substantially enhanced without any overt adverse effects.


Assuntos
Peptídeo 1 Semelhante ao Glucagon/agonistas , Obesidade/tratamento farmacológico , Peptídeos Cíclicos/uso terapêutico , Polietilenoglicóis/química , Receptores de Glucagon/agonistas , Tecido Adiposo/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Peso Corporal/efeitos dos fármacos , AMP Cíclico/biossíntese , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Teste de Tolerância a Glucose , Camundongos , Camundongos Obesos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Conformação Proteica
17.
J Neurosci ; 29(18): 5916-25, 2009 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-19420258

RESUMO

We investigated a possible role of the central glucagon-like peptide (GLP-1) receptor system as an essential brain circuit regulating adiposity through effects on nutrient partitioning and lipid metabolism independent from feeding behavior. Both lean and diet-induced obesity mice were used for our experiments. GLP-1 (7-36) amide was infused in the brain for 2 or 7 d. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR or Western blot. To test the hypothesis that the sympathetic nervous system may be responsible for informing adipocytes about changes in CNS GLP-1 tone, we have performed direct recording of sympathetic nerve activity combined with experiments in genetically manipulated mice lacking beta-adrenergic receptors. Intracerebroventricular infusion of GLP-1 in mice directly and potently decreases lipid storage in white adipose tissue. These effects are independent from nutrient intake. Such CNS control of adipocyte metabolism was found to depend partially on a functional sympathetic nervous system. Furthermore, the effects of CNS GLP-1 on adipocyte metabolism were blunted in diet-induced obese mice. The CNS GLP-1 system decreases fat storage via direct modulation of adipocyte metabolism. This CNS GLP-1 control of adipocyte lipid metabolism appears to be mediated at least in part by the sympathetic nervous system and is independent of parallel changes in food intake and body weight. Importantly, the CNS GLP-1 system loses the capacity to modulate adipocyte metabolism in obese states, suggesting an obesity-induced adipocyte resistance to CNS GLP-1.


Assuntos
Sistema Nervoso Central/metabolismo , Metabolismo dos Lipídeos/fisiologia , Obesidade/fisiopatologia , Receptores de Glucagon/fisiologia , Transdução de Sinais/fisiologia , Sistema Nervoso Simpático/fisiologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Análise de Variância , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/genética , Composição Corporal/fisiologia , Sistema Nervoso Central/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1 , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Obesidade/etiologia , Obesidade/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores Adrenérgicos beta/deficiência , Receptores de Glucagon/antagonistas & inibidores , Transdução de Sinais/genética , Sistema Nervoso Simpático/efeitos dos fármacos , Fatores de Tempo
18.
Front Biosci ; 11: 89-112, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16146716

RESUMO

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.


Assuntos
Fármacos Anti-HIV/farmacologia , Regulação Viral da Expressão Gênica , HIV-1/metabolismo , Ácidos Nucleicos/química , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/virologia , Animais , Antivirais/química , Sequência de Bases , DNA de Cadeia Simples/genética , Produtos do Gene gag/metabolismo , Produtos do Gene rev/metabolismo , Produtos do Gene tat/metabolismo , Genes env , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Integrase de HIV/metabolismo , Repetição Terminal Longa de HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Nucleocapsídeo , Peptídeo Hidrolases/química , Conformação Proteica , Precursores de Proteínas/metabolismo , Montagem de Vírus , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana
19.
RNA Biol ; 3(4): 163-9, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17396357

RESUMO

Pausing by reverse transcriptase (RT) during retroviral replication increases the frequency of homologous strand transfer, nucleotide misincorporation, and non-templated nucleotide addition. Pausing frequency increases at sites of DNA damage or upon incorporation of nucleotide analogs with steric barriers. These lesions thus likely stimulate mutations leading to resistant viral strains that escape drug treatments or immune surveillance. To study the response of retroviral RTs to bulky 2' adducts, a ribozyme-catalyzed reaction was used to generate an RNA template strand containing a thiophosphate adduct at a specific 2'-hydroxyl located upstream from a polyadenosine sequence. Subsequent alkylation increased the size of the adduct. Polymerization readthrough efficiencies were compared for mature RTs derived from HIV-1 (p66/p51), AMV (p95/p63), MMLV (p80 monomer), and a truncated version of HIV-1 RT lacking the RNase H domain (p51/p51 homodimer). Readthrough at the 2' lesion was markedly greater for the p51/p51 homodimer of HIV-1 RT than for the other enzymes, suggesting that the presence of the RNase H domain increases the probability that the modified primer/template will encounter a barrier to translocation. Comparison to published structures suggests potential unfavorable interactions between the 2' adduct and W24, F61, I63, D76, and R78 in the fingers domain of the RT. We propose that the enhanced readthrough observed upon RNase H domain deletion alters the trajectory of the primer/template in this region that diminishes steric and electrostatic clash with these residues. The template also included a penta-adenosine sequence that induced pausing in the order MMLV > HIV-1 (p66/p51) > AMV ~ HIV-1 (p51/p51).


Assuntos
Adutos de DNA/química , Transcriptase Reversa do HIV/química , RNA Viral/química , Ribonuclease H/química , Deleção de Sequência , Sequência de Bases , Adutos de DNA/genética , Transcriptase Reversa do HIV/genética , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , RNA Viral/genética , Ribonuclease H/genética , Eletricidade Estática
20.
RNA ; 9(9): 1029-33, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12923252

RESUMO

A better understanding of aptamer function in bacteria would help to establish simple model systems for screening RNA-protein interactions within an intracellular context. Escherichia coli DNA polymerase I mutants (Pol I(ts)) fail to grow at 37 degrees C unless an exogenous DNA polymerase such as HIV-1 reverse transcriptase (RT) is expressed within the cell. Here, we show that four RNA aptamers that inhibit HIV-1 RT in vitro block complementation by HIV-1 RT when expressed in vivo. No other essential functions are impaired by aptamer expression at either temperature. Intracellular aptamer RNA concentrations from induced cultures were measured to range from 76 to 180 nM, which is comparable with exogenously expressed HIV-1 RT levels in these cells. RT polymerase activity was reduced to background levels in cell-free extracts prepared from cultures expressing both HIV-1 RT and the 70.28 aptamer, compared with extracts from cultures expressing HIV-1 RT alone. Intracellularly expressed RNA aptamers can thus be used to generate conditional null mutants in bacteria by titrating an essential protein.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , RNA/metabolismo , Escherichia coli/metabolismo , Temperatura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...