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1.
J Nurs Educ ; 59(2): 68-75, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003845

RESUMO

BACKGROUND: Although there is an abundance of empirical evidence on principals' leadership practices and teacher satisfaction in K-12 settings, a paucity of publications explore nursing faculty job satisfaction as influenced by leadership styles of academic deans. This research examined leadership styles of nursing deans to determine whether they correlate with nursing faculty job satisfaction at public U.S. universities with high research activity. METHOD: A descriptive, correlational study was conducted with electronic self-administered questionnaires. The sample was recruited from 24 universities; 303 questionnaires were returned. RESULTS: Faculty perceived that nursing deans displayed transformational leadership style more frequently. Nursing faculty were moderately satisfied in their jobs, and they were more satisfied with nursing deans who practiced attributed idealized influence. CONCLUSION: Three types of leadership behaviors explained significant variance in faculty job satisfaction, indicating the need for training and development focused on effective leadership behaviors. [J Nurs Educ. 2020;59(2):68-75.].

2.
Elife ; 92020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31958057

RESUMO

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here we characterize RAS in live human and mouse cells using single molecule tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein's globular domain. To understand the altered mobility of an oncogenic KRAS4b we used complementary experimental and molecular dynamic simulation approaches to reveal a detailed mechanism.

3.
Bioorg Med Chem Lett ; 19(10): 2693-8, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19362470

RESUMO

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor-bound protein 2 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low micromolar range binding affinity, such as cyclic peptide 15 with an K(d)=0.359microM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.


Assuntos
Proteína Adaptadora GRB2/metabolismo , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Simulação por Computador , Descoberta de Drogas , Proteína Adaptadora GRB2/antagonistas & inibidores , Biblioteca de Peptídeos , Peptídeos Cíclicos/síntese química , Ligação Proteica , Relação Estrutura-Atividade , Domínios de Homologia de src
4.
J Med Chem ; 52(6): 1612-8, 2009 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-19226165

RESUMO

A fluorescence anisotropy (FA) competition-based Shc Src homology 2 (SH2) domain-binding was established using the high affinity fluorescein isothiocyanate (FITC) containing peptide, FITC-NH-(CH2)4-CO-pY-Q-G-L-S-amide (8; Kd = 0.35 microM). Examination of a series of open-chain bis-alkenylamide containing peptides, prepared as ring-closing metathesis precursors, showed that the highest affinities were obtained by replacement of the original Gly residue with N alpha-substituted Gly (NSG) "peptoid" residues. This provided peptoid-peptide hybrids of the form "Ac-pY-Q-[NSG]-L-amide." Depending on the NSG substituent, certain of these hybrids exhibited up to 40-fold higher Shc SH2 domain-binding affinity than the parent Gly-containing peptide (IC50 = 248 microM) (for example, for N-homoallyl analogue 50, IC50 = 6 microM). To our knowledge, this work represents the first successful example of the application of peptoid-peptide hybrids in the design of SH2 domain-binding antagonists. These results could provide a foundation for further structural optimization of Shc SH2 domain-binding peptide mimetics.


Assuntos
Peptídeos/metabolismo , Peptoides/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Fluoresceína-5-Isotiocianato/química , Polarização de Fluorescência , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peptídeos/química , Peptoides/química , Receptores Proteína Tirosina Quinases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
5.
J Med Chem ; 51(23): 7459-68, 2008 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-18989951

RESUMO

We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.


Assuntos
Biotina/farmacologia , Proteína Adaptadora GRB2/antagonistas & inibidores , Domínios de Homologia de src/efeitos dos fármacos , Sítios de Ligação , Biotina/análogos & derivados , Biotina/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
6.
J Org Chem ; 72(25): 9635-42, 2007 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-17990895

RESUMO

Ring-closing metathesis (RCM) was employed to join carboxy-terminal alkenyl glycine side chains together with vinyl- and allyl-functionality appended to the beta-methylene of amino-terminal phosphotyrosyl (pTyr) mimetics. This required the synthesis of a variety of new pTyr mimetics, including a novel aza-containing analogue. Many of the resulting 15-member macrocyclic tetrapeptide mimetics exhibited low nanomolar Grb2 SH2 domain-binding affinities in spite of the fact that differing ring junction stereochemistries and geometries of the RCM-derived double bond were employed. The finding that significant latitude exists in the structural requirements for ring closure may facilitate the development of therapeutically relevant macrocyle-based Grb2 SH2 domain-binding antagonists. The synthetic approaches used in this study may also find application to peptide mimetics directed at other biological targets.


Assuntos
Proteína Adaptadora GRB2/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/síntese química , Oligopeptídeos/química , Oligopeptídeos/síntese química , Sítios de Ligação , Ciclização , Conformação Molecular , Mimetismo Molecular , Estereoisomerismo , Domínios de Homologia de src
7.
J Biomol Tech ; 18(4): 259-66, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17916799

RESUMO

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.


Assuntos
Produtos do Gene gag/metabolismo , HIV-1/metabolismo , Oligonucleotídeos/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Sequência de Bases , Primers do DNA , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier
8.
J Med Chem ; 50(8): 1978-82, 2007 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-17371004

RESUMO

A 4-aminopiperidine-4-carboxylic acid residue was placed in the pTyr+1 position of a Grb2 SH2 domain-binding peptide to form a general platform, which was then acylated with a variety of groups to yield a library of compounds designed to explore potential binding interactions, with protein features lying below the betaD strand. The highest affinities were obtained using phenylethyl carbamate and phenylbutyrylamide functionalities.


Assuntos
Proteína Adaptadora GRB2/química , Oligopeptídeos/química , Fosfotirosina/química , Piperidinas/síntese química , Domínios de Homologia de src , Acilação , Sítios de Ligação , Modelos Moleculares , Conformação Molecular , Piperidinas/química
9.
Org Biomol Chem ; 5(2): 367-72, 2007 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-17205182

RESUMO

A family of previously reported ring-closing metathesis (RCM)-derived macrocycles that exhibit potent Grb2 SH2 domain-binding affinity is characterized by stereoselectively-introduced upper ring junctions that bear bicyclic aryl substituents. However, the synthetic complexity of these macrocycles presents a potential limit to their therapeutic application. Therefore, the current study was undertaken to simplify these macrocycles through the use of achiral 4-pentenylamides as ring-forming components. A series of macrocycles (5a-f) was prepared bearing both open and cyclic constructs at the upper ring junction. The Grb2 SH2 domain-binding affinities of these macrocycles varied, with higher affinities being obtained with cyclo-substituents. The most potent analogue (5d) contained a cyclohexyl group and exhibited Grb2 SH2 domain-binding affinity (K(D) = 1.3 nM) that was nearly equal to the parent macrocycle (2), which bore a stereoselectively-introduced naphthylmethyl substituent at the upper ring junction (K(D) = 0.9 nM). The results of this study advance design considerations that should facilitate the development of Grb2 SH2 domain-binding antagonists.


Assuntos
Amidas/química , Química Orgânica/métodos , Receptores ErbB/química , Proteína Adaptadora GRB2/química , Aminas/química , Animais , Cinética , Modelos Químicos , Peptídeos/química , Fosfotirosina , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de Superfície , Domínios de Homologia de src
10.
Biochem Biophys Res Commun ; 349(2): 497-503, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16945340

RESUMO

Development of Grb2-SH2 domain antagonists is considered to be an effective and non-cytotoxic strategy to develop new antiproliferative agents because of their potential to shut down the Ras signaling pathway. We developed a concise route for the efficient synthesis of G1TE analogs on solid phase. Using this route, a series of cyclic peptides that do not rely on phosphotyrosine or its mimics were designed and synthesized based upon the phage library-derived cyclopeptide, G1TE. Considering that Gly7 plays prominent roles for G1TE binding to the Grb2-SH2 domain, we introduced different amino acids in the 7th position. The D-Ala7-containing peptide 3 demonstrates improved binding affinity by adopting favorable conformation for protein binding. This can be rationalized by molecular modeling. The optimization at the Leu2 position was also studied, and the resulting cyclopeptides exhibited remarkably improved binding affinity. Based upon these global modifications, a highly potent peptide ligand 9 was discovered with a Kd = 17 nM, evaluated by Biacore binding assay. This new analog is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. This potent peptidomimetic provides a new template for the development of non-pTyr containing Grb2-SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.


Assuntos
Química Farmacêutica/métodos , Desenho de Drogas , Proteína Adaptadora GRB2/antagonistas & inibidores , Fosfotirosina/química , Alanina/química , Animais , Humanos , Concentração Inibidora 50 , Camundongos , Peptídeos/química , Ligação Proteica , Proteínas Tirosina Quinases/química , Domínios de Homologia de src
11.
Bioorg Med Chem Lett ; 16(20): 5265-9, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-16908148

RESUMO

Copper (I) promoted [3+2] Huisgen cycloaddition of azides with terminal alkynes was used to prepare triazole-containing macrocycles based on the Grb2 SH2 domain-binding motif, 'Pmp-Ac(6)c-Asn', where Pmp and Ac(6)c stand for 4-phosphonomethylphenylalanine and 1-aminocyclohexanecarboxylic acid, respectively. When cycloaddition reactions were conducted at 1mM substrate concentrations, cyclization of monomeric units occurred. At 2mM substrate concentrations the predominant products were macrocyclic dimers. In Grb2 SH2 domain-binding assays the monomeric (S)-Pmp-containing macrocycle exhibited a K(d) value of 0.23microM, while the corresponding dimeric macrocycle was found to have greater than 50-fold higher affinity. The open-chain dimer was also found to have affinity equal to the dimeric macrocycle. This work represents the first application of 'click chemistry' to the synthesis of SH2 domain-binding inhibitors and indicates its potential utility.


Assuntos
Alquinos/química , Azidas/síntese química , Proteína Adaptadora GRB2/química , Compostos Macrocíclicos/síntese química , Domínios de Homologia de src , Azidas/química , Sítios de Ligação , Cobre/química , Ciclização , Proteína Adaptadora GRB2/efeitos dos fármacos , Ligantes , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Sensibilidade e Especificidade , Estereoisomerismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Triazóis/química , Domínios de Homologia de src/efeitos dos fármacos
12.
Nucleic Acids Res ; 34(2): 472-84, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16434700

RESUMO

The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.


Assuntos
Proteínas do Capsídeo/química , Produtos do Gene gag/química , Oligodesoxirribonucleotídeos/química , Proteínas Virais/química , Sítios de Ligação , Ligação Competitiva , Calorimetria , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Fluorescência , Polarização de Fluorescência , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Mutação , Espectrometria de Massas por Ionização por Electrospray , Ressonância de Plasmônio de Superfície , Triptofano/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana
13.
J Med Chem ; 48(16): 5369-72, 2005 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16078854

RESUMO

A new phosphotyrosyl mimetic 4-(alpha-hydroxymalonyl)phenylalanine and its incorporation into a Grb2 SH2 domain-binding tripeptide are presented. In whole-cell studies using malonyl ethyl ester prodrug derivatives, it was observed that the 4-(alpha-hydroxymalonyl)phenylalanyl-containing peptide exhibited greater efficacy than the nonhydroxylated 4-(malonyl)phenylalanyl-containing congener in blocking the association of Grb2 with activated erbB-2 tyrosine kinase. These results are consistent with de-esterification and at least partial intracellular decarboxylation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Malonatos/síntese química , Oligopeptídeos/síntese química , Fenilalanina/análogos & derivados , Fosfotirosina/química , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Linhagem Celular Tumoral , Desenho de Drogas , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Proteína Adaptadora GRB2 , Humanos , Malonatos/química , Malonatos/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fenilalanina/síntese química , Fenilalanina/química , Fenilalanina/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
14.
J Med Chem ; 48(12): 3945-8, 2005 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-15943469

RESUMO

Reported herein are the design, synthesis, and Grb2 SH2 domain-binding affinities of several phosphoryl-mimicking groups displayed within the context of a conformationally constrained macrocyclic platform. With use of surface plasmon resonance techniques, single-digit nanomolar affinities were exhibited by phosphonic acid and malonyl-containing diacidic phosphoryl mimetics (for 4h and 4g, K(D) = 1.47 and 3.62 nM, respectively). Analogues containing monoacidic phosphoryl mimetics provided affinities of K(D) = 16-67 nM. Neutral phosphoryl-mimicking groups did not show appreciable binding.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Compostos Macrocíclicos/síntese química , Organofosfatos/química , Domínios de Homologia de src , Sítios de Ligação , Ligação Competitiva , Ensaio de Imunoadsorção Enzimática , Proteína Adaptadora GRB2 , Compostos Macrocíclicos/química , Mimetismo Molecular , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
15.
Bioorg Med Chem ; 13(13): 4200-8, 2005 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15893931

RESUMO

Although considerable effort has been devoted to developing Grb2 SH2 domain-binding antagonists, important questions related to ligand specificity, and identification of intracellular targets remain unanswered. In order to begin addressing these issues, the design, synthesis, and evaluation of a novel biotinylated macrocycle are reported that bears biotin functionality at a C-terminal rather than the traditional N-terminal position. With a Grb2 SH2 domain-binding K(eq) value of 3.4 nM, the title macrocycle (5) is among the most potent biotinylated SH2 domain-binding ligands yet disclosed. This should be a useful tool for elucidating physiological targets of certain Grb2 SH2 domain-binding antagonists.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mimetismo Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fosfotirosina/química , Receptor ErbB-2/metabolismo , Domínios de Homologia de src , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Biotinilação , Neoplasias da Mama/metabolismo , Ciclização , Feminino , Proteína Adaptadora GRB2 , Humanos , Ligantes , Modelos Moleculares , Fragmentos de Peptídeos/química , Ligação Proteica , Relação Estrutura-Atividade , Células Tumorais Cultivadas
16.
Bioorg Med Chem ; 13(7): 2431-8, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15755645

RESUMO

Preferential binding of ligands to Grb2 SH2 domains in beta-bend conformations has made peptide cyclization a logical means of effecting affinity enhancement. This is based on the concept that constraint of open-chain sequences to bend geometries may reduce entropy penalties of binding. The current study extends this approach by undertaking ring-closing metathesis (RCM) macrocyclization between i and i+3 residues through a process involving allylglycines and beta-vinyl-functionalized residues. Ring closure in this fashion results in minimal macrocyclic tetrapeptide mimetics. The predominant effects of such macrocyclization on Grb2 SH2 domain binding affinity were increases in rates of association (from 7- to 16-fold) relative to an open-chain congener, while decreases in dissociation rates were less pronounced (approximately 2-fold). The significant increases in association rates were consistent with pre-ordering of solution conformations to near those required for binding. Data from NMR experiments and molecular modeling simulations were used to interpret the binding results. An understanding of the conformational consequences of such i to i+3 ring closure may facilitate its application to other systems where bend geometries are desired.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Alilglicina/química , Compostos Macrocíclicos/química , Mimetismo Molecular , Peptídeos/síntese química , Fosfotirosina/química , Ciclização , Proteína Adaptadora GRB2 , Compostos Macrocíclicos/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Estrutura Terciária de Proteína , Estereoisomerismo , Relação Estrutura-Atividade , Domínios de Homologia de src
17.
J Med Chem ; 48(3): 764-72, 2005 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-15689160

RESUMO

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or alpha-methyl-p-phosphonophenylalanine ((alpha-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously presents structural features of both (alpha-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (alpha-Me)Ppp residues when incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide platform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (alpha-Me)Ppp-containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent (KD = 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate. Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold decrease in binding affinity (KD = 97.8 nM), which was the result of a further 6-fold increase in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of the key structural components found in (alpha-Me)Ppp into the Ac6c residue, the tetralin hybrid does enhance binding on-rate. However, net binding affinity is decreased due to an associated increase in binding off-rate. Alternatively, global conformational constraint of an (alpha-Me)Ppp-containing peptide by beta-macrocyclization did result in pronounced elevation of binding affinity, which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM. However this did not closely approximate the data obtained. Rather, use of a complex model that assumed two binding sites resulted in a very close fit of data and provided KD values of 97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational constraint in the pY + 1 residue proved to be deleterious, global conformational constraint through beta-macrocyclization achieved higher affinity. Similar beta-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Organofosfonatos/química , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Fosfopeptídeos/síntese química , Fosfotirosina/química , Domínios de Homologia de src , Sítios de Ligação , Ciclização , Proteína Adaptadora GRB2 , Modelos Moleculares , Conformação Molecular , Mimetismo Molecular , Fenilalanina/química , Fosfopeptídeos/química , Ligação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/química
18.
Bioorg Med Chem Lett ; 15(5): 1385-8, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15713392

RESUMO

Fluorescence labeling has become a general technique for studying the intracellular accumulation and localization of exogenously administered materials. Reported herein is a low nanomolar affinity Grb2 SH2 domain-binding antagonist that utilizes the environmentally-sensitive nitrobenzoxadiazole (NBD) fluorophore as a naphthyl replacement. This novel agent should serve as a useful tool to visualize the actions of this class of Grb2 SH2 domain-binding antagonists in whole cell systems.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Corantes Fluorescentes/química , Oxidiazóis/química , Peptídeos/química , Peptídeos/farmacologia , Domínios de Homologia de src/efeitos dos fármacos , Ligação Competitiva , Desenho de Drogas , Corantes Fluorescentes/síntese química , Proteína Adaptadora GRB2 , Ligantes , Conformação Molecular , Mimetismo Molecular , Oxidiazóis/síntese química , Ligação Proteica/efeitos dos fármacos , Coloração e Rotulagem/métodos , Relação Estrutura-Atividade
19.
Chembiochem ; 6(4): 668-74, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15719347

RESUMO

Ring-closing metathesis (RCM) of peptides often requires insertion of allylglycines at the intended sites of ring juncture, which can result in the displacement of residues that are needed for biological activity. This type of side-chain deletion can be avoided by appending beta-vinyl substituents onto the parent residues at the intended sites of ring juncture, thereby effectively converting them into functionalized allylglycine equivalents. Such an approach has been previously applied in modified form to growth-factor receptor bound 2 (Grb2) SH2 domain-binding peptides by using an N-terminal beta-vinyl-functionalized phosphotyrosyl mimetic and C-terminal 2-allyl-3-aryl-1-propanamides that lacked the alpha-carboxyl portion of allylglycine residues. These C-terminal moieties involved lengthy synthesis and once prepared, required an individual total synthesis of each final macrocycle. Work reported herein significantly enhances the versatility of the original approach through the use of C-terminal allylglycine amides that can be prepared from commercially available L- and D-allylglycines and suitable amines. This methodology could be generally useful where macrocylization is desired with maintenance of functionality at a site of ring juncture.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Alilglicina/química , Compostos Macrocíclicos/síntese química , Mimetismo Molecular , Fosfotirosina/química , Domínios de Homologia de src , Antineoplásicos/síntese química , Ciclização , Proteína Adaptadora GRB2 , Humanos , Estrutura Molecular , Ligação Proteica , Ressonância de Plasmônio de Superfície
20.
Chem Biodivers ; 2(4): 447-56, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17191992

RESUMO

As typified by 2-{(9S,10S,14R,18S)-18-(2-amino-2-oxoethyl)-14-[(5-methyl-1H-indol-1-yl)methyl]-8,17,20-trioxo-10-[4-(phosphonomethyl)phenyl]-7,16,19-triazaspiro[5.14]icos-11-en-9-yl}acetic acid ((14R)-1b), ring-closing methathesis-derived macrocyclic tetrapeptide mimetics have recently been reported that bind with high affinity to Grb2 SH2 domains in both extracellular and whole-cell assays. The synthetic complexity of this class of agents limits further therapeutic development. Although a significant component of this synthetic complexity arises from the presence of three stereogenic centers, C(9) (S), C(10) (S), and C(14) (R), it is unclear whether stereoselective introduction of defined configuration at C(14) is required for high-affinity binding. Reported herein is a synthetic route to these macrocycles lacking stereoselectivity in the formation of the C(14) ring junction, which is four synthetic steps shorter than the original stereoselective synthesis. Separation of C(14)-epimers obtained by this approach was achieved by preparative HPLC. Molecular-dynamics studies of ligands bound to the Grb2 SH2 domain protein indicated that the (14R)-configuration should display more-favorable interactions with the protein relative to the (14S)-epimer. Indeed, although surface-plasmon-resonance-derived binding constants to Grb2 SH2 domain protein indicated that the affinity of the (14R)-epimer (KD = 4.8 nM) is greater than that of the (14S)-epimer (KD = 11 nM), it is only marginally so. Therefore, little affinity would be lost through a non-stereoselective synthesis of the C(14)-center. Further studies are in progress to explore reduced structural complexity at the C(14)-center.


Assuntos
Proteína Adaptadora GRB2/metabolismo , Compostos Macrocíclicos/síntese química , Domínios de Homologia de src , Proteína Adaptadora GRB2/química , Estrutura Molecular , Peptídeos , Estrutura Terciária de Proteína
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