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2.
Methods Mol Biol ; 805: 349-65, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22094816

RESUMO

The identification of peptide and protein ligands by directed evolution in vitro has been of enormous utility in molecular biology and biotechnology. However, the translation step in almost all polypeptide selection methods is performed in vivo or in crude extracts, restricting applications. These restrictions include a limited library size due to transformation efficiency, unwanted competing reactions in translation, and an inability to incorporate multiple unnatural amino acids (AAs) with high fidelity and efficiency. These restrictions can be addressed by "pure translation display" where the translation step is performed in a purified system. To date, all pure translation display selections have coupled genotype to phenotype in a ribosome display format, though other formats also should be practical. Here, we detail the original, proof-of-principle, pure-translation-display method because this version should be the most suitable for encoding multiple unnatural AAs per peptide product toward the goal of "peptidomimetic evolution." Challenges and progress toward this ultimate goal are discussed and are mainly associated with improving the efficiency of ribosomal polymerization of multiple unnatural AAs.


Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Modelos Moleculares , Peptídeos/genética , Ribossomos/genética , Ribossomos/metabolismo
3.
J Neurooncol ; 103(2): 207-19, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20824305

RESUMO

Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glioblastoma/patologia , Isoxazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Imunofluorescência , Glioblastoma/tratamento farmacológico , Humanos , Marcação In Situ das Extremidades Cortadas , Isoxazóis/química , Camundongos , Radiossensibilizantes/química , Radiossensibilizantes/farmacologia
4.
Biochemistry ; 49(10): 2177-85, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20141197

RESUMO

Ribosomal incorporations of N-alkyl amino acids including proline are slower than incorporations of non-N-alkyl l-amino acids. The chemical reactivity hypothesis proposes that these results and the exclusion of nonproline N-alkyl amino acids from the genetic code are explained by intrinsic chemical reactivities of the amino acid nucleophiles. However, there is little data on the reactivities relevant to physiological conditions. Here, we use nonenzymatic, aqueous-based, buffered reactions with formylmethionine-N-hydroxysuccinimide ester to model 11 amino acid nucleophiles in dipeptide formation. The relative rates in the nonenzymatic and translation systems correlate well, supporting the chemical reactivity hypothesis and arguing that peptide bond formation, not accommodation, is rate limiting for natural Pro-tRNA(Pro) isoacceptors. The effects of N-substitution sterics, side chain sterics, induction, and pK(a) were evaluated in the chemical model. The dominant factor affecting relative rates was found to be N-substitution sterics.


Assuntos
Modelos Químicos , Peptídeos/química , Peptídeos/metabolismo , Biossíntese de Proteínas , Aminoácidos/química , Aminoácidos/metabolismo , Dipeptídeos/biossíntese , Dipeptídeos/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Biológicos , Ribossomos/metabolismo
5.
PLoS One ; 3(3): e1861, 2008 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-18365016

RESUMO

Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.


Assuntos
Transglutaminases/metabolismo , Sítios de Ligação , Catálise , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Oxazóis/farmacologia , Transglutaminases/antagonistas & inibidores
6.
Biochemistry ; 46(11): 3385-93, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17315981

RESUMO

The epothilone synthetase is a decamodular megasynthase responsible for the biosynthesis of a class of polyketide natural products with clinically promising antitumor activity. Recently, we developed a system comprised of modules 6-9 of the epothilone synthetase for the precursor-directed biosynthesis of epothilones in Escherichia coli [Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004) J. Am. Chem. Soc. 126, 7436-7437]. To systematically explore the biosynthetic potential of this system, we have now investigated the ability of the crucial first module in this engineered pathway, EpoD-M6, to accept, elongate, and process unnatural substrates. EpoD-M6 was expressed, purified, and demonstrated to accept both acyl-CoA and acylSNAC substrates. Of the substrates that were tested, octanoylSNAC and 3-octenoylSNAC proved to be excellent substrates in addition to the more complex natural substrate. Thus, this polyketide synthase module showed considerable tolerance, a feature that bodes well for the precursor-directed biosynthesis of epothilone analogues and related complex polyketides.


Assuntos
Policetídeo Sintases/metabolismo , Subunidades Proteicas/metabolismo , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Myxococcales/enzimologia , Policetídeo Sintases/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Especificidade por Substrato
7.
J Med Chem ; 49(25): 7493-501, 2006 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-17149878

RESUMO

Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, and some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-,5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here, we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity and have led to the discovery of an inhibitor with about 50-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S)-dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R) stereoisomer.


Assuntos
Proteínas de Ligação ao GTP/antagonistas & inibidores , Isoxazóis/síntese química , Transglutaminases/antagonistas & inibidores , Sítios de Ligação , Proteínas de Ligação ao GTP/química , Humanos , Isoxazóis/química , Proteína 2 Glutamina gama-Glutamiltransferase , Estereoisomerismo , Relação Estrutura-Atividade , Transglutaminases/química
8.
J Am Chem Soc ; 126(24): 7436-7, 2004 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-15198579

RESUMO

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.


Assuntos
Epotilonas/biossíntese , Escherichia coli/metabolismo , Complexos Multienzimáticos/biossíntese , Precursores de Proteínas/fisiologia , Antibióticos Antineoplásicos/análise , Antibióticos Antineoplásicos/biossíntese , Epotilonas/análise , Epotilonas/genética , Escherichia coli/genética , Estrutura Molecular , Complexos Multienzimáticos/genética
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