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1.
Sci Rep ; 13(1): 13408, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37591971

RESUMO

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody's activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.


Assuntos
Anticorpos Biespecíficos , Receptores de Enterotoxina , Linfócitos T , Humanos , Epitopos , Reconhecimento Psicológico
2.
Sci Rep ; 12(1): 8699, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610505

RESUMO

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology "Augmented Binary Substitution" (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.


Assuntos
Anticorpos , Células Germinativas , Sequência de Aminoácidos , Aminoácidos , Sítios de Ligação de Anticorpos , Humanos , Imidazóis , Sulfonamidas , Tiofenos
3.
MAbs ; 13(1): 1850395, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33459147

RESUMO

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.


Assuntos
Anticorpos Biespecíficos/imunologia , Complexo CD3/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Enterotoxina/imunologia , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Biespecíficos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Hibridomas , Macaca fascicularis/imunologia , Macaca fascicularis/metabolismo , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo
4.
Antibodies (Basel) ; 5(1)2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-31557987

RESUMO

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

5.
J Mol Biol ; 425(18): 3429-45, 2013 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-23827136

RESUMO

MntC is a metal-binding protein component of the Mn²âº-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn²âº-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn²âº-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium-hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn²âº.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Manganês/metabolismo , Proteínas Periplásmicas de Ligação/química , Staphylococcus aureus , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Fenômenos Biofísicos , Calorimetria/métodos , Dicroísmo Circular , Cristalografia por Raios X , Medição da Troca de Deutério , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Conformação Proteica , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
6.
J Biol Chem ; 288(24): 17408-19, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23632026

RESUMO

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.


Assuntos
Anticorpos Monoclonais Humanizados/química , Tubarões/imunologia , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Proteínas de Peixes , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Albumina Sérica/química
7.
J Biol Chem ; 288(23): 16529-16537, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23615911

RESUMO

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C(H)3-C(H)3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.


Assuntos
Fragmentos Fc das Imunoglobulinas , Engenharia de Proteínas , Animais , Linhagem Celular , Glicosilação , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Receptores Fc/genética , Receptores Fc/metabolismo
8.
ACS Med Chem Lett ; 4(1): 118-23, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24900572

RESUMO

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) catalyzes the conversion of inactive glucocorticoid cortisone to its active form, cortisol. The glucocorticoid receptor (GR) signaling pathway has been linked to the pathophysiology of diabetes and metabolic syndrome. Herein, the structure-activity relationship of a series of piperazine sulfonamide-based 11ß-HSD1 inhibitors is described. (R)-3,3,3-Trifluoro-2-(5-(((R)-4-(4-fluoro-2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-yl)sulfonyl)thiophen-2-yl)-2-hydroxypropanamide 18a (HSD-621) was identified as a potent and selective 11ß-HSD1 inhibitor and was ultimately selected as a clinical development candidate. HSD-621 has an attractive overall pharmaceutical profile and demonstrates good oral bioavailability in mouse, rat, and dog. When orally dosed in C57/BL6 diet-induced obesity (DIO) mice, HSD-621 was efficacious and showed a significant reduction in both fed and fasting glucose and insulin levels. Furthermore, HSD-621 was well tolerated in drug safety assessment studies.

9.
Bioorg Med Chem Lett ; 20(2): 632-5, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19959359

RESUMO

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S(1) to the S(3) pocket.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/química , Hidantoínas/química , Imidazóis/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Hidantoínas/síntese química , Hidantoínas/farmacologia , Ligação de Hidrogênio , Imidazóis/síntese química , Imidazóis/farmacologia
10.
J Med Chem ; 52(17): 5449-61, 2009 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-19673466

RESUMO

Cortisol and the glucocorticoid receptor signaling pathway have been implicated in the development of diabetes and obesity. The reduction of cortisone to cortisol is catalyzed by 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1). 2,4-Disubsituted benzenesulfonamides were identified as potent inhibitors of both the human and mouse enzymes. The lead compounds displayed good pharmacokinetics and ex vivo inhibition of the target in mice. Cocrystal structures of compounds 1 and 20 bound to human 11beta-HSD1 were obtained. Compound 20 was found to achieve high concentrations in target tissues, resulting in 95% inhibition in the ex vivo assay when dosed with a food mix (0.5 mg of drug per g of food) after 4 days. Compound 20 was efficacious in a mouse diet-induced obesity model and significantly reduced fed glucose and fasted insulin levels. Our findings suggest that 11beta-HSD1 inhibition may be a valid target for the treatment of diabetes.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Dieta/efeitos adversos , Inibidores Enzimáticos/farmacologia , Obesidade/enzimologia , Obesidade/etiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Conformação Molecular , Obesidade/tratamento farmacológico , Relação Estrutura-Atividade
11.
J Med Chem ; 52(16): 5013-6, 2009 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-19645448

RESUMO

The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.


Assuntos
Trifosfato de Adenosina/fisiologia , Antineoplásicos/síntese química , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/síntese química , Pirimidinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular Tumoral , Classe Ib de Fosfatidilinositol 3-Quinase , Cristalografia por Raios X , Desenho de Fármacos , Peptídeos e Proteínas de Sinalização Intracelular/química , Isoenzimas/química , Camundongos , Camundongos Nus , Microssomos/metabolismo , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Proteínas Serina-Treonina Quinases/química , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR , Ureia/análogos & derivados , Ureia/síntese química , Ureia/química , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Bioorg Med Chem Lett ; 18(3): 1063-6, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18162398

RESUMO

Proteolytic cleavage of amyloid precursor protein by beta-secretase (BACE-1) and gamma-secretase leads to formation of beta-amyloid (A beta) a key component of amyloid plaques, which are considered the hallmark of Alzheimer's disease. Small molecule inhibitors of BACE-1 may reduce levels of A beta and thus have therapeutic potential for treating Alzheimer's disease. We recently reported the identification of a novel small molecule BACE-1 inhibitor N-[2-(2,5-diphenyl-pyrrol-1-yl)-acetyl]guanidine (3.a.1). We report here the initial hit-to-lead optimization of this hit and the SAR around the aryl groups occupying the S(1) and S(2') pockets leading to submicromolar BACE-1 inhibitors.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Técnicas de Química Combinatória , Guanidinas/síntese química , Guanidinas/farmacologia , Pirróis/química , Cristalografia por Raios X , Guanidinas/química , Conformação Molecular , Estrutura Molecular , Pirróis/farmacologia , Relação Estrutura-Atividade
13.
Protein Sci ; 17(1): 16-21, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18042673

RESUMO

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.


Assuntos
Proteínas ADAM/química , Pró-Colágeno N-Endopeptidase/química , Proteína ADAMTS4 , Proteína ADAMTS5 , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformação Proteica
15.
J Med Chem ; 49(21): 6158-61, 2006 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17034121

RESUMO

BACE1 is an aspartyl protease responsible for cleaving amyloid precursor protein to liberate Abeta, which aggregates leading to plaque deposits implicated in Alzheimer's disease. We have identified small-molecule acylguanidine inhibitors of BACE1. Crystallographic studies show that these compounds form unique hydrogen-bonding interactions with the catalytic site aspartic acids and stabilize the protein in a flap-open conformation. Structure-based optimization led to the identification of potent analogs, such as 10d (BACE1 IC(50) = 110 nM).


Assuntos
Secretases da Proteína Precursora do Amiloide/química , Guanidinas/síntese química , Peptídeos/química , Inibidores de Proteases/síntese química , Domínio Catalítico , Cristalografia por Raios X , Guanidinas/química , Ligação de Hidrogênio , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Inibidores de Proteases/química , Relação Estrutura-Atividade
16.
J Med Chem ; 49(11): 3052-5, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16722622

RESUMO

Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor. Optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity. The crystal structure of 6 and NS5b polymerase complex confirmed the binding near the active site region. The optimization approach and SAR are discussed in detail.


Assuntos
Antivirais/síntese química , Prolina/análogos & derivados , Prolina/síntese química , Sulfonamidas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Antivirais/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Prolina/química , Relação Estrutura-Atividade , Sulfonamidas/química
17.
Peptides ; 27(7): 1877-85, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16574278

RESUMO

Beta-secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel "bis-statine" inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.


Assuntos
Aminoácidos/química , Bioquímica/métodos , Endopeptidases/química , Peptídeos/química , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Biotinilação , Células CHO , Cricetinae , Cristalização , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
18.
Antimicrob Agents Chemother ; 50(2): 556-64, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436710

RESUMO

A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 microM, 4.3 to 10.3 microM, and 6.8 to 29.4 microM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 microM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 A resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 microM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 microg/ml) and 4 (MICs, 4 to 8 microg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.


Assuntos
Antibacterianos/farmacologia , Desidrogenases de Carboidrato/antagonistas & inibidores , Bactérias Gram-Positivas/efeitos dos fármacos , Pirazóis/farmacologia , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/metabolismo , Cristalografia , Fluorescência , Testes de Sensibilidade Microbiana , Peptidoglicano/biossíntese , Ligação Proteica
19.
J Med Chem ; 48(16): 5092-5, 2005 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-16078826
20.
Methods ; 34(3): 329-47, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15325651

RESUMO

Growth of high quality crystals is often the most difficult step in the determination of protein structures by X-ray diffraction. Automation can improve the success of this process both by reducing the amount of protein required for each screen and by relieving the tedium of setting up crystallization experiments by hand. We have been using an automated system for the design and execution of hanging drop crystallization experiments for the last two years. The system includes robots for the preparation of solutions, setup of hanging drops, and automated imaging, as well as a new software package (RoCKS) for managing all phases of the crystallization process. Here, we review the fundamentals of automated protein crystallization and present results from our comparisons of various approaches to screening.


Assuntos
Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Proteínas/química , Cristalização , Bases de Dados de Proteínas , Projetos de Pesquisa , Robótica , Software , Solventes
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