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1.
Nucleic Acids Res ; 52(12): 7305-7320, 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38842936

RESUMO

The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Microscopia Crioeletrônica , Modelos Moleculares , Proteínas Repressoras , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Ligação Proteica , Multimerização Proteica , DNA/química , DNA/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , DNA Bacteriano/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Óperon/genética , Frutosedifosfatos
2.
Biomacromolecules ; 20(1): 412-421, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30485077

RESUMO

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.


Assuntos
Antineoplásicos/química , Gangliosídeos/imunologia , Nanoconjugados/química , Anticorpos de Cadeia Única/química , Células 3T3 , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Bungarotoxinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacologia , Camundongos , Ácidos Polimetacrílicos/química , Ligação Proteica , Anticorpos de Cadeia Única/imunologia
3.
J Biol Chem ; 291(40): 21234-21245, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27510031

RESUMO

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.


Assuntos
Antígenos CD/química , Fator de Crescimento Insulin-Like II/química , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Substituição de Aminoácidos , Antígenos CD/genética , Antígenos CD/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Biomacromolecules ; 14(3): 881-9, 2013 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-23373696

RESUMO

The specificity of polymer conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) bearing cytostatic drugs for cancer cells could be significantly increased by the incorporation of a suitable targeting ligand, such as a monoclonal antibody (mAb). However, direct binding of the protein to the polymer carrier could cause considerable problems, such as decreasing the binding capacity of mAb to its target. Here, we introduce a novel strategy of joining a targeting moiety to a polymeric conjugate with cytostatic drug. The scFv of B1 mAb (specific for BCL1 leukemia cells) was tagged with peptide K ((VAALKEK)4). Peptide E ((VAALEKE)4), which forms a stable coiled coil structure heterodimer with peptide K, was assembled with the HPMA copolymers bearing doxorubicin. Such targeted polymeric conjugates possess very selective and high binding activity toward BCL1 cells. Similarly, targeted polymeric conjugates exert approximately 100 times higher cytostatic activity toward BCL1 cells in comparison to nontargeted conjugates in vitro. At the same time, the conjugates have comparable and rather low cytostatic activity for 38C13 cells, which are used as a negative control, in vitro.


Assuntos
Acrilamidas/farmacologia , Materiais Biocompatíveis/farmacologia , Citostáticos/farmacologia , Leucemia/tratamento farmacológico , Polímeros/farmacologia , Acrilamidas/síntese química , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Anticorpos Monoclonais/química , Materiais Biocompatíveis/síntese química , Linhagem Celular Tumoral , Proliferação de Células , Citostáticos/química , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Metacrilatos/química , Camundongos , Camundongos Endogâmicos BALB C , Polímeros/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Acta Crystallogr D Biol Crystallogr ; 68(Pt 2): 176-85, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22281747

RESUMO

In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector L-arabinose has been determined at 2.2 Šresolution. The L-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K(d) value was 8.4 ± 0.4 µM. The effect of L-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas Repressoras/química , Arabinose/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo
6.
Biomacromolecules ; 12(10): 3645-55, 2011 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-21863890

RESUMO

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.


Assuntos
Acrilamidas/química , Anticorpos Monoclonais/química , Química Click/métodos , Portadores de Fármacos/síntese química , Imunoconjugados/química , Oligopeptídeos/síntese química , Proteínas Recombinantes/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/imunologia , Anidrases Carbônicas/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/enzimologia , Carcinoma/imunologia , Carcinoma/patologia , Dicroísmo Circular , Clonagem Molecular , Dimerização , Portadores de Fármacos/farmacologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacologia , Conformação Molecular , Oligopeptídeos/imunologia , Oligopeptídeos/farmacologia , Plasmídeos , Polietilenoglicóis/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transformação Bacteriana
7.
Acta Crystallogr D Struct Biol ; 77(Pt 11): 1411-1424, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726169

RESUMO

The SorC/DeoR family is a large family of bacterial transcription regulators that are involved in the control of carbohydrate metabolism and quorum sensing. To understand the structural basis of DNA recognition, structural studies of two functionally characterized SorC/DeoR family members from Bacillus subtilis were performed: the deoxyribonucleoside regulator bsDeoR and the central glycolytic genes regulator bsCggR. Each selected protein represents one of the subgroups that are recognized within the family. Crystal structures were determined of the N-terminal DNA-binding domains of bsDeoR and bsCggR in complex with DNA duplexes representing the minimal operator sequence at resolutions of 2.3 and 2.1 Å, respectively. While bsDeoRDBD contains a homeodomain-like HTH-type domain, bsCggRDBD contains a winged helix-turn-helix-type motif. Both proteins form C2-symmetric dimers that recognize two consecutive major grooves, and the protein-DNA interactions have been analyzed in detail. The crystal structures were used to model the interactions of the proteins with the full DNA operators, and a common mode of DNA recognition is proposed that is most likely to be shared by other members of the SorC/DeoR family.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica
8.
Biomedicines ; 9(11)2021 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-34829829

RESUMO

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugated with immunologically active protein B7-H6 via complementary coiled coil VAALEKE (peptide E) and VAALKEK (peptide K) sequences. Receptor B7-H6 was described as a binding partner of NKp30, and its expression has been proven for various tumor cell lines. The binding of B7-H6 to NKp30 activates NK cells and results in Fas ligand or granzyme-mediated apoptosis of target tumor cells. In this work, we optimized the expression of coiled coil tagged B7-H6, its ability to bind activating receptor NKp30 has been confirmed by isothermal titration calorimetry, and the binding stoichiometry of prepared chimeric biopolymer has been characterized by analytical ultracentrifugation. Furthermore, this coiled coil B7-H6-loaded polymer conjugate activates NK cells in vitro and, in combination with coiled coil scFv, enables their targeting towards a model tumor cell line. Prepared chimeric biopolymer represents a promising precursor for targeted cancer immunotherapy by activating the cytotoxic activity of natural killer cells.

9.
J Struct Biol ; 167(2): 145-52, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19401235

RESUMO

Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.


Assuntos
Ácido Aspártico Endopeptidases/química , Candida/enzimologia , Proteínas Fúngicas/química , Pepstatinas/química , Sítios de Ligação , Candida/patogenicidade , Domínio Catalítico , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
10.
Mol Microbiol ; 69(4): 895-910, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18554327

RESUMO

Expression of genes in the gapA operon encoding five enzymes for triose phosphate interconversion in Bacillus subtilis is negatively regulated by the Central glycolytic genes Regulator (CggR). CggR belongs to the large SorC/DeoR family of prokaryotic transcriptional regulators, characterized by an N-terminal DNA-binding domain and a large C-terminal effector-binding domain. When no glucose is present in growth media, CggR binds to its target DNA sequence and blocks the transcription of genes in the gapA operon. In the presence of glucose, binding of the known effector molecule fructose-1,6-bisphosphate abolishes this interaction. We have identified dihydroxyacetone phosphate, glucose-6-phosphate and fructose-6-phosphate as additional CggR ligands that can bind to the effector-binding site. Crystal structures of C-CggR, the C-terminal effector-binding domain of CggR, both unliganded as well as in complex with the four ligands at resolutions between 1.65 and 1.80 A reveal unique ligand-specific structural changes in the binding site that affect the dimer interface. Binding affinities of these ligands were determined by isothermal titration calorimetry. Chemical cross-linking shows that CggR oligomerization is mediated through its effector-binding domain, and that binding of the different ligands differentially affects the distribution of oligomers. Electrophoretic mobility shift assays (EMSAs) confirmed a destabilizing effect of fructose-1,6-bisphosphate on the CggR/DNA complex, and also showed similar effects for dihydroxyacetone phosphate. Our results suggest that CggR stability and function may be modulated by various effectors in a complex fashion.


Assuntos
Bacillus subtilis/enzimologia , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glicólise/genética , Proteínas Repressoras/química , Bacillus subtilis/genética , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Fosfato de Di-Hidroxiacetona/química , Fosfato de Di-Hidroxiacetona/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Frutosefosfatos/química , Frutosefosfatos/metabolismo , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Ligantes , Óperon , Estrutura Terciária de Proteína , Proteínas Repressoras/metabolismo
11.
Biochim Biophys Acta Proteins Proteom ; 1867(4): 376-381, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639426

RESUMO

Germline mutations in NUDT15 cause thiopurine intolerance during treatment of leukemia or autoimmune diseases. Previously, it has been shown that the mutations affect the enzymatic activity of the NUDT15 hydrolase due to decreased protein stability in vivo. Here we provide structural insights into protein destabilization in R139C and V18I mutants using thermolysin-based proteolysis and H/D exchange followed by mass spectrometry. Both mutants exhibited destabilization of the catalytic site, which was more pronounced at higher temperature. This structural perturbation is shared by the mutations despite their different positions within the protein structure. Reaction products of NUDT15 reverted these conformational abnormalities, demonstrating the importance of ligands for stabilization of a native state of the mutants. This study shows the action of pharmacogenetic variants in NUDT15 in a context of protein structure, which might open novel directions in personalized chemotherapy.


Assuntos
Nucleotídeos de Desoxiguanina/química , Pirofosfatases/química , Pirofosfatases/genética , Domínio Catalítico , Mutagênese Sítio-Dirigida , Mutação , Estabilidade Proteica , Temperatura , Termolisina/química
12.
Appl Radiat Isot ; 143: 87-97, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30391716

RESUMO

Human carbonic anhydrase IX is a membrane enzyme that is significantly expressed in some types of cancer cells, while copper radioisotopes offer wide range of diagnostic, therapeutic and theranostic properties. The work was focused on a new approach to the labelling of antibody IgG M75 for epitope human carbonic anhydrase IX with copper radioisotopes 61Cu and 64Cu and its in vivo testing in mice with inoculated colorectal cancer. Monoclonal antibody IgG M75 for epitope human carbonic anhydrase IX was successfully conjugated with copper-specific chelator "phosphinate" and labelled with 61Cu and 64Cu The obtained molecule has considerable potential as a radioimmuno pharmaceutical suitable for imaging of tumours expressing carbonic anhydrase IX by positron emission tomography (PET).


Assuntos
Anticorpos Monoclonais/química , Antígenos de Neoplasias/imunologia , Anidrase Carbônica IX/imunologia , Radioisótopos de Cobre/química , Animais , Anticorpos Monoclonais/farmacocinética , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/enzimologia , Radioisótopos de Cobre/farmacocinética , Células HT29 , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoglobulina G/química , Masculino , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons , Radioimunodetecção , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual
13.
Antiviral Res ; 78(3): 275-7, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18329737

RESUMO

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.


Assuntos
Anticorpos Monoclonais/farmacologia , Farmacorresistência Viral/genética , Variação Genética , Protease de HIV/efeitos dos fármacos , HIV-1/enzimologia , Fragmentos de Imunoglobulinas/farmacologia , Proteínas Recombinantes/farmacologia , Anticorpos Monoclonais/imunologia , Terapia Antirretroviral de Alta Atividade , Dimerização , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Protease de HIV/genética , Protease de HIV/imunologia , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Fragmentos de Imunoglobulinas/imunologia , Modelos Moleculares , Mutação , Proteínas Recombinantes/imunologia
14.
Macromol Biosci ; 18(1)2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28921867

RESUMO

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.


Assuntos
Ciclina D1/imunologia , Imunoconjugados/administração & dosagem , Fragmentos de Imunoglobulinas/administração & dosagem , Leucemia/terapia , Acrilamidas/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Química Click , Ciclina D1/antagonistas & inibidores , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Imunoconjugados/química , Fragmentos de Imunoglobulinas/imunologia , Leucemia/imunologia , Leucemia/patologia , Camundongos , Terapia de Alvo Molecular , Peptídeos/química , Peptídeos/imunologia , Polímeros/administração & dosagem , Polímeros/química
15.
J Med Chem ; 52(22): 7132-41, 2009 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-19874035

RESUMO

HIV protease (HIV PR) is a primary target for anti-HIV drug design. We have previously identified and characterized substituted metallacarboranes as a new class of HIV protease inhibitors. In a structure-guided drug design effort, we connected the two cobalt bis(dicarbollide) clusters with a linker to substituted ammonium group and obtained a set of compounds based on a lead formula [H(2)N-(8-(C(2)H(4)O)(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))-3,3'-Co)(2)]Na. We explored inhibition properties of these compounds with various substitutions, determined the HIV PR:inhibitor crystal structure, and computationally explored the conformational space of the linker. Our results prove the capacity of linker-substituted dual-cage cobalt bis(dicarbollides) as lead compounds for design of more potent inhibitors of HIV PR.


Assuntos
Compostos de Boro/química , Compostos de Boro/farmacologia , Carbono/química , Cobalto/química , Desenho de Fármacos , Protease de HIV/metabolismo , Compostos de Boro/síntese química , Compostos de Boro/metabolismo , Cristalografia por Raios X , Elétrons , Protease de HIV/química , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Modelos Moleculares , Conformação Molecular
16.
J Struct Biol ; 149(3): 332-7, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15721587

RESUMO

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.


Assuntos
Anticorpos Monoclonais/química , Ácido Aspártico Endopeptidases/química , Reações Cruzadas , Anticorpos Anti-HIV/química , Protease de HIV/imunologia , Peptídeos/química , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/imunologia , Cristalografia por Raios X , Anticorpos Anti-HIV/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/imunologia , Conformação Proteica , Alinhamento de Sequência
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