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1.
Proteins ; 89(9): 1216-1225, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33983654

RESUMO

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.


Assuntos
Proteases 3C de Coronavírus/química , SARS-CoV-2/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Inibidores de Proteases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
2.
Nature ; 550(7677): 481-486, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045389

RESUMO

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.


Assuntos
Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Future Med Chem ; 9(13): 1465-1481, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28795598

RESUMO

AIM: DNA methyltransferases (DNMTs) are important drug targets for epigenetic therapy of cancer. Nowadays, non-nucleoside DNMT inhibitors are in development to address high toxicity of nucleoside analogs. However, these compounds still have low activity in cancer cells and mode of action of these compounds remains unclear. MATERIALS & METHODS: In this work, we studied maleimide derivatives of RG108 by biochemical, structural and computational approaches to highlight their inhibition mechanism on DNMTs. RESULTS: Findings demonstrated a correlation between cytotoxicity on mesothelioma cells of these compounds and their inhibitory potency against DNMTs. Noncovalent and covalent docking studies, supported by crystallographic (apo structure of M.HhaI) and differential scanning fluorimetry assays, provided detailed insights into their mode of action and revealed essential residues for the stabilization of such compounds inside DNMTs. [Formula: see text].


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Maleimidas/química , Ftalimidas/química , Triptofano/análogos & derivados , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/toxicidade , Fluorometria , Humanos , Concentração Inibidora 50 , Camundongos , Simulação de Acoplamento Molecular , Ftalimidas/metabolismo , Ftalimidas/toxicidade , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Triptofano/química , Triptofano/metabolismo , Triptofano/toxicidade
4.
Biochemistry ; 56(6): 824-832, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28121423

RESUMO

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.


Assuntos
Modelos Moleculares , Nicotinamida N-Metiltransferase/metabolismo , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Biocatálise/efeitos dos fármacos , Calibragem , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Limite de Detecção , Metilação/efeitos dos fármacos , Nicotinamida N-Metiltransferase/antagonistas & inibidores , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/genética , Conformação Proteica , Redobramento de Proteína/efeitos dos fármacos , Compostos de Quinolínio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , S-Adenosilmetionina/metabolismo , Espectrometria de Fluorescência
5.
Biochemistry ; 56(6): 886-895, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28098447

RESUMO

Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-ß-d-manno-heptose to Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Glicosiltransferases/metabolismo , Lipídeo A/metabolismo , Modelos Moleculares , Acilação , Substituição de Aminoácidos , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sítios de Ligação , Biocatálise , Dicroísmo Circular , Estabilidade Enzimática , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/química , Glicosiltransferases/genética , Lipídeo A/química , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Solventes/química , Espectrometria de Fluorescência , Propriedades de Superfície , Triptofano/química
6.
Biochemistry ; 55(19): 2760-71, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27100912

RESUMO

The structure of formate dehydrogenase from Candida boidinii (CbFDH) is of both academic and practical interests. First, this enzyme represents a unique model system for studies on the role of protein dynamics in catalysis, but so far these studies have been limited by the availability of structural information. Second, CbFDH and its mutants can be used in various industrial applications (e.g., CO2 fixation or nicotinamide recycling systems), and the lack of structural information has been a limiting factor in commercial development. Here, we report the crystallization and structural determination of both holo- and apo-CbFDH. The free-energy barrier for the catalyzed reaction was computed and indicates that this structure indeed represents a catalytically competent form of the enzyme. Complementing kinetic examinations demonstrate that the recombinant CbFDH has a well-organized reactive state. Finally, a fortuitous observation has been made: the apoenzyme crystal was obtained under cocrystallization conditions with a saturating concentration of both the cofactor (NAD(+)) and inhibitor (azide), which has a nanomolar dissociation constant. It was found that the fraction of the apoenzyme present in the solution is less than 1.7 × 10(-7) (i.e., the solution is 99.9999% holoenzyme). This is an extreme case where the crystal structure represents an insignificant fraction of the enzyme in solution, and a mechanism rationalizing this phenomenon is presented.


Assuntos
Candida/enzimologia , Formiato Desidrogenases/química , Proteínas Fúngicas/química , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Candida/genética , Formiato Desidrogenases/antagonistas & inibidores , Formiato Desidrogenases/genética , Formiato Desidrogenases/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , NAD/química , NAD/metabolismo , Azida Sódica/química
7.
J Biol Chem ; 291(3): 1175-97, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26507654

RESUMO

The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage ß-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Endo-1,3(4)-beta-Glucanase/metabolismo , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Prevotella/enzimologia , Substituição de Aminoácidos , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Celulase/antagonistas & inibidores , Celulase/química , Celulase/genética , Celulose/química , Celulose/metabolismo , Endo-1,3(4)-beta-Glucanase/antagonistas & inibidores , Endo-1,3(4)-beta-Glucanase/química , Endo-1,3(4)-beta-Glucanase/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glucanos/química , Glucanos/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Mutação , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xilanos/química , Xilanos/metabolismo
8.
Biochemistry ; 54(33): 5209-24, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26249842

RESUMO

The Fic domain was recently shown to catalyze AMPylation-the transfer of AMP from ATP to hydroxyl side chains of diverse eukaryotic proteins, ranging from RhoGTPases to chaperon BiP. We have carried out a series of explicit solvent molecular dynamics (MD) simulations up to 1 µs duration on the apo, holo, and substrate/product bound IbpA Fic domain (IbpAFic2). Simulations on holo-IbpAFic2 revealed that binding of Mg(2+) to α and ß phosphates is crucial for preserving catalytically important contacts involving ATP. Comparative analysis of the MD trajectories demonstrated how binding of ATP allosterically induces conformational changes in the distal switch II binding region of Fic domains thereby aiding in substrate recognition. Our simulations have also identified crucial aromatic-aromatic interactions which stabilize the orientation of the catalytic histidine for inline nucleophilic attack during AMPylation, thus providing a structural basis for the evolutionary conservation of these aromatic residue pairs in Fic domains. On the basis of analysis of interacting interface residue pairs that persist over the microsecond trajectory, we identified a tetrapeptide stretch involved in substrate recognition. The structure-based genome-wide search revealed a distinct conservation pattern for this segment in different Fic subfamilies, further supporting its proposed role in substrate recognition. In addition, combined use of simulations and phylogenetic analysis has helped in the discovery of a new subfamily of Fic proteins that harbor a conserved Lys/Arg in place of the inhibitory Glu of the regulatory helix. We propose the novel possibility of auto-enhancement of AMPylation activity in this new subfamily via the movement of regulatory helix, in contrast to auto-inhibition seen in most Fic proteins.


Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Simulação de Dinâmica Molecular , Filogenia , Processamento de Proteína Pós-Traducional , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Biocatálise , Sequência Conservada , Proteínas de Choque Térmico/antagonistas & inibidores , Magnésio/metabolismo , Estrutura Terciária de Proteína , Solventes/química
9.
PLoS One ; 9(8): e103638, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25111382

RESUMO

Protein kinase C related kinase 1 (PRK1) is a component of Rho-GTPase, androgen receptor, histone demethylase and histone deacetylase signaling pathways implicated in prostate and ovarian cancer. Herein we describe the crystal structure of PRK1 in apo form, and also in complex with a panel of literature inhibitors including the clinical candidates lestaurtinib and tofacitinib, as well as the staurosporine analog Ro-31-8220. PRK1 is a member of the AGC-kinase class, and as such exhibits the characteristic regulatory sequence at the C-terminus of the catalytic domain--the 'C-tail'. The C-tail fully encircles the catalytic domain placing a phenylalanine in the ATP-binding site. Our inhibitor structures include examples of molecules which both interact with, and displace the C-tail from the active site. This information may assist in the design of inhibitors targeting both PRK and other members of the AGC kinase family.


Assuntos
Carbazóis/metabolismo , Carbazóis/farmacologia , Piperidinas/metabolismo , Piperidinas/farmacologia , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Pirróis/metabolismo , Pirróis/farmacologia , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Cristalografia por Raios X , Furanos , Humanos , Ligantes , Conformação Proteica/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia
10.
PLoS One ; 8(6): e63828, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23755111

RESUMO

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²âº) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²âº site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²âº ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.


Assuntos
Álcool Desidrogenase/química , Proteínas Arqueais/química , Pyrobaculum/enzimologia , Álcool Desidrogenase/antagonistas & inibidores , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Proteínas Arqueais/antagonistas & inibidores , Biocatálise , Domínio Catalítico , Cobalto/química , Complexos de Coordenação/química , Cristalografia por Raios X , Ensaios Enzimáticos , Estabilidade Enzimática , Flunarizina/química , Ensaios de Triagem em Larga Escala , Ligação de Hidrogênio , Modelos Moleculares , NADP/química , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , Tetralonas/química , Zinco/química
11.
PLoS One ; 7(9): e44828, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028635

RESUMO

Human calcium/calmodulin-dependent protein kinase I (CaMKI) plays pivotal roles in the nervous system. The activity of human CaMKI is regulated by a regulatory region including an autoinhibitory segment and a CaM-binding segment. We report here four structures of three CaMKIα truncates in apo form and in complexes with ATP. In an apo, autoinhibited structure, the activation segment adopts a unique helical conformation which together with the autoinhibitory segment constrains helices αC and αD in inactive conformations, sequesters Thr177 from being phosphorylated, and occludes the substrate-binding site. In an ATP-bound, inactive structure, the activation segment is largely disordered and the CaM-binding segment protrudes out ready for CaM binding. In an ATP-bound, active structure, the regulatory region is dissociated from the catalytic core and the catalytic site assumes an active conformation. Detailed structural analyses reveal the interplay of the regulatory region, the activation segment, and the nucleotide-binding site in the regulation of CaMKI.


Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Calmodulina/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Ratos , Deleção de Sequência
12.
Biochemistry ; 51(31): 6148-63, 2012 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-22788966

RESUMO

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the first unique step of the GMP branch of the purine nucleotide biosynthetic pathway. This enzyme is found in organisms of all three kingdoms. IMPDH inhibitors have broad clinical applications in cancer treatment, as antiviral drugs and as immunosuppressants, and have also displayed antibiotic activity. We have determined three crystal structures of Bacillus anthracis IMPDH, in a phosphate ion-bound (termed "apo") form and in complex with its substrate, inosine 5'-monophosphate (IMP), and product, xanthosine 5'-monophosphate (XMP). This is the first example of a bacterial IMPDH in more than one state from the same organism. Furthermore, for the first time for a prokaryotic enzyme, the entire active site flap, containing the conserved Arg-Tyr dyad, is clearly visible in the structure of the apoenzyme. Kinetic parameters for the enzymatic reaction were also determined, and the inhibitory effect of XMP and mycophenolic acid (MPA) has been studied. In addition, the inhibitory potential of two known Cryptosporidium parvum IMPDH inhibitors was examined for the B. anthracis enzyme and compared with those of three bacterial IMPDHs from Campylobacter jejuni, Clostridium perfringens, and Vibrio cholerae. The structures contribute to the characterization of the active site and design of inhibitors that specifically target B. anthracis and other microbial IMPDH enzymes.


Assuntos
Bacillus anthracis/enzimologia , IMP Desidrogenase/química , IMP Desidrogenase/metabolismo , Inosina Monofosfato/metabolismo , Ribonucleotídeos/metabolismo , Sequência de Aminoácidos , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Modelos Moleculares , Dados de Sequência Molecular , Ácido Micofenólico/metabolismo , NAD/metabolismo , Ligação Proteica , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologia , Xantina
13.
J Med Chem ; 55(2): 956-60, 2012 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-22168824

RESUMO

Casein kinase 1 delta (CK1δ) and its closest homologue CK1ε are key regulators of diverse cellular growth and survival processes such as Wnt signaling, DNA repair, and circadian rhythms. We report three crystal structures of the kinase domain of human CK1δ, one apo and two complexed with a potent and selective CK1δ/ε inhibitor PF670462 in two different crystal forms. These structures provide a molecular basis for the strong and specific inhibitor interactions and suggest clues for further development of CK1δ/ε inhibitors.


Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Imidazóis/química , Modelos Moleculares , Pirimidinas/química , Sequência de Aminoácidos , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase 1 épsilon/química , Caseína Quinase Idelta/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Ligantes , Dados de Sequência Molecular , Conformação Proteica , Relação Quantitativa Estrutura-Atividade
14.
Pac Symp Biocomput ; : 181-92, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21121046

RESUMO

The p38 MAP kinases play a critical role in regulating stress-activated pathways, and serve as molecular targets for controlling inflammatory diseases. Computer-aided efforts for developing p38 inhibitors have been hampered by the necessity to include the enzyme conformational flexibility in ligand docking simulations. A useful strategy in such complicated cases is to perform ensemble-docking provided that a representative set of conformers is available for the target protein either from computations or experiments. We explore here the abilities of two computational approaches, molecular dynamics (MD) simulations and anisotropic network model (ANM) normal mode analysis, for generating potential ligand-bound conformers starting from the apo state of p38, and benchmark them against the space of conformers (or the reference modes of structural changes) inferred from principal component analysis of 134 experimentally resolved p38 kinase structures. ANM-generated conformations are found to provide a significantly better coverage of the inhibitor-bound conformational space observed experimentally, compared to MD simulations performed in explicit water, suggesting that ANM-based sampling of conformations can be advantageously employed as input structural models in docking simulations.


Assuntos
Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química , Anisotropia , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Biologia Computacional , Simulação por Computador , Cristalografia por Raios X , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Inibidores de Proteínas Quinases/química , Água , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Chem Inf Model ; 50(1): 136-45, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19928754

RESUMO

The combination of docking and molecular dynamics simulation is used to explain the isoform-specific selectivity between PI3Kalpha and PI3Kgamma, which are two lipid kinases in the class I PI3Ks. The protein flexibility is incorporated in docking the ligands to the ensemble of representative structures extracted from a clustering analysis of the molecular dynamics simulation in explicit aqueous solution. The reported most potent PI3Kalpha inhibitor PIK-75 was studied, and we predicted three possible PIK-75-bound conformations for PI3Kalpha and two for PI3Kgamma. Comparative analysis between the PI3Kalpha and PI3Kgamma docking experiments indicates that the residue Trp780 and Asn782 in PI3Kalpha and the corresponding residues Trp812 and Glu814 in PI3Kgamma in the solvent-accessible region can confer the PI3Kalpha and PI3Kgamma isoform specificity. The predicted bound conformations are further studied in aqueous solution by molecular dynamics simulation. The work provides a possible effective pharmacophore model for PI3Kalpha inhibitor. The dynamic behaviors of the LY294002-bound PI3Ks are studied too.


Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Cromonas/química , Cromonas/metabolismo , Cromonas/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Morfolinas/química , Morfolinas/metabolismo , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/química , Estrutura Terciária de Proteína , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Homologia de Sequência de Aminoácidos , Soluções , Especificidade por Substrato , Água/química
16.
J Biol Chem ; 284(1): 284-291, 2009 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-18984583

RESUMO

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.


Assuntos
Adenilil Imidodifosfato/química , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Tiazóis/química , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Adenilil Imidodifosfato/uso terapêutico , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linfócitos B/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Dasatinibe , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Terciária de Proteína , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Quinases da Família src/metabolismo
17.
Protein Pept Lett ; 15(8): 843-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18855758

RESUMO

This work describes for the first time a model of Purine Nucleoside Phosphorylase from Listeria monocytogenes (LmPNP). We modeled the complexes of LmPNP with ligands in order to determine the structural basis for specificity. Comparative analysis of the model of LmPNP allowed identification of structural features responsible for ligand affinities.


Assuntos
Biologia Computacional , Listeria monocytogenes/enzimologia , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Desenho de Fármacos , Humanos , Ligantes , Listeria monocytogenes/efeitos dos fármacos , Listeriose/tratamento farmacológico , Modelos Moleculares , Estrutura Terciária de Proteína , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/metabolismo , Especificidade por Substrato
18.
Proteins ; 72(4): 1308-19, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18384081

RESUMO

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.


Assuntos
Ácido Aspártico Endopeptidases/química , Candida albicans/enzimologia , Proteínas Fúngicas/química , Pepstatinas/química , Proteínas Recombinantes/química , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Recombinantes/antagonistas & inibidores
19.
Biochemistry ; 47(11): 3484-92, 2008 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-18293939

RESUMO

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.


Assuntos
Amidoidrolases/química , Ácido Aspártico/análogos & derivados , Encéfalo/enzimologia , Compostos Organofosforados/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Amidoidrolases/metabolismo , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Glicosilação , Humanos , Mutagênese Sítio-Dirigida , Compostos Organofosforados/química , Ligação Proteica/genética
20.
Arch Biochem Biophys ; 471(1): 32-41, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18155152

RESUMO

Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]2+ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe2+ ions and the remaining S2- ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]2+ or [2Fe-2S]2+ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Dobramento de Proteína , Sulfurtransferases/química , Sulfurtransferases/metabolismo , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Catálise , Meios de Cultura/metabolismo , Dimerização , Estabilidade Enzimática , Proteínas de Escherichia coli/antagonistas & inibidores , Deleção de Genes , Marcação de Genes , Proteínas Ferro-Enxofre/antagonistas & inibidores , Desnaturação Proteica , Estrutura Quaternária de Proteína , Sulfurtransferases/antagonistas & inibidores , Termodinâmica
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