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1.
Dev Biol ; 481: 148-159, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34599906

RESUMO

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.


Assuntos
Padronização Corporal , Movimento Celular , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário , Proteínas dos Microfilamentos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Proteínas dos Microfilamentos/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
2.
Biochemistry ; 59(11): 1202-1215, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32133840

RESUMO

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families (a) to bind to CP, (b) to allosterically inhibit barbed-end capping by CP, and (c) to allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs across all protein families [Hernandez-Valladares, M., et al. (2010) Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. Nat. Struct. Mol. Biol. 17, 497-503; Bruck, S., et al. (2006) Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein. J. Biol. Chem. 281, 19196-19203]. These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.


Assuntos
Proteína de Capeamento de Actina CapZ/química , Proteína de Capeamento de Actina CapZ/metabolismo , Actinas/química , Actinas/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Animais , Proteína de Capeamento de Actina CapZ/genética , Dimerização , Eucariotos/classificação , Eucariotos/genética , Eucariotos/metabolismo , Humanos , Cinética , Peptídeos/química , Peptídeos/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
3.
Proteins ; 74(2): 489-96, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18655058

RESUMO

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.


Assuntos
Bacillus/enzimologia , Subtilisinas/química , Animais , Cálcio/metabolismo , Domínio Catalítico , Temperatura Baixa , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Subtilisinas/metabolismo
4.
J Struct Biol ; 162(1): 152-69, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18086534

RESUMO

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.


Assuntos
Proteínas de Bactérias/química , Fenilalanina-tRNA Ligase/química , Staphylococcus haemolyticus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Fenilalanina-tRNA Ligase/metabolismo , Engenharia de Proteínas/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Staphylococcus haemolyticus/genética
5.
Cell Rep ; 23(9): 2795-2804, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29847807

RESUMO

Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ßß subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites.


Assuntos
Proteínas de Capeamento de Actina/metabolismo , Medição da Troca de Deutério , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Regulação Alostérica , Animais , Camundongos , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Solventes
6.
Proteins ; 66(3): 538-46, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17120228

RESUMO

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.


Assuntos
Bactérias/enzimologia , Inibidores de Proteases/farmacologia , Aminopeptidases/química , Aminopeptidases/isolamento & purificação , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Cristalização , Cristalografia por Raios X , Cinética , Espectroscopia de Ressonância Magnética , Metionil Aminopeptidases , Modelos Moleculares , Inibidores de Proteases/química , Conformação Proteica , Teoria Quântica
7.
J Med Chem ; 47(11): 2724-7, 2004 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-15139749

RESUMO

2-Aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-alpha) inhibitors, which contain a novel bicyclic pyrazolone core, are described. Many showed low-nanomolar activity against lipopolysaccharide-induced TNF-alpha production in monocytic cells. Secondary screening data are presented for the pyrimidinyl bicyclic pyrazolones. Several of these analogues showed good oral bioavailability in rat and efficacy in the rat iodoacetate in vivo model.


Assuntos
Pirazóis/síntese química , Pirimidinas/síntese química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Humanos , Técnicas In Vitro , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese
8.
J Invest Dermatol ; 127(9): 2138-46, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17460728

RESUMO

Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology.


Assuntos
Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lipase/genética , Lipase/metabolismo , Malassezia/enzimologia , Couro Cabeludo/microbiologia , Clonagem Molecular , Diglicerídeos/química , Regulação Fúngica da Expressão Gênica , Glicerídeos/química , Humanos , Lipídeos/química , Modelos Biológicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trioleína/química
9.
Bioorg Med Chem Lett ; 16(21): 5687-90, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16908145

RESUMO

Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.


Assuntos
Desenho de Fármacos , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Linhagem Celular , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia
10.
Bioorg Med Chem Lett ; 16(21): 5598-601, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16962772

RESUMO

Utilizing modeling information from a recently resolved structure of human HIF-1alpha prolyl hydroxylase (EGLN1) and structure-based design, a novel series of imidazo[1,2-a]pyridine derivatives was prepared. The activity of these compounds was determined in a human EGLN1 assay and a limited SAR was developed.


Assuntos
Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Piridinas/farmacologia , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Relação Estrutura-Atividade
11.
Bioorg Med Chem Lett ; 16(21): 5517-22, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16931007

RESUMO

A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.


Assuntos
Desenho de Fármacos , Oxiquinolina/análogos & derivados , Oxiquinolina/farmacologia , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Prolina Dioxigenases do Fator Induzível por Hipóxia , Oxiquinolina/síntese química , Oxiquinolina/química , Pró-Colágeno-Prolina Dioxigenase/química , Relação Estrutura-Atividade
12.
Acta Crystallogr D Biol Crystallogr ; 62(Pt 12): 1435-45, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17139078

RESUMO

Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.


Assuntos
Domínio Catalítico , Desenho de Fármacos , Engenharia de Proteínas , Proteínas Tirosina Fosfatases/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
13.
Bioorg Med Chem Lett ; 16(21): 5616-20, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16908149

RESUMO

Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.


Assuntos
Desenho de Fármacos , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia
15.
Bioorg Med Chem Lett ; 16(21): 5659-63, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16919947

RESUMO

The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.


Assuntos
Amidas/farmacologia , Desenho de Fármacos , Indóis/farmacologia , Osteoporose/tratamento farmacológico , PPAR gama/metabolismo , Células 3T3 , Amidas/síntese química , Animais , Indóis/síntese química , Camundongos
16.
Bioorg Med Chem Lett ; 16(13): 3510-3, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16632356

RESUMO

A new class of tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors based on an N-2,4-pyrimidine-N-phenyl-N'-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated TNF-alpha production. X-ray co-crystallization studies with mutated p38alpha showed that these trisubstituted ureas interact with the ATP-binding pocket in a pseudo-bicyclic conformation brought about by the presence of an intramolecular hydrogen bonding interaction.


Assuntos
Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Trifosfato de Adenosina/química , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Estrutura Molecular , Compostos de Fenilureia/classificação , Pirimidinas/classificação , Estereoisomerismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese
17.
Bioorg Med Chem Lett ; 16(16): 4360-5, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16750367

RESUMO

A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-alpha production are described. The most potent analogs showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell based assay. The SAR of the series was optimized with the aid of X-ray co-crystal structures of these inhibitors bound with mutated p38 (mp38).


Assuntos
Purinas/química , Fator de Necrose Tumoral alfa/química , Linhagem Celular , Química Farmacêutica , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Lipopolissacarídeos/química , Modelos Químicos , Modelos Moleculares , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
Bioorg Med Chem Lett ; 15(9): 2285-9, 2005 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-15837310

RESUMO

4-Aryl-5-pyrimidyl based cytokine synthesis inhibitors that contain a novel monocyclic, pyrazolone heterocyclic core are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-alpha production. One of the compounds (6e) was found to be efficacious in the rat iodoacetate (RIA) in vivo model of osteoarthritis. The X-ray crystal structure of a pyrazolone inhibitor cocrystallized with mutated p38 (mp38) is presented.


Assuntos
Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Pirazolonas/síntese química , Pirazolonas/farmacologia , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Cinética , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Conformação Molecular , Osteoartrite/prevenção & controle , Pirazolonas/química , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
20.
Bioorg Med Chem Lett ; 14(16): 4267-72, 2004 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-15261284

RESUMO

Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-alpha (TNF-alpha) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-alpha production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.


Assuntos
Pirazolonas/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Lipopolissacarídeos/farmacologia , Modelos Moleculares , Pirazolonas/administração & dosagem , Pirazolonas/farmacocinética , Pirazolonas/farmacologia , Ratos , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese
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