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1.
Nature ; 609(7926): 416-423, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35830882

RESUMO

RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation6-14-and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions.


Assuntos
Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos , Proteína Fosfatase 1 , Proteínas ras , Proteínas 14-3-3 , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Complexos Multiproteicos/química , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Quinases raf , Proteínas ras/química , Proteínas ras/metabolismo
2.
Protein Expr Purif ; 147: 38-48, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29475084

RESUMO

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.


Assuntos
Humor Aquoso/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Olho/química , Glicoproteínas/química , Multimerização Proteica , Animais , Sítios de Ligação/genética , Western Blotting , Células COS , Linhagem Celular , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutação , Conformação Proteica
3.
Bioorg Med Chem Lett ; 21(21): 6440-5, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21937229

RESUMO

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.


Assuntos
Ácidos Hidroxâmicos/farmacologia , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Sulfonamidas/química , Cristalografia por Raios X , Ácidos Hidroxâmicos/química , Modelos Moleculares , Inibidores de Proteases/química , Relação Estrutura-Atividade
4.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 1): 58-66, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19153467

RESUMO

The inhibitor of apoptosis protein (IAP) family of molecules inhibit apoptosis through the suppression of caspase activity. It is known that the XIAP protein regulates both caspase-3 and caspase-9 through direct protein-protein interactions. Specifically, the BIR3 domain of XIAP binds to caspase-9 via a ;hotspot' interaction in which the N-terminal residues of caspase-9 bind in a shallow groove on the surface of XIAP. This interaction is regulated via SMAC, the N-terminus of which binds in the same groove, thus displacing caspase-9. The mechanism of suppression of apoptosis by cIAP1 is less clear. The structure of the BIR3 domain of cIAP1 (cIAP1-BIR3) in complex with N-terminal peptides from both SMAC and caspase-9 has been determined. The binding constants of these peptides to cIAP1-BIR3 have also been determined using the surface plasmon resonance technique. The structures show that the peptides interact with cIAP1 in the same way that they interact with XIAP: both peptides bind in a similar shallow groove in the BIR3 surface, anchored at the N-terminus by a charge-stabilized hydrogen bond. The binding data show that the SMAC and caspase-9 peptides bind with comparable affinities (85 and 48 nM, respectively).


Assuntos
Caspase 9/química , Complexos Multiproteicos/química , Oligopeptídeos/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Animais , Apoptose , Sítios de Ligação , Caspase 9/metabolismo , Cristalização , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Complexos Multiproteicos/metabolismo , Oligopeptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Ressonância de Plasmônio de Superfície , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
5.
Bioorg Med Chem Lett ; 18(24): 6568-72, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19008098

RESUMO

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Antituberculosos/química , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Resistência a Múltiplos Medicamentos , Fluoroquinolonas/farmacologia , Gatifloxacina , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Químicos , Conformação Molecular , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo
6.
J Mol Med (Berl) ; 96(1): 9-19, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28669027

RESUMO

The contributions of structural biology to drug discovery have expanded over the last 20 years from structure-based ligand optimization to a broad range of clinically relevant topics including the understanding of disease, target discovery, screening for new types of ligands, discovery of new modes of action, addressing clinical challenges such as side effects or resistance, and providing data to support drug registration. This expansion of scope is due to breakthroughs in the technology, which allow structural information to be obtained rapidly and for more complex molecular systems, but also due to the combination of different technologies such as X-ray, NMR, and other biophysical methods, which allows one to get a more complete molecular understanding of disease and ways to treat it. In this review, we provide examples of the types of impact molecular structure information can have in the clinic for both low molecular weight and biologic drug discovery and describe several case studies from our own work to illustrate some of these contributions.


Assuntos
Descoberta de Drogas , Animais , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Humanos , Imunoterapia , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/terapia , Conformação Proteica , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo
7.
Structure ; 24(4): 502-508, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27050687

RESUMO

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.


Assuntos
Bases de Dados de Proteínas , Proteínas/química , Cristalografia por Raios X , Curadoria de Dados , Guias como Assunto , Ligantes , Modelos Moleculares , Conformação Proteica
8.
J Med Chem ; 52(11): 3523-38, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19422229

RESUMO

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would therefore be a novel disease modifying therapy for the treatment of arthritis. Our efforts have resulted in the discovery of a series of carboxylic acid inhibitors of MMP-13 that do not significantly inhibit the related MMP-1 (collagenase-1) or tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE). It has previously been suggested (but not proven) that inhibition of the latter two enzymes could lead to side effects. A promising carboxylic acid lead 9 was identified and a convergent synthesis developed. This paper describes the optimization of 9 and the identification of a compound 24f for further development. Compound 24f is a subnanomolar inhibitor of MMP-13 (IC(50) value 0.5 nM and K(i) of 0.19 nM) having no activity against MMP-1 or TACE (IC(50) of >10000 nM). Furthermore, in a rat model of MMP-13-induced cartilage degradation, 24f significantly reduced proteoglycan release following oral dosing at 30 mg/kg (75% inhibition, p < 0.05) and at 10 mg/kg (40% inhibition, p < 0.05).


Assuntos
Cartilagem/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Piperidinas/farmacologia , Inibidores de Proteases/síntese química , Sulfonamidas/farmacologia , Animais , Cartilagem/metabolismo , Bovinos , Colágeno Tipo II/metabolismo , Cristalografia por Raios X , Concentração Inibidora 50 , Piperidinas/administração & dosagem , Piperidinas/síntese química , Piperidinas/farmacocinética , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Proteoglicanas/metabolismo , Ratos , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética
9.
Aesthetic Plast Surg ; 28(5): 301-6, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15529199

RESUMO

BACKGROUND: First impressions are greatly influenced by facial appearance. Clinical data from psychiatric medicine overwhelming indicate that attractive individuals receive better judgment, treatment and behavior [1,2,10]. This study aimed to determine whether cosmetic alteration of facial features projects a better first impression. METHODS: Random reviewers were asked independently to grade standardized preoperative and postoperative photographs of patients who underwent facial plastic surgery. The reviewers were blinded to the pre- or postoperative status of the photograph. The questions posed to the reviewers were based on first-impression studies used in the past. RESULTS: The findings indicate that postoperative cosmetic surgery patients were graded as 31% more attractive, 27% better in social skills, 22% more successful in dating, 19% better in athletic skills, 15% better in relationship skills, and 13% more financially successful. CONCLUSION: Facial cosmetic surgery can improve the first impression an individual creates.


Assuntos
Estética , Face , Relações Interpessoais , Procedimentos de Cirurgia Plástica , Percepção Social , Blefaroplastia , Face/cirurgia , Humanos , Fotografação , Rinoplastia , Ritidoplastia , Inquéritos e Questionários , Fatores de Tempo
10.
Clin Sci (Lond) ; 103 Suppl 48: 94S-97S, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12193063

RESUMO

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five(TM) insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM and 4.9 min(-1) respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.


Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Isoenzimas/isolamento & purificação , Fosfatase Alcalina/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Baculoviridae/genética , Bovinos , Linhagem Celular , Cromatografia , Clonagem Molecular , Ácido Edético/farmacologia , Endotelina-1/metabolismo , Enzimas Conversoras de Endotelina , Endotelinas/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Glicopeptídeos/farmacologia , Concentração de Íons de Hidrogênio , Insetos , Isoenzimas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Precursores de Proteínas/metabolismo , Cloreto de Sódio/farmacologia , Transfecção
11.
Antimicrob Agents Chemother ; 46(9): 2752-64, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12183225

RESUMO

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.


Assuntos
Amidoidrolases , Aminopeptidases/antagonistas & inibidores , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Ureia/análogos & derivados , Animais , Bactérias/efeitos dos fármacos , Biotransformação , Cristalografia por Raios X , Primers do DNA , Resistência a Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/metabolismo , Feminino , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Humanos , Ácidos Hidroxâmicos/farmacocinética , Técnicas In Vitro , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Conformação Molecular , Inibidores de Proteases/farmacocinética , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológico , Sepse/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/genética , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Ureia/síntese química , Ureia/farmacocinética , Ureia/farmacologia
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