RESUMO
Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.
Assuntos
Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Proteína Son Of Sevenless de Drosófila/ultraestrutura , Proteínas ras/ultraestrutura , Animais , Biomimética , Cristalografia por Raios X , Descoberta de Drogas , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/ultraestrutura , Células HCT116 , Sequências Hélice-Alça-Hélice/genética , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteoma/genética , Transdução de Sinais/genética , Proteína Son Of Sevenless de Drosófila/química , Proteína Son Of Sevenless de Drosófila/genética , Proteínas ras/química , Proteínas ras/genéticaRESUMO
Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, ß-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.
Assuntos
Neoplasias/química , Peptídeos/química , Peptidomiméticos/química , Pinocitose , Linhagem Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Neoplasias/patologia , Ligação Proteica , Conformação ProteicaRESUMO
OBJECTIVES: To evaluate the influence of instrumentation technique on the early osseointegration histomorphometrics and biomechanical fixation of fully laser-etched microgrooves implant surfaces in a sheep model. MATERIAL AND METHODS: Six sheep were subjected to bilateral hip surgeries 3 and 6 weeks before euthanasia. A total of 48 implants (∅4.5 mm, 8 mm in length) were distributed among four sites (8 per animal) and placed in bone sites drilled to 4.6 mm (reamer), 4.1 mm (loose), 3.7 mm (medium) and 3.2 mm (tight) in diameter. After healing, the animals were euthanized and half of the implants were biomechanically tested, while the remainder was subjected to non-decalcified histologic processing. The histomorphometric parameters assessed were bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO). Statistical analysis was performed using a mixed-model analysis of variance with significance level set at P < 0.05. RESULTS: A general increasing trend is present from 3 to 6 weeks for most of the variables. The groups prepared to be press fit seemed to present higher values, which were maintained throughout the observation period. The reamer group presented the lowest BIC probably due to the drilling technique; however qualitatively, more new bone seemed to be in contact to the implant surface, at 3 weeks, whereas the implants placed in press-fit situations were mainly supported by cortical bone. CONCLUSION: The laser-etched microgrooved implant presented osteoconductive and biocompatible properties for all surgical procedures tested. However, procedures providing increasingly higher press-fit scenarios presented the strongest histomorphometric and biomechanical responses at 3 and 6 weeks.
Assuntos
Implantação Dentária Endóssea/métodos , Implantes Dentários , Implantes Experimentais , Osseointegração , Ossos Pélvicos/cirurgia , Animais , Fenômenos Biomecânicos , Lasers , Carneiro Doméstico , Propriedades de Superfície , TorqueRESUMO
Protein-protein interactions (PPIs) play a critical role in fundamental biological processes. Competitive inhibition of these interfaces requires compounds that can access discontinuous binding epitopes along a large, shallow binding surface area. Conformationally defined protein surface mimics present a viable route to target these interactions. However, the development of minimal protein mimics that engage intracellular targets with high affinity remains a major challenge because mimicry of a portion of the binding interface is often associated with the loss of critical binding interactions. Covalent targeting provides an attractive approach to overcome the loss of noncovalent contacts but have the inherent risk of dominating noncovalent contacts and increasing the likelihood of nonselective binding. Here, we report the iterative design of a proteolytically stable α3ß chimeric helix mimic that covalently targets oncogenic Ras G12C as a model system. We explored several electrophiles to optimize preferential alkylation with the desired C12 on Ras. The designed lead peptide modulates nucleotide exchange, inhibits activation of the Ras-mediated signaling cascade, and is selectively toxic toward mutant Ras G12C cancer cells. The relatively high frequency of acquired cysteines as missense mutations in cancer and other diseases suggests that covalent peptides may offer an untapped therapeutic approach for targeting aberrant protein interactions.
Assuntos
Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Peptidomiméticos/farmacologia , Proteínas ras/efeitos dos fármacos , Fenômenos Biofísicos , Linhagem Celular Tumoral , Humanos , Ligantes , Peptidomiméticos/química , Conformação Proteica , Mapas de Interação de Proteínas , Proteólise , Transdução de SinaisRESUMO
The drilling technique and the surface characteristics are known to influence the healing times of oral implants. The influence of osteotomy dimension on osseointegration of microroughned implant surfaces treated with resorbable blasting media was tested in an in vivo model. Ninety-six implants (ø4.5 mm, 8 mm in length) with resorbable blasting media-treated surfaces were placed in the ileum of six sheep. The final osteotomy diameters were 4.6 mm (reamer), 4.1 mm (loose), 3.7 mm (medium), and 3.2 mm (tight). After three and six weeks of healing, the implants were biomechanically tested and histologically evaluated. Statistical analysis was performed using Page L trend test for ordered and paired sample and linear regression, with significance level at p < 0.05. An overall increase in all dependent variables was observed with the reduction of osteotomy diameter. In addition, all osseointegration scores increased over time. At three weeks, the retention was significantly higher for smaller osteotomies. The histological sections depicted intimate contact of bone with all the implant surfaces and osteoblast lines were visible in all sections. The resorbable blasting media microroughed surfaces achieved successful osseointegration for all the instrumentation procedures tested, with higher osseointegration scores for the high insertion torque group.