Biophysical analysis of protein-protein and protein-small molecule interactions

The validation of small molecule inhibitors identified by high throughput screening requires a set of robust assays for interaction analysis. Here I describe the implementation of three methods: bioluminescence resonance energy transfer (BRET) for the analysis of protein/protein interaction in cells, surface plasmon resonance (SPR) for the measurement of binding kinetics in vitro and capture compound mass spectrometry (CCMS) for the determination of binding specificity in proteome. All the methods described were tested on cytohesins, a family of guanine nucleotide exchange factors. The very recent discovery of their additional role in the regulation of receptor tyrosine kinases (RTKs) signalling and the availability of specific small molecule inhibitors (the Secins) made them an interesting target. BRET was applied to the analysis of a possible binding of the cytohesin ARNO to the RTK EGF receptor (EGFR). Two strategies were devised: a direct, where the interaction of ARNO and EGFR was monitored, and an indirect, which followed the changes in the EGFR/EGFR interaction upon overexpression of ARNO. Two SPR approaches were developed to analyse the interaction between ARNO and the EGFR on the one hand, and to determine the kinetic parameters of binding of ARNO to its inhibitor Secin16 on the other hand. For CCMS, a photoreactive affinity based SecinH3 probe (SecinH3-TPD) was synthesised and its ability to specifically label ARNO was shown, although the labelling yield was limited by low solubility. A protocol for the enrichment, digestion and MS-analysis of the labelled proteins was established

Implantação de classe hospitalar em um hospital público universitário de São Paulo

OBJETIVO: descrever a implantação de classe hospitalar em hospital público universitário na cidade de São Paulo. MÉTODO: trata-se de descrever o processo, no período compreendido entre 2007 e o primeiro trimestre de 2011, nas respectivas etapas de implantação. RESULTADOS: verificamos através de relatos dos familiares, profissionais da saúde e das escolas de origem dos alunos que a implantação da classe hospitalar colaborou na atenção às necessidades educacionais dos aluno e na inclusão destes na escola regular. Nota-se a importância da celebração dos convênios com as Secretarias da Educação do Estado e do Município e de novas medidas humanizadoras pós-implantação da classe hospitalar. CONCLUSÃO: a hospitalização pode significar uma experiência difícil para o paciente e sua família, porém a implantação da classe hospitalar principalmente em hospitais públicos representa um recurso potencial no desenvolvimento biopsicossocial da criança e do adolescente, colaborando na diminuição dos índices de fracasso e evasão escolar e na inclusão escolar dos alunos após a alta hospitalar.OBJECTIVE: the goal of this article is to describe the hospital grade deployment in a university hospital in the city of São Paulo. METHOD: we described the process between 2007 and 2010, in their respective deployment steps. RESULTS AND DISCUSSION: we verified through reports and actions that the deployment of class hospital collaborated for continued training of teachers, construction of a systematic practice, meet the needs of students and their preparation for inclusion in regular school, integration of pedagogue in multidisciplinary team. We noted the importance of the conclusion of agreements with the secretariats of State education and of Town and new post-implementation hospital measures. CONCLUSION: hospitalization can mean a difficult experience for the patient and his/her family. The hospital grade deployment on a public university hospital represents a resource potential in the bio psychosocial development of children and adolescents, reducing the incidence of failure and dropout, and the inclusion of students after discharge

OXavidin for Tissue Targeting Biotinylated Therapeutics

Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an “artificial receptor” for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications

AvidinOX (TM) for highly efficient tissue-pretargeted radionuclide therapy

Avidin is widely used in vitro for its capacity to bind biotin. However, avidiÅ\u84s in vivo use is limited by its short residence in blood and tissues. An avidin variant, named AvidinOX,â\u84¢ has been recently described. This product is obtained by 4-hydroxyazobenzene-2-carboxylic acid-assisted sodium periodate oxidation of avidin. This method generates aldehyde groups from avidin carbohydrates, sparing biotin-binding sites from inactivation. AvidinOX binds cellular and interstitial protein amino groups through Schiff's bases, resulting in a tissue half-life of 2 weeks, compared with 2 hours of native avidin. Binding of AvidinOX occurs in normal and neoplastic tissues. Data show that AvidinOX, administered intranipple in the breast of transgenic BALB/neuT mice, is highly efficient for capturing 90Y-biotinDOTA, intravenously injected after 48 hours, leading to eradication of multifocal cancer lesions. Efficacy data, together with good tolerability results, indicate that AvidinOX is a highly innovative reagent for tissue-pretargeted radionuclide therapy. © Mary Ann Liebert, Inc

Improved tumor targeting by combined use of two antitenascin antibodies

Purpose: In the pretargeted antibody-guided radioimmunotherapy (PAGRIT) system, the combined use of two different antibodies directed against the same tumor antigen could represent a valid approach for improving tumor targeting and therapeutic efficacy. We developed a novel monoclonal antitenascin antibody, ST2485, and studied its biochemical and functional properties by in vitro and in vivo assays. We then investigated the first of the three-step therapy combining ST2485 with another antitenascin antibody, ST2146, previously described, to increase accumulation of biotinylated antibodies at the tumor site. Experimental Design: Studies of immunoreactivity, affinity, immunohistochemistry, and biodistribution in xenograft model were carried out on ST2485. Analysis of the ST2485 and ST2146 combination was preliminary carried out by ELISA and BiaCore tests and then by in vivo distribution studies after administration of the radiolabeled biotinylated antibodies, followed by a chase with avidin as clearing agent. Results: ST2485 was found to be a suitable antibody for therapeutic applications. Indeed, for its behavior in all tests, it was comparable with ST2146 and better than BC2, an antibody already used for clinical trials. The additivity of ST2146 and ST2485 in tenascin C binding, shown by in vitro tests, was confirmed by biodistribution studies in a xenograft model where tumor localization of the antibodies was near the sum of each antibody alone, with a tumor-to-blood ratio higher than 24. Conclusion: The results reported in this study suggest that a monoclonal antitenascin antibody mixture can improve tumor targeting. This strategy could represent progress for therapeutic approaches such as PAGRIT. © 2005 American Association for Cancer Research

Therapeutic use of avidin is not hampered by antiavidin antibodies in humans

Hen egg white avidin is increasingly used in the clinic as part of multifactor treatments such as pretargeted radionuclide therapy of cancer or as an antidote of biotinylated drugs. Taking into account that naturally occurring human antiavidin antibodies (HAVA) are common in humans, the present work investigates avidin immunogenicity as part of risk/benefit evaluations. Sera from 139 oncology patients naive to avidin were confirmed to exhibit HAVA with lognormally distributed titers. HAVA were boosted after avidin treatment, with no correlation with the avidin dose or with the basal titer. No antibody-related clinical symptoms were observed in 21 HAVA-positive patients treated with avidin. In mouse models, high mouse antiavidin antibody titers, induced to simulate the worst human condition, neither reduced the biotin uptake of intratissue-injected avidin nor affected the capacity of intravenously injected avidin to clear a biotinylated drug from circulation. In both models the avidin treatment was well tolerated. Results indicate that avidin immunogenicity does not affect its safety and efficacy, thus encouraging its further use in clinical applications. Copyright 2010, Mary Ann Liebert, Inc

Preclinical pharmacology and safety of a novel avidin derivative for tissue-targeted delivery of radiolabelled biotin

We recently described an oxidized avidin variant, named AvidinOX ®, which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX ®, is stable for 2weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX ® (up to 10μg/5Ã\u9710 5 cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX ® up to the highest concentration compatible with its solubility (about 12mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX ® to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX ®, performed to mimic an accidental injection of the dose intended for a local administration (15μL of 3.3mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX ® is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society