The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation.

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.

Antimicrobial, Antitumor and Side Effects Assessment of a Newly Synthesized Tamoxifen Analog.

BACKGROUND: Tamoxifen citrate is a very prevalent drug marketed under several trade names like Apo-Tamox, Nolvadex, Tamec, Tamizam, and Tamoplex. This molecule is approved by the FDA for breast cancer treatment. Some studies have shown that tamoxifen has anti-tuberculosis and antiparasitic activities. Like any drug, tamoxifen possesses side effects, more or less dangerous. AIMS: Basically, this work is a comparative study that aims to: primarily compare the antimicrobial and antitumor activities of tamoxifen and a newly synthesized tamoxifen analog; and to determine the molecule with lesser side effects. METHODS: Three groups of mice were injected with tamoxifen citrate and compound 2(1,1-bis[4-(3- dimethylaminopropoxy)phenyl]-2-phenyl-but-1-ene dihydrochloride) at doses corresponding to C1 (1/10), C2 (1/50), and C3 (1/100) to compound 2 lethal dose (LD50 = 75 mg/kg) administered to adult mice. A group of noninjected mice served as a study control. RESULTS: Experimental results suggest that compound 2 has better antitumor and antimicrobial activity than tamoxifen citrate besides its lower toxicity effects. CONCLUSION: The results obtained from the present study confirmed the antitumor and antimicrobial effect of tamoxifen citrate and its hematological side effects. Compound 2 seems to be more effective than tamoxifen citrate for antitumor and antimicrobial treatment while having less hematological side effects and less disruption of the blood biochemical parameters. These findings encourage us to perform further studies on compound 2 and test it for other therapeutic uses for which tamoxifen was found effective.

A premature termination of human epidermal growth factor receptor transcription in Escherichia coli.

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

Antibacterial properties and mode of action of new triaryl butene citrate compounds.

The aim of this study was to evaluate the antibacterial activity of newly synthesized triaryl butene analogues of tamoxifen. Several compounds were synthesized and converted to citrate salts to ensure greater solubility. Four compounds showed significant antibacterial activity at micromolar concentrations against Gram-positive and Gram-negative foodborne pathogens including Listeria monocytogenes, Listeria ivanovii, Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Two compounds at 50 µM, caused only 7.8 and 11% hemolysis. One of these as well as the remaining two caused high K(+) and Na(+) efflux from bacterial cells. Ultrastructural alterations were also visible using transmission electron microscopy, which revealed severe damage of the inner or outer membrane of E. coli. L. ivanovii showed swelling, corrugations and similar damage indicating a loss of cell-wall integrity. Organometallic compounds may offer interesting opportunities for the design of novel classes of antimicrobial compounds.

In vitro activation and inhibition of recombinant EGFR tyrosine kinase expressed in Escherichia coli.

The present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinant proteins were produced as a glutathione S-transferase (GST) fusion in Escherichia coli, and the solubilisation was performed by sarkosyl addition during extraction. The produced proteins spontaneously dimerize allowing the activation of the tyrosine kinase domain in the presence of [γ-(32)P]ATP. The activity assay has revealed the autophosphorylation of EGFR proteins which was decreased in the presence of genistein. Our system could facilitate the screening of EGFR inhibitors without the need of adding an exogenous substrate.

Effect of the amino chain length and the transformation into citric acid salts of aryl-diphenyl-butenes and ferrocenyl-diphenyl-butenes bearing two dimethylaminoalkyl chains on their antimicrobial activities.

In a previous work we have demonstrated the antimicrobial activity of ferrocenyl or phenyl derivatives of diphenyl butene series. This finding has opened a new area of applications of organometallic compounds. In order to improve these activities, we have synthesized new organic and organometallic diaryl butene compounds with different lengths of their amino chains. These new compounds, and also their ammonium salts, were tested against man pathogenic microorganisms Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538) and Enterococcus hirae (ATCC 10541). It emerged from the tests that the Gram+ bacteria are more sensitive to the compounds than Gram-, and the compounds with 3 carbon amino chains have a better antimicrobial activity than the one having a chain of 2 or 4 carbons. The transformation of compounds to citrate salts was accompanied by a significant regression of antibacterial activity against Pseudomonas aeruginosa, for both organic and ferrocenic molecules. This resistance problem has been solved using hydrochlorides salts rather than citrates one.

Targeting the calmodulin-regulated ErbB/Grb7 signaling axis in cancer therapy.

Signal transduction pathways essential for the survival and viability of the cell and that frequently present aberrant expression or function in tumors are attractive targets for pharmacological intervention in human cancers. In this short review we will describe the regulation exerted by the calcium-receptor protein calmodulin (CaM) on signaling routes involving the family of ErbB receptors - highlighting the epidermal growth factor receptor (EGFR/ErbB1) and ErbB2 - and the adaptor protein Grb7, a downstream signaling component of these receptors. The signaling mechanism of the ErbB/Grb7 axis and the regulation exerted by CaM on this pathway will be described. We will present a brief overview of the current efforts to inhibit the hyperactivity of ErbB receptors and Grb7 in tumors. The currently available information on targeting the CaM-binding site of these signaling proteins will be analyzed, and the pros and cons of directly targeting CaM versus the CaM-binding domain of the ErbB receptors and Grb7 as potential anti-cancer therapy will be discussed.

Monoclonal antibodies as cancer therapeutics.

Three main targets were subjected for the most approved monoclonal antibodies (mAbs) in cancer therapy: EGFR in solid cancer, the clusters of differentiation in blood cancer and VEGF in angiogenesis. Meanwhile side effects, the elevated costs and resistance problems are limiting the efficiency of mAbs as targeted therapy. The combinatory therapy with chemo or radiotherapy has improved the efficiency of mAbs. The present review aims to shed more light on the immunotherapy and the related patents that were developed for cancer treatment.

The offer of chemistry to targeted therapy in cancer.

Cancer therapy is facing the big challenge of destroying selectively tumour cells without harming the normal tissues. Chemotherapy was trying from the beginning to kill malignant cells because of their proliferative activity since normal cells are in general quiescent. Meanwhile side effects were produced due to the destruction of some normal cells that need regular proliferation. The discovery of biomarkers led to the identification of molecular targets within tumour cells in order to kill them selectively. Chemistry followed the progress of biomarkers biotechnology by the production of target specific antagonists which were the subject of many patents. Meanwhile novel problems of tumour resistance appeared and made the battle against cancer a non stop development of new strategies and new weapons. As a consequence, paralleled activities of patenting biomarkers and chemical antagonists are continuously generated. The offer of chemistry does not actually limit the efficiency of Targeted therapy but the identification of biomarkers is still missing the exclusive specificity to tumour cells.

Calmodulin-mediated regulation of the epidermal growth factor receptor.

In this review, we first describe the mechanisms by which the epidermal growth factor receptor generates a Ca(2+) signal and, subsequently, we compile the available experimental evidence regarding the role that the Ca(2+)/calmodulin complex, formed after the rise in cytosolic free Ca(2+) concentration, exerts on the receptor. We focus not only on the indirect action that Ca(2+)/calmodulin exerts on the epidermal growth factor receptor, as a result of the activation of distinct calmodulin-dependent kinases, but also, and more extensively, on the direct interaction of Ca(2+)/calmodulin with the receptor. We also describe several mechanistic models that could account for the Ca(2+)/calmodulin-mediated regulation of epidermal growth factor receptor activity. The control exerted by calmodulin on distinct epidermal growth factor receptor-mediated cellular functions is also discussed. Finally, the phosphorylation of this Ca(2+) sensor by the epidermal growth factor receptor is highlighted.