Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: pharmacological and conformational studies.

Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other alpha-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.

Induction of antioxidant and detoxification response by oxidants in cardiomyocytes: evidence from gene expression profiling and activation of Nrf2 transcription factor.

Mild or low doses of oxidants are known to prime cells towards resistance against further damage. In cardiomyocytes, we found that pretreatment with 100 microM H(2)O(2) prevents the cells from apoptosis induced by doxorubicin (Dox). Affymetrix microarray analyses of 28,000 genes reveal that H(2)O(2) treated cells reduced expression of genes encoding cytochrome c, mitochondrial complex I, III, IV and V and several contractile proteins. Elevated expression of antioxidant and detoxification genes appears as a dominant feature of the gene expression profile of H(2)O(2) treated cells. Most of the genes in this category contain an Antioxidant Response Element (ARE) in their promoters. Measurements of ARE promoter-reporter gene activity indicate a dose- and time-dependent activation of the ARE by H(2)O(2). Since the Nrf2 transcription factor regulates ARE-mediated gene expression, we overexpressed Nrf2 to test whether activation of Nrf2 is sufficient to induce cytoprotection. High levels of Nrf2 expression were achieved via adenovirus mediated gene delivery. Transduced Nrf2 was present in the nuclei and caused an increase in the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1), a representative downstream target of Nrf2. Unlike H(2)O(2) pretreated cells, the cells expressing high levels of Nrf2 were not resistant to Dox-induced apoptosis. Therefore, the cytoprotective effect of H(2)O(2) pretreatment is not reliant upon Nrf2 activation alone as measured by resistance against Dox-induced apoptosis.