Medicinal chemistry of Gq proteins and their inhibitors: synthesis, computational calculations and (bio)analytical studies

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The natural product FR900359, a depsipeptide which selectively inhibits Galphaq proteins, was hydrogenated using tritium gas to provide a new compound designated [3H]PSB 15900. We established a radioligand binding assay, and found that the radioligand [3H]PSB 15900 displays high affinity in the low nanomolar range for native Galphaq proteins. In the present project the four human Galphaq protein subtypes will be stably overexpressed in Chinese hamster ovary (CHO) cells which were shown to express very low levels of Gq proteins. These cell lines will be used for investigating the selectivity of the radioligand and of other, newly isolated and synthesized compounds. Modulation of radioligand binding by ions, nucleotides, lipids and proteins will be studied. The radioligand will be used to determine Galphaq expression levels in various cells, tissues and organs under physiological and pathological conditions. With a screening approach utilizing compound libraries we want to identify structurally novel Galphaq inhibitors. Moreover we will optimize an LCMS method to quantify FR900359 in biological samples obtained from in vivo studies, and determine its metabolites.