Fast Mek1 Hit Identification with TRIC Technology Correlates Well with Other Biophysical Methods.

The variety and complexity of drug targets are expanding rapidly. At the same time, there is significant interest in exploring a larger chemical space to identify new candidates. Fragment-based screening (FBS) has emerged as a popular alternative to traditional high-throughput screening campaigns to identify such drug candidates. FBS identifies hit fragments that exhibit weak interactions with the target of interest, thereby enabling the rational design of small-molecule compounds from the identified hit fragments, which serve as building blocks. This strategy reduces the number of molecules to screen while also allowing the exploration of a greater chemical space.Here we use temperature-related intensity change (TRIC) technology to perform FBS against the target MAPK/ERK kinase-1 (Mek1). TRIC describes the change in fluorescence intensity of a fluorescently labeled molecule upon a change in temperature. This intensity variation is dependent on the physicochemical environment in the vicinity of the dye and strongly affected by binding events. Thus, the detection of binding events is independent of mass, making TRIC an ideal tool for FBS.Using only 150 pmol of labeled Mek1, the authors screened 193 fragments from a prescreened library in less than 1 h of measurement time, leading to 66 hits. Among those hits, they identified more than 80% of the published top hits found using orthogonal techniques. Furthermore, TRIC allowed the identification of fragments that were of poor solubility but could be mistaken as false-positive hits in other methods.

Expression patterns of anoctamin 1 and anoctamin 2 chloride channels in the mammalian nose.

Calcium-activated chloride channels are expressed in chemosensory neurons of the nose and contribute to secretory processes and sensory signal transduction. These channels are thought to be members of the family of anoctamins (alternative name: TMEM16 proteins), which are opened by micromolar concentrations of intracellular Ca(2+). Two family members,ANO 1 (TMEM16A) and ANO 2 (TMEM16B), are expressed in the various sensory and respiratory tissues of the nose.We have examined the tissue specificity and sub-cellular localization of these channels in the nasal respiratory epithelium and in the five chemosensory organs of the nose: the main olfactory epithelium, the septal organ of Masera, the vomeronasal organ, the Grueneberg ganglion and the trigeminal system. We have found that the two channels show mutually exclusive expression patterns. ANO 1 is present in the apical membranes of various secretory epithelia in which it is co-localized with the water channel aquaporin 5. It has also been detected in acinar cells and duct cells of subepithelial glands and in the supporting cells of sensory epithelia. In contrast, ANO 2 expression is restricted to chemosensory neurons in which it has been detected in microvillar and ciliary surface structures. The different expression patterns of ANO 1 and ANO 2 have been observed in the olfactory, vomeronasal and respiratory epithelia. No expression has been detected in the Grueneberg ganglion or trigeminal sensory fibers. On the basis of this differential expression, we derive the main functional features of ANO 1 and ANO 2 chloride channels in the nose and suggest their significance for nasal physiology.