The proposed screen is to identify inhibitors for the 2' phosphotransferase Tpt1 a gene conserved throughout all phylogeny with well defined and essential roles in RNA end-joining reactions in yeast such as non-conventional splicing during the unfolded protein response (UPR) and tRNA splicing. The UPR is a feedback pathway by which cells regulates the levels of proteins involved in secretory pathway protein folding by inducing transcription of their genes in response to detected shortfalls in their levels. Non-conventional splicing of the transcription factor Hac1/XBP1 mRNA that regulates gene expression during the UPR is initiated by excision of an intron by the conserved endoribonuclease IRE1. In yeast the subsequent RNA end-joining reactions required the sequential actions of two essential genes tRNA ligase Trl1p and 2' phosphotransferase Tpt1p. tRNA ligase joins the ends leaving a 2'PO42+ at the splice junction and Tpt1p removes the 2'PO42+ by transfer to NAD+ to form the unique by-product ADP-2'-3'cyclic ribose (Appr>p). However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin RNA cleaved by IRE1 or tRNA endonuclease. Remarkably, inactivation of the Trpt1 gene, encoding the mammalian homologue of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on splicing during the UPR or tRNA splicing indicating that their ligation proceeds by distinct pathways in yeast and mammals. Furthermore, young Trpt1 mutant mice are apparently healthy and fertile. This raises the question of whether 2' phosphotransferase activity has been conserved throughout phylogeny for alternative processes such as production of Appr>p a potential signaling molecule, or new rolls in RNA metabolism a possibility suggested by the neurological degeneration seen in mice lacking CNP, encoding a functional domain provided by in tRNA ligase in yeast. The isolation of inhibitors will prove useful in three areas of research: creating reagents useful for structural studies studying Tpt1s unique reaction chemistry; exploration of 2'phosphotransferase function in mammals, and isolating lead compounds for new anti-fungal agents that take advantage of the differential requirement of 2'phosphotransferase for viability in fungi and mammals. We have developed robust homogenous fluorescence polarization based screening method that measures 2' phosphotransferase activity in a HTS-ready 384 well plate format and eliminates false positives through secondary screens. The assay was validated through the screening the ~1900 compound NCI diversity library in which a IC50 ~100um inhibitor was identified. We propose to apply the screen to search the~200fold larger chemical libraries available at the MLPCN centers to identify additional higher affinity inhibitors.
The identification of new antifungal agents that may be used independently or in combination with existing therapies is needed to combat the growing problem of topical and invasive/systemic fungal infections due to the increased number of diseases and treatments that lead to an immuno-compromised state. The enzyme that will be targeted in the proposed molecular probe screen Tpt1 is essential in fungi yet dispensable in mammals suggesting it may be possible new drug target for antifungal therapy. The probes identified in the proposed screen will allow us to test this hypothesis and provide tools to further explore the Tpt1 catalytic mechanism. ? ? ?
