Profiling a Selective Probe for RTG Branch of Yeast TORC1 Signaling Pathway
In this report, we describe a high throughput flow cytometry based multiplexed screen for molecules that either functionally mimic rapamycin (e.g. those that are transducer of regulated CREB 1 [TORC1] pathway selective, but non-discriminating of TORC1 branchpoints) or compounds that selectively target individual branches of the yeast TORC1 pathway. The high throughput screening (HTS) and the subsequent follow-up structure activity relationship (SAR) studies identified a chemotype that contains analogs of both functional types. Obtaining structurally related analogs with differential selectivity assisted in the characterization of the probe and the pathway in which it operates. Here we report ML231 as a TORC1 Retrograde signaling (RTG) branch selective probe (CIT2 EC50 = 3.8 μM). ML231 is selective on the RTG branch and only affects expression of a small subset of rapamycin-responsive genes. This selectivity appears unrelated to solubility as we showed that a more soluble analog was also selective on the RTG branch. ML231 and its analogs inhibited Sch9 phosphorylation, the major substrate and downstream effector of the TORC1 pathway. This result indicates that this chemotype affects TORC1 activity. ML231 inhibited TORC1 activity incompletely, as compared to a related analog, SID 96099781, which mimicked rapamycin and demonstrated complete TORC1 inhibition. This feature may be partially attributable to the branch selectivity of ML231. ML231 behaved very differently from SID 96099781 in the TORC1 bypass cell growth assay, showing that selective compounds may function downstream of TORC1 while SID 96099781 functions upstream or at the level of TORC1. This result suggests that the ML231 targets components downstream of TORC1, thus executing a partial function of TORC1. Collectively, this suggests that ML231 targets the RTG branch selectively. We further showed that ML231 induced the nuclear translocation of Rtg1p/Rtg3p transcription factors and the activity of ML231 on the CIT2 gene expression was dependent on the Rtg2 protein. These results further confirmed the activity of ML231 on the RTG branch. Although ML231 possesses structural and solubility challenges, this probe represents an achievement in target of rapamycin (TOR) selectivity that to our knowledge has not been previously described. ML231 will be used by investigators focusing on TORC1 signaling, RTG pathways and regulatory signaling between mitochondria and the nucleus.