The unusually high MAO-B activity consistently observed in Parkinson's disease (PD) patients has been proposed as a biomarker; however, this has not been realized due to the lack of probes suitable for MAO-B-specific detection in live cells/tissues. Here we report the first two-photon, small molecule fluorogenic probe (U1) that enables highly sensitive/specific and real-time imaging of endogenous MAO-B activities across biological samples. We also used U1 to confirm the reported inverse relationship between parkin and MAO-B in PD models. With no apparent toxicity, U1 may be used to monitor MAO-B activities in small animals during disease development. In clinical samples, we find elevated MAO-B activities only in B lymphocytes (not in fibroblasts), hinting that MAO-B activity in peripheral blood cells might be an accessible biomarker for rapid detection of PD. Our results provide important starting points for using small molecule imaging techniques to explore MAO-B at the organism level.
DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small-molecule inhibitors of DOT1L such as FED1 are potential anti-cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo-reactive and "clickable" affinity-based probes (AfBPs), P1 and P2, which were cell-permeable and structural mimics of FED1. The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub-cellular localization properties of the probes were subsequently studied in live-cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1. Finally with P1/P2, large-scale cell-based proteome profiling, followed by quantitative LC-MS/MS, was carried out to identify potential cellular off-targets of FED1. Amongst the more than 100 candidate off-targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off-target of FED1 by preliminary validation experiments including pull-down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).
A hydrogel-functionalized small molecule microarray has been developed, on which PC-3 cancer cells were selectively grown. Subsequent controlled release of immobilized bioactive compounds enabled cell-based screening to be directly carried out on this platform.
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