Systematic analysis of nonprogrammed frameshift suppression in via translational tiling proteomics.

The synthesis of proteins as encoded in the genome depends critically on translational fidelity. Nevertheless, errors inevitably occur, and those that result in reading frame shifts are particularly consequential because the resulting polypeptides are typically nonfunctional. Despite the generally maladaptive impact of such errors, the proper decoding of certain mRNAs, including many viral mRNAs, depends on a process known as programmed ribosomal frameshifting.

Phenotype-based screening rediscovered benzopyran-embedded microtubule inhibitors as anti-neuroinflammatory agents by modulating the tubulin-p65 interaction.

Neuroinflammation is one of the critical processes implicated in central nervous system (CNS) diseases. Therefore, alleviating neuroinflammation has been highlighted as a therapeutic strategy for treating CNS disorders. However, the complexity of neuroinflammatory processes and poor drug transport to the brain are considerable hurdles to the efficient control of neuroinflammation using small-molecule therapeutics.

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action.

Recent advances in identifying protein targets in drug discovery.

Phenotype-based screening has emerged as an alternative route for discovering new chemical entities toward first-in-class therapeutics. However, clarifying their mode of action has been a significant bottleneck for drug discovery. For target protein identification, conventionally bioactive small molecules are conjugated onto solid supports and then applied to isolate target proteins from whole proteome.

Phenotypic Discovery of an Antivirulence Agent against via Modulation of Quorum-Sensing Regulator SmcR.

An antivirulence agent against named quoromycin (QM) was discovered by a phenotype-based elastase inhibitor screening. Using the fluorescence difference in two-dimensional gel electrophoresis (FITGE) approach, SmcR, a quorum-sensing master regulator and homologue of LuxR, was identified as the target protein of QM. We confirmed that the direct binding of QM to SmcR inhibits the quorum-sensing signaling pathway by controlling the DNA-binding affinity of SmcR and thus effectively alleviates the virulence of and .

Label-free target identification reveals oxidative DNA damage as the mechanism of a selective cytotoxic agent.

Phenotypic screening can not only identify promising first-in-class drug candidates, but can also reveal potential therapeutic targets or neomorphic functions of known proteins. In this study, we identified target proteins of SB2001, a cytotoxic agent that acts specifically against HeLa human cervical cancer cells. Because SB2001 lacks chemical modification sites, label-free target identification methods including thermal stability shift-based fluorescence difference in two-dimensional gel electrophoresis (TS-FITGE) and thermal proteome profiling (TPP) were applied to characterize its mechanism of action.

Label-free target identification in drug discovery via phenotypic screening.

Phenotypic screening has demonstrated its advantage in the discovery of first-in-class therapeutics, whereas target-based screening has showed strength for follower drugs. Owing to the unbiased nature of phenotypic screening, novel druggable proteins can be uncovered by target identification. Chemical label-free target identification methods can eliminate the functionalization step of an original bioactive compound.

Label-free target identification using in-gel fluorescence difference thermal stability shift.

Target engagement is a prerequisite for the therapeutic effects of bioactive small molecules, and unbiased identification of their target proteins can facilitate drug discovery and chemical biology research. Structural modifications of bioactive natural products for target identification exhibit potential limitations such as synthetic difficulties, limited supplies from natural sources, and loss of original efficacy. Herein, we developed a label-free method for proteome-wide target identification using in-gel fluorescence difference caused by thermal stability shift, namely TS-FITGE.

Discovery of a Small-Molecule Inhibitor of Protein-MicroRNA Interaction Using Binding Assay with a Site-Specifically Labeled Lin28.

MicroRNAs (miRNAs) regulate gene expression by targeting protein-coding transcripts that are involved in various cellular processes. Thus, miRNA biogenesis has been recognized as a novel therapeutic target. Especially, the let-7 miRNA family is well-known for its tumor suppressor functions and is downregulated in many cancer cells.

Nonspecific protein labeling of photoaffinity linkers correlates with their molecular shapes in living cells.

Herein we report molecular shape-dependent nonspecific labeling of photoaffinity linkers (PLs) in the cellular proteome. Linear PLs have a greater tendency to engage in nonspecific binding than branched PLs. Exploiting this property, we discovered a smaller branched diazirine-based PL as the best photoaffinity probe with minimal nonspecific binding characteristics from among 5 probes with different PLs.