Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification

Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification

Tetrahydroxy Stilbene Glucoside Alleviates Ischemic Stroke by Regulating Conformation-Dependent Intracellular Distribution of PKM2 for M2 Macrophage Polarization

Tetrahydroxy stilbene glucoside (TSG) is a bioactive ingredient with powerful anti-inflammatory and neuroprotective properties. However, the detailed mechanisms concerning the neuroprotective effect of TSG are not fully understood. This study aims to address the molecular mechanism involved in the protective effects of TSG on murine ischemic stroke. We found that TSG meliorated the phenotypes of ischemic stroke in vivo, which was correlated with the increased percentage of infiltrated M2 macrophages in brain after stroke. Mechanistically, TSG regulated macrophage polarization by significantly downregulating the transcriptional levels of M1 marker genes (iNOS and IL-1β) but upregulating that of the M2 marker genes (arg-1 and IL-4) following lipopolysaccharide/interferon-γ stimulation. Consistently, TSG reversed the metabolic profiling of M1 macrophage toward the M2 status at intracellular energy levels. Surprisingly, the knockdown of an established metabolic enzyme pyruvate kinase M2 (PKM2) that is important for M1 switch in macrophages abolished the promotive effect of TSG on the M2 polarization. Further investigation revealed that TSG markedly downregulated the intracellular ratio of dimer/monomer to the tetramer of PKM2 without affecting its total protein expression, leading to a suppressed nuclear translocation of functioning PKM2 in macrophages for M1 differentiation. Taken together, we identified a novel mechanism for macrophage M2 polarization regulation by a small-molecule chemical that controls the quality (conformation) rather than the quantity (expression) of an intracellular M1-promoting metabolic enzyme, which offers a better understanding of the mechanisms of macrophage plasticity and has serious implication in translational strategies for the treatment of macrophage-mediated neurological diseases with natural bioactive products