Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA.

BACKGROUND: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine (CQ) has played an indispensable role, however, its mechanism of action (MoA) is not fully understood. METHODS: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling (ABPP) and mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays. RESULTS: We developed a novel clickable, photo-affinity chloroquine analog probe (CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photo-crosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods. CONCLUSIONS: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled (ABPP) and label-free (MS-CETSA) methods.

Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect.

BACKGROUND: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol (Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis. METHODS: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide (LPS). We screened for potential protein targets of Cel using activity-based protein profiling (ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays (CETSA) and surface plasmon resonance (SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown. RESULTS: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS (P < 0.05). Cel bound to Cys424 of pyruvate kinase M2 (PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis (Warburg effect). Cel also bound to Cys106 in high mobility group box 1 (HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin (IL)-1ß. Cel bound to the Cys residues in lactate dehydrogenase A (LDHA). CONCLUSION: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

Effects of taurine and housing density on renal function in laying hens.

This study investigated the putative protective effects of supplemental 2-aminoethane sulfonic acid (taurine) and reduced housing density on renal function in laying hens. We randomly assigned fifteen thousand green-shell laying hens into three groups: a free range group, a low-density caged group, and a high-density caged group. Each group was further divided equally into a control group (C) and a taurine treatment group (T). After 15 d, we analyzed histological changes in kidney cells, inflammatory mediator levels, oxidation and anti-oxidation levels. Experimental data revealed taurine supplementation, and rearing free range or in low-density housing can lessen morphological renal damage, inflammatory mediator levels, and oxidation levels and increase anti-oxidation levels. Our data demonstrate that taurine supplementation and a reduction in housing density can ameliorate renal impairment, increase productivity, enhance health, and promote welfare in laying hens.

Influence of dietary taurine and housing density on oviduct function in laying hens.

Experiments were conducted to study the effects of dietary taurine and housing density on oviduct function in laying hens. Green-shell laying hens were randomly assigned to a free range group and two caged groups, one with low-density and the other with high-density housing. Each group was further divided into control (C) and taurine treatment (T) groups. All hens were fed the same basic diet except that the T groups' diet was supplemented with 0.1% taurine. The experiment lasted 15 d. Survival rates, laying rates, daily feed consumption, and daily weight gain were recorded. Histological changes, inflammatory mediator levels, and oxidation and anti-oxidation levels were determined. The results show that dietary taurine supplementation and reduced housing density significantly attenuated pathophysiological changes in the oviduct. Nuclear factor-κB (NF-κB) DNA binding activity increased significantly in the high-density housing group compared with the two other housing groups and was reduced by taurine supplementation. Tumor necrosis factor-α (TNF-α) mRNA expression in the high-density and low-density C and T groups increased significantly. In the free range and low-density groups, dietary taurine significantly reduced the expression of TNF-α mRNA. Supplementation with taurine decreased interferon-γ (IFN-γ) mRNA expression significantly in the low-density groups. Interleukin 4 (IL-4) mRNA expression was significantly higher in caged hens. IL-10 mRNA expression was higher in the high-density C group than in the free range and low-density C groups. Supplementation with taurine decreased IL-10 mRNA expression significantly in the high-density group and increased superoxide dismutase (SOD) activity in the free range hens. We conclude that taurine has important protective effects against oviduct damage. Reducing housing density also results in less oxidative stress, less inflammatory cell infiltration, and lower levels of inflammatory mediators in the oviduct. Therefore, both dietary taurine and reduced housing density can ameliorate oviduct injury, enhance oviduct health, and promote egg production in laying hens.