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

Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine Nucleotide Translocases to Impair Mitochondrial Function

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na⁺-K⁺-2Cl^– cotransporter in the thick ascending limb of Henle’s loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA’s targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment

Chemoproteomic Profiling Reveals Ethacrynic Acid Targets Adenine Nucleotide Translocases to Impair Mitochondrial Function

Ethacrynic acid (EA) is a diuretic drug that is widely used to treat high-blood pressure and swelling caused by congestive heart failure or kidney failure. It acts through noncovalent inhibition of the Na⁺-K⁺-2Cl^– cotransporter in the thick ascending limb of Henle’s loop. Chemically, EA contains a Michael acceptor group that can react covalently with nucleophilic residues in proteins; however, the proteome reactivity of EA remains unexplored. Herein, we took a quantitative chemoproteomic approach to globally profile EA’s targets in cancer cells. We discovered that EA induces impaired mitochondrial function accompanied by increased ROS production. Our profiling revealed that EA targets functional proteins on mitochondrial membranes, including adenine nucleotide translocases (ANTs). Site-specific mapping identified that EA covalently modifies a functional cysteine in ANTs, a mutation of which resulted in the rescuing effect on EA-induced mitochondrial dysfunction. The newly discovered modes of action offer valuable information to repurpose EA for cancer treatment

(C&D) Coronal CT examinations.

<p>it showed their relationship. (FR: foramen rotundum; PSC: Palatosphenoidal Canal (Palatovaginal Canal); VC: vidian nerve).</p

The anatomy step of Meckel Cave.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091444#pone-0091444-g004" target="_blank">Figure 4</a>.4: the position of the front door of vidian canal and vidian nerve; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091444#pone-0091444-g004" target="_blank">Figure 4</a>.5: after removal anterior and lateral part of the bone of Meckel cave, Meckel cave which was covered by the dura mater was exposed, the interior and inferior margin of which is the internal carotid artery, lateral side is V2 and V3 branches; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091444#pone-0091444-g004" target="_blank">Figure 4</a>.6: Dura mater was resected, Meckel cave was opened. (C: clivis; ICA: internal carotid artery; M: meckel cave; QS: quadrilateral space; SS: sphenoid sinus; V: trigeminal nerve; V2: maxillary branch of the trigeminal nerve; V3: mandibular branch of the trigeminal nerve; VI: abducent nerve; VN: vidian nerve).</p

(A&B) Axial CT examinations.

<p>It showed relationship of foramen rotundum, vidian canal and palatosphenoidal canal (palatovaginal canal). (PPF: pterygomandibular fossa; PC: vidian canal; PSC: Palatosphenoidal Canal (Palatovaginal Canal); VC: vidian nerve).</p

Additional file 2 of TOP2A deficit-induced abnormal decidualization leads to recurrent implantation failure via the NF-κB signaling pathway

Additional file 2: Figure S2. TOP2A inhibition enhances decidualization in T-HESCs. A Western blotting of TOP2A in DMSO-treated group and Etoposide-treated group (1μM) of T-HESCs. B IGFBP1 protein expression in T-HESCs following treatment with DMSO or Etoposide. The cultures either remained untreated or were decidualized for 4 days. C IGFBP1 and PRL mRNA expression in DMSO and Etoposide group. The cultures either remained untreated or were decidualized for 4 days. D CytoPainter phalloidin-iFluor 488 reagent was used to label filamentous actin, and immunofluorescence was used to analyze the morphological transformation of T-HESCs. Decidualized cells were treated with both 8-bromo-cAMP and MPA for 4days. (F-actin, green fluorescence signals; DAPI, blue signals; scale bar = 200μm). One-way ANOVA or Mann-Whitney U test. Data are the mean ± SEM of 3 biological replicates unless stated otherwise. *p < 0.05, **p < 0.01,***p < 0.001. ns, nonsignificant

Additional file 1 of TOP2A deficit-induced abnormal decidualization leads to recurrent implantation failure via the NF-κB signaling pathway

Additional file 1: Figure S1. Bioinformatics analysis of differentially expressed genes in endometrium with recurrent implantation failure from GSE111974. A Volcano plot of all expressed genes in endometrial tissues of the patients with RIF and healthy controls from GSE111974. B The PPI network of the DEGs and the hub gene screening map from GSE111974

MOESM1 of Metagenomic analysis reveals potential interactions in an artificial coculture

Additional file 1. Additional figures and tables