Quantitative Chemical Proteomic Profiling of the <i>in Vivo</i> Targets of Reactive Drug Metabolites

Idiosyncratic liver toxicity represents an important problem in drug research and pharmacotherapy. Reactive drug metabolites that modify proteins are thought to be a principal factor in drug-induced liver injury. Here, we describe a quantitative chemical proteomic method to identify the targets of reactive drug metabolites <i>in vivo</i>. Treating mice with clickable analogues of four representative hepatotoxic drugs, we demonstrate extensive covalent binding that is confined primarily to the liver. Each drug exhibited a distinct target profile that, in certain cases, showed strong enrichment for specific metabolic pathways (<i>e.g.</i>, lipid/sterol pathways for troglitazone). Site-specific proteomics revealed that acetaminophen reacts with high stoichiometry with several conserved, functional (seleno)­cysteine residues throughout the liver proteome. Our findings thus provide an advanced experimental framework to characterize the proteomic reactivity of drug metabolites <i>in vivo</i>, revealing target profiles that may help to explain mechanisms and identify risk factors for drug-induced liver injury

Quantitative Chemical Proteomic Profiling of the <i>in Vivo</i> Targets of Reactive Drug Metabolites

Idiosyncratic liver toxicity represents an important problem in drug research and pharmacotherapy. Reactive drug metabolites that modify proteins are thought to be a principal factor in drug-induced liver injury. Here, we describe a quantitative chemical proteomic method to identify the targets of reactive drug metabolites <i>in vivo</i>. Treating mice with clickable analogues of four representative hepatotoxic drugs, we demonstrate extensive covalent binding that is confined primarily to the liver. Each drug exhibited a distinct target profile that, in certain cases, showed strong enrichment for specific metabolic pathways (<i>e.g.</i>, lipid/sterol pathways for troglitazone). Site-specific proteomics revealed that acetaminophen reacts with high stoichiometry with several conserved, functional (seleno)­cysteine residues throughout the liver proteome. Our findings thus provide an advanced experimental framework to characterize the proteomic reactivity of drug metabolites <i>in vivo</i>, revealing target profiles that may help to explain mechanisms and identify risk factors for drug-induced liver injury

Quantitative Chemical Proteomic Profiling of the <i>in Vivo</i> Targets of Reactive Drug Metabolites

Idiosyncratic liver toxicity represents an important problem in drug research and pharmacotherapy. Reactive drug metabolites that modify proteins are thought to be a principal factor in drug-induced liver injury. Here, we describe a quantitative chemical proteomic method to identify the targets of reactive drug metabolites <i>in vivo</i>. Treating mice with clickable analogues of four representative hepatotoxic drugs, we demonstrate extensive covalent binding that is confined primarily to the liver. Each drug exhibited a distinct target profile that, in certain cases, showed strong enrichment for specific metabolic pathways (<i>e.g.</i>, lipid/sterol pathways for troglitazone). Site-specific proteomics revealed that acetaminophen reacts with high stoichiometry with several conserved, functional (seleno)­cysteine residues throughout the liver proteome. Our findings thus provide an advanced experimental framework to characterize the proteomic reactivity of drug metabolites <i>in vivo</i>, revealing target profiles that may help to explain mechanisms and identify risk factors for drug-induced liver injury

<i>Onchocerca flexuosa</i> sequences related to <i>de novo</i> purine and pyrimidine biosynthesis.

<p>KEGG pathway modules are outlined for inosine monophosphate (A) and uridine monophosphate (B) biosynthesis (module entries M00048 and M00051, respectively). Boxes representing enzymes present in nematodes (e.g., <i>C. elegans</i>), <i>Wolbachia</i>, or both are colored blue, red and purple, respectively. Enzymes sharing sequence identity with <i>O. flexuosa</i> peptide translations are highlighted in yellow. Enzymes identified from <i>B. malayi</i> are marked with an asterisk.</p

Levels of Aβ species in brain samples of mice treated with JZL184 or vehicle.

<p>A: Levels of Aβ40 were significantly increased in the vehicle-treated Ts65Dn vs. 2N mice. JZL184-treatment reduced the levels of Aβ40 in both Ts65Dn and 2N samples. B: Levels of Aβ42 were also greater in Ts65Dn vs 2N samples and were reduced by the JZL184-treatment.</p

Long-term potentiation in the CA1 region of hippocampal slices of Ts65Dn and 2N mice.

<p>A: In the vehicle-treated animals, LTP was smaller in Ts65Dn vs. 2N slices. Scale bars: 1 mV; 2 ms. B: In the JZL184-treated mice, there was no difference between the Ts65Dn vs. 2N slices. C: Quantification of the data. JZL184-treatment increased LTP in Ts65Dn mice.</p

Long-term memory: Novel object recognition with the retention period of 24 hours.

<p>A: Time of object exploration during acquisition. There was no difference between the groups, and no effect of JZL184-treatment on the exploration time. B: Testing phase. Left: Time of object exploration during testing was smaller in vehicle-treated Ts65Dn vs. 2N mice. There was no such difference between the JZL184-treated Ts65Dn and 2N groups. Right: Discrimination index was smaller in the vehicle-treated Ts65Dn vs. 2N mice. JZL184-treatment significantly increased the discrimination index in Ts65Dn mice, but had no effect on the performance of their 2N littermates.</p

Localization of the putative LolC peptide by immunohistology and <i>in situ</i> hybridization in <i>Onchocerca flexuosa</i>.

<p>The putative LolC peptide and transcript were localized in adult worm tissues via immunohistology (A-G) and <i>in situ</i> hybridization (H-K), respectively. Negative control antibodies against keyhole limpit hemocyanin (carrier protein) showed no specific staining in <i>O. flexuosa</i> (A). Antibodies against a peptide from MS protein 1591, a putative homolog of <i>Wolbachia</i> LolC, stained the fibrillar portions of the somatic muscles (arrow) in a cross section of an adult male worm (B). The somatic muscles (solid arrows) and excretory cell (dashed arrow) are stained in cross sections of young adult females (C, D). Magnification of the excretory cell shows intense staining of the cell membrane (dashed arrow) and more diffuse staining in adjacent muscles (solid arrow) (E). The uterine muscles of an older female (arrow) are clearly stained (F), as are the intrauterine stretched microfilaria (F, G). The LolC sense RNA probe produced no signal in <i>in situ</i> hybridizations (H), while the antisense RNA probe labeled the lateral chords (arrows) and developing sperm within the male testes (I, J). The antisense RNA probe also labeled the lateral chords (arrow), intestine, and uteri of a young adult female (K). Abbreviations: m, muscle; lc, lateral chords; i, intestine; ut, uterus; mf, microfilariae; hy, hypodermis; t, testes; vd, vas deferens. Scale bars = 25 µm.</p

Western blots detecting the putative LolC peptide in <i>Onchocerca flexuosa</i> adult worm lysate.

<p>Affinity-purified rabbit antibodies raised against a peptide from MS protein 1591, a putative LolC homolog, bound a 38 kDa band in Western blots of <i>O. flexuosa</i> adult worm lysate (lane 1). Purified IgG from pre-immune serum (lane 2) and antibodies against the keyhole limpit hemocyanin carrier (lane 3) did not bind to proteins in this size range.</p

Selected KEGG pathway modules in <i>B. malayi</i> and <i>O. flexuosa</i>. Table lists the number of module enzymes represented in each dataset.

<p>Selected KEGG pathway modules in <i>B. malayi</i> and <i>O. flexuosa</i>. Table lists the number of module enzymes represented in each dataset.</p