Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5‑Methylcytosine

Arsenic toxicity is a serious public health problem worldwide that brings more than 100 million people into the risk of arsenic exposure from groundwater and food contamination. Although there is accumulating evidence linking arsenic exposure with aberrant cytosine methylation in the global genome or at specific genomic loci, very few have investigated the impact of arsenic on the oxidation of 5-methylcytosine (5-mC) mediated by the Ten-eleven translocation (Tet) family of proteins. Owing to the high binding affinity of As­(III) toward cysteine residues, we reasoned that the highly conserved C₃H-type zinc fingers situated in Tet proteins may constitute potential targets for arsenic binding. Herein, we found that arsenite could bind directly to the zinc fingers of Tet proteins in vitro and in cells, and this interaction substantially impaired the catalytic efficiency of Tet proteins in oxidizing 5-mC to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Treatments with arsenite also led to a dose-dependent decrease in the level of 5-hmC, but not 5-mC, in DNA isolated from HEK293T cells overexpressing the catalytic domain of any of the three Tet proteins and from mouse embryonic stem cells. Together, our study unveiled, for the first time, that arsenite could alter epigenetic signaling by targeting the zinc fingers of Tet proteins and perturbing the Tet-mediated oxidation of 5-mC in vitro and in cells. Our results offer important mechanistic understanding of arsenic epigenotoxicity and carcinogenesis in mammalian systems and may lead to novel approaches for the chemoprevention of arsenic toxicity

A unified strategy in selection of the best allometric scaling methods to predict human clearance based on drug disposition pathway

<p>1. It is critical to develop a unified strategy to select the best allometric scaling (AS) method for a given group of drugs.</p> <p>2. A total of 446 drugs with known human <i>CL_iv</i>, clear disposition pathway and animal (rat, dog, monkey) <i>CL_iv</i> were analyzed. All drugs were stratified based on their disposition pathway, liver extraction ratio (<i>ER_H</i>) and ratios of unbound clearance to renal glomerular filtration rate (<i>R_GFR</i>). Up to 22 AS methods were applied and compared in prediction of human <i>CL_iv</i> to each group of drugs.</p> <p>3. AS methods that give the best prediction of human <i>CL_iv</i>, were identified for drugs primarily eliminated through liver with a fraction of renal elimination (<i>f_renal</i>) within 0.3–0.5 or <i>ER_H</i> > 0.3, where human <i>CL_iv</i> of more than 80% or 90% drugs could be accurately (within 2- or 3-fold error) predicted. For drugs with <i>ER_H</i> < 0.3, acceptable accuracy could be achieved by a two species method <i>TS_R,D</i> resulting more than 60% or 75% drugs were predicted within 2- or 3-fold error.</p> <p>4. By stratified analysis of drugs, according to their disposition pathway and organ extraction ratio, a unified strategy was developed to select the best AS method in prediction of human <i>CL_iv</i>.</p

Arsenite Binds to the Zinc Finger Motif of TIP60 Histone Acetyltransferase and Induces Its Degradation via the 26S Proteasome

Arsenic is a ubiquitous environmental contaminant with widespread public health concern. Epidemiological studies have revealed that chronic human exposure to arsenic in drinking water is associated with the prevalence of skin, lung, and bladder cancers. Aberrant histone modifications (e.g., methylation, acetylation, and ubiquitination) were previously found to be accompanied by arsenic exposure; thus, perturbation of epigenetic pathways is thought to contribute to arsenic carcinogenesis. Arsenite is known to interact with zinc finger motifs of proteins, and zinc finger motif is present in and indispensable for the enzymatic activities of crucial histone-modifying enzymes especially the MYST family of histone acetyltransferases (e.g., TIP60). Hence, we reasoned that trivalent arsenic may target the zinc finger motif of these enzymes, disturb their enzymatic activities, and alter histone acetylation. Herein, we found that As³⁺ could bind directly to the zinc-finger motif of TIP60 <i>in vitro</i> and in cells. In addition, exposure to As³⁺ could lead to a dose-dependent decrease in TIP60 protein level via the ubiquitin-proteasome pathway. Thus, the results from the present study revealed, for the first time, that arsenite may target cysteine residues in the zinc-finger motif of the TIP60 histone acetyltransferase, thereby altering the H4K16Ac histone epigenetic mark. Our results also shed some new light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans

Image1_Pharmacokinetics, pharmacodynamics and safety of 15 mg-tolvaptan administered orally for 7 consecutive days to Chinese patients with child-Pugh B cirrhosis.pdf

Background: Tolvaptan, a selective vasopressin V2-receptor antagonist, can elicit a diuretic effect without significant electrolyte loss. The aims were to evaluate multiple-dose pharmacokinetics, pharmacodynamics and safety of daily administration of 15 mg tolvaptan in Chinese adult patients with confirmed Child-Pugh Class B cirrhosis accompanied by ascites.Methods: This was an open-label, single-center, single- and multiple-dose study. All patients received a daily 15 mg dose of tolvaptan for 7 consecutive days. The plasma concentrations of tolvaptan and its two metabolites (DM-4103, DM-4107) were measured using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). In addition, various pharmacokinetics parameters were calculated. The pharmacodynamic outcomes evaluated changes in serum sodium and potassium concentrations, daily urine volume, daily water consumption, fluid balance and body weight. Safety profiles, including the incidence of treatment-emergent adverse events (TEAEs), were carefully recorded.Results: Eleven patients with Child-Pugh B cirrhosis were eventually enrolled in the study. Plasma concentrations of tolvaptan and DM-4107 reached steady-states after 7 days of consecutive oral administration. No accumulation of tolvaptan or DM-4107 was found, but DM-4103 accumulated 18.2-fold after multiple-dosing. The daily urine volume and daily water consumption were statistically significantly increased after administration of tolvaptan from Day 1 to Day 7 (all p Conclusion: Tolvaptan at a daily dose of 15 mg had a diuretic effect but did not increase serum sodium excretion or lead to tolvaptan accumulation. It is therefore can be safely used for short-term treatment of Chinese adult patients with confirmed Child-Pugh B cirrhosis.Clinical Trial Registration:https://clinicaltrials.gov/search?term=NCT01359462, identifier NCT01359462.</p

PK/PD Disconnect Observed with a Reversible Endothelial Lipase Inhibitor

Screening of a small set of nonselective lipase inhibitors against endothelial lipase (EL) identified a potent and reversible inhibitor, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamide (<b>5</b>; EL IC₅₀ = 61 nM, EL_HDL IC₅₀ = 454 nM). Deck mining identified a related hit, <i>N</i>-(3-(3,4-dichlorophenyl)­propyl)-4-hydroxy-1-methyl-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>6a</b>; EL IC₅₀ = 41 nM, EL_HDL IC₅₀ = 1760 nM). Both compounds were selective against lipoprotein lipase (LPL) but nonselective versus hepatic lipase (HL). Optimization of compound <b>6a</b> for EL inhibition using HDL as substrate led to <i>N</i>-(4-(3,4<b>-</b>dichlorophenyl)­butan-2-yl)-1-ethyl-4-hydroxy-5-oxo-2,5-dihydro-1<i>H</i>-pyrrole-3-carboxamide (<b>7c</b>; EL IC₅₀ = 148 nM, EL_HDL IC₅₀ = 218 nM) having improved PK over compound <b>6a</b>, providing a tool molecule to test for the ability to increase HDL-cholesterol (HDL-C) levels in vivo using a reversible EL inhibitor. Compound <b>7c</b> did not increase HDL-C in vivo despite achieving plasma exposures targeted on the basis of enzyme activity and protein binding demonstrating the need to develop more physiologically relevant in vitro assays to guide compound progression for in vivo evaluation

Diphenylpyridylethanamine (DPPE) Derivatives as Cholesteryl Ester Transfer Protein (CETP) Inhibitors

A series of diphenylpyridylethanamine (DPPE) derivatives was identified exhibiting potent CETP inhibition. Replacing the labile ester functionality in the initial lead <b>7</b> generated a series of amides and ureas. Further optimization of the DPPE series for potency resulted in the discovery of cyclopentylurea <b>15d</b>, which demonstrated a reduction in cholesterol ester transfer activity (48% of predose level) in hCETP/apoB-100 dual transgenic mice. The PK profile of <b>15d</b> was suboptimal, and further optimization of the N-terminus resulted in the discovery of amide <b>20</b> with an improved PK profile and robust efficacy in transgenic hCETP/apoB-100 mice and in hamsters. Compound <b>20</b> demonstrated no significant changes in either mean arterial blood pressure or heart rate in telemeterized rats despite sustained high exposures