A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors.

Human sirtuin-2 (SIRT2) has emerged as an attractive drug target for a variety of diseases. The enzyme is a deacylase that can remove chemically different acyl modifications from protein lysine residues. Here, we developed a high-throughput screen based on a homogeneous time-resolved fluorescence (HTRF) binding assay to identify inhibitors of SIRT2's demyristoylase activity, which is uncommon among many ligands that only affect its deacetylase activity.

Effects of Dimerization on the Deacylase Activities of Human SIRT2.

Human sirtuin isoform 2 (SIRT2) is an NAD-dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking.

Modeling Biphasic, Non-Sigmoidal Dose-Response Relationships: Comparison of Brain-Cousens and Cedergreen Models for a Biochemical Dataset.

Biphasic, non-sigmoidal dose-response relationships are frequently observed in biochemistry and pharmacology, but they are not always analyzed with appropriate statistical methods. Here, we examine curve fitting methods for "hormetic" dose-response relationships where low and high doses of an effector produce opposite responses. We provide the full dataset used for modeling, and we provide the code for analyzing the dataset in SAS using two established mathematical models of hormesis, the Brain-Cousens model and the Cedergreen model.

Replication Protein A Enhances Kinetics of Uracil DNA Glycosylase on ssDNA and Across DNA Junctions: Explored with a DNA Repair Complex Produced with SpyCatcher/SpyTag Ligation.

DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections.

Assay design for analysis of human uracil DNA glycosylase.

Human uracil DNA glycosylase (UNG2) is an enzyme whose primary function is to remove uracil bases from genomic DNA. UNG2 activity is critical when uracil bases are elevated in DNA during class switch recombination and somatic hypermutation, and additionally, UNG2 affects the efficacy of thymidylate synthase inhibitors that increase genomic uracil levels. Here, we summarize the enzymatic properties of UNG2 and its mitochondrial analog UNG1.

A Conserved Mechanism for Hormesis in Molecular Systems.

Hormesis refers to dose-response phenomena where low dose treatments elicit a response that is opposite the response observed at higher doses. Hormetic dose-response relationships have been observed throughout all of biology, but the underlying determinants of many reported hormetic dose-responses have not been identified. In this report, we describe a conserved mechanism for hormesis on the molecular level where low dose treatments enhance a response that becomes reduced at higher doses.

High-Throughput Screening Identifies Ascorbyl Palmitate as a SIRT2 Deacetylase and Defatty-Acylase Inhibitor.

Small-molecule inhibitors of the human sirtuin SIRT2 are being developed because of their therapeutic potential in a variety of diseases. Here, we developed a high-throughput screen to identify novel SIRT2 inhibitors using a fluorescent SIRT2 probe, 1-aminoanthracene (AMA). AMA has high fluorescence when bound to SIRT2, and its fluorescence reduces >10-fold when it is displaced from SIRT2 by other ligands.

A possible link to uracil DNA glycosylase in the synergistic action of HDAC inhibitors and thymidylate synthase inhibitors.

It is well established that thymidylate synthase inhibitors can cause cellular toxicity through uracil DNA glycosylase (UNG2)-dependent pathways. Additionally, thymidylate synthase inhibitors and HDAC inhibitors are known to act synergistically in a variety of cancer types. A recent article from J.

Substrate-Dependent Modulation of SIRT2 by a Fluorescent Probe, 1-Aminoanthracene.

Sirtuin isoform 2 (SIRT2) is an enzyme that catalyzes the removal of acyl groups from lysine residues. SIRT2's catalytic domain has a hydrophobic tunnel where its substrate acyl groups bind. Here, we report that the fluorescent probe 1-aminoanthracene (AMA) binds within SIRT2's hydrophobic tunnel in a substrate-dependent manner.

Analysis of uracil DNA glycosylase (UNG2) stimulation by replication protein A (RPA) at ssDNA-dsDNA junctions.

Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction.

N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions.

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction.

Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells.

DNA 'sliding' by human repair enzymes is considered to be important for DNA damage detection. Here, we transfected uracil-containing DNA duplexes into human cells and measured the probability that nuclear human uracil DNA glycosylase (hUNG2) excised two uracil lesions spaced 10-80 bp apart in a single encounter without escaping the micro-volume containing the target sites. The two-site transfer probabilities were 100% and 54% at a 10 and 40 bp spacing, but dropped to only 10% at 80 bp.

Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation.

Nuclear human uracil-DNA glycosylase (hUNG2) initiates base excision repair (BER) of genomic uracils generated through misincorporation of dUMP or through deamination of cytosines. Like many human DNA glycosylases, hUNG2 contains an unstructured N-terminal domain that encodes a nuclear localization signal, protein binding motifs, and sites for post-translational modifications. Although the N-terminal domain has minimal effects on DNA binding and uracil excision kinetics, we report that this domain enhances the ability of hUNG2 to translocate on DNA chains as compared to the catalytic domain alone.

Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis.

Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that prevents the stable incorporation of deoxyuridine monophosphate into DNA in the form of U/A basepairs. During S-phase, UNG2 remains associated with the replication fork through its interactions with two proteins, Proliferating Cell Nuclear Antigen (PCNA) and Replication Protein A (RPA), which are critical for DNA replication and repair. In this work, we used protein semisynthesis and fluorescence anisotropy assays to explore the interactions of UNG2 with PCNA and RPA and to determine the effects of two UNG2 phosphorylation sites (Thr and Tyr) located within its PCNA-interacting motif (PIP-box).

Macroscopic and macromolecular specificity of alkylphenol anesthetics for neuronal substrates.

We used a photoactive general anesthetic called meta-azi-propofol (AziPm) to test the selectivity and specificity of alkylphenol anesthetic binding in mammalian brain. Photolabeling of rat brain sections with [(3)H]AziPm revealed widespread but heterogeneous ligand distribution, with [(3)H]AziPm preferentially binding to synapse-dense areas compared to areas composed largely of cell bodies or myelin. With [(3)H]AziPm and propofol, we determined that alkylphenol general anesthetics bind selectively and specifically to multiple synaptic protein targets.

Role for the propofol hydroxyl in anesthetic protein target molecular recognition.

Propofol is a widely used intravenous general anesthetic. We synthesized 2-fluoro-1,3-diisopropylbenzene, a compound that we call "fropofol", to directly assess the significance of the propofol 1-hydroxyl for pharmacologically relevant molecular recognition in vitro and for anesthetic efficacy in vivo. Compared to propofol, fropofol had a similar molecular volume and only a small increase in hydrophobicity.

Propofol inhibits SIRT2 deacetylase through a conformation-specific, allosteric site.

meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [(3)H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr(139), Phe(190), and Met(206)) that are located in a single allosteric protein site, and propofol inhibited [(3)H]AziPm photolabeling of this site in myelin SIRT2.

Discovery of a novel general anesthetic chemotype using high-throughput screening.

Background: The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures.

Methods: Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay.

Sites and functional consequence of VDAC-alkylphenol anesthetic interactions.

General anesthetics have previously been shown to bind mitochondrial VDAC. Here, using a photoactive analog of the anesthetic propofol, we determined that alkylphenol anesthetics bind to Gly56 and Val184 on rat VDAC1. By reconstituting rat VDAC into planar bilayers, we determined that propofol potentiates VDAC gating with asymmetry at the voltage polarities; in contrast, propofol does not affect the conductance of open VDAC.

Computational investigation of cholesterol binding sites on mitochondrial VDAC.

The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs.

Mechanisms revealed through general anesthetic photolabeling.

General anesthetic photolabels are used to reveal molecular targets and molecular binding sites of anesthetic ligands. After identification, the relevance of anesthetic substrates or binding sites can be tested in biological systems. Halothane and photoactive analogs of isoflurane, propofol, etomidate, neurosteroids, anthracene, and long chain alcohols have been used in anesthetic photolabeling experiments.

Direct modulation of microtubule stability contributes to anthracene general anesthesia.

Recently, we identified 1-aminoanthracene as a fluorescent general anesthetic. To investigate the mechanism of action, a photoactive analogue, 1-azidoanthracene, was synthesized. Administration of 1-azidoanthracene to albino stage 40-47 tadpoles was found to immobilize animals upon near-UV irradiation of the forebrain region.

In vivo activation of azipropofol prolongs anesthesia and reveals synaptic targets.

General anesthetic photolabels have been instrumental in discovering and confirming protein binding partners and binding sites of these promiscuous ligands. We report the in vivo photoactivation of meta-azipropofol, a potent analog of propofol, in Xenopus laevis tadpoles. Covalent adduction of meta-azipropofol in vivo prolongs the primary pharmacologic effect of general anesthetics in a behavioral phenotype we termed "optoanesthesia.