Michael D. Scholle

Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

Gcg-XTEN: An Improved Glucagon Capable of Preventing Hypoglycemia without Increasing Baseline Blood Glucose

Objective While the majority of current diabetes treatments focus on reducing blood glucose levels, hypoglycemia represents a significant risk associated with insulin treatment. Glucagon plays a major regulatory role in controlling hypoglycemia in vivo, but its short half-life and hyperglycemic effects prevent its therapeutic use for non-acute applications. The goal of this study was to identify a modified form of glucagon suitable for prophylactic treatment of hypoglycemia without increasing baseline blood glucose levels. Methodology/Principal Findings Through application of the XTEN technology, we report the construction of a glucagon fusion protein with an extended exposure profile (Gcg-XTEN). The in vivo half-life of the construct was tuned to support nightly dosing through design and testing in cynomolgus monkeys. Efficacy of the construct was assessed in beagle dogs using an insulin challenge to induce hypoglycemia. Dose ranging of Gcg-XTEN in fasted beagle dogs demonstrated that the compound was biologically active with a pharmacodynamic profile consistent with the designed half-life. Prophylactic administration of 0.6 nmol/kg Gcg-XTEN to dogs conferred resistance to a hypoglycemic challenge at 6 hours post-dose without affecting baseline blood glucose levels. Consistent with the designed pharmacokinetic profile, hypoglycemia resistance was not observed at 12 hours post-dose. Importantly, the solubility and stability of the glucagon peptide were also significantly improved by fusion to XTEN. Conclusions/Significance The data show that Gcg-XTEN is effective in preventing hypoglycemia without the associated hyperglycemia expected for unmodified glucagon. While the plasma clearance of this Gcg-XTEN has been optimized for overnight dosing, specifically for the treatment of nocturnal hypoglycemia, constructs with significantly longer exposure profiles are feasible. Such constructs may have multiple applications such as allowing for more aggressive insulin treatment regimens, treating hypoglycemia due to insulin-secreting tumors, providing synergistic efficacy in combination therapies with long-acting GLP1 analogs, and as an appetite suppressant for treatment of obesity. The improved physical properties of the Gcg-XTEN molecule may also allow for novel delivery systems not currently possible with native glucagon.

High-Throughput Screening of Small Molecule Libraries using SAMDI Mass Spectrometry

High-throughput screening is a common strategy used to identify compounds that modulate biochemical activities, but many approaches depend on cumbersome fluorescent reporters or antibodies and often produce false-positive hits. The development of “label-free” assays addresses many of these limitations, but current approaches still lack the throughput needed for applications in drug discovery. This paper describes a high-throughput, label-free assay that combines self-assembled monolayers with mass spectrometry, in a technique called SAMDI, as a tool for screening libraries of 100 000 compounds in one day. This method is fast, has high discrimination, and is amenable to a broad range of chemical and biological applications.

The E. coli sirtuin CobB shows no preference for enzymatic and nonenzymatic lysine acetylation substrate sites

Nε‐lysine acetylation is an abundant posttranslational modification of thousands of proteins involved in diverse cellular processes. In the model bacterium Escherichia coli, the ε‐amino group of a lysine residue can be acetylated either catalytically by acetyl‐coenzyme A (acCoA) and lysine acetyltransferases, or nonenzymatically by acetyl phosphate (acP). It is well known that catalytic acCoA‐dependent Nε‐lysine acetylation can be reversed by deacetylases. Here, we provide genetic, mass spectrometric, structural and immunological evidence that CobB, a deacetylase of the sirtuin family of NAD+‐dependent deacetylases, can reverse acetylation regardless of acetyl donor or acetylation mechanism. We analyzed 69 lysines on 51 proteins that we had previously detected as robustly, reproducibly, and significantly more acetylated in a cobB mutant than in its wild‐type parent. Functional and pathway enrichment analyses supported the hypothesis that CobB regulates protein function in diverse and often essential cellular processes, most notably translation. Combined mass spectrometry, bioinformatics, and protein structural data provided evidence that the accessibility and three‐dimensional microenvironment of the target acetyllysine help determine CobB specificity. Finally, we provide evidence that CobB is the predominate deacetylase in E. coli.

Discovery of SIRT3 Inhibitors Using SAMDI Mass Spectrometry

Lysine acetylation plays a critical role in cellular regulation and is implicated in human disease. Sirtuin deacetylases remove acetyl groups from modified lysine residues, and sirtuin 3 (SIRT3) has been identified as a target for cancer therapeutics. Robust and high-throughput screening methods for these targets will be important to the development of therapeutics. This article describes the use of self-assembled monolayer desorption/ionization mass spectrometry, or SAMDI-MS—a label-free drug discovery tool—to characterize SIRT3 activity and discover inhibitors. SAMDI-MS was used to analyze a peptide array having 361 distinct acetylated peptides to identify an active SIRT3 substrate (GYKAcRGC). This peptide was used in a screen of 100,000 small molecules to identify inhibitors of SIRT3. A total of 306 SIRT3 inhibitors were identified, with one compound, SDX-437, having an IC50 of 700 nM with >100-fold selectivity for SIRT3 over SIRT1.

A High-Throughput Mass Spectrometry Assay Coupled with Redox Activity Testing Reduces Artifacts and False Positives in Lysine Demethylase Screening.

Demethylation of histones by lysine demethylases (KDMs) plays a critical role in controlling gene transcription. Aberrant demethylation may play a causal role in diseases such as cancer. Despite the biological significance of these enzymes, there are limited assay technologies for study of KDMs and few quality chemical probes available to interrogate their biology. In this report, we demonstrate the utility of self-assembled monolayer desorption/ionization (SAMDI) mass spectrometry for the investigation of quantitative KDM enzyme kinetics and for high-throughput screening for KDM inhibitors. SAMDI can be performed in 384-well format and rapidly allows reaction components to be purified prior to injection into a mass spectrometer, without a throughput-limiting liquid chromatography step. We developed sensitive and robust assays for KDM1A (LSD1, AOF2) and KDM4C (JMJD2C, GASC1) and screened 13,824 compounds against each enzyme. Hits were rapidly triaged using a redox assay to identify compounds that interfered with the catalytic oxidation chemistry used by the KDMs for the demethylation reaction. We find that overall this high-throughput mass spectrometry platform coupled with the elimination of redox active compounds leads to a hit rate that is manageable for follow-up work.

Identification of Small-Molecule Noncovalent Binders Utilizing SAMDI Technology

In recent years, the ability to unambiguously identify complex mixtures of analytes with high accuracy and resolving power in a label-free format continues to expand the application of mass spectrometry (MS) in the drug discovery process. This advantage combined with improved instrumentation makes MS suitable for targets with limited alternative assays for high-throughput screening (HTS). We describe a novel screening format using Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization (SAMDI) technology. SAMDI enables affinity capture of a target protein for use in a small-molecule–binding assay format. Subsequent ionization enables the inferred identification of noncovalent compound interactions. SAMDI technology overcomes shot-to-shot variability by uniformly saturating the surface with captured protein, thereby minimizing matrix crystallization “hot spots.” Furthermore, the combination with high-resolution matrix-assisted laser desorption/ionization time of flight significantly reduces interference of small-molecule detection from salt, detergent, and matrix. By using a pooled library format, the SAMDI assay can significantly improve the throughput of MS-based screening irrespective of enzyme activity. Finally, we demonstrate binding affinity rank ordering from a pool of compounds that correlates with potency data from a biochemical assay.

Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism

Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depend on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using self-assembled monolayers for matrix-assisted laser desorption ionization (SAMDI) mass spectrometry. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the Fe(II)/Zn(II) HDAC8 activity ratio varies widely (from 2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo that, in turn, may regulate its activity. However, future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.

Abstract 1127: MMSET: Can we flip the switch.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epigenetic regulation of gene expression involves covalent modifications of histones and DNA in a way that does not alter the underlying DNA sequence. Recently, misregulation of epigenetic mechanisms has been linked to a number of diseases, including cancer. These data signify the importance of understanding normal and aberrant epigenetic regulation and for development of tools that allow us to modulate various aspects of epigenetic machinery. One approach to do this is by targeting epigenetic enzymes with small molecule inhibitors. MMSET (Multiple Myeloma SET domain), is a histone methyltransferase overexpressed in many cancers, including a subset of multiple myelomas (MM) harboring the t(4;14) translocation. Overexpression of MMSET leads to a global increase in dimethylation of lysine 36 on histone H3 (H3K36me2). The ability of MMSET to methylate H3 depends on a functional SET (Suppressor of variegation, Enhancer of zeste and Trithorax) domain, found in most histone methyltransferases. Methylation of H3K36 by MMSET affects overall chromatin structure thereby affecting the expression of many genes, including genes that play a role in cellular proliferation and adhesion. Conversely, the loss of MMSET in t(4;14)+ cells suppresses cell growth and induces apoptosis thus supporting the idea that inhibition of MMSET activity is a viable approach to treatment of this particular type of myeloma. Currently there are not any known inhibitors of MMSET. Using the previously identified structure of the SET domain of NSD1, an MMSET homologue, we performed an in silico screen against a library of compounds to identify those that may fit in the substrate binding pocket of MMSET. We tested the in silico hits in a high throughput screen (HTS) that combines Self-Assembled Monolayers and matrix-assisted laser Desorption Ionization time-of-flight (SAMDI). This method allows us to test the ability of the compounds to inhibit MMSETs methylation activity in a label-free format. From this screen two potential hits have been identified. These compounds have IC50 values in the micromolar range and Differential Scanning Fluorimetry (DSF) confirmed the binding of the small molecule to MMSET. Future in vivo work will determine the potency and selectivity of these compounds towards MMSET. Identifying an effective inhibitor of MMSET could make way for new potential therapeutics in the treatment of t(4;14)+ multiple myeloma, as well as other cancers that overexpress this protein, and could also prove to be highly useful in our efforts to understand the various mechanisms of epigenetic regulation. Citation Format: Christine Will, Michael Scholle, Roodolph St. Pierre, Ji Hyun Shim, Zhong Jun Cheng, Relja Popovic, Dinshaw J. Patel, James E. Bradner, Alex D. MacKerell, Milan Mrksich, Jonathan D. Licht. MMSET: Can we flip the switch. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1127. doi:10.1158/1538-7445.AM2013-1127