Synthesis and Evaluation of Eight- and Four-membered Iminosugar Analogues as Inhibitors of Testicular Ceramide-specific Glucosyltransferase, Testicular β-Glucosidase 2, and other Glycosidases

Eight- and four-membered analogues of N-butyldeoxynojirimycin (NB-DNJ), a reversible male contraceptive in mice, were prepared and tested. A chiral pool approach was used for the synthesis of the target compounds. Key steps for the synthesis of the eight-membered analogues involve: ringclosing metathesis and Sharpless asymmetric dihydroxylation, and for the four-membered analogues: Sharpless epoxidation, epoxide ring opening (azide), and Mitsunobu reaction to form the four-membered ring. (3S,4R,5S,6R,7R)-1-Nonylazocane-3,4,5,6,7-pentaol (6), was moderately active against rat-derived ceramide-specific glucosyltransferase and four of the other eight-membered analogues were weakly active against rat-derived β-glucosidase 2. Among the four-membered analogues, ((2R,3s,4S)-3-hydroxy-1-nonylazetidine-2,4-diyl)dimethanol (25), displayed selective inhibitory activity against mouse-derived ceramide-specific glucosyltransferase and was about half as potent as NB-DNJ against the rat-derived enzyme. ((2S,4S)-3-Hydroxy-1-nonyl-azetidine-2,4-diyl)dimethanol (27) was found to be a selective inhibitor of β-glucosidase 2, with potency similar to NB-DNJ. Additional glycosidase assays were performed to identify potential other therapeutic applications. The eight-membered iminosugars exhibited specificity for almond-derived β-glucosidase and the 1-nonylazetidine 25 inhibited α-glucosidase (Saccharomyces cerevisiae) with an IC50 of 600 nM and β-glucosidase (almond) with an IC50 of 20 µM. Only N-nonyl derivatives were active, emphasizing the importance of a long lipophilic side chain for inhibitory activity of the analogues studied

PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS

Significant resources in early drug discovery are spent unknowingly pursuing artifacts and promiscuous bioactive compounds, while understanding the chemical basis for these adverse behaviors often goes unexplored in pursuit of lead compounds. Nearly all the hits from our recent sulfhydryl-scavenging high-throughput screen (HTS) targeting the histone acetyltransferase Rtt109 were such compounds. Herein, we characterize the chemical basis for assay interference and promiscuous enzymatic inhibition for several prominent chemotypes identified by this HTS, including some pan-assay interference compounds (PAINS). Protein mass spectrometry and ALARM NMR confirmed these compounds react covalently with cysteines on multiple proteins. Unfortunately, compounds containing these chemotypes have been published as screening actives in reputable journals and even touted as chemical probes or preclinical candidates. Our detailed characterization and identification of such thiol-reactive chemotypes should accelerate triage of nuisance compounds, guide screening library design, and prevent follow-up on undesirable chemical matter

A Cell-Free Fluorometric High-Throughput Screen for Inhibitors of Rtt109-Catalyzed Histone Acetylation

<div><p>The lysine acetyltransferase (KAT) Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac) in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as <i>Candida albicans</i>. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS) for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length <i>Drosophila</i> histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7%) were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC₅₀ values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation <i>in vivo</i>, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could serve as chemical probes or leads for a new class of antifungals targeting an epigenetic enzyme.</p></div

Rtt109-Vps75 HTS protocol notes.

<p>Rtt109-Vps75 HTS protocol notes.</p

Assay validation using the LOPAC ± detergent.

<p>(A) Z' factors for replicate LOPAC experiments ± detergent. (B–C) Comparison of duplicate runs of the LOPAC ± detergent. Each point represents the activity of a discrete compound from the LOPAC. (D) Comparison of the LOPAC results ± detergent. Percent inhibitions represent the means of the replicate LOPAC experiments. Trend line (solid, red), ideal correlation line (dashed, blue). (E) Percent inhibition distribution of the averaged LOPAC results ± detergent, binned in 5% intervals.</p

Assay design and optimization.

<p>(A) Titration matrix of CoA and CPM in buffer-only conditions to determine the optimal assay levels of acetyl-CoA and CPM. (B) Titration matrix of CPM and acetyl-CoA in buffer-only conditions to verify acetyl-CoA and CPM do not form fluorescent adducts under HTS conditions. (C) Time-course study of CoA titrations with 20 µM CPM in buffer-only conditions to determine the optimal time for the reaction involving CoA and CPM. (D) HTS plate template. Arrows denote chosen HTS conditions.</p

Fluorometric Rtt109-Vps75 HTS schematic.

<p>The Rtt109-Vps75 KAT complex catalyzes the transfer of an acetyl group from acetyl-CoA to specific histone lysine residues within the Asf1-dH3-H4 substrate complex. The resulting CoA contains a free thiol group (-SH), which can react with the sulfhydryl-sensitive probe CPM to form a fluorescent adduct, thereby permitting the quantification of free CoA as a measure of KAT activity.</p

Noteworthy PAINS substructures in the primary Rtt109-Vps75 HTS.

<p><i>Italics</i> denote the original names of published PAINS substructures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078877#pone.0078877-Baell1" target="_blank">[51]</a>. For individual substructures, the ratios denote the number of primary active compounds divided by the number of compounds tested for each HTS production run.</p

Example active compound discovered with the Rtt109 HTS and post-HTS triage.

<p>(A) CPM-based assay dose-response with compound <b>1</b>. Fluconazole = negative control compound. (B) Orthogonal slot blot assay to detect the presence of H3K56ac by Western blot. Equal protein content was verified for each membrane by Ponceau S staining. (C) Dose-response of compound <b>1</b> using a [³H]-acetyl-CoA orthogonal assay.</p