Post-HTS case report and structural alert: Promiscuous 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one actives verified by ALARM NMR

Abstract

Despite its wide use, not every high-throughput screen (HTS) yields chemical matter suitable for drug development campaigns, and seldom are 'go/no-go' decisions in drug discovery described in detail. This case report describes the follow-up of a 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one active from a cell-free HTS to identify small-molecule inhibitors of Rtt109-catalyzed histone acetylation. While this compound and structural analogs inhibited Rtt109-catalyzed histone acetylation in vitro, further work on this series was halted after several risk mitigation strategies were performed. Compounds with this chemotype had a poor structure-activity relationship, exhibited poor selectivity among other histone acetyltransferases, and tested positive in a β-lactamase counter-screen for chemical aggregates. Furthermore, ALARM NMR demonstrated compounds with this chemotype grossly perturbed the conformation of the La protein. In retrospect, this chemotype was flagged as a 'frequent hitter' in an analysis of a large corporate screening deck, yet similar compounds have been published as screening actives or chemical probes versus unrelated biological targets. This report-including the decision-making process behind the 'no-go' decision-should be informative for groups engaged in post-HTS triage and highlight the importance of considering physicochemical properties in early drug discovery.

Keywords: Chemical aggregation; Drug discovery; High-throughput screening; PAINS; Pan-assay interference compounds; Structure–activity relationships.

Figure 1

Inhibitory activity of a 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one screening compound against Rtt109-catalyzed histone acetylation in vitro. (A) Dose–response curves for compound 1a in library cherry-pick (mean for two replicates) and commercial resupply compound samples (mean ± SD for three replicates). Shown is the chemical structure of compound 1a. (B) HAT inhibition of compound 1a in the orthogonal slot blot assay using the commercial resupply sample.

Figure 2

CPM-based assay interference counter-screens for compound 1a. (A) Compound 1a weakly interferes with the HTS assay in a fluorescence quenching counter-screen. BHQ-1 and fluconazole are positive and negative quenching controls, respectively. (B) Absorption profile for compound 1a in assay buffer. (C) Compound 1a does not interfere with the HTS assay readout via CoA-trapping (n.b. compare to panel 2A). PC (positive control; 4-((9H-purin-6-yl)thio)-7-bromo-5-nitrobenzo[c][1,2,5]thiadiazole) and fluconazole are positive and negative thioltrapping controls, respectively. Data are mean ± SD for three replicates.

Figure 3

Assessment of compound 1a and related analogs for chemical aggregation. (A) β-Lactamase counter-screen for chemical aggregators. Compounds were tested for inhibition of the β-lactamase AmpC ± non-ionic detergent (0.01% Triton X-100) in Tris buffer pH 8.0. Rottlerin and lidocaine were included as positive control (PC) and negative control (NC) compounds, respectively. Data are mean ± SD for three replicates. (B) Inhibition of compound 1a against Rtt109–Vps75 as a function of Triton X-100 and BSA concentration using the [³H]-acetyl-CoA-based HAT assay in the presence of 5 mM DTT. Data are mean ± SD for three replicates. Data shown for compound 1a under standard conditions (1 detergent and BSA) is taken from Table 2.

Figure 4

Effect of the 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one active (compound 1a) on La protein conformation using ALARM NMR. Shown are 2D ¹H–¹³C HMQC spectra of selected ¹³C-labelled methyl groups for compound 1a as tested by ALARM NMR. Compounds were incubated with the La protein in either the presence (blue) or absence (red) of 20 mM DTT. 2-Chloro-1,4-naphthoquinone (PC) and fluconazole are shown as positive and negative compound controls, respectively. A scaled-up view of compound 1a is shown (‘1a zoom’). Note the spectra with and without DTT are nearly identical. Compound 2i, an inactive analog, was also included as a negative control compound. Signals were normalized to DMSO controls. Shown are representative results from one of three independent experiments.

Figure 5

Additional mechanistic experiments for compound 1a. (A) Dependence of incubation time on activity of compound 1a against Rtt109–Vps75 activity. Compound 1a was allowed to pre-incubate with Rtt109–Vps75 for 5, 15 or 30 min prior to initiating the HAT reaction with [³H]-acetyl-CoA. Data are mean ± SD for three replicates. (B) Dilution-based experiment to examine reversibility of Rtt109–Vps75 inhibition by compound 1a. Data are mean ± SD for nine replicates.

Figure 6

Publication trend of compound 1a substructure. Shown is the annual number of publications in which compounds containing a compound 1a substructure were reported as being used in ‘biological studies’. The data was obtained from SciFinder using default substructure searching methods. A and B refer to the first reported synthesis and subsequent bioactivity measurements for this substructure, respectively.^, Data accessed 29 December 2014.

Figure 7

Selected examples of bioactive 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-ones in PubChem. The compounds shown are structurally similar to compound 1a and are active in multiple PubChem bioassays versus a variety of protein targets. There is no PubChem bioactivity data reported for compound 1a. Data accessed 2 December 2014.

Figure 8

Summary of in vitro structure–activity/structure–interference relationships for chemotype 1. Shown is compound 1a, the parent compound originally identified in a cell-free fluorometric HTS for inhibitors of Rtt109-catalyzed histone acetylation.