Post-HTS case report and structural alert: Promiscuous 4-aroyl-1,5-disubstituted-3-hydroxy-2 H -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.

Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors

“…ALARM NMR and ALARM MS have been employed in the industrial setting to both validate pre-clinical candidates 22 and to identify nonspecific reactive compounds 23 . We have previously shown that the assay can also flag non-reactive modulators of La antigen conformation; specifically, a class of 4-aroyl-1,5-disubstituted-3-hydroxy-2 H -pyrrol-2-ones we encountered during an HTS campaign targeting the HAT Rtt109 24 . Compounds with this chemotype did not show evidence of thiol reactivity in several experiments, including an ultra-performance LC (UPLC)-MS GSH adduct assay, but were ALARM NMR-positive independent of DTT, a pattern consistent with nonspecific protein perturbation.…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…Actual drug discovery programs do not rely on just one technique but utilize multiple sources of information to rule out non-stoichiometric inhibition. [29,51,55] However, on its own, such a subtractive approach is not always sufficient to unambiguously demonstrate stoichiometric inhibition, which should be proven by positive evidence.…”

“…[49] Another technique to detect aggregation of a compound is nuclear magnetic resonance (NMR), which either analyzes the signal from the compound itself [50] or uses the conformational distortion of the human La antigen as a surrogate readout in what is called ALARM NMR. [18,51] An indirect approach compares compound potency at different concentrations of enzyme [25,52]; bellshaped dose-response curves in cellular assays are another warning signal. One of several possible explanations is the failure of colloidal aggregates to enter intact cells.…”

Repurposing approved drugs on the pathway to novel therapies

“…Other FDA‐approved drugs are not ideal candidates for further development . Many chemotherapy drugs that require infusion, such as the bright blue compound mitroxantrone, an agent which has a variety of activities in cells, have limited solubility and may induce nonspecific aggregation of the protein or assay reagents in high throughput screening .…”