Toxicological Evaluation of Thiol-Reactive Compounds Identified Using a La Assay To Detect Reactive Molecules by Nuclear Magnetic Resonance

Abstract

We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.

New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays

“…145 It is also true that some marketed drugs contain PAIN structures, and these are given in SI Figure S2. However, not only are these percentages relatively small (2.8-6.5% as shown in Table 7) but the substructures recognized by our filters appear to be strikingly linked with metabolic or toxicity-associated deficiencies, 7,[190][191][192][193][194][195][196][197][198][199][200][201][202][203] with few exceptions. 204 We provide an extended discussion on this in the SI.…”

“…Their promiscuity has been noted 40 and their reactivity been subtly alerted to, either directly 39 or indirectly. 7 It has been suggested by Bristol-Myers Squibb that selective protein reactivity may be due in part to strain induced by initial, genuine, noncovalent affinity. 39 Observation of light-induced reactivity further complicates the situation.…”

“…This point has also been made by Abbott, and known drugs that are active in their ALARM-NMR protein-reactivity assay tend to have associated limitations in therapeutic settings. 7 These represent a suboptimum starting point for development, and we are comfortable with the exclusion of compounds bearing assay interference moieties from our screening libraries. However, by far the most important point to make is that, in general, any compound recognized by our filters that arises from a screen is likely to be a false positive or problematic and not yield to conventional SAR and optimization.…”

Validating New Tuberculosis Computational Models with Public Whole Cell Screening Aerobic Activity Datasets

“…Filters can enable removal or flagging of undesirable molecules (thiol traps and redox-active compounds, epoxides, anhydrides, and Michael acceptors that can covalently modify a cysteine moiety in a surrogate protein (13)(14)(15)), false positives and frequent hitters (16). For example, we have previously compared the filtering of malaria hits and datasets screened against Mtb, and three antimalarial datasets had very high failures with the Abbott Alerts (11), while a similar pattern was seen for Mtb (> 81% failure) versus known Mtb drugs (> 50% failure) (10).…”

Cheminformatics aspects of high throughput screening: from robots to models: symposium summary

“…The problem of false positives in HTS has been studied in the past [6-14], and diverse sets of structural filters have been proposed to remove these promiscuous compounds from screening libraries, or from the list of the active hits that will further advance in the project. One question to be asked is whether we use the right filters to select the right chemical scaffolds for probe optimization.…”