PAINS in the assay: chemical mechanisms of assay interference and promiscuous enzymatic inhibition observed during a sulfhydryl-scavenging HTS

Abstract

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.

Figure 1

Susceptibility of CPM-based HTS to screening compound-based interference. (A) Assay schematic for the CPM-based HTS used in this study. The assay measures the HAT activity of the Rtt109–Vps75 complex, which catalyzes the transfer of an acetyl moiety from acetyl-CoA to specific lysine residues on the Asf1–dH3–H4 substrate complex to produce acetylated histone residues and coenzyme A (CoA). Addition of the thiol-scavenging probe CPM leads to a highly fluorescent adduct by reacting with the CoA byproduct, which is used to quantify HAT activity via fluorescence intensity measurement. (B) Representative assay interference chemotypes identified during post-HTS triage.

Figure 2

Dose–responses of select screening compounds in the Rtt109 HTS and an assay interference counter-screen. Shown are representative examples from chemotypes 1, 2, 3, 4, and 6, which displayed promising low micromolar IC₅₀ values by the primary HTS assay (solid lines). A counter-screen that replaced the acetyl-CoA substrate with the CoA reaction product produced similar dose–response curves by the same assay readout (dashed lines). Data are mean ± SD for three replicates.

Figure 3

Compound–GSH adducts detected by qualitative UPLC–MS. (A) Selected interference compounds were incubated with MeOH (black traces), HTS buffer (blue traces), or HTS buffer plus GSH (red traces) and analyzed by UPLC–MS. Shown are overlays of the simultaneous ELS and 254 nm traces. Selected mass spectra are also shown for a select sample in MeOH (black spectrum) and selected adducts (red spectra). Numbers in parentheses represent the predominant ion molecular weight (“–” denotes negative ion mode). Data are representative results from one of at least two independent experiments. (B) Simplified schematics of the proposed reaction mechanisms to generate the observed adducts.

Figure 4

Labile adducts between p-hydroxyarylsulfonamides (6) and GSH detected by qualitative UPLC–MS. (A) Simplified scheme of adduct formation between biological thiols and chemotype 6. (B) UPLC–MS analyses of compound 6a mixed with GSH in HTS buffer. 6a was treated with GSH after varying lengths of incubation in HTS buffer (5, 15, 30 min). After 5 min, reaction aliquots were analyzed by UPLC–MS. Trace (iv) shows the same sample from trace (i) analyzed 15 min later. (C). Summary of experiments described in (B) performed with compounds 6a–6e. All test compounds initially formed the expected adducts (6a′–6e′). A common breakdown product 6″ was detected for all five sulfonamides tested (rt = 3.28 min, m/z = 446). See Supporting Information, Figures S5, S7, and S11, for additional stability studies with chemotype 6. a = compound incubated in HTS buffer for 5 min, then GSH added, then analyzed by UPLC-MS 5 min later; b = same sample from a, but analyzed by UPLC-MS 15 min later.

Figure 5

Selected spectra of compound–peptide adducts detected by peptide mass spectrometry. Prototype compounds were incubated with purified proteins from the Rtt109 HTS, and then samples were subjected to LC-MS/MS analyses after in-gel proteolysis. Shown are peptide MS/MS spectra with assigned y- and b-type fragments. (A) Compound 1a forms a detectable adduct with C94 on yeast Rtt109. (B) Compound 6a forms a detectable adduct with mono-oxidized C21 on yeast Vps75. Shown in each spectra are the sequences for the precursor peptide and a simplified reaction scheme for the adduct formation. See Supporting Information, Table S3, for additional examples of compound–peptide adducts detected by peptide mass spectrometry.

Figure 6

Thiol reactivity of select screening compounds with the La protein as measured by ALARM NMR. (A) 2D ¹H–¹³C HMQC spectra of selected ¹³C-labeled methyl groups for the selected compounds 1a, 2a, 3a, 4a, 6a, and 6b as tested by ALARM NMR for protein reactivity. These methyl groups have been shown to undergo peak shifts and intensity decreases in the presence of many compounds that covalently react with neighboring cysteine residues. Compounds were incubated with the La protein in either the presence or absence of 20 mM DTT. PC denotes the positive control compound, 2-chloro-1,4-naphthoquinone. Fluconazole is shown as a negative compound control. Shown are representative results from one of two independent experiments. (B) Summary of the additional compounds tested by ALARM NMR, including several negative compound controls that were inactive in the Rtt109 HTS and thiol-reactive counter-screen.

Figure 7

Select examples of compounds containing thiol-reactive chemotypes that demonstrate promiscuous PubChem bioassay profiles. Shown are conspicuous examples of compounds containing chemotypes 1, 2, 3, 4, and 6 that have promiscuous bioassay profiles according to a PubChem substructure search (accessed 1 March 2014). Accompanying each structure is the PubChem CID followed by the ratio (number of bioassays where the compound was classified as active/number of bioassays that the compound was tested).

Figure 8

Methods to help identify nonselective cysteine reactivity in compounds from HTS campaigns. Triage of active compounds from HTS (real or virtual) should always include knowledge-based methods to flag potential reactive entities. Flagged compounds should then either be removed from consideration or investigated more rigorously using two or more of the experimental-based methods described above. Notes: Several of these methods have been described in the text and elsewhere.^,, The use of frontier molecular orbital (FMO) calculations has been reported as a gross method of flagging “frequent-hitters”. Certain cysteine proteases (e.g., caspase-1, -8) have been used as probes for reactivity including cysteines oxidation by redox-active compounds.^, MSTI = (E)-2-(4-mercaptostyryl)-1,3,3- trimethyl-3H-indol-1-ium; REOS, rapid elimination of swill.