Identificationof Artifact Chemotypes in a Fluorescence-Based HTS

We previouslyreported the use of a CPM-based method to screen approximately 225Kcompounds for their abilities to inhibit Rtt109-catalyzed histoneacetylation in vitro.⁴² In a HAT reaction,an acetyl group is enzymatically transferred from acetyl-CoA to theε-amino group of a histone lysine side chain, resulting in theproduction of an acetylated lysine and a CoA byproduct. The free thiolon CoA can then react with suitable substrates, such as maleimide-basedprobes like CPM, to form highly fluorescent adducts that can indirectlyassay HAT activity (Figure1A). In practice,we and others have found this method to be low-cost and relativelyrobust.⁴⁶ We were also aware that thismethod is subject to assay interference by thiol-containing compounds.⁴⁷ However, we soon learned that this assay ishighly susceptible to other mechanisms of reactive compound interferenceespecially when testing potentially heterogeneous chemical matterlike HTS libraries. In principle, compounds can interfere with theCPM fluorescence intensity by fluorescence quenching. Also, compoundswith nucleophilic or electrophilic reactivity can react with eitherthe CPM probe or the CoA reaction product, respectively.

Despite identifying 1.5K actives in the primary screen, apost-HTS triage consisting of computational filtering, experimentalcounter-screens, and orthogonal assays demonstrated only three compoundsthat could inhibit Rtt109-catalyzed histone acetylation. Therefore,significant portions of this collection of experimental and computationallyfiltered (“filtrand”) compounds were either false positivesor assay artifacts. In addition, many of the computationally filteredactives were flagged as PAINS, which we believed could inhibit HATactivity by nontherapeutically useful mechanisms. In all, severalprominent chemotypes were identified among the primary actives duringthe course of the post-HTS triage (Figure1B).

On the basis of their chemical structures, it appearedlikely that each chemotype could interfere with our HTS assay readoutthrough different and, perhaps, multiple thiol-trapping mechanisms.Many of these compounds were flagged as PAINS, but still many werenot verified as “bad actors” until relatively late inour triage process. To further confound matters, these electrophiliccompounds could conceivably inhibit enzymatic activity by nonspecificreactivity with the protein components in the assay, further scramblingthe assay readout. To better understand the chemical mechanism(s)behind this form of assay behavior and to assess whether this interferencecould have implications beyond our HTS, we examined five prominentsubclasses of compounds in more detail.

Orthogonal and Counter-ScreensIdentify Inhibitors among Interference Compounds

Severalclasses of compounds demonstrated low micromolar IC₅₀ valuesin the CPM-based HTS method (Figure2). Compoundswith steep Hill slopes (e.g., >2) have been associated with cooperativityand anomalous binding behaviors, such as chemical aggregation.^42,48,49 Most of the compounds showedslightly elevated Hill slopes, but this was not an immediate concern,as we had included a detergent (Triton X-100) in our assay buffersto mitigate micelle formation.

Reaction aliquots from the active compounds (1a,2a,3a,4a,6a, and6b) all showed decreases in histone acetylationat 125 μM when they were analyzed by an orthogonal slot blotassay that uses H3K56ac- and H3K27ac-specific antibodies to probefor the acetylated histone lysine product rather than the CoA byproduct(Tables1–5).We examined both histone modifications because the Rtt109–Vps75complex is capable of acetylating multiple histone H3 residues. Overall,reaction aliquots showed similar levels of histone acetylation, regardlessof whether H3K27ac or H3K56ac was examined, strongly suggesting thatany observed enzymatic inhibition was not specific to one particularhistone modification. This assay also detected decreases in histoneacetylation with garcinol, a natural product previously shown to inhibitRtt109 activity and other HATs in vitro at low micromolar compoundconcentrations.^42,50 When the same assay was usedto examine reaction aliquots at lower compound concentrations (8 μM),we observed more discrepancies between the HTS and slot blot readouts(data not shown). Overall, these observations showed that representativesof these five compound classes could inhibit acetylation activityat high concentrations, but their HTS assay readouts were confoundedby assay interference near their apparent IC₅₀ values.

To further examine the mechanisms underlying this assay interference,we first established a fluorescence-quenching counter-screen to assessfor fluorescence interference. This assay accurately identified thefluorescence quencher BHQ-1, a positive control (Supporting Information, Figure S1). However, none of thesecompounds (1a,2a,3a,4a, and6b) showed evidence of fluorescence quenching,intrinsic fluorescence or the generation of fluorescent adducts witheither CoA or CPM in a set of assays mimicking our HTS procedures(Tables1–5).When the acetyl-CoA reactant was replaced by the CoA reaction byproduct,these compounds provided assay readouts that were strikingly similarto those obtained under the HTS assay conditions (Figure2). The degree of assay signal reduction was alsodependent on the levels of CoA present (data not shown). Similar assaybehavior was observed for a variety of chemical analogues bearingthese chemotypes (Tables1–5). Therefore, the body of evidence strongly implicateda thiol-trapping mechanism of assay interference. Next, we soughtto understand the chemical basis of this interference and potentialsources of enzymatic inhibition.

Further Characterizationof Compound–Thiol Adducts by UPLC-MS and LC-HRMS

Toprovide more direct evidence of the presumed compound–CoA adducts,we incubated the compounds with CoA under HTS-like conditions. Wealso tested for adducts with reducedl-glutathione (GSH),another important biological thiol, to assess if this presumed thiolreactivity was unique to CoA. UPLC–MS and LC–HRMS analysesshowed that the test compounds (1a,2a,3a and4a) form adducts with both CoA and GSH(Figure3 andSupportingInformation, Figure S2). We also observed the similar and expectedadducts by UPLC–MS for multiple other representative compoundsfrom each chemotype (data not shown). While thep-hydroxyarylsulfonamides6a–6e yieldedthe expected compound-GSH adducts (6a′–6e′; Figures4A,B), we alsoobserved that6a′–6e′were really intermediates that converted to a common adduct in situafter only 15 min under the HTS conditions (Figure4B,C).

Together, this data is consistentwith a thiol-trapping mechanism as a major contributor to the CPM-basedassay signal reduction in the compound classes studied, as the testedcompounds reacted with both CoA and GSH. We note the generation ofcompound–GSH adducts is an important consideration for certaincell-based assays, or for in situ or in vivo assays, where xenobiotic–glutathioneconjugation is a major source of Phase II metabolism.

Proposed ChemicalMechanisms of Thiol Reactivity

The selected compounds interferewith the HTS assay readout and form thiol adducts by a variety ofchemical mechanisms (Figures3B and4A). On the basis of the UPLC–MS and chemicalprinciples, we propose the following chemical mechanisms of thiolreactivity for chemotypes1,2,3,4, and6 (Figure1):

Benzo[b]thiophene 1,1-dioxides1 (“benzothiophenes”) interfere via a straightforwardMichael addition–elimination reaction at the electrophilicC3-position through thiolate nucleophilic attack. The compounds withchemotype1 most likely to interfere and form thiol adductsin our experimental conditions were those withS-linkedheteroaromatic substituents (Table1). The UPLC–MS experiments using1a confirmed the presence of the adduct1a′ andthe leaving group1a″ (Figure3A). This proposed mechanism is also supported by the observationsthat several 2,3-dihydro analogues did not show appreciable levelsof apparent Rtt109 inhibition or interference in the CoA–CPMcounter-screen (Supporting Information, FigureS3). The level of assay interference is consistent with theleaving group ability of the C3 substituent, as compounds withN-,O-, orC-linked groupsat this C3-position did not show as significant levels of interferenceor apparent inhibition (Supporting Information,Figure S3). Most of the compounds with chemotype1 showed only partial decreases in histone acetylation at high compoundconcentrations (Table1), demonstrating thesecompounds can weakly inhibit Rtt109 activity in our HTS, most likelyby nonspecific thiol reactivity (Table1).

Thebenzothiadiazole/benzofurazan scaffold2 likely formsthiol adducts via nucleophilic aromatic substitution through a Meisenheimercomplex intermediate between the nucleophilic thiol and the stronglyelectrophilic heteroaromatic core. The benzofurazan core has beenpreviously associated with promiscuous thiol reactivity,^18,43 while some related benzothiadiazoles have been reported as PAINS(e.g., substructures “diazox_B” and “diazox_sulfon_A”).¹ Additionally, similar compounds have been shownto form covalent adducts with proteins.⁵¹ The related compound 4-chloro-7-nitrobenzofurazan (“NBD-Cl”)and its derivatives are widely used as probes for studying thiolsin biological systems.⁵² UPLC–MSanalysis of2a demonstrates that the parent compoundis stable to the HTS buffer but that the addition of a thiol sourceleads to near-complete conversion to the thiol adduct2a′ with the thiopurine serving as the leaving group (Figure3A). The compounds that showed the strongest apparentenzyme inhibition and interference contained electron-withdrawingsubstituents such as nitro groups and halogens, although there wasno apparent reactivity difference between benzothiadiazoles and benzofurazans.Another important feature for interference was the presence of anS-linked aryl substituent, which serves as the leaving groupeven when there are other electron-withdrawing groups present (Table2). Benzothiadiazoleswithout these features did not show significant levels of apparentenzyme inhibition or assay interference (SupportingInformation, Figure S4). While flagged as PAINS, many sulfoxide-substitutedanalogues tested in our system were inactive and noninterfering, anobservation we attribute to the absence of an additional strong electron-withdrawinggroup (e.g., nitro). Many compounds with chemotype2 werecapable of completely inhibiting Rtt109-catalyzed histone acetylationin our HTS (Table2).

Whilethe core 1,2,4-thiadiazole heterocycle3 may appear benign,many such compounds can react quite readily with thiols⁵³⁻⁵⁷ but not other functional groups like alcohols or amines.^58,59 Many properties of 1,2,4-thiadiazoles have been documented.⁶⁰⁻⁶² The 1,2,4-thiadiazole core can be susceptible to ring-opening reactions,recyclization side products, and nonenzymatic reductions.⁶³⁻⁶⁷ Notably, this scaffold is similar to the “het_thio_N_5A”PAINS substructure, although it differs by resonance and substitutionat the N2-position, meaning this chemotype could bypass some PAINSfilters depending on its structural representation and certain chemoinformaticparameters.¹

This speculation aside,the likely chemical mechanism of interference in our assay is sulfhydryl-scavengingby the 1,2,4-thiadiazole core at the S1-position, specifically a ring-openingreaction that generates a disulfide that can then be reduced by anotherthiol or electron source in situ to form the corresponding thiourea.⁵⁷ Indeed, we first observed the formation of thethiourea form (3a″), as evidenced by a major shiftin the UPLC retention time upon the addition of thiols (Figure3A). The parent ions for this entity (i.e.,m/z = 270) were difficult to observe byUPLC–MS, and notably we did not observe any coeluting GSH ions,suggesting this peak was not the3a′ form withan attached GSH moiety. To gain a further structural understandingof the3a adducts, we synthesized it under HTS-like conditionsand characterized its identity and structure in situ by LC–HRMS.This data further pointed toward the detectable “adduct”being the thiourea form3a″ rather than the directcompound–GSH3a′ adduct (Supporting Information), which is consistent with a previousreport on this chemotype.⁵⁷ These data,combined with our findings that compounds3 are stronglyreactive in our thiol-trapping interference screen, suggests the3–GSH adduct forms (3′) are notstable to our characterization procedures and/or our LC-MS conditions.

Examination of close analogues showed the assay interference stronglycorrelates with additional alkylation at the core N2-position to generatea partially cationic nitrogen, which presumably activates the S1–N2bond for thiol-mediated cleavage. Compounds lacking these substituentson the N2-position were inactive and showed minimal interference (Supporting Information, Figure S5). Of note,another related PAINS substructure is “het_5_inium”,which bears resemblance to the charged 1,2,4-thiadiazoles in thischemotype. Neither the nature of the R¹–R⁴ substituents nor the particular salt composition appeared to havesignificant effects on thiol-trapping (Table3). Consistent with thismechanism, many 1,2,4-oxadiazole analogues showed minimal assay activityand interference (Supporting Information, FigureS5). We observed that many of these 1,2,4-thiadiazoles inhibitedRtt109-catalyzed histone acetylation quite effectively (Table3), suggesting these compounds can not only interferewith the HTS assay but can also inhibit enzymatic activity, againpresumably by nonspecific reactivity with protein thiols.

Forthe succinimide chemotype4, our data is consistent withan elimination event followed by a Michael addition of a free thiolto the resulting maleimide. This is the same sulfhydryl-sensitivegroup present in the CPM probe used in our HTS. Elimination is likely,given the slightly alkaline pH of the assay buffer (pH 8.0). The thiolleaving groups (e.g.,4a″) were detected by UPLC–MSwhen the parent compound4a was incubated in HTS buffer.This same proposed leaving group was not detected when4a was incubated in neat MeOH (Figure3A). Severaltrends also support this proposed mechanism. First, the proposed eliminationproduct maleimides5 showed nearly identical IC₅₀ values in both the HTS and interference counter-screens comparedto their parent succinimides (Table4). Second, the apparent enzymatic inhibition andcounter-screen IC₅₀ values correlate well with the presumedleaving-group ability of the succinimide substituent. For instance,succinimides withS-linked aryl groups showed significantassay interference (Table4), while nonaryl,S-linked leaving groups showed no significant activity andinterference (Supporting Information, Figure S6). Third, succinimides withN-linked substituentsdid not appear to inhibit Rtt109 in our HTS or show interference inour counter-screens (Supporting Information, FigureS6). The substituents on the resulting maleimides did not havea noticeable effect on either the HTS or counter-screen IC₅₀ values (Table4). Most of the interferingsuccinimides4 could only partially inhibit Rtt109 activityat higher compound concentrations in our HTS, which may be a reflectionof the kinetics of the succinimide-to-maleimide conversion in buffer.

Thep-hydroxyarylsulfonamide chemotype6 has beenidentified as a PAINS substructure.¹ Othershave shown this scaffold to be redox-active⁹ as well as subject to addition–elimination at the substitutedC3-position.⁴⁴ During our post-HTS triage,we also observed that many of these compounds produced H₂O₂ in our assay buffer, both in the presence and absenceof the reducing agent DTT using a horseradish peroxidase–phenolred assay (HRP-PR;Supporting Information, TableS1, and data not shown). Several of these compounds did notproduce detectable levels of H₂O₂ in our assay,however. This may be related to compound stability in assay buffer,as discussed below. Given these results, we suspected that H₂O₂ production might be another source of assay interferencefor this chemotype (by oxidizing the free thiol on CoA to sulfenicand sulfinic acids). However, we found that even relatively high levelsof H₂O₂ (1 mM final concentrations) did notinterfere with the assay readout in the CoA–CPM counter-screenwhen compared to control reactions (p = 0.61,n = 8). Even H₂O₂ present in levelsgreater than those observed in our HRP-PR redox assay did not appreciablyinhibit the HAT activity of Rtt109–Vps75 or other HATs eitherin the presence or absence of DTT (SupportingInformation, Table S2), suggesting these particular proteinsare not overly susceptible to H₂O₂-mediatedinactivation under our experimental conditions. Additionally, noneof the other prototype compounds used in this report showed evidenceof redox activity when tested (Supporting Information,Table S1). Together, these data suggest H₂O₂ release by these redox-active compounds is not the primaryfactor behind their compound-mediated reductions in HTS signal orenzymatic activity.

UPLC–MS experiments provided importantinsights into the complex nature of this “triple-threat”chemotype. We first observed adducts6a′–6e′, which are consistent with addition–eliminationof a thiol on the parent compounds at the C3-position (Figure4B,C).⁶⁸ Importantly,these sulfonamides and the aforementioned adducts were not stableto our assay conditions. To our surprise, compounds6a–6e (Table5) and adducts6a′–6e′ showed a time-dependent degradation in our HTSbuffer when monitored by UPLC (Figure4B,CandSupporting Information, Figure S7).These data are consistent with and extends a previous report examininga similar screening compound.⁶⁹ On thebasis of this data and plausible chemical mechanisms, we speculatethat the degradation products are arylsulfonamides and naphthoquinonesresulting from imine hydrolysis and perhaps other as yet unidentifiedintermediates. Evidence supporting the complex and subversive reactivityof this class of compounds includes the observation that treatmentof6a–6e with GSH led to a commoncompound adduct (6″) with anm/z of 446. We propose that this GSH adduct is formedby loss of the arylsulfonamide and water, perhaps by imine hydrolysisof6a′–6e′ at the C–Nbond (Figure4C andSupportingInformation, Figure S7). Further characterization of this degradationprocess is ongoing.

In theaqueous and slightly alkaline HTS conditions, it is likely chemotype6 can also undergo imine hydrolysis to generate a reactivenaphthoquinone in situ, although we were unable to observe this compounddirectly by our UPLC–MS setup. Naphthoquinone formation isconsistent with the production of H₂O₂ and ourobservation of a common compound–glutathione adduct. It islikely the thiols could react with a resulting naphthoquinone viaMichael addition–elimination. Interestingly, compounds witha quinone moiety7 in place of a naphthoquinone generallyshowed less interference (Supporting Information,Figure S8) and none of these compounds were active in the slotblot or showed signs of redox activity (SupportingInformation, Table S1). Taken together, these data suggestthe napthoquinone moiety is an important structural factor for bothredox activity and thiol reactivity, at least under our experimentalconditions. Many compounds with the chemotype6 inhibitedRtt109-catalyzed histone acetylation as determined by slot blot (Table2), suggesting these compounds can inhibit enzymaticactivity either by reacting with proteins and/or other nonspecificmechanism(s).

We also examined the ability of the presumed aromaticleaving groups formed from these substrates (e.g.,1a″) to interfere with the assay readout. Many of these leavinggroups did not reduce the HTS or CoA-based counter-screen readouts,especially at the same low micromolar compound concentrations usedfor the prototype compounds (Supporting Information,Figure S9), and none inhibited enzymatic activity in the slotblot orthogonal assay. The severity of interference, however, appearsto increase when they were allowed to incubate longer with CoA (unpublishedobservations). Despite containing a thiol group, none of these leavinggroups formed fluorescent adducts with CPM, suggesting they are notsufficiently nucleophilic to react with the maleimide probe underthe conditions tested. Interestingly, the only leaving groups thatformed fluorescent adducts with CPM were somep-hydroxyarylsulfonamides(e.g.,6a) with thioglycolic or 3-mercaptopropionic acidsubstituents, as these compounds showed profiles consistent with false-negativeenzymatic inhibition (Table5).

Select AssayArtifacts Inhibit HAT-Catalyzed Histone Acetylation

Althoughcompounds with chemotypes1,2,3,4, and6 interfere with the assay readoutby trapping CoA, several of these same compounds were shown to inhibitRtt109-catalyzed histone acetylation at high compound concentrationsby slot blot assay (Tables1–5). We confirmed this inhibition for several compoundsusing a second, lower-throughput orthogonal HAT assay that utilized[³H]-acetyl-CoA. We found that most of these compoundsinhibited Rtt109-catalyzed histone acetylation in the low micromolarrange, particularly scaffolds2,3, and6 (Table6). This shows compounds with these chemotypes can inhibit Rtt109enzymatic activity in vitro, but this is most likely via nonspecificprotein reactivity, given the ability of these compounds to form thioladducts. As expected for compounds with nonspecific thiol reactivity,these same compounds also inhibited the human HAT p300 and the yeastGcn5–Ada2–Ada3 HAT complex at similar concentrations(Table6). This inhibition was profoundly attenuatedby the inclusion of DTT (Table6), which isconsistent with these chemotypes being thiol-reactive agents.

Select AssayArtifacts Form Covalent Bonds with Protein Assay Components

To further examine the thiol reactivity of these problematic compounds,we performed protein mass spectrometry (LC–MS/MS) using trypticdigestions of samples containing select prototype compounds incubatedwith the protein components of the HTS assay. As expected for potentthiol-trapping compounds, we observed several ionized peptides withaccurate mass measurements corresponding to covalently modified cysteineresidues on Rtt109 (Figure5 andSupporting Information, Table S3). Detectableadducts were also observed with select cysteine residues on Vps75and Asf1 (Figure5 andSupporting Information, Table S3). These compounds did notform detectable adducts with all the cysteines in the HTS proteinsunder our experimental conditions. We speculate this may be becausesome of the adducts were particularly labile under the experimentalconditions or were not amenable to ionization or because stericallyinaccessible and/or chemically inactivated cysteines (via sulfur oxidation)were not subject to reactivity. Further studies are needed to assessthese possibilities.

Overall, these data demonstrate the prototype in each ofthe chemical classes can covalently modify the protein componentsof our HAT assays in a promiscuous fashion. Given this data, and thestrong attenuation of enzymatic inhibition by the inclusion of DTTin our radiolabeled HAT assays (Table6), themost likely mechanism of enzymatic inhibition is nonspecific thiolreactivity. Because Rtt109 does not have a known catalytic cysteineresidue,⁷⁰ it is most likely the case thatthis thiol modification alters protein structure and dynamics ratherthan directly inhibiting the catalytic mechanism. The fact that severalof the protein components included in the HTS method were modified(and that HAT inhibition can be significantly attenuated with DTT)further suggests these compounds react with thiols indiscriminatelyand may therefore show promiscuous bioactivity.

Demonstrationof Compound–Protein Adducts by ALARM NMR

It is knownthat the reactivity of thiols in a proteinaceous microenvironmentmay be different than their reactivity with small-molecule thiolslike GSH⁷¹ (and presumably CoA). To completeour study, we investigated whether these interfering compounds couldreact with protein cysteines from a completely unrelated protein,the La antigen, using ALARM NMR.^18,72 Importantly,this assay utilizes a completely orthogonal detection method, thatis, not based on fluorescence, mass spectrometry, antibodies, or radioactivesubstrates. We tested the prototype compounds (1a,2a,3a,4a,6a, and6b) as well as positive and negative control compounds 2-chloro-1,4-naphthoquinoneand fluconazole, respectively (Figure6A).Consistent with the previous findings, all of these prototype compoundsinduced peak shifts in the regions of interest in the absence of DTT.These effects could be prevented by the inclusion of DTT in the assaybuffer, the addition of which does not lead to peak shifts or signalattenuation (Figure6A andSupporting Information, Figure S10). Together, these resultsindicate these prototype compounds (1a,2a,3a,4a,6a, and6b) covalently modify cysteines located on the La antigen. In the caseof the arylsulfonamides6a and6b, it appearsthe protein conformation is strongly perturbed (“denatured”)without the inclusion of DTT. Of possible relevance, related compoundshave been recently reported as disrupters of protein–proteininteractions.⁷³

To further show the utilityof this method and that the results were not exclusive to a selectsubset, we also tested several other analogues of these prototypecompounds, including some negative controls comprised of structuralanalogues that did not show interference in our HTS counter-screensnor inhibition of Rtt109-catalyzed histone acetylation in the slotblot assay. As expected, all of the prototype analogues, but not thenegative controls, were ALARM NMR-positive (Figure6B). As before, including DTT in the sample buffer preventedthe ALARM NMR reactivity. This demonstrates by a non-MS-based methodthat the interfering chemotypes are also susceptible to reactionswith protein cysteines, a known source of nonspecific enzymatic inhibitionand bioassay promiscuity.

Thiol-Reactive Chemotypes Show PromiscuousBehavior in Academic and Industrial Bioassays

As there isconsiderable chemical overlap in many academic screening libraries(unpublished observations), due in part to shared commercial vendorsand the “combiphilic” nature (i.e., amenable to synthesisby combinatorial schemes) of many screening scaffolds, we examinedthe scientific literature and the PubChem database to gauge whetherour findings may be more broadly applicable to other biological systemsand assay formats.⁷⁴⁻⁷⁶ Not surprisingly, compounds with the interferingscaffolds and some closely related derivatives have been reportedin the context of many biological systems with varying degrees ofbiological activity and claims of utility.⁷⁷ Several compounds bearing the scaffolds described in this reportalso showed patterns of bioassay promiscuity in a simple search ofPubChem bioassay records (Figure7). On thebasis of our findings, it is likely much of this bioassay promiscuityis due to nonspecific thiol reactivity.

Finally, we analyzed HTS records from a major pharmaceuticalcompany for evidence of frequent-hitter behavior across the chemotypesthat we have described above. It is commonly understood that academicand corporate libraries vary in size, composition, and chemical diversity,and therefore it is not immediately obvious that the trends seen inacademic data would apply outside of this domain. For the purposeof comparison, we derived frequent-hitter scores for a large subset(>1 M compounds) of AstraZeneca’s corporate compound collection.⁷⁸ The frequent-hitter scores are based on thebody of historical HTS screening data for these compounds, typically,compounds in the corporate screening deck will have been tested inseveral tens to hundreds of HTS campaigns. The frequent-hitter scorewe derive takes into account the anticipated incidence of activityfor an average compound, with high scores suggesting a higher-than-expectedlevel of activity. A score cutoff is defined to identify those compoundswith an unexpectedly high level of activity, thereby designating frequenthitters empirically. Details of the derivation of the frequent-hitterscore (pBSF) have been described previously.⁷⁸ It should be noted that the corporate data set used to identifyfrequent hitters covers a wide range of assay types, and does notsolely encompass assays like those described earlier in this publication.

We examined the incidence of frequent hitters across various categoriesof nuisance chemotypes in the AstraZeneca collection (Table7 andSupporting Information, Table S4). It is clear that some ofthe nuisance chemotypes derived from academic data display an elevatedincidence of frequent-hitter behavior in the corporate data as well,although not all chemotypes showed the same degree of promiscuity.Chemotypes1 (benzothiophene dioxides),2 (benzothiadiazole/benzofurazans), and6 (p-hydroxysulfonamides) exhibit high levels of promiscuous behaviorin the AstraZeneca screening deck, suggesting their indiscriminateand deleterious influence is present in a wide range of assay technologies.For thep-hydoxysulfonamides6, theobservation that they may also cause protein denaturation in the ALARMNMR assay in this study suggests another mode of action along theselines (that is, in addition to their other liabilities of redox activityand thiol reactivity). The AstraZeneca corporate data showed highlevels of assay promiscuity for chemotypes6 and7, which suggests the inactivity and weaker interference ofchemotype7 in our systems may be an assay-specific observation.That is, chemotype7 may still be relatively promiscuousunder other assays conditions,⁷⁹ a speculationthat may be pursued in future investigations. Nonsalt forms of the1,2,4-thiadiazoles3 show only slightly elevated levelsof promiscuous behavior in the corporate data set (Supporting Information, Table S4), while the salt forms wererelatively promiscuous (Table7). The succinimidechemotype4 did not exhibit high promiscuity in the AstraZenecascreening deck, but this may be an indication of problematic behaviorunder specific assay conditions such as alkaline assay buffers, whichwe expect would be needed to generate the reactive maleimides5. We note that some of the assays used for generating thefrequent-hitter scores have been stabilized with additions of DTT,which has the potential to mitigate the effects of reactive behaviordepending on assay specifics. Therefore, the bioassay promiscuityemerging from this set of data may also be indicative of interferencecause by mechanisms other than thiol reactivity. Overall, the observationsderived from the larger set of corporate data corroborate the evidencederived from the academic data in this publication.

MolecularLibraries, Compounds, and Reagents

The chemical library hasbeen described previously.⁴² The followingreagents were obtained from Sigma-Aldrich: DMSO, CPM, CoA (sodiumsalt hydrate), acetyl-CoA (sodium salt), bovine serum albumin (BSA),H₂O₂, and Triton X-100. Compounds tested inpost-HTS assays were repurchased as solid powders from standard chemicalvendors (e.g., eMolecules). In a quality-control sampling of a random5% of the chemical library samples used in this report, greater than90% of the tested commercial samples had acceptable purities (>90%)by UPLC–MS analysis and¹H NMR and LRMS–ESIspectra consistent with their vendor-provided structures.

Rtt109 HTSand Dose–Response Experiments

The CPM-based Rtt109assays have been detailed in a previous report with minor modifications.⁴² Briefly, all compounds studied in this reportwere rescreened in assay buffer containing freshly prepared 0.01%Triton X-100 (v/v) and enzyme concentrations of 50 nM Rtt109–Vps75complex. For IC₅₀ experiments, compounds were tested intriplicate at eight compound concentrations ranging from 200 nM to125 μM final compound concentrations. Slot blots were performedon reaction aliquots using standard techniques with a Bio-Rad Bio-DotSF microfiltration apparatus. Membranes were imaged with a LI-COROdyssey and analyzed using Image Studio (LI-COR Biosciences). Equalprotein loading was verified by Ponceau S staining of each membrane.

Data Analysis and Statistics

Z′factors for each plate were calculated using eq1:⁹⁵

1

where σ and μ represent the standarddeviation and mean of the positive (c⁺) and negative (c^–) plate controls, respectively. All plates tested inthese studies hadZ′ factors ≥0.5.IC₅₀ values were determined by fitting dose–responsedata to the sigmoidal dose–response variable slope four-parameterequation in GraphPad Prism 6.0. Other statistical analyses were alsoperformed in Prism using standard procedures.

Assay Interference Counter-Screens

Compounds interfering with the CPM-based assay readout were identifiedas previously described with minor modifications.⁴² Compounds were tested in triplicate at eight concentrationsranging from 200 nM to 125 μM final compound concentrationsusing an adaption of the Rtt109 HTS assay format. Proteins and assaybuffer were dispensed to assay plates analogously to the HTS procedure,then the acetyl-CoA substrate was replaced with CoA in concentrationstitered to match the fluorescence intensity observed for the uninhibitedenzyme reaction in the HTS assay (approximately 5 μM CoA). Compoundswere incubated with CoA and allowed to react with CPM under conditionsidentical to the HTS procedure. Assay interference was quantifiedby comparing the background-corrected (compound + proteins + CoA +CPM) fluorescence intensities to the (DMSO + proteins + CoA + CPM)controls. To further investigate their fluorescence behavior underthe HTS conditions, select compounds were also incubated with assayreagents (buffer-only, buffer + CoA, buffer + CPM) and their fluorescenceintensity measured. The overall plate layout, controls, protocols,and assay readouts were unchanged from the aforementioned compound–CoA–CPMcounter-screen.

Fluorescence Quenching Counter-Screen

Compounds were tested for evidence of fluorescence quenching usinga modification of our published procedure.⁴² Briefly, CPM and CoA (20 and 5 μM final concentrations, respectively)were allowed to react to completion in assay buffer. Completion wasdefined as a stable signal plateau, usually after 5 min reaction time.The CPM–CoA adduct solution (20 μL per well) was thendispensed into assay plates preplated with DMSO and test compounds.Compounds were dispensed using an ECHO 550 contactless liquid dispenser(Labcyte). Microplates were shaken for 5 min and allowed to equilibratefor another 5 min at room temperature. Fluorescence intensity wasmeasured, and the data was analyzed as percent signal reduction comparedto DMSO controls.

Compound–Thiol Adduct Characterization

Selected compounds (1 equiv) and either CoA or reducedl-glutathione (2 equiv) were incubated under HTS-like conditions,except with 5% DMSO (v/v) and no detergent in the assay buffer.⁴² Compounds were also tested in HTS buffer ormethanol minus the addition of biological thiols. Compounds were typicallyincubated at 0.5 mM final concentrations. Samples with visible precipitateswere passed through 0.25 μm syringe filters to remove particulates.Sample injections were typically 1.0 μL in volume performedby an autosampler and were analyzed on a Waters UPLC system usinga BEH C18 2.1 mm × 50 mm column. The flow rate was 0.250 mL/minwith a standard gradient starting at 95% Solution A (950 mL H₂O, 50 mL MeCN, 1 mL formic acid) and ending with 100% solutionB (1000 mL MeCN plus 1 mL formic acid) over 6.5 min. The samples weremonitored simultaneously using an ELS detector, a diode array detector(214, 220, 244, and 254 nm), and a ZQ mass spectrometer (ESI positiveand negative modes).

Redox-Activity Assay

Selected compoundswere assessed for redox activity using published protocols.^9,42,96 Freshly prepared 100 μMH₂O₂ (Sigma) was included as a positive platecontrol, while NSC-663284 and 4-amino-1-naphthol were used as positiveredox-active controls for DTT and DTT-free assay conditions, respectively.Fluconazole and DMSO were used as negative compound and plate controls,respectively. Compounds were tested in triplicate at eight final concentrations(200 nM to 125 μM via 2.5-fold dilutions) in either the presenceor absence of 1 mM DTT final concentration. All active compounds didnot interfere with the assay readout at A₆₁₀ (data notshown).

[³H]-Acetyl-CoA HAT Assays

For selectedcompounds, inhibition of HAT activity was also checked with an orthogonalin vitro radiolabeled substrate assay. Rtt109 inhibition was testedat eight compound concentrations (200 nM to 125 μM final compoundconcentrations via 2.5-fold dilutions) in an adaptation of a previousprocedure.⁴² Briefly, reactions were performedin standard volume 384-well microplates using 45 μL total reactionvolumes containing the following in final concentrations: 50 mM TrisHCl, pH 8.0, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.01% Triton X-100(v/v), 50 ng/μL BSA, and 2.5 μM [³H]-acetyl-CoA(PerkinElmer). Purified recombinant yeast Rtt109–Vps75 wastested at approximately 5 nM final concentrations, while purifiedrecombinant Asf1–dH3–H4 (approximately 250 nM) was usedas acetylation substrate. Compounds and DMSO were plated with a multichannelpipet, followed by a similar addition of a solution containing enzymeand histone substrate (36 μL). Test compounds were allowed toequilibrate with enzyme and histone substrate for 10 min at 30 °Cin an incubator. The HAT reaction was initiated by adding [³H]-acetyl-CoA solution (7.5 μL). DMSO content was kept constantacross all reactions at 3% (v/v). After 5 min, the reactions werequenched by multichannel pipet transfer of reaction aliquots (35 μL)to adjacent microplate wells each containing 35 μL of 2-propanol.Aliquots (35 μL) of the quenched solutions were carefully spottedonto Whatman P-81 phosphocellulose paper filters (GE Healthcare) andair-dried. Filter papers were washed five times for 5 min per cyclewith 50 mM NaHCO₃, pH 9.0, then rinsed with acetone andthen allowed to air-dry for 30 min. [³H]-Acetate incorporationwas then measured by an LS6500 liquid scintillation counter (Beckman–Coulter).Percent inhibition was calculated as a percentage of DMSO control.Similar reactions minus Rtt109–Vps75 were used as backgroundcontrols. Testing versus p300–BHC and the Gcn5–Ada2–Ada3complex were performed similarly, except that the final enzyme concentrationswere approximately 500 pM and the substrate was purified recombinantdH3–H4 tetramers.⁹⁷⁻⁹⁹

Protein Mass Spectrometry

Test compounds were incubated with purified Rtt109–Vps75or Asf1 complexes. Compounds and proteins were incubated togetherat 30 °C for 60 min at 100 μM and 10 μM final concentrations,respectively. Reaction mixtures were denatured with gentle heatingand then further resolved by SDS-PAGE. Protein bands were excisedafter staining with Coomassie blue. In-gel protease digestions wereperformed in an adaption of published procedures.¹⁰⁰ Peptide extracts were dried in vacuo and reconstitutedin 98:2:0.1 H₂O:acetonitrile:TFA; approximately 0.2 μgof each gel band was analyzed by capillary LC–MS on a VelosOrbitrap mass spectrometer (Thermo Fisher) with higher energy collisioninduced dissociation activation.¹⁰¹ PeaksStudio 6.0 build 20120620 (Bioinformatics Solutions) software packagewas used for interpretation of tandem MS and protein inference.¹⁰² Search parameters for Rtt109, Vps75, and Asf1proteins were UniProt database (Sacharomyces cerevisiae strain ATCC 204508/S288c, taxonomy ID 559292, accessed 19 May 2014)concatenated with the common lab contaminant proteins (www.thegpm.org); parent mass error tolerance = 20.0 ppm; fragment mass error tolerance= 0.1 Da; precursor mass search type = monoisotopic; enzyme trypsinwith max missed cleavages = 2 and nonspecific trypsin cleavage; variablemodifications = methionine oxidation and dioxidation, cysteine oxidation,and dioxidation, and suspected compound adducts; maximum variablemodifications per peptide = 5; false discovery rate calculation =on; spectra merge options = 0.2 min within 10.0 ppm mass window; chargecorrection = on for charge states 2–8; spectral filter quality>0.65. Support for the detection of peptides plus adducts fromeach supporting tandem MS data was based on: (1) high confidence peakspeptide score (minimum −10 logP 35), (2)a minimum of five consecutive b- or y-type peptide fragment ions,(3) high precursor mass accuracy (<7 ppm), and (4) supporting signatureion peaks for the site localization of the pertinent cysteine modificationon one or more peptide fragments.

Cheminformatics

Incidence of frequent-hitting behavior was checked in the AstraZenecacorporate screening deck by mining the historical screening data.We calculate a descriptor, pBSF, for each compound to determine whetherit is more active than expected.⁷⁸ ThepBSF score is the negative logarithm of the probability that the observedpattern of activity and inactivity is observed by chance, given theknown “average” behavior across all compounds in thescreening deck and across the historical set of screening campaignsthey have been measured in. If the likelihood of seeing the patternat hand is high, the compound is likely not a frequent hitter andall is fine. However, if the probability of seeing the pattern islow, the resulting pBSF score will be high and the pattern shouldbe regarded as anomalous. A cutoff of pBSF > 2 was used to designatecompounds exhibiting suspicious binding behavior. To check the incidenceof frequent-hitting behavior, we searched the corporate collectionusing substructures (with in-house tools), collated pBSF scores forthe set, and counted the number of frequent-hitting compounds usingthe pBSF threshold stated in the above. For reference, the averagefraction of compounds displaying frequent-hitting behavior acrossthe collection of compounds with historical HTS data is 6%.⁷⁸ The number of HTS data points is variable foreach compound, as it depends on the number of times a compound hasbeen screened. The median number of data points per compound in thedata set is approximately 200, with only 10% of the compounds havingless than 50 data points. Only biochemical assay data, and not cell-basedassay data, were used to derive the frequent hitter score

ALARM NMR

ALARM NMR was performed as previously described with minor modifications.^18,72 The gene encoding amino acids 100–324 of the human La antigenwas cloned into pET-28b+ vector (Novagen) such that it contained bothan N- and C-terminal His tag. The plasmid was freshly transformedintoEscherichia coli Rosetta cells(Novagen) and cultured in M9 minimal media supplemented with¹⁵NH₄Cl (CIL) in an adaption of published procedures.^103,104 The La antigen was enriched with¹³C at the δ-methylgroups of leucine, the δ-methyl group of isoleucine, and theγ-methyl groups of valine by the addition [3-¹³C]-α-ketobutyrateand [3,3′-¹³C]-α-ketoisovalerate (sodium salts,CIL) to the culture medium 30 min before inducing in the presenceof 1 mM IPTG for 8 h at 25 °C (OD₆₀₀ was approximately0.8 at time of induction). Harvested cells were lysed by French pressin ice-cold lysis buffer consisting of 50 mM Tris, pH 7.6, 300 mMNaCl, 10% glycerol (v/v), 5 mM β-mercaptoethanol (BME), 5 mMimidazole, 2 mM MgCl₂, benzonase (Sigma), and proteaseinhibitor cocktail. This solution containing the lysed cells was sonicatedbriefly (3 × 15 s pulse sequence) on ice, then loaded onto aprewashed Ni-bead column (GE Healthcare) kept at 4 °C. Proteinswere eluted from the beads with an elution buffer consisting of 50mM Tris, pH 7.6, 300 mM NaCl, 10% glycerol (v/v), 5 mM BME ,and animidazole gradient ranging from 5 mM to 0.5 M. Pooled elution fractionscontaining the La antigen were dialyzed overnight (25 mM sodium phosphate,pH 7.0, 5 mM DTT), flash-frozen in liquid N₂, and storedat −80 °C until further use. Prior to use, aliquots of500 μM protein was incubated in the presence of 20 mM DTT at37 °C for 1 h, then dialyzed versus 2 × 2 L of 25 mM sodiumphosphate buffer, pH 7.0 (no DTT) at 4 °C with constant N₂ bubbling. The¹H/¹³C-HMQC spectra wereacquired in 25 mM sodium phosphate buffer, pH 7.0, 10% D₂O (v/v; CIL) ± 200 μM test compounds delivered from 10mM DMSO stock solutions, and ± 20 mM DTT. Compounds were incubatedwith proteins at 37 °C for 1 h and then 30 °C for 15 h priorto data collection. Data were recorded at 25 °C on a Bruker 700MHz NMR spectrometer equipped with a cryoprobe (Bruker) and autosampler.Samples were loaded into Bruker 1.7 mM SampleJet tubes with 40 μLtotal sample volumes and stored at 4 °C while in queue. The ALARMNMR samples were tested at 50 μM protein concentrations using16 scans, 2048 complex points in F2, and 80 points in F1 using standardprotein HMQC and water suppression pulse sequences. Nonreactive compoundswere identified by the absence of chemical shifts (¹³C-methyl)± 20 mM DTT. Reactive compounds induced chemical shifts in certaindiagnostic peaks in the absence of DTT, and this effect was significantlyattenuated when 20 mM DTT was included in an otherwise identical sample.¹⁸ As an additional precaution against trace reactivecontaminants, compounds tested by ALARM NMR were repurified in-houseby standard HPLC procedures using mass-directed collection.

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