the growing use of high - throughput screening 
 ( hts ) as a discovery tool in academic translational centers has resulted 
 in the pursuit of assay artifacts , promiscuous bioactive compounds , 
 and screening actives with major absorption , distribution , metabolism , 
 excretion , and toxicological ( admet ) liabilities .
a similar situation 
 may exist in industry , and this observation may simply be a reflection 
 of academic pressures to publish . in either case , the follow - up of 
 such compounds can significantly burden the post - hts triage and hit - to - lead 
 stages of the discovery process . therefore , chasing assay artifacts 
 and promiscuous screening compounds can waste both time and other 
 valuable resources , and failure to triage these compounds has led 
 to many artifacts and  frequent hitters  making their 
 way into the scientific literature , patent applications , and research 
 funding applications . as an example , pan - assay interference 
 compounds ( pains ) can display apparent bioactivity and/or interfere 
 with assay readouts across unrelated biological targets and testing 
 methods .
multiple sources for promiscuous behavior or assay 
 interference have been described , including : chemical aggregation , chelation , singlet 
 oxygen production , compound fluorescence 
 effects , redox activity , sample impurities , membrane disruption , cysteine oxidation , and nonselective compound reactivity with proteins .
reporter interference as 
 the most likely source of biological assay readouts in a compound 
 that has progressed to human clinical trials .
an important point with these luciferase experiments is that confounding 
 readouts are not isolated to cell - free assays .
cell - based assays with 
 perturbations in cell proliferation may be particularly susceptible 
 to assay interference or off - target and confounding effects .
misleading 
 readouts can have clinical relevance , as a recent study suggests the 
 pharmacological activity of acamprosate ( an fda - approved drug for 
 relapse prevention in alcoholism ) may be due to the calcium cation 
 component of its formulation rather than the long - presumed bioactive 
 ingredient , n - acetylhomotaurinate . despite the risks associated with pursuing these 
 types of undesirable compounds , their identities and the chemical 
 mechanisms by which they can mislead even seasoned researchers often 
 go uncharacterized in the pursuit of identifying lead compounds .
unfortunately , 
 this leaves open the possibility for other groups to fall into the 
 same scientific maelstrom that most often results in costly failure . 
 in an effort to alert the uninitiated , we describe herein the structure  interference 
 relationships ( sir ) in five series of problematic compounds we encountered 
 in a recent hts campaign .
epigenetic enzymes , such as histone 
 deacetylases , methyltransferases , and histone acetyltransferases ( hats ) , 
 are an important emerging class of therapeutic targets .
epigenetic 
 chemical probes and enzymatic modulators are sought for a variety 
 of human diseases including cancers .
our 
 group and others have focused on a series of enzymes unique to fungi , 
 rtt109 hats , that are critical for dna replication - coupled nucleosome 
 assembly and genomic stability and therefore may represent a novel 
 antifungal therapeutic approach .
antibody - based 
 approaches can probe for specific histone modifications such as methylation 
 and acetylation .
another more indirect 
 approach probes for reaction byproducts via chemical probes or reporter 
 enzymes .
one chemical probe , n-[4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl]maleimide 
 ( cpm ) , readily reacts with free thiols to form highly fluorescent 
 adducts .
several cpm - based assays and screens have 
 been reported for multiple biological targets , including some epigenetic 
 enzymes .
recently , our group screened approximately 225k small molecules 
 for their ability to inhibit rtt109-catalyzed histone acetylation 
 using a cell - free cpm - based hts .
pains 
 were computationally filtered at the beginning of our triage and were 
 not initially evaluated in our post - hts counter - screens .
post - hts 
 triage of approximately 1.5k primary screening hits demonstrated only 
 a few confirmed actives . in retrospect , this indicated a significant 
 portion of the screening hits were either false positives or assay 
 artifacts resulting from fluorescence quenching , compound 
reagent 
 interference , and/or other mechanisms . on the basis of the chemical 
 structures of the triaged compounds
, we speculated many chemotypes 
 from the primary hts campaign were reacting with the coa byproduct 
 to produce an interfering assay readout mimicking enzymatic inhibition . 

we also speculated many of these thiol - reactive compounds , including 
 several series of pains we had previously triaged , could also inhibit 
 enzymatic activity by reacting with protein cysteines , a recognized 
 source of promiscuous enzyme inhibition and metabolic liability .
we 
 observed chemotypes that were enriched among the actives that appeared 
 chemically similar to many of the published pains substructures but 
 were not flagged by our cheminformatic pains filters .
there is a growing interest in both assay interference and 
 promiscuous enzymatic inhibition , including nonspecific thiol reactivity .
therefore , identifying thiol - reactive 
 chemotypes in compound screening libraries is important for enhancing 
 library design and post - hts decision - making
. additionally , the characterization 
 of the chemical mechanisms of thiol reactivity may also be useful 
 for reactivity prediction , compound optimization , and the avoidance 
 of follow - up on compounds that may have metabolic or selectivity liabilities 
 further downstream in the drug discovery pipeline .
the observation 
 that some pains - like compounds may escape cheminformatics filters 
 may have significant consequences for screening centers without experienced 
 hts triage personnel , especially if those performing hts triage are 
 overly reliant on cheminformatics filters .
herein , we report the identification of multiple chemotypes 
 (  chemical structural motifs  ) that showed publication - quality 
 ic50 values in our primary assay but through a series of 
 orthogonal assays and counter - screens showed thiol - reactive assay 
 interference and also promiscuous enzymatic inhibition . using sir 
 and structure  activity relationships ( sar ) from our rtt109 
 post - hts triage , along with multiple analytical techniques , we provide 
 evidence supporting several chemical mechanisms of assay interference 
 relating to thiol reactivity .
we show these chemotypes can form covalent 
 adducts with other biologically relevant thiols such as glutathione 
 ( gsh ) and cysteine residues on multiple structurally unrelated proteins . 

the chemical mechanisms we propose contributing to assay promiscuity 
 include addition  elimination reactions , nucleophlic aromatic 
 substitution , buffer instability , disulfide bond formation , and h2o2 production .
our findings may be more broadly 
 applicable , as several compounds containing these chemotypes formed 
 covalent adducts with the la antigen ( alarm nmr ) , demonstrating potentially 
 broad - spectrum thiol reactivity .
furthermore , compounds with these 
 chemotypes showed evidence of assay promiscuity in analyses of pubchem 
 and the hts database of a major pharmaceutical company . despite these 
 red flags ,
several such compounds have been reported in the patent 
 literature and reputable scientific journals with varying claims of 
 target specificity and utilities as either chemical probes or therapeutic 
 leads .
it is hoped that the identification and detailed characterization 
 of these thiol - reactive chemotypes can accelerate post - hts triage , 
 enhance lead identification , and prevent follow - up on unpromising 
 chemical matter by other researchers .
we previously 
 reported the use of a cpm - based method to screen approximately 225k 
 compounds for their abilities to inhibit rtt109-catalyzed histone 
 acetylation in vitro . in a hat reaction , 

an acetyl group is enzymatically transferred from acetyl - coa to the 
 -amino group of a histone lysine side chain , resulting in the 
 production of an acetylated lysine and a coa byproduct .
the free thiol 
 on coa can then react with suitable substrates , such as maleimide - based 
 probes like cpm , to form highly fluorescent adducts that can indirectly 
 assay hat activity ( figure 1a ) . in practice
, 
 we and others have found this method to be low - cost and relatively 
 robust .
we were also aware that this 
 method is subject to assay interference by thiol - containing compounds .
however , we soon learned that this assay is 
 highly susceptible to other mechanisms of reactive compound interference 
 especially when testing potentially heterogeneous chemical matter 
 like hts libraries . in principle , compounds can interfere with the 
 cpm fluorescence intensity by fluorescence quenching .
also , compounds 
 with nucleophilic or electrophilic reactivity can react with either 
 the cpm probe or the coa reaction product , respectively .
( a ) assay 
 schematic for the cpm - based hts used in this study .
the assay measures 
 the hat activity of the rtt109vps75 complex , which catalyzes 
 the transfer of an acetyl moiety from acetyl - coa to specific lysine 
 residues on the asf1dh3h4 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 .
despite identifying 1.5k actives in the primary screen , a 
 post - hts triage consisting of computational filtering , experimental 
 counter - screens , and orthogonal assays demonstrated only three compounds 
 that could inhibit rtt109-catalyzed histone acetylation .
therefore , 
 significant portions of this collection of experimental and computationally 
 filtered (  filtrand  ) compounds were either false positives 
 or assay artifacts .
in addition , many of the computationally filtered 
 actives were flagged as pains , which we believed could inhibit hat 
 activity by nontherapeutically useful mechanisms . in all
, several 
 prominent chemotypes were identified among the primary actives during 
 the course of the post - hts triage ( figure 1b ) . on the basis of their chemical structures
, it appeared 
 likely that each chemotype could interfere with our hts assay readout 
 through different and , perhaps , multiple thiol - trapping mechanisms . 

many of these compounds were flagged as pains , but still many were 
 not verified as  bad actors  until relatively late in 
 our triage process . to further confound matters
, these electrophilic 
 compounds could conceivably inhibit enzymatic activity by nonspecific 
 reactivity with the protein components in the assay , further scrambling 
 the assay readout . to better understand the chemical mechanism(s ) 
 behind this form of assay behavior and to assess whether this interference 
 could have implications beyond our hts , we examined five prominent 
 subclasses of compounds in more detail .
several 
 classes of compounds demonstrated low micromolar ic50 values 
 in the cpm - based hts method ( figure 2 ) .
2 ) have been associated with cooperativity 
 and anomalous binding behaviors , such as chemical aggregation .
most of the compounds showed 
 slightly elevated hill slopes , but this was not an immediate concern , 
 as we had included a detergent ( triton x-100 ) in our assay buffers 
 to mitigate micelle formation . 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 
reaction aliquots from the active compounds ( 1a , 2a , 3a , 4a , 6a , and 6b ) all showed decreases in histone acetylation 
 at 125 m when they were analyzed by an orthogonal slot blot 
 assay that uses h3k56ac- and h3k27ac - specific antibodies to probe 
 for the acetylated histone lysine product rather than the coa byproduct 
 ( tables 15 ) . 

we examined both histone modifications because the rtt109vps75 
 complex is capable of acetylating multiple histone h3 residues .
overall , 
 reaction aliquots showed similar levels of histone acetylation , regardless 
 of whether h3k27ac or h3k56ac was examined , strongly suggesting that 
 any observed enzymatic inhibition was not specific to one particular 
 histone modification .
this assay also detected decreases in histone 
 acetylation with garcinol , a natural product previously shown to inhibit 
 rtt109 activity and other hats in vitro at low micromolar compound 
 concentrations .
when the same assay was used 
 to examine reaction aliquots at lower compound concentrations ( 8 m ) , 
 we observed more discrepancies between the hts and slot blot readouts 
 ( data not shown ) .
overall , these observations showed that representatives 
 of these five compound classes could inhibit acetylation activity 
 at high concentrations , but their hts assay readouts were confounded 
 by 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 assess 
 for fluorescence interference .
this assay accurately identified the 
 fluorescence quencher bhq-1 , a positive control ( supporting information , figure s1 ) .
however , none of these 
 compounds ( 1a , 2a , 3a , 4a , and 6b ) showed evidence of fluorescence quenching , 
 intrinsic fluorescence or the generation of fluorescent adducts with 
 either coa or cpm in a set of assays mimicking our hts procedures 
 ( tables 15 ) . 
 when the acetyl - coa reactant was replaced by the coa reaction byproduct , 
 these compounds provided assay readouts that were strikingly similar 
 to those obtained under the hts assay conditions ( figure 2 ) .
the degree of assay signal reduction was also 
 dependent on the levels of coa present ( data not shown ) .
similar assay 
 behavior was observed for a variety of chemical analogues bearing 
 these chemotypes ( tables 15 ) .
therefore , the body of evidence strongly implicated 
 a thiol - trapping mechanism of assay interference .
next , we sought 
 to understand the chemical basis of this interference and potential 
 sources of enzymatic inhibition .
coa adducts , 
 we incubated the compounds with coa under hts - like conditions .
we 
 also tested for adducts with reduced l - glutathione ( gsh ) , 
 another important biological thiol , to assess if this presumed thiol 
 reactivity was unique to coa .
ms and lc  hrms analyses 
 showed that the test compounds ( 1a , 2a , 3a and 4a ) form adducts with both coa and gsh 
 ( figure 3 and supporting 
 information , figure s2 ) .
ms for multiple other representative compounds 
 from each chemotype ( data not shown ) .
while the p - hydroxyarylsulfonamides 6a6e yielded 
 the expected compound - gsh adducts ( 6a6e ; figures 4a , b ) , we also 
 observed that 6a6e 
 were really intermediates that converted to a common adduct in situ 
 after only 15 min under the hts conditions ( figure 4b , c ) .
( 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
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 ) .
( b ) simplified 
 schematics of the proposed reaction mechanisms to generate the observed 
 adducts .
labile adducts between p - hydroxyarylsulfonamides 
 ( 6 ) and gsh detected by qualitative uplc 
( 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 ) .
trace ( iv ) shows the same sample from trace ( i ) analyzed 15 min later . 

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 . together , this data is consistent 
 with a thiol - trapping mechanism as a major contributor to the cpm - based 
 assay signal reduction in the compound classes studied , as the tested 
 compounds reacted with both coa and gsh .
gsh adducts is an important consideration for certain 
 cell - based assays , or for in situ or in vivo assays , where xenobiotic 
the selected compounds interfere 
 with the hts assay readout and form thiol adducts by a variety of 
 chemical mechanisms ( figures 3b and 4a ) . on the basis of the uplc  ms and chemical 
 principles , we propose the following chemical mechanisms of thiol 
 reactivity for chemotypes 1 , 2 , 3 , 4 , and 6 ( figure 1 ) : benzo[b]thiophene 1,1-dioxides 1 (  benzothiophenes  ) interfere via a straightforward 
 michael addition  elimination reaction at the electrophilic 
 c3-position through thiolate nucleophilic attack .
the compounds with 
 chemotype 1 most likely to interfere and form thiol adducts 
 in our experimental conditions were those with s - linked 
 heteroaromatic substituents ( table 1 ) .
the uplc  ms experiments using 1a confirmed the presence of the adduct 1a and 
 the leaving group 1a ( figure 3a ) .
this proposed mechanism is also supported by the observations 
 that several 2,3-dihydro analogues did not show appreciable levels 
 of apparent rtt109 inhibition or interference in the coa 
the level of assay interference is consistent with the 
 leaving group ability of the c3 substituent , as compounds with n- , o- , or c - linked groups 
 at this c3-position did not show as significant levels of interference 
 or apparent inhibition ( supporting information , 
 figure s3 ) .
most of the compounds with chemotype 1 showed only partial decreases in histone acetylation at high compound 
 concentrations ( table 1 ) , demonstrating these 
 compounds can weakly inhibit rtt109 activity in our hts , most likely 
 by nonspecific thiol reactivity ( table 1 ) .
hts refers to ic50 values 
 calculated from the cpm - based rtt109 hts method , coa - cpm refers to 
 ic50 values calculated from the coa - based hts counter - screen .
hts reaction aliquots from 
 compounds tested at 125 m final concentrations ; yes (  y  ) , 
 no (  n  ) , or partial (  p  ) .
compounds flagged as quenchers if greater 
 than 20% assay signal reduction at 10 m final concentrations .
compounds flagged if fluorescence 
 intensity greater than 20% assay signal at 125 m final concentrations ; 
 compounds tested in either hts buffer (  buffer  ) , hts 
 buffer plus 20 m cpm (  + cpm  ) , or hts buffer 
 plus 7.5 m coa (  + coa  ) .
the 
 benzothiadiazole / benzofurazan scaffold 2 likely forms 
 thiol adducts via nucleophilic aromatic substitution through a meisenheimer 
 complex intermediate between the nucleophilic thiol and the strongly 
 electrophilic heteroaromatic core .
the benzofurazan core has been 
 previously associated with promiscuous thiol reactivity , while some related benzothiadiazoles have been reported as pains 
 ( e.g. , substructures  diazox_b  and  diazox_sulfon_a  ) .
the related compound 4-chloro-7-nitrobenzofurazan (  nbd - cl  ) 
 and its derivatives are widely used as probes for studying thiols 
 in biological systems .
ms 
 analysis of 2a demonstrates that the parent compound 
 is stable to the hts buffer but that the addition of a thiol source 
 leads to near - complete conversion to the thiol adduct 2a with the thiopurine serving as the leaving group ( figure 3a ) .
the compounds that showed the strongest apparent 
 enzyme inhibition and interference contained electron - withdrawing 
 substituents such as nitro groups and halogens , although there was 
 no apparent reactivity difference between benzothiadiazoles and benzofurazans . 

another important feature for interference was the presence of an s - linked aryl substituent , which serves as the leaving group 
 even when there are other electron - withdrawing groups present ( table 2 ) .
benzothiadiazoles 
 without these features did not show significant levels of apparent 
 enzyme inhibition or assay interference ( supporting 
 information , figure s4 ) .
while flagged as pains , many sulfoxide - substituted 
 analogues tested in our system were inactive and noninterfering , an 
 observation we attribute to the absence of an additional strong electron - withdrawing 
 group ( e.g. , nitro ) .
many compounds with chemotype 2 were 
 capable of completely inhibiting rtt109-catalyzed histone acetylation 
 in our hts ( table 2 ) .
hts refers to ic50 values calculated from 
 the cpm - based rtt109 hts method , coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen .
hts reaction aliquots from compounds 
 tested at 125 m final concentrations ; yes (  y  ) , 
 no (  n  ) or partial (  p  ) .
compounds flagged as quenchers if greater 
 than 20% assay signal reduction at 10 m final concentrations .
compounds flagged if fluorescence 
 intensity greater than 20% assay signal at 125 m final concentrations ; 
 compounds tested in either hts buffer (  buffer  ) , hts 
 buffer plus 20 m cpm (  + cpm  ) . or hts buffer 
 plus 7.5 m coa (  + coa  ) .
while 
 the core 1,2,4-thiadiazole heterocycle 3 may appear benign , 
 many such compounds can react quite readily with thiols but not other functional groups like alcohols or amines .
the 1,2,4-thiadiazole core can be susceptible to ring - opening reactions , 
 recyclization side products , and nonenzymatic reductions .
pains substructure , although it differs by resonance and substitution 
 at the n2-position , meaning this chemotype could bypass some pains 
 filters depending on its structural representation and certain chemoinformatic 
 parameters .
this speculation aside , 
 the likely chemical mechanism of interference in our assay is sulfhydryl - scavenging 
 by the 1,2,4-thiadiazole core at the s1-position , specifically a ring - opening 
 reaction that generates a disulfide that can then be reduced by another 
 thiol or electron source in situ to form the corresponding thiourea .
indeed , we first observed the formation of the 
 thiourea form ( 3a ) , as evidenced by a major shift 
 in the uplc retention time upon the addition of thiols ( figure 3a ) . the parent ions for this entity ( i.e.
ms , and notably we did not observe any coeluting gsh ions , 
 suggesting this peak was not the 3a form with 
 an attached gsh moiety . to gain a further structural understanding 
 of the 3a adducts , we synthesized it under hts - like conditions 
 and characterized its identity and structure in situ by lc
gsh 3a adduct ( supporting information ) , which is consistent with a previous 
 report on this chemotype .
these data , 
 combined with our findings that compounds 3 are strongly 
 reactive in our thiol - trapping interference screen , suggests the 3gsh adduct forms ( 3 ) are not 
 stable to our characterization procedures and/or our lc - ms conditions .
examination of close analogues showed the assay interference strongly 
 correlates with additional alkylation at the core n2-position to generate 
 a partially cationic nitrogen , which presumably activates the s1n2 
 bond for thiol - mediated cleavage .
compounds lacking these substituents 
 on the n2-position were inactive and showed minimal interference ( supporting information , figure s5 ) . of note , 

het_5_inium  , 
 which bears resemblance to the charged 1,2,4-thiadiazoles in this 
 chemotype . neither the nature of the r 
r substituents nor the particular salt composition appeared to have 
 significant effects on thiol - trapping ( table 3 ) .
consistent with this 
 mechanism , many 1,2,4-oxadiazole analogues showed minimal assay activity 
 and interference ( supporting information , figure 
 s5 ) .
we observed that many of these 1,2,4-thiadiazoles inhibited 
 rtt109-catalyzed histone acetylation quite effectively ( table 3 ) , suggesting these compounds can not only interfere 
 with the hts assay but can also inhibit enzymatic activity , again 
 presumably by nonspecific reactivity with protein thiols .
hts refers to ic50 values calculated from the cpm - based rtt109 hts method , 
 coa - cpm refers to ic50 values calculated from the coa - based 
 hts counter - screen .
hts 
 reaction aliquots from compounds tested at 125 m final concentrations ; 
 yes (  y  ) , no (  n  ) or partial (  p  ) . compounds flagged as quenchers 
 if greater than 20% assay signal reduction at 10 m final concentrations .
compounds flagged if fluorescence 
 intensity greater than 20% assay signal at 125 m final concentrations ; 
 compounds tested in either hts buffer (  buffer  ) , hts 
 buffer plus 20 m cpm (  + cpm  ) , or hts buffer 
 plus 7.5 m coa (  + coa  ) .
our data is consistent with 
 an elimination event followed by a michael addition of a free thiol 
 to the resulting maleimide .
this is the same sulfhydryl - sensitive 
 group present in the cpm probe used in our hts .
elimination is likely , 
 given the slightly alkaline ph of the assay buffer ( ph 8.0 ) .
this same proposed leaving group was not detected when 4a was incubated in neat meoh ( figure 3a ) .
first , the proposed elimination 
 product maleimides 5 showed nearly identical ic50 values in both the hts and interference counter - screens compared 
 to their parent succinimides ( table 4 ) .
second , the apparent enzymatic inhibition and 
 counter - screen ic50 values correlate well with the presumed 
 leaving - group ability of the succinimide substituent . for instance , 
 succinimides with s - linked aryl groups showed significant 
 assay interference ( table 4 ) , while nonaryl , s - linked leaving groups showed no significant activity and 
 interference ( supporting information , figure s6 ) .
third , succinimides with n - linked substituents 
 did not appear to inhibit rtt109 in our hts or show interference in 
 our counter - screens ( supporting information , figure 
 s6 ) .
the substituents on the resulting maleimides did not have 
 a noticeable effect on either the hts or counter - screen ic50 values ( table 4 ) .
most of the interfering 
 succinimides 4 could only partially inhibit rtt109 activity 
 at higher compound concentrations in our hts , which may be a reflection 
 of the kinetics of the succinimide - to - maleimide conversion in buffer .
hts refers to ic50 values calculated 
 from the cpm - based rtt109 hts method , coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen hts reaction aliquots from compounds 
 tested at 125 m final concentrations ; yes (  y  ) , 
 no (  n  ) or partial (  p  ) .
compounds flagged as quenchers if greater 
 than 20% assay signal reduction at 10 m final concentrations .
compounds flagged if fluorescence 
 intensity greater than 20% assay signal at 125 m final concentrations ; 
 compounds tested in either hts buffer (  buffer  ) , hts 
 buffer plus 20 m cpm (  + cpm  ) , or hts buffer 
 plus 7.5 m coa (  + coa  ) .
others 
 have shown this scaffold to be redox - active as well as subject to addition  elimination at the substituted 
 c3-position . during our post - hts triage
, 
 we also observed that many of these compounds produced h2o2 in our assay buffer , both in the presence and absence 
 of the reducing agent dtt using a horseradish peroxidase 
phenol 
 red assay ( hrp - pr ; supporting information , table 
 s1 , and data not shown ) .
several of these compounds did not 
 produce 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 interference 
 for this chemotype ( by oxidizing the free thiol on coa to sulfenic 
 and sulfinic acids ) .
however , we found that even relatively high levels 
 of h2o2 ( 1 mm final concentrations ) did not 
 interfere with the assay readout in the coa 
cpm counter - screen 
 when compared to control reactions ( p = 0.61 , n = 8) .
even h2o2 present in levels 
 greater than those observed in our hrp - pr redox assay did not appreciably 
 inhibit the hat activity of rtt109vps75 or other hats either 
 in the presence or absence of dtt ( supporting 
 information , table s2 ) , suggesting these particular proteins 
 are not overly susceptible to h2o2-mediated 
 inactivation under our experimental conditions . additionally , none 
 of the other prototype compounds used in this report showed evidence 
 of redox activity when tested ( supporting information , 
 table s1 ) .
together , these data suggest h2o2 release by these redox - active compounds is not the primary 
 factor behind their compound - mediated reductions in hts signal or 
 enzymatic activity .
ms experiments provided important 
 insights into the complex nature of this  triple - threat  
 chemotype .
elimination 
 of a thiol on the parent compounds at the c3-position ( figure 4b , c ) .
importantly , 
 these sulfonamides and the aforementioned adducts were not stable 
 to our assay conditions . to our surprise ,
compounds 6a6e ( table 5 ) and adducts 6a6e showed a time - dependent degradation in our hts 
 buffer when monitored by uplc ( figure 4b , c 
 and supporting information , figure s7 ) . 

these data are consistent with and extends a previous report examining 
 a similar screening compound . on the 
 basis of this data and plausible chemical mechanisms , we speculate 
 that the degradation products are arylsulfonamides and naphthoquinones 
 resulting from imine hydrolysis and perhaps other as yet unidentified 
 intermediates .
evidence supporting the complex and subversive reactivity 
 of this class of compounds includes the observation that treatment 
 of 6a6e with gsh led to a common 
 compound adduct ( 6 ) with an m / z of 446 .
we propose that this gsh adduct is formed 
 by loss of the arylsulfonamide and water , perhaps by imine hydrolysis 
 of 6a6e at the c 
hts refers to ic50 values calculated 
 from the cpm - based rtt109 hts method , coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen hts reaction aliquots from compounds 
 tested at 125 m final concentrations ; yes (  y  ) , 
 no (  n  ) or partial (  p  ) .
compounds flagged as quenchers if greater 
 than 20% assay signal reduction at 10 m final concentrations .
compounds flagged if fluorescence 
 intensity greater than 20% assay signal at 125 m final concentrations ; 
 compounds tested in either hts buffer (  buffer  ) , hts 
 buffer plus 20 m cpm (  + cpm  ) , or hts buffer 
 plus 7.5 m coa (  + coa  ) .
ic50 values shown are means  sd for 
 three replicates . in the 
 aqueous and slightly alkaline hts conditions
, it is likely chemotype 6 can also undergo imine hydrolysis to generate a reactive 
 naphthoquinone in situ , although we were unable to observe this compound 
 directly by our uplc  ms setup .
naphthoquinone formation is 
 consistent with the production of h2o2 and our 
 observation of a common compound  glutathione adduct .
it is 
 likely the thiols could react with a resulting naphthoquinone via 
 michael addition  elimination .
interestingly , compounds with 
 a quinone moiety 7 in place of a naphthoquinone generally 
 showed less interference ( supporting information , 
 figure s8 ) and none of these compounds were active in the slot 
 blot or showed signs of redox activity ( supporting 
 information , table s1 ) . taken together , these data suggest 
 the napthoquinone moiety is an important structural factor for both 
 redox activity and thiol reactivity , at least under our experimental 
 conditions .
many compounds with the chemotype 6 inhibited 
 rtt109-catalyzed histone acetylation as determined by slot blot ( table 2 ) , suggesting these compounds can inhibit enzymatic 
 activity either by reacting with proteins and/or other nonspecific 
 mechanism(s ) .
we also examined the ability of the presumed aromatic 
 leaving groups formed from these substrates ( e.g. , 1a ) to interfere with the assay readout .
many of these leaving 
 groups did not reduce the hts or coa - based counter - screen readouts , 
 especially at the same low micromolar compound concentrations used 
 for the prototype compounds ( supporting information , 
 figure s9 ) , and none inhibited enzymatic activity in the slot 
 blot orthogonal assay . the severity of interference , however , appears 
 to increase when they were allowed to incubate longer with coa ( unpublished 
 observations ) . despite containing a thiol group , none of these leaving 
 groups formed fluorescent adducts with cpm , suggesting they are not 
 sufficiently nucleophilic to react with the maleimide probe under 
 the conditions tested .
interestingly , the only leaving groups that 
 formed fluorescent adducts with cpm were some p - hydroxyarylsulfonamides 
 ( e.g. , 6a ) with thioglycolic or 3-mercaptopropionic acid 
 substituents , as these compounds showed profiles consistent with false - negative 
 enzymatic inhibition ( table 5 ) .
although 
 compounds with chemotypes 1 , 2 , 3 , 4 , and 6 interfere with the assay readout 
 by trapping coa , several of these same compounds were shown to inhibit 
 rtt109-catalyzed histone acetylation at high compound concentrations 
 by slot blot assay ( tables 15 ) .
we confirmed this inhibition for several compounds 
 using a second , lower - throughput orthogonal hat assay that utilized 
 [ h]-acetyl - coa .
we found that most of these compounds 
 inhibited rtt109-catalyzed histone acetylation in the low micromolar 
 range , particularly scaffolds 2 , 3 , and 6 ( table 6 ) .
this shows compounds with these chemotypes can inhibit rtt109 
 enzymatic activity in vitro , but this is most likely via nonspecific 
 protein reactivity , given the ability of these compounds to form thiol 
 adducts .
as expected for compounds with nonspecific thiol reactivity , 
 these same compounds also inhibited the human hat p300 and the yeast 
 gcn5ada2ada3 hat complex at similar concentrations 
 ( table 6 ) .
this inhibition was profoundly attenuated 
 by the inclusion of dtt ( table 6 ) , which is 
 consistent with these chemotypes being thiol - reactive agents . in parentheses 

compounds tested identically in the presence of 1 mm 
 dtt ; results similar versus rtt109-vps75 , p300 , and gcn5 ; typically 
 < 20% inhibition was observed at 125 m final compound concentrations . previously published value . to further examine the thiol reactivity of these problematic compounds , 
 we performed protein mass spectrometry ( lc  ms / ms ) using tryptic 
 digestions of samples containing select prototype compounds incubated 
 with the protein components of the hts assay . as expected for potent 
 thiol - trapping compounds , we observed several ionized peptides with 
 accurate mass measurements corresponding to covalently modified cysteine 
 residues on rtt109 ( figure 5 and supporting information , table s3 ) .
detectable 
 adducts were also observed with select cysteine residues on vps75 
 and asf1 ( figure 5 and supporting information , table s3 ) .
these compounds did not 
 form detectable adducts with all the cysteines in the hts proteins 
 under our experimental conditions .
we speculate this may be because 
 some of the adducts were particularly labile under the experimental 
 conditions or were not amenable to ionization or because sterically 
 inaccessible and/or chemically inactivated cysteines ( via sulfur oxidation ) 
 were not subject to reactivity .
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 .
( 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 .
overall , these data demonstrate the prototype in each of 
 the chemical classes can covalently modify the protein components 
 of our hat assays in a promiscuous fashion . given this data , and the 
 strong attenuation of enzymatic inhibition by the inclusion of dtt 
 in our radiolabeled hat assays ( table 6 ) ,
the 
 most likely mechanism of enzymatic inhibition is nonspecific thiol 
 reactivity . because rtt109 does not have a known catalytic cysteine 
 residue ,
it is most likely the case that 
 this thiol modification alters protein structure and dynamics rather 
 than directly inhibiting the catalytic mechanism .
the fact that several 
 of 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 indiscriminately 
 and may therefore show promiscuous bioactivity .
it is known 
 that the reactivity of thiols in a proteinaceous microenvironment 
 may be different than their reactivity with small - molecule thiols 
 like gsh ( and presumably coa ) . to complete 
 our study , we investigated whether these interfering compounds could 
 react with protein cysteines from a completely unrelated protein , 
 the la antigen , using alarm nmr . importantly , 
 this assay utilizes a completely orthogonal detection method , that 
 is , not based on fluorescence , mass spectrometry , antibodies , or radioactive 
 substrates .
we tested the prototype compounds ( 1a , 2a , 3a , 4a , 6a , and 6b ) as well as positive and negative control compounds 2-chloro-1,4-naphthoquinone 
 and fluconazole , respectively ( figure 6a ) . 

consistent with the previous findings , all of these prototype compounds 
 induced peak shifts in the regions of interest in the absence of dtt . 
 these effects could be prevented by the inclusion of dtt in the assay 
 buffer , the addition of which does not lead to peak shifts or signal 
 attenuation ( figure 6a and supporting information , figure s10 ) .
together , these results 
 indicate these prototype compounds ( 1a , 2a , 3a , 4a , 6a , and 6b ) covalently modify cysteines located on the la antigen . in the case 
 of the arylsulfonamides 6a and 6b
, it appears 
 the protein conformation is strongly perturbed (  denatured  ) 
 without the inclusion of dtt . of possible relevance , related compounds 

thiol reactivity of select 
 screening compounds with the la protein as measured by alarm nmr . 

 c hmqc spectra of selected c - 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 . 

( 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 . to further show the utility 
 of this method and that the results were not exclusive to a select 
 subset , we also tested several other analogues of these prototype 
 compounds , including some negative controls comprised of structural 
 analogues that did not show interference in our hts counter - screens 
 nor inhibition of rtt109-catalyzed histone acetylation in the slot 
 blot assay .
as expected , all of the prototype analogues , but not the 
 negative controls , were alarm nmr - positive ( figure 6b ) .
as before , including dtt in the sample buffer prevented 
 the alarm nmr reactivity .
this demonstrates by a non - ms - based method 
 that the interfering chemotypes are also susceptible to reactions 
 with protein cysteines , a known source of nonspecific enzymatic inhibition 
 and bioassay promiscuity . as there is 
 considerable chemical overlap in many academic screening libraries 
 ( unpublished observations ) , due in part to shared commercial vendors 
 and the  combiphilic  nature ( i.e. , amenable to synthesis 
 by combinatorial schemes ) of many screening scaffolds , we examined 
 the scientific literature and the pubchem database to gauge whether 
 our findings may be more broadly applicable to other biological systems 
 and assay formats .
not surprisingly , compounds with the interfering 
 scaffolds and some closely related derivatives have been reported 
 in the context of many biological systems with varying degrees of 
 biological activity and claims of utility .
several compounds bearing the scaffolds described in this report 
 also showed patterns of bioassay promiscuity in a simple search of 
 pubchem bioassay records ( figure 7 ) . on the 
 basis of our findings ,
it is likely much of this bioassay promiscuity 
 is due to nonspecific thiol reactivity .
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 ) . finally , we analyzed hts records from a major pharmaceutical 
 company for evidence of frequent - hitter behavior across the chemotypes 
 that we have described above .
it is commonly understood that academic 
 and corporate libraries vary in size , composition , and chemical diversity , 
 and therefore it is not immediately obvious that the trends seen in 
 academic data would apply outside of this domain . for the purpose 
 of comparison
, we derived frequent - hitter scores for a large subset 
 ( > 1 m compounds ) of astrazeneca s corporate compound collection .
the frequent - hitter scores are based on the 
 body of historical hts screening data for these compounds , typically , 
 compounds in the corporate screening deck will have been tested in 
 several tens to hundreds of hts campaigns .
the frequent - hitter score 
 we derive takes into account the anticipated incidence of activity 
 for an average compound , with high scores suggesting a higher - than - expected 
 level of activity .
a score cutoff is defined to identify those compounds 
 with an unexpectedly high level of activity , thereby designating frequent 
 hitters empirically .
details of the derivation of the frequent - hitter 
 score ( pbsf ) have been described previously .
it should be noted that the corporate data set used to identify 
 frequent hitters covers a wide range of assay types , and does not 
 solely encompass assays like those described earlier in this publication .
we examined the incidence of frequent hitters across various categories 
 of nuisance chemotypes in the astrazeneca collection ( table 7 and supporting information , table s4 ) .
it is clear that some of 
 the nuisance chemotypes derived from academic data display an elevated 
 incidence of frequent - hitter behavior in the corporate data as well , 
 although not all chemotypes showed the same degree of promiscuity . 

chemotypes 1 ( benzothiophene dioxides ) , 2 ( benzothiadiazole / benzofurazans ) , and 6 ( p - hydroxysulfonamides ) exhibit high levels of promiscuous behavior 
 in the astrazeneca screening deck , suggesting their indiscriminate 
 and deleterious influence is present in a wide range of assay technologies . 
 for the p - hydoxysulfonamides 6 , the 
 observation that they may also cause protein denaturation in the alarm 

nmr assay in this study suggests another mode of action along these 
 lines ( that is , in addition to their other liabilities of redox activity 
 and thiol reactivity ) .
the astrazeneca corporate data showed high 
 levels of assay promiscuity for chemotypes 6 and 7 , which suggests the inactivity and weaker interference of 
 chemotype 7 in our systems may be an assay - specific observation . 

that is , chemotype 7 may still be relatively promiscuous 
 under other assays conditions , a speculation 
 that may be pursued in future investigations .
nonsalt forms of the 
 1,2,4-thiadiazoles 3 show only slightly elevated levels 
 of promiscuous behavior in the corporate data set ( supporting information , table s4 ) , while the salt forms were 
 relatively promiscuous ( table 7 ) .
the succinimide 
 chemotype 4 did not exhibit high promiscuity in the astrazeneca 
 screening deck , but this may be an indication of problematic behavior 
 under specific assay conditions such as alkaline assay buffers , which 
 we expect would be needed to generate the reactive maleimides 5 .
we note that some of the assays used for generating the 
 frequent - hitter scores have been stabilized with additions of dtt , 
 which has the potential to mitigate the effects of reactive behavior 
 depending on assay specifics .
therefore , the bioassay promiscuity 
 emerging from this set of data may also be indicative of interference 
 cause by mechanisms other than thiol reactivity .
overall , the observations 
 derived from the larger set of corporate data corroborate the evidence 
 derived from the academic data in this publication .
structure 
 annotations : a , any atom ; ns , number of substituents 
 ( e.g. ,  2s  ) ; nr , number of connected 
 ring bonds ( e.g. ,  2r  ) ; x , halogen .
ndata designates the subset 
 of compounds for which a pbsf score had been derived .
expected incidence of anomalous binders is 6% ( averaged 
 over all compounds ) . observed fractions of frequent hitters for structural classes ( chemotypes ) . 

note only biochemical assay data , and not cell - based assay data , were 
 used to derive the frequent hitter score .
in this article , we characterized 
 the chemical basis of assay interference for five problematic chemotypes 
 ( 1 , 2 , 3 , 4 , and 6 ) identified during the course of a recent triage of a sulfhydryl - scavenging 
 hts for inhibitors of rtt109-catalyzed histone acetylation . these 
 chemotypes were flagged as pains or have close chemical structural 
 similarities to certain pains substructures .
we first showed that 
 while compounds containing any of these five scaffolds are capable 
 of inhibiting rtt109-catalyzed histone acetylation , this inhibition 
 was confounded by the ability of these compounds to interfere with 
 the hts assay readout by reacting with free coa in the rtt109 hts . 

ms and lc  hrms that these 
 compounds can form adducts with other biological thiols such as gsh , 
 and in another orthogonal enzymatic assay , can inhibit several different 
 hats in vitro only when dtt is absent in the reaction mixture .
protein 
 mass spectrometry confirmed several of these compounds could covalently 
 modify multiple cysteines in the hts . using alarm nmr , yet another 
 orthogonal detection method
, we showed that the majority of these 
 compounds can covalently modify cysteines on a completely unrelated 
 protein system .
the findings described herein strongly suggest 
 investigators ( and reviewers ) flag these problematic compounds and 
 avoid their follow up .
it is particularly troubling that many of these 
 compound classes are still being reported in the patent literature 
 and reputable scientific journals , some with dubious claims of biological 
 utility ( see supporting information ) .
perhaps 
 not coincidentally , these compound types were active in other bioassays 
 according to pubchem queries .
the propagation of these nuisance compounds 
 in reputable journals suggests that many academicians and reviewers 
 alike are not fully aware of nuisance compounds such as pains and 
 perhaps not appreciative of their potential to sidetrack early drug 
 discovery projects . on the basis of our studies ,
we highly recommend 
 the problematic chemotypes described in this report be pursued as 
 chemical leads with high levels of skepticism and that investigators 
 currently working with these compounds carefully re - evaluate the interpretation 
 of their results when there are claims of biological utility , including 
 apparent enzymatic and cell - based selectivity , bioactivity , and mechanism - of - action 
 studies .
for instance , we posit that much of the selectivity observed 
 for these chemotypes ( a common defense for those publishing pains 
 as bioactive compounds ) is due to different susceptibilities of assay 
 components ( e.g. , enzymes , cell lines ) to thiol - reactive compounds 
 or other nonspecific mechanisms and that observed bioactivity is likely 
 attributable to off - target effects . with regards to mechanistic studies , 

it is likely some key component is missing from the experimental design 
 ( e.g. , assessing the effect of dtt or rigorously testing for irreversibility ) . 
 a recommended list of assays for evaluating the potential for compound 
we recommend 
 that knowledge - based methods be supplemented by more than one of the 
 experimental - based methods .
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 .
the emergence of epigenetic targets such as hats has led 
 to the development of several types of hts assays to study epigenetic 
 modifications like acetylation .
hat activity can be probed with antibody - based 
 methods ( e.g. , western blots , amplified luminescent proximity homogeneous 
 assays , tr - fret ) or sulfhydryl - scavenging methods ( e.g. , fluorescent 
 probes or coupled - enzyme reporters ) .
other methods such as radiolabeled substrates , mass 
 spectrometry , and electrophoretic mobility , have been used to assay 
 the status of protein acetylation , including cell - based adaptations .
while each method has distinct advantages and disadvantages , each 
 is still susceptible to false positives , assay artifacts , and identifying 
 promiscuous 
the maleimide - based 
 screens are subject to several mechanisms of chemical interference . 

we determined thiol - trapping compounds represented a significant source 
 of assay artifacts in our cpm - based rtt109 hts , especially chemotypes 1 , 2 , 3 , 4 , and 6 .
these compounds interfered with our hts readout by forming 
 covalent adducts with the coa produced by the hat reaction , creating 
 a convoluted readout of enzymatic inhibition .
nucleophilic screening 
 compounds can also form adducts with cpm that can create either a 
 false - positive or false - negative readout pattern , depending on the 
 fluorescent characteristics of the adduct .
another artifact source 
 is fluorescence quenching , although we did not encounter many examples 
 of fluorescence quenchers in our post - hts triage ( data not shown ) . 
 on the basis of the chemotypes in this report , along with the other 
 triaged compounds , the majority of assay artifacts from our cpm - based 
 hts resulted from thiol - trapping rather than compound 
, a major ( though not insurmountable ) disadvantage 
 of this screening method is the high levels of assay interference 
 and the time and resources needed in the post - hts phase to triage 
 these artifacts . in fairness , it is worth noting that this method 
 is capable of identifying compounds that inhibit enzymatic activity 
 and has distinct advantages such as low cost and robustness . to prevent follow - up on bad chemical matter discovered by 
 maleimide - based screens ,
first , we 
 strongly recommend having a validated , robust orthogonal assay in 
 place prior to conducting an hts with this method .
relying solely 
 on the cpm - based method could lead to the selection of thiol - trapping 
 compounds , and if used for the basis of compound optimization could 
 lead to the unfortunate case of optimizing for thiol reactivity rather 
 than the desired enzymatic inhibition .
this could conceivably happen 
 if one were to view the apparent enzymatic inhibition data in tables 15 as evidence of 
 a preliminary sar , when in fact it would be more appropriately called 
 sir ( i.e. ,  structure  interference relationship  ) . 

as the nature of the cpm - based format contraindicates the use of dtt 
 and other biological reducing agents , it would be advisible to have 
 an orthogonal assay that can test candidate compounds in both the 
 presence and absence of dtt or similar reducing agent to further rule 
 out thiol reactivity ( e.g. , table 6 ) .
this assay can identify assay 
 artifacts , and if used in parallel with an orthogonal assay , can identify 
 potentially problematic thiol - reactive enzymatic inhibitors .
third , 
 the ratio of acetyl - coa to test compound should be kept as high as 
 possible , although this must be balanced with other important factors 
 such as the acetyl - coa km .
if fluorescence 
 quenching is a concern , we recommend the facile counter - screen used 
 in this manuscript , as it should not be easily susceptible to interference 
 from thiol - trapping compounds . a deeper understanding and appreciation 
 for the chemical mechanisms of assay interference and thiol reactivity 
 can have important implications for early analogue selection and screening 
 library design ( see figure 1 for the general 
 chemotypes discussed here ) .
elimination reactions , 
 and on the basis of our results , we recommend a leaving group analysis 
 for such compounds . therefore ,
selecting and/or testing analogues 
 with weaker leaving groups or a reduction at the c2c3 position 
 ( supporting information , figure s2 ) may 
 be a potential strategy to overcome thiol reactivity in this chemotype . 

the former strategy may be useful for certain succinimides 4 with good leaving groups ( e.g. , s - linked heteroaromatics ; supporting information , table s4 ) .
certain 1,2,4-thiadiazoles 3 are susceptible to attack by thiol nucleophiles at the s1-position , 
 specifically when the n2-position is positively charged .
we note our 
 findings with this chemotype are consistent with other previous mechanistic 
 work on these compounds . should investigators 
 choose to pursue compounds bearing chemotype 3 , it may 
 be useful to assess the effect of switching to 1,2,4-oxadiazole analogues , 
 as well as testing the nonsalt forms of 1,2,4-thiadiazoles .
the benzothiadiazoles / benzofurazans 2 interfere by nucleophilic aromatic substitution , and this 
 interference correlated with the apparent strength of the presumed 
 leaving group . as with chemotypes 1 , 4 , 
 and 6 , a strategy for navigating away from this problematic 
 chemotype would be to select analogues with weaker leaving groups 
 or with less electron - withdrawing functional groups on the heteroaromatic 
 core .
substructure to include additional strong electron - withdrawing moieties 
 such as nitro groups ( table 2 ) .
the sar / sir 
 of these chemotypes also raises important questions about the ability 
 of certain pains to be converted to non - pains .
it is interesting to 
 note that even in the cases where incidence of anomalous behavior 
 is high , presence of the offending substructure does not predispose 
 all compounds to anomalous behavior .
this suggests that it may be 
 possible to  design out  such behavior if the activity 
 seen in the assay is true after all .
nonetheless , presence of the 
 nuisance chemotype does suggest that there is a very significant risk 
 of failure in attempting such optimization , as chances are high the 
  active
 
 is acting via a therapeutically uninteresting mechanism , thereby rendering 
 such hits unattractive start points for hts follow - up .
both 
 the succinimides 4 and the p - hydroxyarylsulfonamides 6 illustrate the susceptibility of screening compounds to 
 undergo chemical transformations under certain assay conditions .
the 
 decomposition of chemotype 6 was unexpected , and it will 
 be interesting to examine the conditions critical for this conversion 
 as well as more detailed characterization of this decomposition process . 
 for instance , it appears several of these problematic arylsulfonamides 
 were also unstable in alarm nmr buffer , suggesting this scaffold is 
 likely unstable in many other biologically relevant aqueous buffers 
 and not an isolated phenomenon ( supporting information , 
 figure s11 ) .
ms / ms , 
 further attesting to the instability of this scaffold in our assay 
 conditions ( supporting information , table s3 ) .
given the instability of scaffolds 4 and 6 , we recommend assessing the stability of any promising compounds 
 in assay buffer by analytical techniques to verify the structure of 
 the active chemical entity in the biological context .
this is a rather 
 straightforward experiment , and in light of our findings , it may be 
 an important confirmatory experiment to perform before proceeding 
 to more extensive experiments , such as molecular modeling , that are 
 based on correct structure identification and integrity . there 

has been a recent resurgence of interest in covalent drugs ( e.g. , 
 ibrutinib and dimethyl fumarate ) .
this renewed interest has been used as a line of defense in the 
 reporting of known reactive compounds , including pains , as viable 
 drug leads .
we have shown in this manuscript 
 that all interference compounds are not created equal and that they 
 can exhibit a distinct sir . however , we suggest it is highly unlikely 
 that compounds that show indiscriminate protein reactivity , as do 
 pains , will ever be useful drug or probe leads .
while there may be 
 exceptions , we expect that most of the recently developed covalent 
 drugs have either been purposefully designed as such or have undergone 
 extensive mechanistic studies and medicinal chemistry optimization . 

they are usually not the outcome of the optimization of nonselective , 
 reactive , and promiscuous compounds that sometimes are reported from 
 hts .
therefore , we recommend that the scientific community apply an 
 extremely high standard of rigor to the review and publication of 
 manuscripts that claim any drug- or probe - like potential for these 
 types of compounds . additionally , we caution researchers that commercially 
 available  probes  that feature known thiol - reactive 
 moieties , including but not limited to those chemotypes discussed 
 herein , may be less selective versus the proteome than their  probe - like  
 status suggests ( supporting information , table 
 s5 ) . our findings highlight the importance of taking 
 a chemocentric approach to hts triage and hit prioritization and highlight 
 the need for carefully planned counter - screens and orthogonal assays 
 in a well - validated cascade of hit - triaging assays ( figure 8) .
we believe our investigation also demonstrates 
 the importance of partnering with medicinal chemists in the post - hts 
 triage process and should serve as caution for lead selection based 
 primarily on initial potency and sar data without confirmation of 
 activity by orthogonal methods . the continued growth of cheminformatics 
 and the incorporation of pains filters into both commercial software 
 suites ( e.g. , sybyl , schrodinger canvas ) and freeware is undoubtedly 
 a positive advancement for the field .
however , many compounds with 
 chemotypes 14 were not flagged by 
 our cheminformatics pains filters .
this raises important concerns 
 about the potential for overreliance on cheminformatics filters . for 
 instance
, unseasoned researchers ( and reviewers ) may fall into the 
 trap that because pains were removed by substructure filters , that 
 they no longer have to consider any related nuisance compounds .
strategies 
 to mitigate this risk are to ( 1 ) take a chemocentric approach to hts 
 triage using a well - validated cascade 
 of deconvoluting assays , ( 2 ) encourage more mechanistic studies of 
 nuisance compounds to further the understanding of their behavior , 
 ( 3 ) periodically update pains filters as more data is made available , 
 and ( 4 ) mine the ever - increasing amount of hts data for insights into 
 pains substructures ( e.g. , along the lines shown in a recent report ) .
such undertakings also raise important follow - up 
 questions for those in hts triage about what exactly should constitute 
 a pains and what criteria should form the basis for classifying a 
 compound as  promiscuous  and/or  pan - assay  . 

likely , these definitions will have to be dependent on the screening 
 context , at least in part , and guided by those with sufficient expertise 
 in hts triage .
the following 
 reagents were obtained from sigma - aldrich : dmso , cpm , coa ( sodium 
 salt hydrate ) , acetyl - coa ( sodium salt ) , bovine serum albumin ( bsa ) , 
 h2o2 , and triton x-100 .
compounds tested in 
 post - hts assays were repurchased as solid powders from standard chemical 
 vendors ( e.g. , emolecules ) . in a quality - control sampling of a random 
 5% of the chemical library samples used in this report
, greater than 
 90% of the tested commercial samples had acceptable purities ( > 90% ) 
 by uplc 
the cpm - based rtt109 
 assays have been detailed in a previous report with minor modifications .
briefly , all compounds studied in this report 
 were rescreened in assay buffer containing freshly prepared 0.01% 
 triton x-100 ( v / v ) and enzyme concentrations of 50 nm rtt109vps75 
 complex . for ic50 experiments
, compounds were tested in 
 triplicate at eight compound concentrations ranging from 200 nm to 
 125 m final compound concentrations .
slot blots were performed 
 on reaction aliquots using standard techniques with a bio - rad bio - dot 
 sf microfiltration apparatus .
membranes were imaged with a li - cor 
 odyssey and analyzed using image studio ( li - cor biosciences ) .
z 
 factors for each plate were calculated using eq 1:1where  and  represent the standard 
 deviation and mean of the positive ( c ) and negative ( c ) plate controls , respectively .
response 
 data to the sigmoidal dose  response variable slope four - parameter 
 equation in graphpad prism 6.0 .
compounds interfering with the cpm - based assay readout were identified 
 as previously described with minor modifications .
compounds were tested in triplicate at eight concentrations 
 ranging from 200 nm to 125 m final compound concentrations 
 using an adaption of the rtt109 hts assay format .
proteins and assay 
 buffer were dispensed to assay plates analogously to the hts procedure , 
 then the acetyl - coa substrate was replaced with coa in concentrations 
 titered to match the fluorescence intensity observed for the uninhibited 
 enzyme reaction in the hts assay ( approximately 5 m coa ) .
compounds 
 were incubated with coa and allowed to react with cpm under conditions 
 identical to the hts procedure .
assay interference was quantified 
 by comparing the background - corrected ( compound + proteins + coa + 
 cpm ) fluorescence intensities to the ( dmso + proteins + coa + cpm ) 
 controls . to further investigate their fluorescence behavior under 
 the hts conditions , select compounds were also incubated with assay 
 reagents ( buffer - only , buffer + coa , buffer + cpm ) and their fluorescence 
 intensity measured . the overall plate layout , controls , protocols , 
 and assay readouts were unchanged from the aforementioned compound  coa 
compounds were tested for evidence of fluorescence quenching using 
 a modification of our published procedure .
briefly , cpm and coa ( 20 and 5 m final concentrations , respectively ) 
 were allowed to react to completion in assay buffer .
completion was 
 defined as a stable signal plateau , usually after 5 min reaction time . 

the cpm  coa adduct solution ( 20 l per well ) was then 
 dispensed into assay plates preplated with dmso and test compounds . 

microplates were shaken for 5 min and allowed to equilibrate 
 for another 5 min at room temperature .
fluorescence intensity was 
 measured , and the data was analyzed as percent signal reduction compared 
 to dmso controls .
selected compounds ( 1 equiv ) and either coa or reduced l - glutathione ( 2 equiv ) were incubated under hts - like conditions , 
 except with 5% dmso ( v / v ) and no detergent in the assay buffer .
compounds were also tested in hts buffer or 
 methanol minus the addition of biological thiols .
. samples with visible precipitates 
 were passed through 0.25 m syringe filters to remove particulates . 

sample injections were typically 1.0 l in volume performed 
 by an autosampler and were analyzed on a waters uplc system using 
 a beh c18 2.1 mm  50 mm column .
the flow rate was 0.250 ml / min 
 with a standard gradient starting at 95% solution a ( 950 ml h2o , 50 ml mecn , 1 ml formic acid ) and ending with 100% solution 
 b ( 1000 ml mecn plus 1 ml formic acid ) over 6.5 min .
the samples were 
 monitored simultaneously using an els detector , a diode array detector 
 ( 214 , 220 , 244 , and 254 nm ) , and a zq mass spectrometer ( esi positive 
 and negative modes ) . selected compounds 
 were assessed for redox activity using published protocols .
freshly prepared 100 m 
 h2o2 ( sigma ) was included as a positive plate 
 control , while nsc-663284 and 4-amino-1-naphthol were used as positive 
 redox - active controls for dtt and dtt - free assay conditions , respectively . 

compounds were tested in triplicate at eight final concentrations 
 ( 200 nm to 125 m via 2.5-fold dilutions ) in either the presence 
 or absence of 1 mm dtt final concentration .
all active compounds did 
 not interfere with the assay readout at a610 ( data not 
 shown ) . for selected 
 compounds ,
inhibition of hat activity was also checked with an orthogonal 
 in vitro radiolabeled substrate assay .
rtt109 inhibition was tested 
 at eight compound concentrations ( 200 nm to 125 m final compound 
 concentrations via 2.5-fold dilutions ) in an adaptation of a previous 
 procedure .
briefly , reactions were performed 
 in standard volume 384-well microplates using 45 l total reaction 
 volumes containing the following in final concentrations : 50 mm tris 
 hcl , ph 8.0 , 50 mm kcl , 0.1 mm edta , 1 mm dtt , 0.01% triton x-100 
 ( v / v ) , 50 ng/l bsa , and 2.5 m [ h]-acetyl - coa 
 ( perkinelmer ) .
purified recombinant yeast rtt109vps75 was 
 tested at approximately 5 nm final concentrations , while purified 
 recombinant asf1dh3h4 ( approximately 250 nm ) was used 
 as acetylation substrate .
compounds and dmso were plated with a multichannel 
 pipet , followed by a similar addition of a solution containing enzyme 
 and histone substrate ( 36 l ) .
test compounds were allowed to 
 equilibrate with enzyme and histone substrate for 10 min at 30 c 
 in an incubator .
the hat reaction was initiated by adding [ h]-acetyl - coa solution ( 7.5 l ) .
dmso content was kept constant 
 across all reactions at 3% ( v / v ) .
after 5 min , the reactions were 
 quenched by multichannel pipet transfer of reaction aliquots ( 35 l ) 
 to adjacent microplate wells each containing 35 l of 2-propanol . 

aliquots ( 35 l ) of the quenched solutions were carefully spotted 
 onto whatman p-81 phosphocellulose paper filters ( ge healthcare ) and 
 air - dried .
filter papers were washed five times for 5 min per cycle 
 with 50 mm nahco3 , ph 9.0 , then rinsed with acetone and 
 then allowed to air - dry for 30 min .
[ h]-acetate incorporation 
 was then measured by an ls6500 liquid scintillation counter ( beckman  coulter ) . 

testing versus p300bhc and the gcn5ada2ada3 
 complex were performed similarly , except that the final enzyme concentrations 
 were approximately 500 pm and the substrate was purified recombinant 
 dh3h4 tetramers .
compounds and proteins were incubated together 
 at 30 c for 60 min at 100 m and 10 m final concentrations , 
 respectively .
reaction mixtures were denatured with gentle heating 
 and then further resolved by sds - page .
peptide extracts were dried in vacuo and reconstituted 
 in 98:2:0.1 h2o : acetonitrile : tfa ; approximately 0.2 g 
 of each gel band was analyzed by capillary lc 
ms on a velos 
 orbitrap mass spectrometer ( thermo fisher ) with higher energy collision 
 induced dissociation activation .
peaks 
 studio 6.0 build 20120620 ( bioinformatics solutions ) software package 
 was used for interpretation of tandem ms and protein inference .
search parameters for rtt109 , vps75 , and asf1 
 proteins were uniprot database ( sacharomyces cerevisiae strain atcc 204508/s288c , taxonomy i d 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 trypsin 
 with max missed cleavages = 2 and nonspecific trypsin cleavage ; variable 
 modifications = methionine oxidation and dioxidation , cysteine oxidation , 
 and dioxidation , and suspected compound adducts ; maximum variable 
 modifications per peptide = 5 ; false discovery rate calculation = 
 on ; spectra merge options = 0.2 min within 10.0 ppm mass window ; charge 
 correction = on for charge states 28 ; spectral filter quality 
 > 0.65 .
support for the detection of peptides plus adducts from 
 each supporting tandem ms data was based on : ( 1 ) high confidence peaks 
 peptide score ( minimum 10 log p 35 ) , ( 2 ) 
 a minimum of five consecutive b- or y - type peptide fragment ions , 
 ( 3 ) high precursor mass accuracy ( < 7 ppm ) , and ( 4 ) supporting signature 
 ion peaks for the site localization of the pertinent cysteine modification 
 on one or more peptide fragments .
incidence of frequent - hitting behavior was checked in the astrazeneca 
 corporate screening deck by mining the historical screening data . 

we calculate a descriptor , pbsf , for each compound to determine whether 
 it is more active than expected .
the 
 pbsf score is the negative logarithm of the probability that the observed 
 pattern of activity and inactivity is observed by chance , given the 
 known  average  behavior across all compounds in the 
 screening deck and across the historical set of screening campaigns 
 they have been measured in . if the likelihood of seeing the pattern 
 at hand is high , the compound is likely not a frequent hitter and 
 all is fine .
however , if the probability of seeing the pattern is 
 low , the resulting pbsf score will be high and the pattern should 
 be regarded as anomalous . a cutoff of pbsf > 2 was used to designate 
 compounds exhibiting suspicious binding behavior . to check the incidence 
 of frequent - hitting behavior ,
we searched the corporate collection 
 using substructures ( with in - house tools ) , collated pbsf scores for 
 the set , and counted the number of frequent - hitting compounds using 
 the pbsf threshold stated in the above . for reference ,
the average 
 fraction of compounds displaying frequent - hitting behavior across 
 the collection of compounds with historical hts data is 6% .
the number of hts data points is variable for 
 each compound , as it depends on the number of times a compound has 
 been screened .
the median number of data points per compound in the 
 data set is approximately 200 , with only 10% of the compounds having 
 less than 50 data points .
only biochemical assay data , and not cell - based 
 assay data , were used to derive the frequent hitter score alarm nmr was performed as previously described with minor modifications .
the gene encoding amino acids 100324 of the human la antigen 
 was cloned into pet-28b+ vector ( novagen ) such that it contained both 
 an n- and c - terminal his tag .
the plasmid was freshly transformed 
 into escherichia coli rosetta cells 
 ( novagen ) and cultured in m9 minimal media supplemented with nh4cl ( cil ) in an adaption of published procedures .
the la antigen was enriched with c at the -methyl 
 groups of leucine , the -methyl group of isoleucine , and the 
 -methyl groups of valine by the addition [ 3-c]--ketobutyrate 
 and [ 3,3-c]--ketoisovalerate ( sodium salts , 
 cil ) to the culture medium 30 min before inducing in the presence 
 of 1 mm iptg for 8 h at 25 c ( od600 was approximately 
 0.8 at time of induction ) .
harvested cells were lysed by french press 
 in ice - cold lysis buffer consisting of 50 mm tris , ph 7.6 , 300 mm 
 nacl , 10% glycerol ( v / v ) , 5 mm -mercaptoethanol ( bme ) , 5 mm 
 imidazole , 2 mm mgcl2 , benzonase ( sigma ) , and protease 
 inhibitor cocktail .
this solution containing the lysed cells was sonicated 
 briefly ( 3  15 s pulse sequence ) on ice , then loaded onto a 
 prewashed ni - bead column ( ge healthcare ) kept at 4 c .
proteins 
 were eluted from the beads with an elution buffer consisting of 50 
 mm tris , ph 7.6 , 300 mm nacl , 10% glycerol ( v / v ) , 5 mm bme , and an 
 imidazole gradient ranging from 5 mm to 0.5 m. pooled elution fractions 
 containing the la antigen were dialyzed overnight ( 25 mm sodium phosphate , 
 ph 7.0 , 5 mm dtt ) , flash - frozen in liquid n2 , and stored 
 at 80 c until further use .
prior to use , aliquots of 
 500 m protein was incubated in the presence of 20 mm dtt at 
 37 c for 1 h , then dialyzed versus 2  2 l of 25 mm sodium 
 phosphate buffer , ph 7.0 ( no dtt ) at 4 c with constant n2 bubbling .
the h / c - hmqc spectra were 
 acquired in 25 mm sodium phosphate buffer , ph 7.0 , 10% d2o ( v / v ; cil )  200 m test compounds delivered from 10 
 mm dmso stock solutions , and  20 mm dtt .
compounds were incubated 
 with proteins at 37 c for 1 h and then 30 c for 15 h prior 
 to data collection .
data were recorded at 25 c on a bruker 700 
 mhz nmr spectrometer equipped with a cryoprobe ( bruker ) and autosampler . 

samples were loaded into bruker 1.7 mm samplejet tubes with 40 l 
 total sample volumes and stored at 4 c while in queue .
the alarm 
 nmr samples were tested at 50 m protein concentrations using 
 16 scans , 2048 complex points in f2 , and 80 points in f1 using standard 
 protein hmqc and water suppression pulse sequences .
nonreactive compounds 
 were identified by the absence of chemical shifts ( c - methyl ) 
  20 mm dtt .
reactive compounds induced chemical shifts in certain 
 diagnostic peaks in the absence of dtt , and this effect was significantly 
 attenuated when 20 mm dtt was included in an otherwise identical sample .
as an additional precaution against trace reactive 
 contaminants , compounds tested by alarm nmr were repurified in - house 
 by standard hplc procedures using mass - directed collection .
detailed adduct synthetic procedures 
 and chemical characterization can be found in the supporting information .
the gene encoding amino acids 100324 of the human la antigen 
 was cloned into pet-28b+ vector ( novagen ) such that it contained both 
 an n- and c - terminal his tag .
the plasmid was freshly transformed 
 into escherichia coli rosetta cells 
 ( novagen ) and cultured in m9 minimal media supplemented with nh4cl ( cil ) in an adaption of published procedures .
the la antigen was enriched with c at the -methyl 
 groups of leucine , the -methyl group of isoleucine , and the 
 -methyl groups of valine by the addition [ 3-c]--ketobutyrate 
 and [ 3,3-c]--ketoisovalerate ( sodium salts , 
 cil ) to the culture medium 30 min before inducing in the presence 
 of 1 mm iptg for 8 h at 25 c ( od600 was approximately 
 0.8 at time of induction ) .
harvested cells were lysed by french press 
 in ice - cold lysis buffer consisting of 50 mm tris , ph 7.6 , 300 mm 
 nacl , 10% glycerol ( v / v ) , 5 mm -mercaptoethanol ( bme ) , 5 mm 
 imidazole , 2 mm mgcl2 , benzonase ( sigma ) , and protease 
 inhibitor cocktail .
this solution containing the lysed cells was sonicated 
 briefly ( 3  15 s pulse sequence ) on ice , then loaded onto a 
 prewashed ni - bead column ( ge healthcare ) kept at 4 c .
proteins 
 were eluted from the beads with an elution buffer consisting of 50 
 mm tris , ph 7.6 , 300 mm nacl , 10% glycerol ( v / v ) , 5 mm bme , and an 
 imidazole gradient ranging from 5 mm to 0.5
m. pooled elution fractions 
 containing the la antigen were dialyzed overnight ( 25 mm sodium phosphate , 
 ph 7.0 , 5 mm dtt ) , flash - frozen in liquid n2 , and stored 
 at 80 c until further use .
prior to use , aliquots of 
 500 m protein was incubated in the presence of 20 mm dtt at 
 37 c for 1 h , then dialyzed versus 2  2 l of 25 mm sodium 
 phosphate buffer , ph 7.0 ( no dtt ) at 4 c with constant n2 bubbling .
the h / c - hmqc spectra were 
 acquired in 25 mm sodium phosphate buffer , ph 7.0 , 10% d2o ( v / v ; cil )  200 m test compounds delivered from 10 
 mm dmso stock solutions , and  20 mm dtt .
compounds were incubated 
 with proteins at 37 c for 1 h and then 30 c for 15 h prior 
 to data collection .
data were recorded at 25 c on a bruker 700 
 mhz nmr spectrometer equipped with a cryoprobe ( bruker ) and autosampler . 

samples were loaded into bruker 1.7 mm samplejet tubes with 40 l 
 total sample volumes and stored at 4 c while in queue .
the alarm 
 nmr samples were tested at 50 m protein concentrations using 
 16 scans , 2048 complex points in f2 , and 80 points in f1 using standard 
 protein hmqc and water suppression pulse sequences .
nonreactive compounds 
 were identified by the absence of chemical shifts ( c - methyl ) 
  20 mm dtt .
reactive compounds induced chemical shifts in certain 
 diagnostic peaks in the absence of dtt , and this effect was significantly 
 attenuated when 20 mm dtt was included in an otherwise identical sample . as an additional precaution against trace reactive 
 contaminants , compounds tested by alarm nmr
were repurified in - house 
 by standard hplc procedures using mass - directed collection .
detailed adduct synthetic procedures 
 and chemical characterization can be found in the supporting information .
 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 . 