Natural Products, Natural Product Drugs, and Pan ... - ACS Publications

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Feeling Nature’s PAINS: Natural Products, Natural Product Drugs, and Pan Assay Interference Compounds (PAINS) Jonathan B. Baell* Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3084, Australia

ABSTRACT: We have previously reported on classes of compounds that can interfere with bioassays via a number of different mechanisms and termed such compounds Pan Assay INterference compoundS, or PAINS. These compounds were defined on the basis of high-throughput data derived from vendor-supplied synthetics. The question therefore arises whether the concept of PAINS is relevant to compounds of natural origin. Here, it is shown that this is indeed the case, but that the context of the biological readout is an important factor that must be brought into consideration.

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INTRODUCTION In my former Institute, a high-throughput screening (HTS) capability was established in 2003. This was subsequent to a period of some 10 months in 2002 during which a battery of selection criteria was developed with the intention to exclude all but small, simple, and optimizable analogues. The resulting HTS library contained exclusively compounds with a molecular weight between 150 and 400 Da, with no fewer than one ring and no more than four rings, and the vast majority had no chiral centers. Considerable effort was made to exclude all chemotypes that might be protein-reactive. All compounds were sourced from vendor-supplied catalogs of screening synthetics. In the ensuing years, it was realized that the HTS library did, in fact, contain a relatively small but not insignificant proportion (ca. 10%) of promiscuous compound classes that recurred as screening hits. These subversive compounds attracted considerable attention, as they often appeared to have selectivity over counterscreens and with signs of early structure−activity relationship (SAR), yet they inevitably ended up wasting all resources that were transferred to optimization efforts. Scrutiny of the literature provided evidence that in many cases such compounds may exhibit multiple behaviors that could interfere in assay readouts, such as metal chelation, redox cycling, and protein reactivity. For this reason such molecules were termed Pan Assay INterference compoundS, or PAINS. Since the time that these findings1 were published in the Journal of Medicinal Chemistry in 2010, we have commented extensively on2−13 and have received considerable interest in this area, in particular from research groups being hindered by unsuccessful optimization campaigns based around such compound classes. More recently, we have been increasingly involved in conversations about whether or not the concept of PAINS has any relevance to natural products. © XXXX American Chemical Society and American Society of Pharmacognosy

After all, natural products enjoy a privileged position in drug discovery,14,15 and few would resemble the compounds in our inaugural (stage 1) screening library as defined above and from which PAINS definitions were derived. This matter is now discussed herein.

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OVERVIEW OF NATURAL PRODUCT PAINS COMPOUNDS It is instructive to consider this hypothetical scenario: a set of several hundred natural products and natural product-like compounds are screened in a medium-throughput patch clamp assay to detect those that inhibit the voltage-gated ion channel Kv1.3, a target of interest in autoimmune disease.16,17 Scrutiny of the hypothetical hits reveals the following compounds as low-micromolar-potency hits: apomorphine, artemisinin, capsaicin, cephalexin, chaetocin, chelerythrine, curcumin, daunorubicin, droxidopa, epigallocatechin gallate (EGCG), epothilones, epoxomicin, fluticasone, geldanamycin, genistein, an unnamed glycoside, gossypol, khellinone, menadione, menaquinone, mitomycin C, mitoxantrone, resveratrol, rifampicin, rohitukine, sanguinarine, thymoquinone, topotecan, and toxoflavin. Most of these compounds are known to display a variety of biological activities, with the depth of some such as curcumin, EGCG, genistein, gossypol, resveratrol, and thymoquinone so great that they are often viewed as privileged natural products. Others in this list such as mitomycin C, daunorubicin, mitoxantrone, geldanamycin, topotecan, rifampiSpecial Issue: Special Issue in Honor of John Blunt and Murray Munro Received: October 23, 2015

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DOI: 10.1021/acs.jnatprod.5b00947 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Some natural products typically found as actives in bioassays, containing a catechol that is recognized as PAINS motifs (red). In blue is an additional PAINS motif, as a pyrogallol is effectively a bis-catechol.

hit one screen, while 36 were “clean” in the particular screens investigated. As has been explained elsewhere,1 when the total number of analogues that hit between two and six screens is expressed as a percentage of those that were clean, the resulting enrichment value of 97% is extremely high compared with a benign chemistry such as any compound in our library with an amide functional group, where the percentage enrichment is 8%. Why might catechols frequently signal in bioassays? As we have reviewed elsewhere,1 catechols have a high propensity to be redox active and can also variously chelate metals as well as being reactive in the oxidized form to nucleophiles present in the side chains of proteins such as cysteine and lysine (Figure 2).

cin, cephalexin, artemisinin, epothilones, epoxomicin, and fluticasone are themselves or have given rise to compounds approved for clinical use. As hypothetical screening hits for Kv1.3, do these traits therefore make these attractive and useful templates for progression as autoimmune disease therapeutics, especially as they are natural products and not synthetic in origin? Here we show that the precise opposite is the case. Natural Products that Contain PAINS: Catechols. Let us consider the group of hypothetical hits, these being EGCG (1), gossypol (2), apomorphine (3), and droxidopa (4), as shown in Figure 1. To the inexperienced researcher, there may appear to be nothing disadvantageous concerning these compounds, but we would be on high alert, because they all contain the recognizable PAINS motif, catechol. Compounds with such substructures have registered frequently as hits in our assays, yet all attempts at optimizing some of the most promising of these have proven fruitless. It is useful to briefly explain how we analyzed the HTS data to conclude that such compounds were frequent “hitters”. Shown in Table 1 is the PAINS substructure that recognizes catechols and which numbered some 92 analogues in our approximately 100 000-strong HTS inaugural library. Analysis of a set of six selected HTS campaigns showed four analogues to hit all six screens, seven hit five screens, 10 hit four screens, four hit three screens, 10 hit two screens, and 21

Figure 2. Catechols are recognizable PAINS and can interfere in bioassays via different mechanisms.

It was concluded that it is this subversive behavior that led to such compounds signaling so frequently in our assays and that such compounds were not optimizable. As has been discussed previously,1 literature searching revealed many other examples of such compounds appearing as bioactives in many different assays, independent of assay technology and with no evidence of optimizability. Thus, it was concluded that these compounds would be able to provide readouts in assays all the way from target to cell without any common mechanism involved, yet fool the inexperienced researcheror reviewerinto thinking otherwise. While our HTS library contained no natural products, it would be wrong to assume that catechol-containing natural products are somehow exempt from promiscuous behavior simply on the basis that they are natural products, and indeed the prevalence of these compounds in the bioactive literature without evidence of SAR or optimization to highly potent derivatives would suggest otherwise. For example, EGCG has been, and unfortunately continues to be, published on extensively and yet is clearly active promiscuously in unrelated target-based assays, cell-based assays, and in vivo assays.18 Just like any synthetic catechol, it is known to form readily reactive orthoquinones that covalently modify proteins, yet this seldom is taken into account when reporting apparent targets.18,19 Furtherand just like any synthetic catecholit has been shown to chelate metals20 and, in addition to this, to

Table 1. Screening Data Showing that Catechols are Frequent Hitters

a

The exact substructure definitions for all PAINS are defined in Tables S6, S7, and S8 (pp S23−S37) in the Supporting Information of the original Baell and Holloway PAINS publication.1 This is best used as follows, shown using catechol as an example. Since all the PAINS definitions are alphabetically listed in Figure S1 in this Supporting Information (pp 61−84), one can find “catechol_A” on page S64, where the number in parentheses indicates the number of analogues (92). One can then to go to Table S7 (p S24), which contains the filter for substructures with 15−150 analogues, to readily find the definition written in Sybyl line notation. One can then to go to Table S9, p 38, in the Supporting Information) to find the enrichment calculation. b These were six robust assays against protein−protein interactions and hence not biased by enzyme active sites. cTotal number of analogues recognized by the substructure shown. dTotal number of analoguess that hit between two and six screens is expressed as a percentage of those that were clean. B



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Figure 3. Some typical natural products that contain a quinone PAINS motif (shown in red). Mitomycin C (8) also contains a reactive aziridine (shown in pink).

nonspecifically peturb membranes.21 Ironically, in the latter context it has been shown to nonspecifically interfere in ion channel assays,21 the example being used specifically here. In plants, phenolic substances may function as antibiotics, antifeedants, antioxidants, and absorbers of ultraviolet irradiation.22 Since EGCG has been consumed for centuries in green tea and shown to be not acutely toxic, any reports on potentially useful biological activity, which mostly seem to relate to potential anti-inflammatory or antioxidant effects, attracts the construction of an evolutionary link that leads to the notion of EGCG having evolved to be a natural medicine of some sort. Since the barrier to testing hypotheses in humans is obviously very low, EGCG, like many phenolic plant secondary metabolites, has been subjected to extensive clinical trials. However, accumulation of confusing results18 is currently suggesting that this low barrier is simply making it easier to undertake ill-considered clinical investigations. Our remarks on EGCG can be applied to many other plant phenolic catechols. For example, the flavone quercetin displays many of these same promiscuous behaviors.23,24 As will be made clear later, it is useful to briefly consider the discovery of other compounds we discuss. Gossypol (2) is present in cottonseed oil, where it acts as a natural defensive agent against predators, provoking infertility. It was serendipitously and surprisingly found to be efficacious as a male contraceptive and during the 1970s was deliberately consumed in mainland China for this purpose.25 Gossypol readily forms reactive orthoquinones, undergoes redox cycling, chelates metals, and perturbs membranes.26 Further, it contains an aldehydea known toxicophorebut which additionally in this case readily covalently labels proteins through Schiff base formation. Unsurprisingly cardiotoxicity, hepatotoxicity, and DNA fragmentation are observed.26 Intriguingly, combination therapies with the R-(−)-enantiomer of gossypol, called AT101, are currently in various stages of clinical trials for the treatment of various cancers on the basis that AT-101 induces apoptosis in cancer cells as a BH3 mimetic.27 Ironically, it has been shown that gossypol does not induce apoptosis via the Bax/Bak pathway and does not therefore act as a BH3 mimetic.28,29 Should AT-101 eventually reach the marketplace as an anticancer agent, the lesson will be that exhaustive clinical assessment may eventually find an application, even if

somewhat serendipitous and unrelated to supposed mechanisms of action. Apomorphine (3), which is essentially a catecholamine structural mimetic, is a decomposition product from the treatment of morphine with strong acid that was discovered in 1869. It is useful to class apomorphine as natural product-like for the purposes of this discussion, though clearly we are being relatively lenient with such a definition. Potential anticonvulsant properties of apomorphine were observed for this compound in human patients in 1884.20 It progressed subsequently from an emetic drug to its current use as a rescue medication for Parkinson’s disease. Droxidopa (4) is clinically approved for human use and is representative of the catetcholamine drugs, which include levodopa, adrenaline, dopamine, isoprenaline, noradrenaline, dobutamine, carbidopa, and methyldopa. These compounds are not immune from orthoquinoid formation simply because they are “natural”, and indeed they covalently modify proteins and chelate metals.20,30 The PAINS behavior of catechol-containing drugs not only may contribute to toxicity but in certain instances may contribute to their therapeutic efficacy. This apparent paradox could inappropriately make catechol screening hits appear more attractive to development. This would be a mistake because catechol-containing drugs were not discovered through today’s rational and methodical progression from upstream assays to clinical use, but ratheras have been discussedthrough more traditional drug discovery after the observation of early in vivo efficacy at relatively low doses. This is why the history of the discovery of these compounds has been outlined briefly. It is also important to make the distinction between random screening and focused screening. For example, screening a library of catecholamine analogues in a target-based assay relevant to catecholamine targets is inherently biased toward mechanism-based activity, and assay readouts may be taken more seriously, albeit with great caution. The message is that low-micromolar-potency catechol screening hits from a random screen, whether natural or synthetic and whether the assay is target-based or cell-based, are unlikely to be progressable to therapeutically relevant nanomolar levels of potentcy, and no succor can be gained from knowledge that catechol-containing compounds may be clinically approved for use in humans. EGCG, for example, is C



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a major bioactive ingredient of a clinically effective topical treatment of venereal wartsvia mechanisms unknownand sold as Veregen, but this does not mean it should be regarded as privileged in any sense were it to be unearthed as a lowmicromolar-potency hit against a given target. Natural Products that Contain PAINS: Quinones. Let us now consider the next group of hypothetical screening hits, these being vitamin K2 (5), menadione (6), thymoquinone (7), mitomycin C (8), daunorubicin (9), mitoxantrone (10), and geldanamycins (11), as shown in Figure 3. All of these contain the quinone PAINS motif.1 Shown in Table 2 is the PAINS substructure that recognizes quinones

Thymoquinone (7) was isolated in 1963, and its ingestion as a component in plant seeds dates at least back to the Old Testament.34 It exhibits an array of biological activities that are largely associated with its high redox activity, but it can also form adducts as a Michael acceptor.35 Despite sustained and intense focus as a potential anticancer agent, including progression to clinical trials, it has failed to deliver a useful drug, and there is confusion about its various mechanisms of action.34 Mitomycins are potent antibiotics also derived from species of the bacterium genus Streptomyces. Mitomycin C (8) derives from S. caespitosus and has been used in combination chemotherapy since 1974.36 Its potent antitumor activity was first reported in 1958, but its absolute structural identity was not reported until 1983. Its mechanism of action was teased out through the 1990s and thought principally to involve DNA cross-linking, but more recently thioredoxin reductase deactivation has been implicated.37 Mitomycin C (8) is redox active through its quinone and also reacts with biological nucleophiles through its aziridine.36 Daunorubicin (9) is prototypical of the anthracycline anticancer drugs that include doxorubicin, epirubicin, idarubicin, and mitoxantrone (10). Such compounds are redox active and also react with biological nucleophiles,38,39 although, interestingly, doxorubicin is significantly less active than menadione in biochemical redox assays in vitro.40 The discovery of daunorubicin originated from the observation of potent antitumor activity in mice of a component isolated from species of the genus Streptomyces in 1963, and its structure was elucidated a few years later.41 Many years later, it is now known that it exerts its activity at least in part as a topoisomerase II poison via DNA intercalation, but as for many natural products and indeed many synthetic drugs, the mechanism of action of this class is still not fully understood.42 Its discovery spurred the development of variants in an effort to seek improved properties, such as doxorubuicin (= hydroxydaunorubicin = Adriamycin) and mitoxantrone (10).43 Intriguingly, many of the natural product quinones, such as the anthracyclines, are both deeply colored (“rubis” is French and describes the ruby color of doxorubicin) and fluorescent, offering additional means of interfering in assay signaling. Geldanamycin (11a) was originally discovered as an antibiotic in 1970 but in the late 1980s was found to possess antiproliferative activity against a wide range of tumor cell lines.44 A mechanism involving binding to HSP90 aroused significant interest, but therapeutic development was hindered by the redox-active and reactive quinone moiety present. Modification led to 17-AAG (11b), the first HSP90 inhibitor to proceed to clinical trials.44 As was the case for catechol-containing drugs, the quinone PAINS moiety can still display PAINS behavior even if it is embedded in a drug and whether or not it is a natural product, and this behavior may contribute paradoxically to both efficacy and toxicity. Discovery of quinone-containing drugs once again arose from early observation of useful in vivo efficacy or potent cell-based activity at therapeutically relevant concentrations or close to thereof, prior to knowledge of mechanism of action. So once again, the utility of these compounds in humans does not represent a modern and rational progression from upstream assays to downstream assays and eventually to clinical use. No connection should be made between a quinone-containing screening hit and a quinone-containing drug. The PAINS behavior more or less universally exhibited in quinones should

Table 2. Data Showing That Quinones Are Frequent Hitters

a The exact substructure definitions for all PAINS are defined in Tables S6, S7, and S8 (pp S23−S37) in the Supporting Information of the original Baell and Holloway PAINS publication.1 bThese were six robust assays against protein−protein interactions and hence not biased by enzyme active sites. cTotal number of analogues recognized by the substructure shown. dTotal number of analogues that hit between two and six screens is expressed as a percentage of those that were clean.

and which numbered some 370 analogues in our 100 000strong HTS inaugural library. Analysis of a set of six selected HTS campaigns showed 40 analogues to hit all six screens, 57 hit five screens, 48 hit four screens, 41 hit three screens, 42 hit two screens, and 56 hit one screen, while 86 were clean in those particular screens investigated. The resulting enrichment value of 265% is extremely high.1 Why might quinones signal promiscuously in bioassays? As we have reviewed elsewhere,1 quinones have a high propensity to be redox active as well as being reactive to nucleophiles present in the side chains of proteins such as cysteine and lysine (Figure 4). For this reason, we consider quinone screening hits to be PAINS and nonprogressable.

Figure 4. Quinones are recognizable PAINS and can interfere in bioassays via different mechanisms.

This behavior is not absent in 5−11 simply because they are natural products. Indeed the endogenous role of menaquinone (5), as vitamin K2, is to redox cycle with its cognate enzymes, vitamin K epoxide reductase and vitamin K hydroquinone, to allow carboxylation of glutamic acid. Menadione (6) is redox active31 and has an unsubstituted β-carbon open to nucleophilic attack.32 In the context of the ontology of their discovery, it may be noted that clinical investigation of vitamin K and analogues in humans was undertaken in the first half of the 20th century.33 D



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render them deemed to be unprogressable as low-micromolarpotency screening hits, whether in a target-based assay such as Kv1.3 described here or a phenotypic assay. The vitamin K analogues are particularly problematic, as they are present in many available and popular screening libraries, giving rise to numerous publications of likely limited utility. Although screening an existing drug library unearthed menadione (as well as vitamin K3) as low-micromolar-potency inhibitors of human tissue transglutaminase,45 for example, this does not mean these are useful and progressable hits, and in fact the opposite is more likely. The only caveat might be specific drug design involving a cognate host, such as screening a library of analogues of vitamin K2 for modulators of vitamin K epoxide reductase or vitamin K hydroquinone. Nevertheless, while Nature has evolved vitamin K2 to be entirely nontoxic,46 attempting to progress any such quinone-based compounds would be ill-advised because one may encounter unpredictably problematic quinone PAINS behavior at any stage. Natural Products that Contain PAINS: Phenolic Mannich Bases and Hydroxyphenylhydrazones. In Figure 5, two other natural products are shown that contain

Table 3. Phenolic Mannich Bases and Hydroxyphenylhydrazones Are Frequent Hitters

a The exact substructure definitions for all PAINS are defined in Tables S6, S7, and S8 (pp S23−S37) in the Supporting Information of the original Baell and Holloway PAINS publication.1 bThese were six robust assays against protein−protein interactions and hence not biased by enzyme active sites. cTotal number of analogues recognized by the substructure shown. dTotal number of analogues that hit between two and six screens is expressed as a percentage of those that were clean.

Figure 6. Phenolic Mannich bases and hydrophenylhydrazones can form reactive quinone methides.

mechanism of action was proposed in 1985, involving entrapment of topoisomerase I with DNA.49 The antibiotic rifampicin is a derivative of the rifamycins, which were founded on the 1957 observations of antimicrobial activity produced by Nocardia mediterranei. Intensive optimization eventually led to rifampicin in 1965 with improved oral efficacy.50 The target of the rifamycins is a bacterial DNA-dependent RNA polymerase. So once again, these drugs were not discovered via methodological progression, but rather on the basis of the observation of early cell-based activity and early efficacy in vivo, and although they are clinically used, they might not be investigated further were they to be discovered as micromolarpotency screening hits in a random assay. In fact, it has been independently shown that the PAINScontaining drugs dopamine, benserazide, apomorphine, idebenone, clofazimine, and doxorubicin can be regarded as frequent hitters in the context of HTS.51,52 Natural Products with Other Reactive Groups. There are other natural product drugs containing reactive functional groups that may not be recognized by the PAINS electronic filters even though they can reasonably be viewed as PAINS reactives. As explained in detail elsewhere,5 this may be simply because they were excluded from our compound library in the first place. By way of example, in Figure 7 the structures are shown of cephalexin (14), artemisinin (15), epothilones (17), epoxomicin (18), carfilzomib (19), chaetocin (20), fluticasone (21a), mometasone (21b), and curcumin (22). These compounds variously contain a β-lactam, peroxide, epoxide, disulfide, or enone. As low-micromolar-potency screening hits, these compounds would rightly be rejected as progression candidates. Indeed,

Figure 5. Topotecan (12) contains a phenolic Mannich base, and rifampicin (13) contains a hydroxyphenylhydrazone. Both are recognized PAINS motifs.

recognizable PAINS motifs. These are topotecan (12) and rifampicin (13), which contain a phenolic Mannich base and hydroxyphenylhydrazone, respectively. The screening data that led our group to conclude that these were problematic functional groups are shown in Table 3, where it can be seen that these compounds are numerous and give rise to high enrichment values. As has been discussed previously,1 and as shown in Figure 6, these compounds can form reactive quinone methides and additionally are excellent chelators, and it is these properties that likely cause them to be frequent hitters. Indeed, the phenolic Mannich base of topotecan is relatively unstable, and it is thought that it can readily form a reactive quinone methide.47 While there is no evidence of the hydoxyphenylhydrazone of rifampicin reacting with biological nucleophiles via a quinone methide, it is certainly a strong chelator.48 Again, the discovery of these drugs is relevant to this discussion. Topotecan is a semisynthetic derivative of camptothecin designed to increase water solubility. Camptothecin was discovered after the testing of 1000 ethanolic plant extracts in 1958 revealed potent cytotoxic and in vivo antitumor activity for the extract from the ornamental tree Camptotheca acuminata.49 The structure of camptothecin was elucidated in 1966. Clinical trials were initiated in 1972, well before its E



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Figure 7. Natural products drugs, or those derived therefrom, with reactive groups (in red) but not specifically recognized by PAINS filters.

treatment of aggressive metastatic or locally advanced breast cancer.58 Interestingly, the epoxide moiety can be replaced and appears to have only a structural role and not one involving reactivity against its target, even though reactivity to other biological nucleophiles is likely.58 Epoxomicin (18) was isolated from an unidentified actinomycete strain, and its potent murine antitumor activity was reported in 1992. At the time there was a reduced interest in natural products, concern about peptide-like starting points, increased focused on mechanisms of actions (about which for epoxomicin nothing was known), concern about toxicophores, and competition with other more interesting compounds, and thus epoxomicin was not pursued. This changed in 2000, when the Crews group at Yale University reported its crystal structure when complexed with 20S proteosome.61 Medicinal chemistry optimization led to the proteasome inhibitor carfilzomib (19), approved in 2012 by the FDA for the treatment of recurrent multiple myeloma (Figure 7).62 Here the reactivity of the epoxide group plays a key role in its efficacy. Chaetocin (20) is not an FDA-approved drug. It contains a disulfide bond, and “hit-to-lead” medicinal chemists would be on high alert if such a compound were to be unearthed as a screening hit because of the likelihood of redox behavior and covalent modification. Indeed, while chaetocin was published in 2005 as a selective inhibitor of the Drosophila histone methyltransferase SU(VAR)3-9, it has since been shown to be a nonselective and protein-reactive compound.3 Fluticasone (and budenoside) furoate (FF, 21a) and mometasone furoate (MF, 21b) are glucocorticoids, for which their origin essentially lies in the 1948 observation of the beneficial anti-inflammatory effects of the natural hormone cortisone in arthritis patients.63 FF and MF were introduced much more recently as topical treatments for the inhaled treatment of asthma and related indications. The furoate ester is

protein reactivity is the basis of their mechanism of action in some cases, such as β-lactams, epoxomicin, and carfilzomib. Yet, most are FDA-approved drugs. Once again, consideration of their route of discovery is relevant to this apparent anomalous observation, which is now briefly summarized. Cephalexin (14) is a β-lactam, and the discovery of this class was somewhat serendipitous, based on the observations by Fleming in 1929 of a bacterial clear zone in the proximity of a contaminating mold, which turned out to be Penicillium notatum. It was not until about 15 years later that the structure of penicillin was determined and about 35 years later that the mode of action was established, involving covalent inactivation of what we now call penicillin-binding proteins (PBPs).53 Artemisinin (15), the subject of a 2015 Nobel Prize in Physiology and Medicine,54 was essentially discovered in 1971 based on the observation of in vivo efficacy in animal models of malaria when treated with an extract from the plant Artemisia annua, and its structure was published in 1977.55 Even today there is debate about its antimalarial mechanism of action, although recent work suggests that no single mechanism is involved but that activation by heme results in various reactions with more than 100 protein targets.56 In an example of heroic medicinal chemistry, the concept of artemisinin was completely reworked to deliver the synthetic peroxide OZ439 (16), which is looking very promising in phase II clinical trials as a new breakthrough treatment of malaria.57 A series of epothilones (17) was discovered subsequent to observation in the late 1980s and early 1990s of potent antifungal activity in extracts of Sorangium cellulosum, followed by identification of potent cytotoxic activity.58,59 The structures of epothilones A and B were published60 in 1996, and the mechanism of action involved a paclitaxel-like microtubule stabilization. About a decade later, a semisynthetic epothilone, ixabepilone, became the first FDA-approved epothilone, for the F



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Figure 8. Natural products that could signal in assays via a variety of nonspecific mechanisms.

Figure 9. Resorcinols rohitukine (30), flavopiridol (31), cercosporamide (32), radicicol (33), and phenol khellinone (34).

phenolic substances shown to nonspecifically signal through membrane perturbation.21 Clearly, such behavior does not therefore require a compound to contain a catechol group. Genistein and resveratrol, again like many plant phenols, are readily oxidized and can form reactive quinones.70,71 Capsaicin displays many of these properties too: it also possesses a phydroxybenzylamino moiety similar to those used as linkers in antibody−drug conjugates that self-immolate to produce a reactive quinone methide.72,73 Indeed, even though capsaicin has been subjected to various clinical trials, there is concern that capsaicin may be carcinogenic74 possibly through its reactive quinone methide metabolites.24,73 Just because a compound may have been ingested for centuries, this does not mean that it cannot be carcinogenic: evolution has little relevance to a situation where morbidity and mortality are manifest long after genes have been passed on. Perhaps this is being alarmist and that any such quinone methide generated is so reactive that it becomes disabled in a harmless manner or is in sufficiently small quantities that the body’s natural clearance mechanisms are able to cope. Capsaicin is additionally surfactant-like, and indeed Nature is an abundant producer of surfactants,75 in particular glycolipids, of which 26 would be an example. N-Alkylpyridinium compounds frequently appear in screening sets for reasons that are currently unclear (Ducrot, P., Servier Laboratories, Paris, personal communication 2014), and we too have observed them in certain classes of PAINS (called het_pyridiniums_A, het_pyridiniums_B, and het_pyridiniums_C in the original PAINS publication1). It is possible that these lipophilic cations have a propensity for hydrophobic burial or even nucleophilic attack, and clearly there is polypharmacology of some sort involved in their various activities.40 Interestingly, sanguinarine (27) and chelerythrine (28) might appear innocuous enough, but these alkylpyridinium compounds have been shown to be redox active; this

stable and indeed contributes to binding with the glucocorticoid receptor, as does the fluoromethyl thioester in FF and the chloromethyl ketone in MF.64,65 The thioester in FF was designed as a metabolic “soft spot” to quickly render the systemic FF inactive. Hence, just because this group is present in a known drug, it does not mean it is useful in a screening hit. Oddly, little mention appears to be made of the chloromethyl ketone reactivity in MF, and perhaps partly because of this, it is hard to find evidence that it is an issue. Both FF and MF carry an extra enone unit that is not present in cortisone, but there do not seem to be problems with Michael acceptor reactivity. It is possible that steric hindrance renders these enones and the chloromethyl ketone relatively inert, and one should not draw the conclusion that either an unhindered chloromethyl ketone or enone is acceptable in a screening hit just because such groups are present in drugs intended for human use. Curcumin (22) is a major component of turmeric, and hence has been ingested for millennia. It continues to be intensively interrogated in clinical trials for a myriad of indications yet is still not an FDA-approved drug.66 It is hard to fathom the amount of attention that curcumin has received, but unfortunately much of this might be attributed to promiscuous biological activity being misconstrued as “privileged” biological activity.21,67,68 Curcumin displays essentially all known PAINStype behavior. It labels proteins, it chelates metals, it is redox active, and it perturbs membranes.21,67−69 Curcumin is actually quite unstable, but its oxidized degradation products are seldom if ever taken into account when interpreting curcumin bioassay data.68 Natural Products with Nonspecific Global Interference Properties. Shown in Figure 8 are some of the last of our hypothetical screening hits. This collection of natural products will be familiar to many hit-to-lead chemists, as many are known to signal promiscuously in different assays. Genistein (23), resveratrol (24), and capsaicin (25) are among the plant G



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Another aspect of the biological readout that has also been mentioned is how downstream it is in the drug discovery process. For example, if rohitukine displayed anti-inflammatory activity in an animal model after oral administration with an ED50 of 2.5 mg/kg, essentially all aforementioned concerns that it might be in some way artifactual should be dispelled. We now move from speculation to reality, and in fact it was based on such an observation that rohitukine was optimized to become flavopiridol (31, alvocidib), approved by the FDA in 2014 for the treatment of acute myelogenous leukemia and shown to act via potent inhibition of CDKs79,80 (Figure 9). In its capacity as a resorcinol of therapeutic interest it should be noted that flavopiridol is not alone. Cercosporamide (32) is a known antifungal natural product that was later identified by HTS to act as an inhibitor of Pkc1 kinase.81 It was then found to be a potent Mnk inhibitor and a weaker inhibitor of other kinases such as Jak3, GSK3β, ALK4, and Pim1. It has antitumor activity in vivo, possibly mediated via inhibition of Mnk1 and Mnk2, and without doubt represents a genuine ligand, making specific interactions with the binding site of these kinases.82 Radicicol (33) was isolated from a culture broth of Monosporidium bonorden in 1953 as an antifungal antibiotic but later received interest as an anticancer agent acting through Hsp90.44 The resorcinol group is important for binding in radicicol, but intriguingly, and like the epothilones, the epoxide is structural and its reactivity is only manifested by the compound having poor pharmacokinetic properties.44 The resorcinol group is also present in several small-molecule and genuine synthetic inhibitors of Hsp90.83 The intriguing situation is evident that this moiety is strongly associated with promiscuous behavior in some compounds such as EGCG, genistein, and resveratrol. Yet, in other compounds a resorcinol could almost be considered a privileged group for binding to the active site of proteins, in particular the ATP site in kinases, although it may be difficult to impossible to transform this into an orally available compound. Flavopiridol has some oral availability (20%) but is administered intravenously in the clinic.79 Hence, the aversion of medicinal chemists to phenols and in particular compounds with several phenolic groups, because of PK liability, is not without merit. However, it needs to be remembered that some diseases are so severe, such as many cancers, that oral administration is not necessary and further intravenous administration in a clinical setting might actually be preferred in certain cases. This takes us to khellinone (34). How should one view such a compound? Khellinone is phenolic, and so some might be wary of it as a screening hit. However, there is really no literature that flags it as being a promiscuous bioactive substance. Further, it is simple and synthetically highly versatile. Were this compound to register as an inhibitor of Kv1.3 via patch clamp, one might be tempted to undertake preliminary SAR elaboration, even if the activity was weak with an IC50 of around 40 μM, for example. In fact, this situation largely describes our successful discovery of modified khellinones as potent blockers of Kv1.3 (Figure 10) in a program ultimately licensed to Merck-Serono in 2008, of which some has been published16,17 and which is underpinned by seven granted patents. While the starting point for this program was not a medium-throughput patch clamp assay of a random natural product librarythe hypothetical example used for the purposes of this reportthe discovery of khellinone as a weak inhibitor of Kv1.3 did arise through patch clamp testing of a focused benzofuran-based natural product library.

has been attributed to their frequent rediscovery as nonspecific assay hits.40,76 These compounds are deeply colored and brightly fluorescent and so possess this additional potential mechanism of assay signaling interference.77 In a similar vein, the bacterial product toxoflavin (29) is redox active40 and indeed is attracting significant publicity as a false positive, to the extent toxoflavin-based screening manuscripts are likely to become increasingly unpublishable as reviewers become wise to its false behavior. Therefore, it does not make sense to progress such compounds as low-micromolar-potency hits from a random screen. However, by analogy to the discussion on catecholamines, it is important to note that the situation is different if high-affinity targets are discovered. Capsaicin, for example, is a potent ligand of mammalian TRV1 channels, and ligand-based development of TRPV1 agonists and antagonists based on capsaicin is an entirely reasonable endeavor.78 Natural Product Structures That May Be Inappropriately Viewed with Suspicion. This takes us to our last two examples of hypothetical screening hits, rohitukine and khellinone. How should one regard rohitukine (30, Figure 9) if obtained as a low-micromolar-potency screening hit? Rohitukine is a phenol and resorcinol, and like any phenol, this would not be favored by hit-to-lead medicinal chemists because of an association with poor pharmacokinetic properties. Given the association of phenolic substances with membrane perturbation, one might have reason to be further concerned. Further, this compound is a chromone, a moiety that many pharmaceutical company medicinal chemists dislike for reasons that are not entirely clear to us when the electrophilic β-carbon is blocked, as it is in this particular compound. Finally, while aspects of the rohitukine molecule look amenable to rapid SAR elaboration, the complexity associated with the two chiral centers may lead to such a compound being excluded from any further investigation. Would such a negative judgment be appropriate? The main concern would be that the molecule acts on cellular signaling by being a membrane-active polyphenol and therefore unoptimizable. However, it is human nature to be increasingly dogmatic in the absence of evidence, and it might be a mistake to form a firm view here because the rules of phenolic membrane perturbation are still not completely understood. For example, the cLogP of the membrane-active phenols reported by Ingolfsson et al.21 is in the range 3.1−4.1, yet that of rohitukine is 1.4. Simply being a phenol does not necessary render rohitukine promiscuous, and other physicochemical properties such a molecular lipophilicity may be important in membrane perturbation. Indeed, unlike the other phenolic secondary metabolites discussed here, from scrutiny of the literature, rohitukine does not appear to be a commonly reported screening hit. While care should be taken in the interpretation of this finding for compounds that may be less commonly investigated than others, rohitukine could be deemed worthy of further investigation after considered thought. It has already been mentioned that potency is an important factor in judging hits. In general, the more potent a hit is, the more likely that it might turn out to be specific and optimizable. If rohitukine was found to be relatively much more potent than other hits, perhaps with an EC50 of 200 nM rather than 6 μM in our hypothetical screen against Kv1.3, and given that it seems to have little potential for being reactive, one might undertake the next step of synthetic elaboration to ensure that there was clarity in early SAR. H



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synthetics. This is one of two groups of natural products we discuss herein. For this first group, coupled with the reasonable notion that evolution has biased natural products to modulate cellular activity, it is not surprising that Nature has provided many useful drugs for human use. What may seem surprising, however, is that many of these drugs contain reactive groups and toxicophores that would generally be avoided by hit-to-lead medicinal chemists. Some examples of these compounds have been outlined herein, with typical functional groups including catechols, quinones, and other Michael acceptors, epoxides, and aziridines, but many more are known.90 Behavior that might often be regarded as nonprogressable includes chelation, redox activity, and covalent reaction with biological targets, and in many cases, such attributes may be important for the drug’s efficacy. Many of these compounds are recognized as PAINS or being PAINS-like, and their behavior is therefore not surprising. However, the relevance of the PAINS concept to natural product drug discovery may therefore be challenged. Smallmolecule synthetic drugs also contain PAINS motifs, and many such drugs also react with biological targets as part of their efficacious mechanism. The relevance of the PAINS concept to small-molecule drug discovery may therefore be similarly challenged. The latter charge is easily countered with the observation that the reactivity of small-molecule drugs is introduced longer after benign optimization has taken place or was a serendipitous discovery in older drugs usually well after clinical trials had taken place.91−93 These processes are not relevant to hit-to-lead discovery to which the PAINS philosophy should be applied. There are a few apparent exceptions. For example, ezetimibe is a small-molecule synthetic drug that contains a β-lactam and was discovered ostensibly through a more modern, target-based approach but really was progressed successfully after observation of inexplicably potent activity in vivo with an unanticipated pathway being involved.94 Furtherand reminiscent of the hindered and relatively less reactive epoxide in epothilones the β-lactam core is hindered and relatively less reactive. However, the former charge requires more thought, because, as has been shown herein, the PAINS and PAINS-like groups in natural product drugs were in place at their first discovery. How could such compounds therefore progress to the marketplace if the PAINS philosophy is valid? To this charge there are several relevant observations. The first is a caution: the barrier to market entry today is much higher than it was when most of these natural product-based drugs were first approved for human use, and also one cannot assume the same sort of compounds could end up being FDA-approved today. The second is a qualification, which partly relates to the first observation: these natural product drugs were not actually evolved to be human therapeutics and are therefore by no means necessarily perfect. Many, such as droxidopa, mitoxantrone, doxorubicin, mitomycin C, carfilzomib, geldanamycin, and camptothecin are not regarded as having useful oral bioavailability. Further, the natural products that contain reactive moieties tend to be concentrated in the anticancer and antibiotic arenas as cytotoxics and so are not as broadly useful as may be imagined. Moreover, reactivity that in the context of Nature’s intention is useful and that may also play an important role in human therapeutic relevance may also be associated with unacceptable toxicity. The author wishes to emphasize that there are of course many natural products with benign structures that are highly useful as medicaments or pharmacological probes and that are not discussed here because

Figure 10. Development of a potent blocker of the voltage-gated potassium channel, Kv1.3, starting with khellinone as a weak hit.

The discussion to date has focused on the nonprogressability of PAINS and PAINS-like compounds in a hypothetical, targetbased electrophysiology assay. However, there is increasing thought that these issues are just as relevant to compounds unearthed from cell-based assays, not just because of membrane perturbation but also uninterpretable reactivity profiles.84 One could argue that the reason that a reactive target-based hit would not be progressed into a cell-based assaybecause of the likelihood of finding a more dominant intracellular functional readoutis the very reason that one might further investigate a reactive phenotypic hit. Thus, the readout from a phenotypic hit plausibly may already have found its most reactive biological partner and readout. This thought process is fraught with danger. How could one ever trust the meaning of cell-based assay results for an analogue set based on an inherently promiscuous compound when different reactivityderived profiles may be dialed in at any instant for any given analogue? Also, how could one trust the readout of the hit itself when the signal may relate to neither normal nor pathological cellular responses? The Rishton Papers: Warnings Unheeded? Almost 20 years ago the initialism “SRR” for “structure−reactivity relationships” for nonprogressable compounds was coined by Gilbert Rishton.85 In a series of subsequent seminal papers, related discussion on the importance of recognizing “good” hits from “bad” hits followed.86−88 In these papers Rishton also touched on the disconnection between the proud history of natural product drugs and how they were discovered and the focus on target-based biochemical assays that was strongly emerging 20 years or so ago. There are strong parallels between his message and that espoused herein. Why did the warnings of Rishton not take stronger root in academic drug discovery? It is possible that his publications did not reach the most appropriate target audience or that the audience at that time was too inexperienced to appreciate the importance of his observations, or a combination of the two. For this reason, it was significant that the journal Nature published a commentary on PAINS,89 thereby bringing the issue to the attention of mainstream scientists. In that commentary, curcumin, EGCG, genistein, resveratrol, and toxoflavin were all mentioned, but space limitation did not allow for detailed reasoning. Part of the purpose of this review is to provide those details.

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CONCLUSIONS Nature makes available a panoply of compounds with structures often too complicated to be readily envisaged, too difficult to easily synthesize chemically, and of a diversity overwhelmingly greater than is possible in any HTS library of vendor-supplied I



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(although the microbes relevant to human disease might be different). It makes sense for Nature to evolve these complex ligands with numerous binding interactions not wholly reliant on high reactivity in order to impart an affinity and selectivity sufficient for a functionally useful effect. An extreme example of finetuning of reactivity might be the hindered epoxide in some epothilones that does not actually react with the functionally effecting target, tubulin. As ligands, high affinity of all these relatively complex natural products allows for identification of specific targets (although care must be taken for reactive natural products that a labeled protein is indeed the functionally relevant targets).90 This, in turn, allows for more focused assays for ligand-based development and development of target-based HTS screens to identify new chemotypical starting points. However, such reactive natural products are not necessarily privileged regardless of context. The point that is stresssed here is that were these compounds to be unearthed as micromolarpotency screening hits in our hypothetical patch clamp assay, it is reasonable to assume this arises via reactivity or membrane perturbation mechanisms that are neither progressable nor optimizable and it should make no difference whether they have previously been clinically investigated or validated in different contexts. Once again, there are many synthetic drugs such as the neuroleptics that display dual behavior: specific binding at low concentrations but nonspecific behavior at micromolar concentrations such as membrane perturbation, and just because one might obtain an FDA-approved drug as a screening hit, it does not mean it cannot be a nonspecific and false hit. This brings us to the second major group of natural products that we have discussed, which is less specific in its effects on biological systems. The structures here are typically less sophisticated, comprising relatively simple quinones and phenols. Typical protagonists include curcumin, genistein, resveratrol, capsaicin, sanguinarine, chelerythrine, and toxoflavin, but it would be wrong to focus just on these better known compounds; there are many others such as cinnamaldehyde and other reactive compounds discussed in detail elsewhere.90 Interestingly, as distinct from the first group of more complex natural products, this second group tends to be dominated by plant secondary metabolites, such that this fact alone might serve as an alert. As we have documented above, these compounds variously display protein reactivity, metal chelation, redox cycling, and membrane perturbing behaviors. Some of this behavior may be incidental, while in many cases it is plausibly useful in its local and evolutionary context, and so it is not unreasonable to think that relative to small-molecule synthetics, natural products may be greatly overrepresented by compounds with such behavior. It is certainly the case that the literature is replete with reports of catechins, curcumins, and a variety of simple phenolics in a myriad of biological settings. Activity is typically only in the low-micromolar-potency range in target-based or cell-based assays, and yet there seems to be almost no barrier to expensive clinical investigation. A complicating factor is that some of the simple reactives (simple catechols, quinones, and enones, for example) can give a relatively potent cell-based readout (with IC50 values, for example, as low as 100 nM) so as to seem more specific. Here, the simplicity of the structures weighs against them, but ultimately it is the absence of clear SAR that can be used to indict.

that is not the point of this review. In the case of the hypothetical example used for the purposes of this discussion, for example, Nature produces a wonderful array of exquisitely potent and selective pharmacologically active toxins, often polypeptides, which block ion channels. Indeed, a derivative of the sea anemone peptide ShK is a highly potent and selective blocker of Kv1.3 and is progressing through clinical trials as a treatment for autoimmune diseases such as multiple sclerosis and rheumatoid arthritis.95 Alternatively, tetrodotoxin is a small-molecule natural product that is a potent and selective blocker of voltage-gated sodium channels and, while highly poisonous, is being considered in the context of a potential analgesic when administered at subtoxic concentrations.96 As for most natural products and natural product drugs, these pharmacologically active venoms and toxins exhibited potent activity from the start. The third observation is more of a concession: that regardless of the above, the point still stands that PAINS motifs present in natural products from the beginning can remain so in versions that reach the marketplace. The key explanation is this: such natural product-based drugs were not discovered via the route relevant to the PAINS philosophy. That is, they did not progress through the more modern discovery route from upstream weaker activity with hit-to-lead optimization to downstream potent activity and efficacy in vivo. Rather, as has been shown throughout this review, almost without exception such natural product-based drugs were discovered through more traditional means, involving initial observation of potent anticancer or antibiotic activity at close to therapeutically relevant concentrations. Relatively little optimization, if required, may result in an acceptable therapeutic index, particularly for diseases as severe as cancer, that may also be more tolerant of what may be regarded as less attractive routes of administration, such as intravenous dosing. It contributes to this discussion to ask how it is that such complex natural products can fortunately be so potent in cellular and even in vivo settings in the first place? Here, the use of the word fortunate is probably not appropriate, because the more complicated natural products represent a diversity so great that their exquisite activity in a different setting would seem to be beyond chance and is likely attributable to orthologous evolution of related proteins and pathways. Such a case might be the discovery of geldanamycin (11a), plausibly intended by Nature to be antimicrobial via action on Hsp90 proteins and later found to be an inhibitor of mammalian Hsp90 proteins with anticancer activity. Similar remarks apply to the other natural products in this category we have discussed, such as anticancer mitomycin C cytotoxicity and DNA/thioredoxin reductase as targets, the anticancer anthracyclines/camptothecin cytotoxicity and DNA as a major target, the anticancer epothilone cytotoxicity and tubulin as a target, anticancer epoxomicin cytotoxicity and the proteasome as a target, and many other examples too numerous to list here. In other cases the evolutionary link may be more direct, such as that of capsaicin, perhaps intentionally designed by Nature to bind to mammalian TRPV1 channels to elicit a pungency to discourage nonpropagative consumption of fruiting bodies while allowing avian-mediated propagation to continue.97 Glucocorticoids and catecholamine drugs can also be included in this group, in terms of direct action on intended human receptors, as can the β-lactam and rifampicin antibiotics in terms of direct action on the intended antimicrobial targets J



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one could screen a library of synthetic catechols to almost certainly discover numerous compounds with anti-inflammatory, antiproliferative, and antioxidant activity. It is suggested that manuscripts reporting such uninteresting activity coupled with uninteresting chemistry should, in the absence of clear SAR and remarkable optimization, be viewed with relatively little enthusiasm by respectable journals and their reviewers. The worth in the clinical progression of such compounds is also questioned, in particular where there is overwhelming evidence of nonspecific behavior, an absence of SAR, and an absence of potent efficacy in vivo, and in the case of curcumin, for example, widespread ignorance of its chemical instability.68 Apart from specific examples involving topical application, the continued clinical investigations into compounds such as EGCG, curcumin, and resveratrol is hard to explain. The unfortunate possibility remains that through brute force alone eventually a clinical use could conceivably be unearthed, perhaps partly because it relates in some way to Nature’s intended behavior for such compounds. However, the risk to benefit ratio for such an ad hoc approach to drug discovery seems too high to justify, especially based on compounds where clear SAR and optimization in a set of demonstrably stable analogues with the observation of potent and relevant cellbased activity and meaningful PK/PD3,101 seems to be essentially absent. It is certainly the case that modern and methodical drug development can be too focused on pathway selectivity, and there is an argument to progress compounds in vivo as early as ethically possible even if only loosely tied to a pathway on the basis of hoping to capture efficacious polypharmacology that might be missed in a more advanced and selective compound. However, there has to be some sort of rationale that is more stringent than appears to be applied currently to the plant secondary metabolites in particular. Throughout this discussion, and as has been emphasized elsewhere,7 it is a key point that ultimately it is demonstration of clear SAR in a comprehensive set of well-chosen structural variants, preferentially devoid of the promiscuous properties of the parent compound, that is required to prove that any given screening hit is progressable and optimizable. On the other hand, a low-micromolar-potency natural product hit in a target-based screen may amount to an excellent starting point for medicinal chemistry optimization where structural alerts that encode for promiscuity are absent, and the example of khellinone was used in this context. Structure-based exclusion of the poorly optimizable screening hit allows one to focus on the more optimizable compounds, even if they are initially weaker in their apparent activity. This can save significant time and money in successful hit-to-lead optimization. To end on another constructive note, how can one recognize these nonprogressable screening hits? Apart from structure alone, previously our group 1,4,6,7 and more recently others102,103 have provided some practical tips to help determine whether a compound displays PAINS-like behavior. So in conclusion, when the question is put, “in what context can PAINS be relevant to drug discovery when mitoxantrone is an FDA-approved drug and contains the quinone PAINS motif, and that we ingest EGCG and that this compound has undergone numerous clinical trials and contains the catechol PAINS motif?”, the author hopes this discussion will provide an answer that, while perhaps more complicated than might be

The abundance of literature around such compounds should fill a hit-to-lead medicinal chemist with alarm. A very interesting recent study quantitatively defined nuisance natural products, and among the list of 39 compounds were quercetin, gossypol, genistein, curcumin, (+)-catechin, caffeic acid, (−)-epicatechin, resveratrol, gallic acid, EGCG, and capsaicin, all compounds discussed herein in the same context.98 Yet, it seems to be that the more a compound is reported as a bioactive, the more it attracts attention by certain circles of researchers. These compounds are also frequently used as positive controls for bioassays, and this has little validity if acting through nonspecific mechanisms. The author has, at times, received points of view that if such compounds may not be progressable as drugs, then they may be useful as tool compounds. However, the opposite is the case, and the demands of a tool compound, where one wishes to meaningfully interpret biological readouts in terms of mechanism(s), are greater in this context than those of a drug, which simply needs to be safe and efficacious.3,99 The lesson learned to this point is that one should be much more excited by screening hits that do not continually reappear in all sorts of assays, even if weak, and particularly if they show signs of early, sharp, positive, and particularly negative SAR. One cannot overemphasize how dire the situation is in terms of wasted resources, a case in point being a recent review on the current status of inhibitors of sortase A as potential antibacterial virulence agents.100 It is hard to find evidence that any one of the dozens of natural products and synthetics is a genuine hit and not a promiscuous bioactive. In terms of natural products, the “usual suspects” are there, including quercetin, curcumin, berberine, and caffeic acid and its analogues. In terms of the synthetics, enones, β-aminoketones, rhodanines, and isothiazolones, all well-known PAINS,1 are all listed as actives. Locating this review was not the result of exhaustive searching. While the author was composing this review, a biologist colleague enquired about the availability of small-molecule sortase inhibitors, and this was the first review that was retrieved from the literature. This is a very typical situation for many new or “undrugged” targets. Publications of promiscuous compounds appear to beget more publications of the same, and the situation is worsened by wastage of resources spent in patenting compounds or uses thereof that are of little value. Unrewarding student projects may be spawned and fruitless pharmacokinetic studies undertaken, eventually even leading to unjustifiable clinical trials. Journals and research granting agencies play a key role in governing the situation by ensuring correct reviewers are in place. It is also important to point out that screening libraries themselves may contain only a small proportiontypically around 10%of PAINS or PAINS-like compounds.1 That is, the majority of screening compounds are entirely acceptable in terms of their structural characteristics. It is simply that the process of HTS concentrates promiscuous compounds, and a combination of improper hit triage and insufficient rigor in the review process furnishes outcomes such as those summarized in the review of sortase A inhibitors mentioned above.100 In summary, we argue that the concept of PAINS applies to natural products particularly in the context of low-micromolarpotency screening hits. Indeed, reports of promiscuous phenolic natural products with weak biological activity are particularly widespread. A major problem is that apparent target-based activity is linked to cell-based activity and activity in vivo, but there may be no common mechanism involved. It needs to be more widely realized how easy it is to perturb cellular systems with promiscuous compounds. For example, K



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(19) Ishii, T.; Mori, T.; Tanaka, T.; Mizuno, D.; Yamaji, R.; Kumazawa, S.; Nakayama, T.; Akagawa, M. Free Radical Biol. Med. 2008, 45, 1384−1394. (20) Subramony, J. A. Mol. Pharmaceutics 2006, 3, 380−385. (21) Ingolfsson, H. I.; Thakur, P.; Herold, K. F.; Hobart, E. A.; Ramsey, N. B.; Periole, X.; de Jong, D. H.; Zwama, M.; Yilmaz, D.; Hall, K.; Maretzky, T.; Hemmings, H. C., Jr.; Blobel, C.; Marrink, S. J.; Kocer, A.; Sack, J. T.; Andersen, O. S. ACS Chem. Biol. 2014, 9, 1788− 1798. (22) Quideau, S.; Deffieux, D.; Douat-Casassus, C.; Pouysegu, L. Angew. Chem., Int. Ed. 2011, 50, 586−621. (23) Awad, H. M.; Boersma, M. G.; Boeren, S.; van der Woude, H.; van Zanden, J.; van Bladeren, P. J.; Vervoort, J.; Rietjens, I. M. C. M. FEBS Lett. 2002, 520, 30−34. (24) Bolton, J. L. Curr. Org. Chem. 2014, 18, 61−69. (25) Coutinho, E. M. Contraception 2002, 65, 259−263. (26) Kovacic, P. Curr. Med. Chem. 2003, 10, 2711−2718. (27) Baggstrom, M. Q.; Qi, Y.; Koczywas, M.; Argiris, A.; Johnson, E. A.; Millward, M. J.; Murphy, S. C.; Erlichman, C.; Rudin, C. M.; Govindan, R.; Mayo Phase, C.; California, C. J. Thorac. Oncol. 2011, 6, 1757−1760. (28) van Delft, M. F.; Wei, A. H.; Mason, K. D.; Vandenberg, C. J.; Chen, L.; Czabotar, P. E.; Willis, S. N.; Scott, C. L.; Day, C. L.; Cory, S.; Adams, J. M.; Roberts, A. W.; Huang, D. C. S. Cancer Cell 2006, 10, 389−399. (29) Vogler, M.; Weber, K.; Dinsdale, D.; Schmitz, I.; SchulzeOsthoff, K.; Dyer, M. J. S.; Cohen, G. M. Cell Death Differ. 2009, 16, 1030−1039. (30) LaVoie, M. J.; Ostaszewski, B. L.; Weihofen, A.; Schlossmacher, M. G.; Selkoe, D. J. Nat. Med. 2005, 11, 1214−1221. (31) Criddle, D. N.; Gillies, S.; Baumgartner-Wilson, H. K.; Jaffar, M.; Chinje, E. C.; Passmore, S.; Chvanov, M.; Barrow, S.; Gerasimenko, O. V.; Tepikin, A. V.; Sutton, R.; Petersen, O. H. J. Biol. Chem. 2006, 281, 40485−40492. (32) Mack, D. O.; Wolfensberger, M.; Girardot, J. M.; Miller, J. A.; Johnson, B. C. J. Biol. Chem. 1979, 254, 2656−2664. (33) Ferland, G. Ann. Nutr. Metab. 2012, 61, 213−218. (34) Schneider-Stock, R.; Fakhoury, I. H.; Zaki, A. M.; El-Baba, C. O.; Gali-Muhtasib, H. U. Drug Discovery Today 2014, 19, 18−30. (35) Mihara, S.; Shibamoto, T. Allergy, Asthma, Clin. Immunol. 2015, 11, 11. (36) Verweij, J.; Pinedo, H. M. Anti-Cancer Drugs 1990, 1, 5−13. (37) Paz, M. M.; Zhang, X.; Lu, J.; Holmgren, A. Chem. Res. Toxicol. 2012, 25, 1502−1511. (38) Thorn, C. F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.; McLeod, H.; Klein, T. E.; Altman, R. B. Pharmacogenet. Genomics 2011, 21, 440−446. (39) Scheulen, M. E.; Kappus, H.; Nienhaus, A.; Schmidt, C. G. J. Cancer Res. Clin. Oncol. 1982, 103, 39−48. (40) Rana, P.; Naven, R.; Narayanan, A.; Will, Y.; Jones, L. H. MedChemComm 2013, 4, 1175−1180. (41) Lichtman, M. A. Blood Cells, Mol., Dis. 2013, 50, 119−130. (42) Patel, A. G.; Kaufmann, S. H. eLife 2012, 1, e00387. (43) Fox, E. J. Neurology 2004, 63, S15−S18. (44) Hadden, M. K.; Lubbers, D. J.; Blagg, B. S. Curr. Top. Med. Chem. 2006, 6, 1173−1182. (45) Lai, T. S.; Liu, Y. S.; Tucker, T.; Daniel, K. R.; Sane, D. C.; Toone, E.; Burke, J. R.; Strittmatter, W. J.; Greenberg, C. S. Chem. Biol. 2008, 15, 969−978. (46) Pucaj, K.; Rasmussen, H.; Moller, M.; Preston, T. Toxicol. Mech. Methods 2011, 21, 520−532. (47) Kearney, A. S.; Patel, K.; Palepu, N. R. Int. J. Pharm. 1996, 127, 229−237. (48) Sadeghi, S.; Karimi, E. Chem. Pharm. Bull. 2006, 54, 1107−1112. (49) Wall, M. E.; Wani, M. C. Ann. N. Y. Acad. Sci. 1996, 803, 1−12. (50) Sensi, P. Clin. Infect. Dis. 1983, 5, S402−S406. (51) Huth, J. R.; Song, D.; Mendoza, R. R.; Black-Schaefer, C. L.; Mack, J. C.; Dorwin, S. A.; Ladror, U. S.; Severin, J. M.; Walter, K. A.; Bartley, D. M.; Hajduk, P. J. Chem. Res. Toxicol. 2007, 20, 1752−1759.

anticipated, contains the necessary detail that is required for a most thorough and logical response.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+61) 3 99039044. Fax: (+61) 3 99039582. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The author thanks the National Health and Medical Research Council of Australia (NHMRC) for a Senior Research Fellowship (#1020411). The author also thanks D. Erlanson for pointing out thymoquinone as a natural product PAIN worthy of discussion in this review.

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DEDICATION Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.

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REFERENCES

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