Wednesday, 21 January 2015

It's a rhodanine... fetch the ducking stool

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So I promised do a blog post on rhodanines

This isn’t really a post on rhodanines or even PAINS.  It’s actually a post on how we make decisions in drug discovery.  More specifically, the post is about how we use data analysis to inform decisions in drug discovery. It was prompted by a Practical Fragments post which I found to be a rather vapid rant that left me with the impression that a bandwagon had been leapt upon with little idea of whence it came or whither it was going.   I commented and suggested that it might be an idea to present some evidence in support of the opinions presented there and my bigger criticism is of the reluctance to provide that evidence.  Opinions are like currencies and to declare one’s opinion to be above question is to risk sending it the way of the Papiermark.

However, the purpose of this post is not to chastise my friends at Practical Fragments although I do hope that they will take it as constructive feedback that I found their post to fall short of the high standards that the drug discovery community has come to expect of PracticalFragments.   I’ll start by saying a bit about PAINS which is an acronym for Pan Assay INterference compoundS and it is probably fair to say that rhodanines are regarded as the prototypical PAINS class.  The hydrogen molecule of PAINS even?  It’s also worth stating that the observation of assay interference does not imply that a compound in question is actually interacting with a protein and I’ll point you towards a useful article on how to quantify assay interference (and even correct for it when it is not too severe).  A corollary of this is that we can’t infer promiscuity (as defined by interacting with many proteins) or reactivity (e.g. with thiols) simply from the observation of a high hit rate.  Before I get into the discussion, I’d like you to think about one question.  What evidence do you think would be sufficient for you to declare the results of a study to be invalid simply on the basis of a substructure being present in the molecular structure of compound(s) that featured in that study?

The term PAINS was introduced in a 2010 JMC article about which I have already blogged.  The article presents a number of substructural filters which are intended to identify compounds that are likely to cause problems when screened and these filters are based on analysis of the results from six high throughput screening (HTS) campaigns.  I believe that the filters are useful and of general interest to the medicinal chemistry community but I would be wary of invoking them when describing somebody’s work as crap or asserting that the literature was being polluted by the offending structures.   One reason for this is that the PAINS study is that it is not reproducible and this limits the scope for using it as a stick with which to beat those who have the temerity to use PAINS in their research.  My basis for asserting that the study is not reproducible is that chemical structures and assay results are not disclosed for the PAINS and neither are the targets for three of the assays used in the analysis.  There are also the questions of why the output from only six HTS campaigns was used in the analysis and how these six were chosen from the 40+ HTS campaigns that had been run.  Given that all six campaigns were directed at protein-protein interactions employing AlphaScreen technology, I would also question the use of the term ‘Pan’ in this context.  It’s also worth remembering that sampling bias is an issue even with large data sets.  For example, one (highly cited) study asserts that pharmacological promiscuity decreases with molecular weight while another (even more highly cited) study asserts that the opposite trend applies.

This is probably a good point for me to state that I’m certainly not saying that PAINS compounds are ‘nice’ in the context of screening (or in any other context).  I’ve not worked up HTS output for a few years now and I can’t say that I miss it.  Generally, I would be wary of any compound whose chemical structure suggested that it would be electrophilic or nucleophilic under assay conditions or that it would absorb strongly in the uv/visible region or have ‘accessible’ redox chemistry. My own experience with problem compounds was that they didn’t usually reveal their nasty sides by hitting large numbers of assays.  For example, the SAR for a series might be ‘flat’ or certain compounds might be observed to hit mechanistically related assays (e.g. cysteine protease and a tyrosine phosphatase).  When analyzing HTS results the problem is not so much deciding that a compound looks ‘funky’ but more in getting hard evidence that allows you to apply the molecular captive bolt with a clear conscience (as opposed to “I didn’t like that compound” or “it was an ugly brute so I put it out of its misery” or “it went off while I was cleaning it”).

This is a good point to talk about rhodanines in a bit more detail and introduce the concept of substructural context which may be unfamiliar to some readers and I'll direct you to the figure above.  Substructural context becomes particularly important if you’re extrapolating bad behavior observed for one or two compounds to all compounds in which a substructure is present. Have a look at the four structures in the figure and think about what they might be saying to you (if they could talk).  Structure 1 is rhodanine itself but a lot of rhodanine derivatives have an exocyclic double bond as is the case for structures 2 to 4.  The rhodanine ring is usually electron-withdrawing which means that a rhodanine with an exocyclic double bond can function as a Michael acceptor and nucleophilic species like thiols can add across the exocyclic double bond.  I pulled structure 3 from the PAINS article and it is also known as WEHI-76490 and I’ve taken the double bond stereochemistry to be as indicated in the article.  Structure 3 has a styryl substituent on the exocyclic double bond which means that it is a diene and has sigmatropic options that are not available to the other structures.  Structure 4, like rhodanine itself, lacks a substituent on the ring nitrogen and this is why I qualified ‘electron-withdrawing’ with ‘usually’ three sentences previously.   I managed to find a pKa of 5.6 for 4 and this means that we’d expect the compound to predominantly deprotonated at neutral pH (bear in mind that some assays are run at low pH).   Any ideas about how deprotonation of a rhodanine like 4 would affect its ability to function as a Michael acceptor?  As an aside, I would still worry about a rhodanine that was likely to deprotonate under assay conditions but that would be going off on a bit of a tangent.

Now is a good time to take a look at how some of the substructural context of rhodanines was captured in the PAINS paper and we need to go into the supplemental information to do this.  Please take a look a the table above.  I’ve reconstituted a couple of rows from the relevant table in the supplemental material that is provided with the PAINS article.  You’ll notice is that there are two rhodanine substructural definitions, only one of which has the exocyclic double bond that would allow it to function as a Michael acceptor.  The first substructure matches the rhodanine definitions for the 2006 BMS screening deck filters although the 2007 Abbott rules for compound reactivity to protein thiols allow the exocylic double bond to be to any atom.  Do you think that the 60 compounds, matching the first substructure, that fail to hit a single assay should be regarded as PAINS?  What about the 39 compounds that hit a single assay?   You’ll also notice that the enrichment (defined as the ratio of the number of compounds hitting two to six assays to the number of compounds hitting no assays) is actually greater for the substructure lacking the exocyclic double bond.   Do you think that it would appropriate to invoke the BMS filters or Abbott rules as additional evidence for bad behavior by compounds in the second class?  As an aside it is worth remembering that forming a covalent bond with a target is a perfectly valid way to modulate its activity although there are some other things that you need to be thinking about

I should point out that the PAINS filters do provide a richer characterization of substructure than what I have summarized here.   If doing HTS, I would certainly (especially if using AlphaScreen) take note if any hits were flagged up as PAINS  but I would not summarily dismiss somebody's work as crap simply on the basis that they were doing assays on compounds that incorporated a rhodanine scaffold.  If I was serious about critiquing a study, I’d look at some of the more specific substructural definitions for rhodanines and try to link these to individual structures in the study.   However, there are limits to how far you can go with this and, depending on the circumstances, there are number of ways that authors of a critiqued study might counter-attack.  If they’d not used AlphaScreen and were not studying protein-protein interactions, they could argue irrelevance on the grounds that the applicability domain of the PAINS analysis is restricted to AlphaScreen being used to study protein-protein interactions.  They could also get the gloves off and state that six screens, the targets for three of which were not disclosed, are not sufficient for this sort of analysis and that the chemical structures of the offending compounds were not provided.  If electing to attack on the grounds that this is the best form of defense, they might also point out that source(s) for the compounds were not disclosed and it is not clear how compounds were stored, how long they spent in DMSO prior to assay and exactly what structure/purity checks were made.

However, I created this defense scenario for a reason and that reason is not that I like rhodanines (I most certainly don’t).   Had it been done differently, the PAINS analysis could have been a much more effective (and heavier) stick with which to beat those who dare to transgress against molecular good taste and decency.   Two things needed to be done to achieve this.  Firstly, using results from a larger number of screens with different screening technologies would have gone a long way to countering applicability domain and sampling bias arguments.  Secondly, disclosing the chemical structures and assay results for the PAINS would make it a lot easier to critique compounds in literature studies since these could be linked by molecular similarity (or even direct match) to the actual ‘assay fingerprints’ without having to worry about the subtleties (and uncertainties) of substructural context. This is what Open Science is about.

So this is probably a good place to leave things.  Even if you don't agree with what I've said,  I hope that this blog post will have at least got you thinking about some things that you might not usually think about. Also have another think about that question I posed earlier. What evidence do you think would be sufficient for you to declare the results of a study to be invalid simply on the basis of a substructure being present in the molecular structure of compound(s) that featured in that study?


Dan Erlanson said...

I have more comments over at Practical Fragments, but since this post nicely summarizes some of the problems with rhodanines I think it is important to mention two more.

First, as was recently reported, at least some rhodanines are hydrolytically unstable and can decompose to form a thioenolate, which is a good metal chelator.

Second, at least some rhodanines are photochemically active: under normal lighting conditions they covalently modify proteins, possibly through a radical mechanism. Although this observation was published in a high profile journal over a decade ago and the paper is open access, it is rarely cited by people who report rhodanines as active in their assays.

It might be worth adding a "literature cited" section to this post (as you have done in the past), where you also add these two publications.

Pete said...

Hi Dan, I think it would be fairer to say that the post speculated about the potential problems that one might encounter with rhodanines rather than summarizing well-characterized problems. It does give an idea of how I might think about a rhodanine if it showed up as an HTS active. I decided to stop listing cited literature at the end of posts and don't currently have plans to restart.

The MBL paper is a very interesting study although I'd not describe the rhodanine as hydrolytically unstable this context since it seems to be getting turned over by the enzyme. Put another way, is the rhodanine less stable (a thermodynamic term) than an arbitrary compound with an amide in its molecular structure? The rhodanine appears to be functioning as a suicide substrate for MBL and could be thought of as a pro-drug for delivering a thiol group (an abomination that would cause many to throw up their hands in horror although I try to keep a more open mind) to the active site of the target. Whether or not a rhodanine could be developed into an efficacious and safe drug is open to question but this looks like an interesting way to tackle hydrolases that feature metals in their catalytic mechanism and I'm glad that the authors didn't get put off by claims that some rhodanines showed high hit rates in a panel of six AlphaScreen assays. If trying to move forward from this starting point I might check if the TZD analogue ( C=S -> C=O) was active and if methyl on the exocyclic C=C was tolerated (reduce risk of Michael addition). Manipulation of the group on nitrogen might even lead to a suicide substrate with greater specificity for the enzyme.

I agree that inhibition by a photochemical mechanism should be regarded as pathological behavior and I would certainly check for similar behavior if working with analogs. To what extent can this behavior be extrapolated to other rhodanines? Are people claiming to have discovered MedChem starting points or tool compounds that do not match PAINS substructures relieved of the responsibilty to cite examples of structural analogs behaving badly?

Matto di tutti Matti said...

This is just a thorough elaboration on one theme, oh whoa if you'do propose a chiral phosphor amide. .. pesticides and warfare agents only, right?

Pete said...

Hi Matti, I'm not quite sure about what types of chiral phosphoamides you mean. If a substructure is strongly linked to toxicity then I'd want to avoid compounds incorporating the substructure regardless of how many HTS assays were hit.

Linking a substructure to behavior of compounds in a useful manner is not always easy and one needs to be mindful of substructural context. In the case of rhodanines I'd be looking very carefully at whether the ring nitrogen is substituted and the substituents on the exocyclic double bond. I'd expect a lot less photochemistry if the substituent(s) is/are alkyl rather than the alkenyl or aryl that you seem to see on every PAINful rhodanine.

Dan Erlanson said...

Pete, in answer to your closing question on January 23, just because a molecule has not been flagged by PAINS filters does not mean it should be regarded uncritically. Indeed, I subscribe to Richard Feynmann's admonition: "The first principle is that you must not fool yourself – and you are the easiest person to fool. So you have to be very careful about that."

Since we both agree that inhibition by a photochemical mechanism is pathological, and since some rhodanines have been shown to exhibit this behavior, can we not also agree that researchers who report alkenyl-substituted rhodanines as hits but fail to consider this mechanism are guilty of - at the very least - sloppy science?

Pete said...

Hi Dan, One theme that I’ve tried to stress with this post is that is not always straightforward to categorize compounds as ‘good’ or ‘bad’ on the presence of substructures in their molecular structures. The greater the molecular similarity to a compound that shows pathological behavior, the more inclined I would be to label somebody’s work as ‘sloppy’ if they don’t cite the study in which the pathological behavior is described. If somebody is claiming that compounds are novel probes or tools, at very least they need to demonstrate that each compound is pure and what it says on the bottle and I would regard a failure to do this as a bigger sin than ignoring reports of pathological behavior by structural analogs. If they’ve performed high quality and informative assays (e.g. direct measurement of affinity; demonstrated reversibility; demonstrated some coherent SAR; demonstrated selectivity; tested for interference) then I would be more inclined to overlook the fact that they had failed to cite the study in which pathological behavior of a structural analog was observed. However, if they were working on the actual compound(s) that had behaved badly or their assays were less convincing, I would be much stricter.

One point that needs to be made is that the PNAS article probably has a much wider relevance than rhodanines or PAINS. The covalent bond actually forms between a backbone amide N and a pendant aromatic ring and I think anybody assaying (potentially) brightly-colored compounds with extended pi-systems should be aware of this article. I would be much less worried about the possibility of ‘pathological photochemistry’ for an ‘ene-rhodanine’ if the carbon atom(s) linking the ‘ene’ to the remainder of the molecule were saturated. Similarly I would hypothesize that structural features that forced a pendant aromatic ring out of complanarity with the rhodanine ring would make ‘adverse photochemical events’ less probable.

willem said...

Agree with Pete's key comment, which is about not tarring everything with the same brush. We've done our own analysis based on HTS data at AZ (paper here), and quantify risk at the compound rather than structure level (ie. we look for evidence that a particular compound misbehaves). While far from perfect and suffering from sampling bias, it is highly useful as a heads-up.

What we would suggest is using frequent-hitter incidence across a class (substructure) as an indication of risk. As Pete notes about the table or rhodanine hitters, even within the class there are plenty that seem alright.

Another example can be found here for aminothiazoles, a moiety present in many drugs. Again the message is more about being prepared than chucking them out (though I note the Monash guys did chuck them out, on the grounds of past experience).

Pete said...

Hi Willem, Thanks for your comment and apologies for not being able to comment on your article (this is not a journal that we have access to here). The key is assessing risk and substructural context is important when trying to do this for classes of compounds. I would certainly be worried about any compound that hit in a large number of assays and would also be worried about close structural analogs. Ask me about how close is close and, since I’m still in Brazil, I’d respond, “Boa pergunta”.
I remember some compounds showing up immediately as frequent hitters when we started doing HTS at Zeneca in the mid-90s and these were soon culled (it was Jeff and Richard B who applied the captive bolt although I was in full agreement). ICI Pharmaceuticals was effectively a spin off (pardon the pun) from the dye industry there were plenty of ‘legacy compounds’ that had been lovingly optimized against the twin objectives of being brightly colored and adhering non-specifically to textile fibres. Like you said, it’s all about assessing risk at the compound level.