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Drug Assays

Colorful Compounds Strike Again

Here’s a paper of the sort that I wish more people would publish: a report of a false positive compound, and why it was a false positive. The authors, from the Helmholtz Institute at Saarland University, were screening for compounds that might directly inhibit bacterial RNA polymerase. That’s certainly a worthwhile target, although (like the other bacterial enzymes), it’s sure not an easy one. Rifampicin is probably the prototype antibiotic with this mechanism, and not many of us have sent things into the screening collection that look like it does.
pyrroleBut in a fragment screen, the Helmholtz folks came across the small molecule shown, with pretty decent potency and ligand efficiency. Isomers of the same structure had already been reported as polymerase inhibitors, as it turned out. But when they made some more of the compound, well. . .it wasn’t active.
On standing, though, the newly synthesized material began to take on some color, gradually turning redder and redder. Now, you don’t get much redder than rifampicin, which can even stain your sweat, but redness is not a necessary property of a polymerase inhibitor. Suspicious, the team aged some of the material by warming it up in an open flask, and sure enough, the activity in the assay was back. And the redder the stuff got, the better it worked. Analysis showed some high-molecular-weight polymeric material forming – their proposal is that the pyrrole is polymerizing and leaving a backbone of carboxyl-substituted aryls poking off in every direction. I can well believe that. I’ve been on the receiving end of some pyrrole-containing false positives myself, and in several of those cases there was some red-purple stuff that, once removed, killed the activity.
This carboxylate-studded gunk apparently is a good nucleic acid mimic. Gel-shift studies showed that it binds tightly to RNA polymerase, with a Ki in the low nanomolar range. (RNApol, of course, is ready and looking for regular repeated negative charges). A quick check, though, showed that the polymer was a strong inhibitor of a bacteriophase RNA polymerase, but not those from some other species. Still, you could expect that something like this could well tangle up your assay results if your target protein has a net-positive-charge region that’s important.
It’s tempting to just say “Fine, no pyrroles” after seeing things like this. But it’s hard to make rules like that when a pyrrole (albeit a more heavily substituted one) has gone into millions of patients (and pulled in tens of billions of dollars in revenue). Probably the best lesson to draw is to be suspicious of lightly substituted pyrroles, especially if the sample has any color whatsoever, and always be ready to resynthesize/repurify them you start high-fiving anybody.
The Helmholtz group, by the way, did go on to find some things that were more robust. But I’m glad that they published their blind alley along the way – it could do someone some good on some totally unrelated project.
Update: in response to questions in the comments, the authors did try their polymeric stuff against actual cells, but with no effect. It surely has severe problems getting across the membrane, but if you could get it in, I’ll bet it would do all kinds of things. . .

22 comments on “Colorful Compounds Strike Again”

  1. MLB pitcher and Medicinal Chemist says:

    Derek Lowe didn’t mention Duke’s win. Derek decided to pitch in the minors instead of being on a collegiate team because he did not want to be a slave on the NCAA plantation.

  2. crni says:

    Color me naive, but they found a small molecule that could penetrate into bacteria, polymerize there and very specifically inhibit the RNA polymerase of a particular species and that’s a bad thing?

  3. Hap says:

    The parent pyrrole might not oxidize predictably (to give the same or similar products) inside the bacteria. If formed outside the bacteria, the polymer formed by oxidation will probably have the ability to get into cells almost as well as the Chrysler Building. In addition, generating a single species (or a reproducible mixture) that could be formed consistently (if they wanted it as a drug) might be a problem as well. Those would be my guesses.

  4. weirdo says:

    Well, an N-aryl pyrrole is just a protected form of an aniline, deprotection being effected by an acid. So, a good medicinal chemist should have pointed to the possibility of decomposition right away.

  5. Anonymous says:

    typo in Helmholtz, by the way the largest scientific organisation in germany.
    also typo in arenes

  6. Kazoochemist says:

    I do not have access to the article, so I can’t confirm whether or not they indicated that the compound had activity in whole cells. Derek’s comments and the abstract refer to polymerase inhibition, certainly an in vitro screening assay.

  7. luysii says:

    I find it rather remarkable that most proteins are NOT colored. There doesn’t seem to be anything particularly spectacular about placing serine next to tyrosine next to glycine in a protein chain. Assuming each occurs in our proteins at the 5% level, and also assuming that, in the total sequence space of our entire proteome, that all 20 amino acids occur randomly (fairly big assumptions) then the above sequence should occur every 8,000 times in total protein sequence space. Yet in green fluorescent protein (GFP), the three react with each other to form the chromophore. Why can’t other amino acid sequences do this as all their non-aliphatic side chains are capable of reacting.
    For more please see –

  8. anchor says:

    @ mean after the fact, right? You say pyrrole, I say Lipitor. Go figure!

  9. anonymous says:

    IMHO, oxygenated pyrroles can also display superb colours! I do not discount the simple polymerization process as the authors discuss, but my experience in dealing with NIR dye molecules suggest to me that oxygenation of pyrroles (activated also by two methyl as in this case) is more likely!

  10. Pete says:

    I look at this molecule and see a pyrrole (electron-rich heterocycle) being forced into proximity with an anion (also electron-rich). I’d expect this to increase the likelihood of an acid-catalysed polymerization reaction. It would be interesting to see if the corresponding carboxamide behaved in a similar manner.

  11. Rock says:

    An analysis of pyrroles in drugs I did a few years ago found that the vast majority of them are conjugated to a carbonyl (like in Lipitor) making them much less electron rich.

  12. VTJ says:

    Luysii – As an aside, a friend of mine spent several years in grad school working on exactly the problem you bring up. He was looking for shorter sequences that would light up like GFP without all of the unnecessary molecular weight, figuring there must be plenty of good sequences out there that could attain the correct spacial orientation. He found a few that were faintly fluorescent but ultimately, nothing even close to GFP.

  13. Canman says:

    N-aryl pyrroles should always be carefully scrutinized. Nothing but trouble. Just a “protected” aniline, with other problems.

  14. weirdo says:

    #8: You can say “Lipitor” all you want, ain’t no N-aryl pyrrole in it. Not sure I see your point.

  15. antibac says:

    #12: These are two different properties. The scaffold within the beta-barrel of GFP is unique in optimizing fluorescence. The chromophore could use form, in theory, without needing to be fluorescent. I do think it is an interesting question why nature decided not to use minimal post-translational modification that form covalent abducts within sidechains of a single polypeptide. The chemistry is certainly easily accomplished by enzymes – just look at non-ribosomal peptides.

  16. milkshake says:

    Simple alkyl substituted pyrroles: a disaster. They darken on air, provide tasty morsel for CYPs and so on. Definitely you don’t want them in a drug. But amides derived from pyrrolecarboxilic acid, especially if you put some additional electron-withdraving groups on it, seem perfectly reasonable. So it is all about pyrrole ring electron density. The situation is very similar to anilines: simple aryl-NH2 is a no-no in a drug, worse than a nitro group. But aminopyrimidine or aminopyridine is fine.

  17. Barry Bunin says:

    Often the unexpected is the most interesting result. Perhaps the polymer has promising properties, especially if one optimized the polydispersity.

  18. luysii says:

    #12 and #15 — Fascinating comments. There are lots of beta barrels around (particularly in bacterial proteomes and don’t forget triose phosphate isomerase and friends in eukaryotes). Probably It’s time to look to see just what tripeptides AREN’T found on the staves of the barrels, like the dog that did nothing during the night-time in the link I submitted in #7.

  19. T says:

    #7 and #18 – these residues aren’t just near eachother, they are precisely positioned by the protein fold to be in exactly the right position to react to make the fluorophore with the help (catalysis) of nearby residues. And once the fluorophore forms, the fluorescence is tuned and amplified by the precisely calibrated barrel (small changes to which change or abolish the fluorescence). Your argument is like saying “The protease trypsin has a catalytic triad of serine, histidine, and apartate; lots of proteins have these residues, why don’t they all act as proteases?”.

  20. T says:

    Derek what is meant by “bacteriophase RNA polymerase, but not those from some other species”. My first thought was bacteriophage with a typo but a) they are not a species and b) they are viruses and the group was a screening against bacterial RNA pol. I think I’ve just missed something obvious…

  21. HT says:

    @20: it is a typo for bacteriophage, specifically T7 RNA polymerase. What the authors did was first screen against E. coli RNAP, then check whether it works against another RNAP, and they selected T7 RNAP for the latter. No other RNAP from other species were tested, but they tested for inhibition of other enzymes, e.g. CYP17.
    It’s not the most sensible experimental design I’ve seen, but if they determined that the compound is not worth further investigations, then it probably doesn’t matter.

  22. luysii says:

    # 19 — “The protease trypsin has a catalytic triad of serine, histidine, and apartate; lots of proteins have these residues, why don’t they all act as proteases?”. This is quite true, but the triad isn’t made from adjacent positions on the peptide chain — chymotrypsin has the serine at position #195 on the peptide chain histidine at #57 and aspartic acid at #102. As you note they must be brought together.
    In contrast, the 3 amino acids making up the fluorophore in GFP are exactly adjacent to each other on the peptide chain, so they are already in position to react. In addition, they lie on a beta strand of the beta barrel, a conformation which occurs again and again in beta barrels.

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