Everyone who's done drug discovery has encountered this situation: you get what looks like a hit in a screening assay, but when you re-check it with fresh material, it turns out to be inactive. So you go back to the original batch, but it's still active. There are several possibilities: if that original batch was a DMSO solution, perhaps the compound has done something funny on standing, and you don't have what you thought you had. Maybe the DMSO stock was made from the wrong compound, or was mislabeled somehow - in which case, good luck figuring out what's really in there. If the original batch was a solid, the first thing to do is a head-to-head analysis (NMR, LC-mass spec) between the two. (That sort of purity check is actually the first thing you should do with interesting screening hits in general, as experienced chemists will have had several chances to learn).
But if those assay numbers repeat for both batches, you're in the realm of the Infinitely Active Impurity. The thinking is, and it's hard to find fault with it, that there must be something in Batch One that's causing the assay to light up, something that's not present in Batch Two. I found myself in this situation one time where the problem turned out to be that Batch One had the right structure, except it was a zinc complex, a fact the original submitters apparently hadn't appreciated. (We had to send out for metals analysis to confirm that one). In that case, the assay could be made to show a hit by adding zinc to most any compound you wanted, which wasn't too useful.
Most of the time, chasing after these things proves futile, which is frustrating for everyone involved. But not always. There's a recent example of a successful impurity hunt in ACS Medicinal Chemistry Letters, from a group at Pfizer searching for inhibitors of kynurenine aminotransferase II.
One of the hits was that compound 6 shown in the figure, but a second batch of it showed no activity at all. They dug into the original sample, and found that there was a touch of the N-hydroxy compound in it, and that was the reason for all the activity. Interestingly, it turns out that the amino group was involved in a covalent interaction with the enzyme's cofactor, pyridoxal-5′-phosphate (PLP). That's one of the things you probably want to suspect when you find such tiny amounts of a compound having such a large effect.
It's not a deal-breaker, but it's something to keep in mind. The whole topic of irreversible inhibitors has come up around here before, but it's worth another post soon, in light of the recent acquisition of Avila Pharmaceuticals, who specialized in this field. In this case, the compound isn't covalently attached to the protein, but rather to its bound cofactor, which would make people breath a bit easier. (And the group responsible for the covalency, an amine, isn't something to worry about, either).
Still, it's interesting to see this part of the paper:
"Although irreversible inhibition was not one of our lead criteria at the outset of the program, maintaining this attribute of 7 was a high priority through our optimization efforts. The potential advantages of irreversible inhibitors include low dose requirements and reduced off-target toxicity."
I say that because increased off-target toxicity has always been the worry with covalent drugs. But there's been a real revival of interest in the last few years - more on this next week.
College chemistry, 1983
The 2002 Model
After 10 years of blogging. . .
1. JAB on February 10, 2012 9:34 AM writes...
I agree about the usual futility of chasing such hits, and it's always an issue with natural product isolation to assure that the impurities aren't the active species. We had a synthetic hit that turned into a reasonably good story (Mol.Cancer Ther. 8:571-581, 2009) where the contents of the well wasn't as advertised, and one of the several components was the active, but that's the exception that proves the rule. All too often it's a wild goose chase.
Permalink to Comment2. Roger Frechette on February 10, 2012 9:39 AM writes...
The concern of 'off-target toxicity' is one of those bits of pharmaceutical R&D folklore that just feels right, without having to try - as Avila have done. Similarly, until Velcade, it seemed counter intuitive to think that a boronic acid could be part of a therapeutic agent. Related questions: How many tight binding non-covalent drugs are on the market that behave essentially like covalent inhibitors? How often does one observe off-target toxicity with reversible inhibitors?
Looking forward to next week's musings on the subject.
Permalink to Comment3. AnalyticalScientist on February 10, 2012 10:12 AM writes...
I'm guessing most of us have war stories. When I worked at Merck, we came across a compound with great GABA activity as a screening hit, with a structure completely unlike anything we'd found before. When resynthesised, the hit was dead. It took my group about a day to determine the actual structure from the very small amount of material left from the original hit, which turned out to be ... diazepam. Go figure how that got into the vial.
Permalink to Comment4. Toad on February 10, 2012 10:32 AM writes...
The first year of my large pharma career provided this experience in another group, which kept me on my toes through every early program based on HTS hits. A hit was obtained, re-synthesized, and the activity confirmed. SAR around the core for several months was flat. Then a prep HPLC was installed in the lab, and all analogs purified by HPLC were inactive, whereas all analogs purified by flash chromatography on silica were active.
It turns out the key step in the synthesis of the core was a Diels-Alder cyclization, and the small amount of polymeric byproducts (which passed through on a flash column) were mucking up the assay. From then on, all HTS hits had to go through a high molecular weight filter during biology confirmation.
Permalink to Comment5. Edge on February 10, 2012 11:12 AM writes...
How many have done semi-prep HPLC on a hit and tested all the fractions? In one of our projects we had a hit that had two main things in in it, neither active when prepared fresh. All activity turned out to be in the early HPLC fractions, coincidentally green colored. Figured it was Cu or Ni salts in there-made sense in terms of the compound as well. Never could convince the biologists to add metals to the biochemical assay to tie up the story.
Permalink to Comment2. I have another story about chasing an infinitely active impurity, and a bet between biologist and chemist on that activity. Unfortunately the sordid details of the bet cannot be mentioned in polite company. The biologist lost... no activity ever found.
6. LittleGreenPills on February 10, 2012 11:24 AM writes...
@1 Most compound libraries are such mess that this is a problem regardless of source (natural or synthetic). Any hit should always be checked for purity and correct structure.
Permalink to Comment7. John Wayne on February 10, 2012 1:32 PM writes...
We've all been here before; what sort of criteria to you all use before you walk away?
For example, I was on a project that had some leads that looked reasonable. Several compounds were independently synthesized and were inactive. The solid from the sample repository was also active in the assay, but lost activity when purified. We took 1H and 13C NMR spectra and analytical LCMS runs of the two samples (active solid from repository and inactive independetly synthesized material) and they were indistinguishable. Just to be extra sure, we mixed the samples together and recharacterized; they were the same compound.
Several of the biologists wanted to fractionate the original solid and look for the activity, but we didn't do it. I figured if I couldn't see the 'active' compound with the usual techniques, we wouldn't be able to identify it even if we had some in hand.
What else could we have done? I like the idea of using metals analysis (something NMR and LCMS would likely miss); anybody have any other success stories?
Permalink to Comment8. Moody Blue on February 10, 2012 2:52 PM writes...
We had a hit from our screening collection that was inactive when resynthesized. Turned out the screening sample was an amine hydrochloride from "deBoc" reaction. The chemist had done the Boc removal (HCl/dioxane) in a vial which had a cap with aluminum liner. Unintended contamination with AlCl3 proved to be the culprit in the kinase assay! Another instance, the chemist had submitted an aromatic nitro to aniline reduction product to our screening collection. It was a hit for a later program. Again resynthesis was inactive. After some detective work, we identified the active compound was the corresponding aryl hydroxylamine from incomplete reduction!
Permalink to Comment9. -=GiMP=- on February 10, 2012 4:28 PM writes...
N-hydroxy aryl... potential genotoxic imp so lights up like a christmas tree?
Permalink to Comment10. gippgig on February 10, 2012 9:05 PM writes...
Formation of a covalent bond (Schiff base) between an amino group and PLP is not irreversible; it is in fact how PLP-containing enzymes usually function (and in the resting state the PLP is often bound to the side chain amino of a lysine in the protein). (Of course, a 2nd irreversible reaction could occur after the amino group has bound to the PLP.)
Permalink to Comment11. gippgig on February 10, 2012 11:17 PM writes...
After looking at the abstract it is clear that after formation of the Schiff base a normal and reversible isomerization occurs, followed by another unnatural and irreversible isomerization.
Permalink to Comment12. Anonymous on February 11, 2012 12:04 AM writes...
Bioactivity due to a very low concentration reactive impurity is often readily identified by a titration of the enzyme in the biochemical assay. This Ackermann-Potter type of analysis can be used to determine quantitatively the fraction of irreversible impurity in the sample e.g. 0.1%, 0.01% etc. which is not uncommon among HTS hits for certain target classes.
Permalink to Comment13. Morten G on February 13, 2012 10:03 AM writes...
Isn't there a TB drug that relies on a covalent reaction with PLP?
Permalink to Comment14. Kerry on February 13, 2012 3:23 PM writes...
Unless Edge is one of my former colleagues and the story is about me, I too made a rude and unmentionable bet with a biologist who insisted the batch activity was due to a fantastically active impurity buried in the LC peaks. No payoff was necessary.
Permalink to CommentI also once offered a $10,000 bet to a junior chemist in a group meeting that his product couldn't possibly exist. I was kinda mean to him.
15. Dave on February 13, 2012 3:23 PM writes...
It's really fun when the 55 gallon drum of stuff works just fine, but the 50,000 gallon rail tanker load doesn't work at all (and has to be disposed of as waste!). :-(
Dave
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