Yesterday's post touched on something that all experienced drug discovery people have been through: the compound that works - until a new batch is made. Then it doesn't work so well. What to do?
You have a fork in the road here: one route is labeled "Blame the Assay" and the other one is "Blame the Compound". Neither can be ruled out at first, but the second alternative is easier to check out, thanks to modern analytical chemistry. A clean (or at least identical) LC/MS, a good NMR, even (gasp!) elemental analysis - all these can reassure you that the compound itself hasn't changed.
But sometimes it has. In my experience, the biggest mistake is to not fully characterize the original batch, particularly if it's a purchased compound, or if it comes from the dusty recesses of the archive. You really, really want to do an analytical check on these things. Labels can be mistaken, purity can be overestimated, compounds can decompose. I've seen all of these derail things. I believe I've mentioned a putative phosphatase inhibitor I worked on once, presented to me as a fine lead right out of the screening files. We resynthesized a batch of it, which promptly made the assay collapse. Despite having been told that the original compound had checked out just fine, I sent some out for elemental analysis, and marked some of the lesser-used boxes on the form while I was at it. This showed that the archive compound was, in fact, about a 1:1 zinc complex, for reasons that were lost in the mists of time, and that this (as you can imagine) did have a bit of an effect on the primary enzyme assay.
And I've seen plenty of things that have fallen apart on storage, and several commercial compounds that were clean as could be, but whose identity had no relation to what was on their labels (or their invoices for payment, dang it all). Always check, and always do that first. But what if you have, and the second lot doesn't work, and it appears to match the first in every way?
Personally, I say run the assay again, with whatever controls you can think of. I think at that point the chances of something odd happening there are greater than the chemical alternative, which is the dreaded Infinitely Active Impurity. Several times over the years, people have tried to convince me that even though some compound may look 99% clean, that all the activity is actually down there in the trace contaminants, and that if we just find it, we'll have something that'll be so potent that it'll make our heads spin. A successful conclusion to one of these snipe hunts is theoretically possible. But I have never witnessed one.
I'm willing to credit the flip side argument, the Infinitely Nasty Impurity, a bit more. It's easier to imagine something that would vigorously mess up an assay, although even then you generally need more than a trace. An equimolar amount of zinc will do. But an incredibly active compound, one that does just what you want, but in quantities so small that you've missed seeing it? Unlikely. Look for it, sure, but don't expect to find anything - and have 'em re-run that assay while you're looking.
Update: I meant to mention this, but a comment brings it up as well. One thing that may not show up so easily is a difference in the physical form of the compound, depending on how it's produced. This will mainly show up if you're (for example) dosing a suspension of powdered drug substance in an animal. A solution assay should cancel these things out (in vitro or in vivo), but you need to make sure that everything's really in solution. . .