Experienced drug discovery folks, particularly those that work early on in the process, will tell you that aggregation is one of the most common sources of false positive “hits”. This happens when the molecule in question bunches up with others of its kind and makes a larger species, particles of something that has different properties than the individual small molecules themselves. Generally, it’s thought that you get into colloidal territory, with the protein target you’re screening against binding to the newly-formed gunk. Your protein (perhaps many molecules of it) ends up tangled in this pond scum and perhaps even denatured by it. (And that’s what gives you the false-positive readout, of course, since from the assay’s perspective the protein, and the signal coming from its activity, has been inactivated, just not the way that you were imagining it).
But this new paper, from Janssen (J&J) shows another way aggregation can trip you up. The authors were screening for small-molecule inhibitors of TNF-alpha, which is a challenging target. Biologics targeting this protein and pathway are out there making billions of dollars, but no one’s ever been able to go after it successfully with traditional medicinal chemistry. When you’re in that sort of space, you have expect that the great, huge, heaping, vast majority of the hits you get from a small-molecule screen are not going to be real. That’s sad, but it’s a fact. If it were straightforward to get real small-molecule hits against TNF-alpha, you’d have seen more of them by now, even failed projects that got published, and that goes for the insulin receptor, or cMyc, or KRAS, or any of the other superstar targets that no one’s been able to get to work with small molecules yet. These things have seen all the standard screening collection stuff and come up wanting. As a side note, that means that you should be very suspicious when you see a paper where someone’s got a hit against one of these things after screening (say) 5,000 commercially available compounds, because the odds are excellent that they’re wrong.
So here’s JNJ525, which really did look like a TNF-alpha-blocking hit. That’s not the most beautiful molecule in the bunch, but none of its parts are ugly. The only thing to worry about is that it’s pretty bulky for a screening hit, and it has the chain-of-sausage-links geometry that’s common to the synthetically common manipulations of aryl groups and amines (SnAr displacements, Buchwald-Hartwig and Suzuki couplings, N-benzylation, etc.) On the other hand, you ‘d have to think (as I’m sure the Janssen folks did) that anything that actually does hit a target like this might well look a bit large and odd, because it’s a protein-protein interface that has never had any evolutionary pressure to bind small molecules.
This compound came in at about 1 micromolar potency, which is certainly worth paying attention to for a screen like this. It was a FRET-based assay, so the first thing to check is that it’s not causing some sort of assay interference, but the compound passed (you could easily imagine a structure like this causing fluorescence artifacts, on the face of it). But the next thing checked was the killer: adding Triton X-100 detergent to the assay affected the binding of positive control a bit (the TNF/antibody fusion protein Enbrel (etanercept), which is one of those compounds that’s been out there raking in the cash all these years). But it certainly didn’t kill it. On the other hand, detergent hammered the binding curves of JNJ525, which is a Bad Sign.
The group went on the characterize the binding of the compound to TNF-alpha, which is very interesting, but makes me wonder a little bit if they did this characterization first, thinking that it was a real hit, and then later went back and found that Triton killed it. I have a suspicious nature, or it might be that I’ve done it in that order a couple of times myself over the years – just throwing that out there. At any rate, ultracentrifugation suggested that what formed was a dimer of TNF-alpha protein, along with 13 molecules of JNJ525, which is a heck of a lot more defined than the usual aggregation hit. Even more unusually, they actually got a crystal structure of the bound species, and I again hope that they didn’t do all that before running the detergent assay, but who knows.
In the crystal structure, they can see a single TNF-alpha protein binding to a conglomerate of at least five separate molecules of JNJ525, which are making various contacts all over it (and to each other). The protein’s quaternary structure is altered by all this, as well it might be, and thus the inhibition seen in the assay. It’s real inhibition, but it’s just not really useful. The paper also mentions SPD-304, first reported in 2005 as a putative small-molecule hit for TNF-alpha (and sold as such by a number of suppliers), but notes that it, too, has its binding killed by addition of detergent. It seems to be having a similar effect on the protein’s structure through a very similar mechanism. There is a crystal structure of SPD-304 bound to TNF-alpha, but the paper mentions that there’s some unresolved/unassigned electron density running around in that structure in the same region as what they see under better resolution.
So aggregation is even more fun than we’d thought – not only do you get huge driftnets of crap forming, you get small, well-defined crap lumps that can send you down the wrong path just as surely. Run detergent controls, is the answer. And if your assay can’t take detergent controls (and some can’t) then you’d better come to terms with the chances that you’re taking if you rely on the assay data that you generate.