Dan provided some useful comments on the last post and I think it’s better to respond with a post since this makes everything more visible the readers of both our blogs. I agree with Dan’s point that there are pitfalls, such as compound aggregation, in addition to interference that Adam and colleagues describe in their article. In an ideal situation one would always have the ability to measure weak affinity directly. Protein-detect NMR is one of my personal favorites but you do need labelled protein and, if you want to get full value for your money (labelled protein is not cheap), you’ll also need resonance assignments. The SPR technology is widely applicable and like the protein-detect NMR will provide a direct measurement of affinity (and a whole bunch of other stuff). Isothermal titration calorimetry (ITC) represents another option although I believe that the technique is relatively sample-hungry and more limited than the other two techniques in the weakness of binding that can be measured. Also you do need heat so to speak even though the experiment is isothermal.
Nevertheless you can get to the point of having crystal structures with bound fragments using only a biochemical assay to measure potency. Given that you may well be screening at concentrations one or two orders of magnitude above what is ‘normal’ in HTS, it does make sense to use the approach that Adam and colleagues describe even if you’re going to follow up with SPR or NMR. I do sometimes wonder if the promiscuous behaviour of some inhibitors is due to this sort of interference rather than aggregation. One intriguing question is whether aggregates can ‘inhibit’ by changing spectroscopic and fluorimetric properties of assay mixtures rather than by interacting with proteins. At least there’s usually the option of running assays with added detergent to check for aggregation.
I won’t say much right now about the structural nasties that Jonathan Baell and Georgina Holloway have identified as PAINS since I’ll be visiting Jonathan at WEHI next Friday. I became acquainted with some of these unsavory structural types during my time in Big Pharma and do not believe that their PAINfulness is specific to the AlphaScreen technology that the WEHI researchers are using. Back in those days we had the Decrapper and a program called Flush...
Dan mentioned the Practical Fragments post on a Cruzain Screen so I thought I’d finish with a couple of papers that show how things can get unstuck when you’ve got a catalytic cysteine with a malicious streak. In the dock is none other than PTP1B, a target that is much-loved by disease area strategists and much-hated by screening groups. I’m not going to review the articles or even comment on them right now. Just read them in the correct order and perhaps we can pick up this theme later.
PTP1B: Read this first
PTP1B: Read this second
Baell & Holloway, New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays. J. Med. Chem. 2010, ASAP | DOI
Liljebris et al, Synthesis and biological activity of a novel class of pyridazine analogues as non-competitive reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). Bioorg. Med. Chem. 2002, 10, 3197-3122 | DOI
Tjernberg et al, Mechanism of action of pyridazine analogues on protein tyrosine phosphatase 1B (PTP1B). Bioorg. Med. Chem. Lett. 2004, 14, 891-897 | DOI