Filtering fluorescent frauds from fragments for PTP1B

Protein tyrosine phosphatase 1B (PTP1B) is a long-standing target for diabetes and other diseases. But as we’ve written previously, it is challenging to drug due to its small and highly charged active site, which is conserved among phosphatases. In a new open-access Drug Design Dev. Ther. paper, Frank Boeckler and colleagues at Eberhard Karls Universität Tübingen screen fragments against PTP1B and demonstrate the importance of distinct, rigorous controls.

 

The Boeckler group previously developed a fragment library enriched in halogens (HEFLib, which we discussed here) and a separate covalent fragment library (CovLib). These, as well as two other small libraries, were screened against PTP1B using a fluorescence activity assay. Compounds were incubated with the enzyme for an hour at 2 mM in the presence of a substrate (DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate) that releases a fluorescent reporter (DiFMU) when hydrolyzed by the enzyme. A molecule that inhibits the enzyme should lead to less fluorescence. Samples were run in triplicate, and detergent was included to avoid aggregation artifacts. A total of 515 fragments were tested, leading to 56 preliminary hits, defined as molecules that decreased fluorescence by more than 50%.

 

A major challenge with fluorescence assays is that the compounds themselves can interfere with the readout. To assess this, the researchers retested their hits both before and after incubation of PTP1B with substrate. A fluorescence artifact should give similar activity in both conditions, while a true inhibitor should only cause a decrease in fluorescence when added at the beginning of the assay, before the protein has had a chance to process substrate. Unfortunately, only 11 hits passed this test.

 

To further confirm mechanism, the researchers developed an HPLC-based assay that separates the substrate DiFMUP from the product DiFMU. Because this assay detects PTP1B activity using separation rather than fluorescence, it should be immune from fluorescence artifacts. Only three of the 56 preliminary hits confirmed in this assay. Dose-response experiments revealed them to be quite weak, with IC₅₀ values above 5 mM. All three are potentially covalent inhibitors, and two showed reactivity with glutathione. PTP1B has a reactive active-site cysteine so covalent inhibitors are not surprising, though it remains to be seen how specific they are.

 

This paper is a useful reminder of the importance of validation: an initial 11% hit rate dropped to just 0.6%. Full experimental details for all the assays are provided, as are the structures of the hits, while SMILES strings of all 515 compounds screened are listed in the supplementary material. If you’re running a fluorescent fragment screen for the first time, this paper is worth consulting.

NMR vs other methods

Fragment-based lead discovery owes much of its popularity to NMR: the SAR by NMR papers published by Abbott in the mid 1990s demonstrated both the power and the practicality of the approach. Recently SPR has also come into its own as a means for screening fragments, and in a paper in this month’s issue of Drug Discovery Today Claudio Dalvit of Novartis and the Italian Institute of Technology compares these two techniques, along with fluorescence spectroscopy. Not surprisingly given the author’s longstanding research interest, NMR comes out favorably, though with the recommendation that the techniques are complementary, so researchers should combine techniques rather than simply selecting one over another.

As I read the paper, I wondered why fluorine-labeled fragments are not used more widely; Dalvit’s group published another paper about this approach recently in JACS. Fluorine has a strong NMR signal and is very sensitive to the local environment, so when a fluorine-containing fragment binds to a protein this can be easily detected. In fact, the dynamic range for this type of assay is so great that fragment binding can be detected at concentrations several orders of magnitude lower than their dissociation binding constants.

This seems like a very powerful approach, but I haven’t seen many other people using it. Are folks concerned about the need for fluorine in every fragment (although many are commercially available) or is there something else I’m missing?