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.

Virtual phosphate fragments

Phosphate groups are handy little things: easy for enzymes to put on and take off, they pack a lot of charge in a small volume, thereby providing plenty of binding energy for electrostatic interactions. Not surprisingly, they are ubiquitous in biology. Unfortunately, the same things that make them attractive for an organism make them problematic for drugs: they are easily removed, and their highly negative charge gives molecules containing phosphates a real problem getting across membranes. What’s a chemist to do?

This was the dilemma faced by Ruth Brenk, Ian Gilbert, and colleagues at the University of Dundee. They were interested in inhibiting the enzyme 6-phosphogluconate dehydrogenase (6PGDH) from the parasite that causes sleeping sickness. (See here for previous work from the same group using fragment methods to discover inhibitors against a different enzyme from the same organism.) The enzyme 6PGDH, as its name suggests, binds phosphate-containing substrates and has a very polar active site. Nanomolar inhibitors have been reported in the literature, but these contain phosphates and are not active in cell assays.

As reported in a recent issue of Bioorganic and Medicinal Chemistry, the researchers computationally filtered a set of commercially available compounds to find those that were less than 320 Da and were negatively charged, thereby potentially mimicking a phosphate. They then used DOCK 3.5.54 to see which of the resulting 64,000 molecules might bind in the active site of 6PGDH, resulting in 5836 possible hits. Subsequent triaging led to the purchase of 71 compounds. These were tested for inhibition of the enzyme at 200 micromolar concentration. Ten of these compounds inhibited the enzyme more than 80% at this concentration, of which 3 gave clean IC50 curves. These three molecules are all 5-membered carboxylic-acid-containing heterocycles, and although the IC50s are modest (ranging from 28 to 45 micromolar), they have good ligand efficiencies (up to 0.66 (kcal/mol)/atom). A computational search for analogs resulted in a few more active molecules with similar properties.

Whether these fragments can be advanced remains to be seen. The calculated solubilites, Log P, total polar surface area, and intestinal absorption parameters are more attractive than previous inhibitors, but the history of phosphate mimics is not encouraging. Most prominently, the protein PTP-1B, which recognizes phosphotyrosine residues, was once one of the hottest drug targets around, spawning a cottage industry of groups developing phosphotyrosine mimetics. Fragment methods were particularly effective, and numerous potent small molecules were published. But none of them were sufficiently drug-like, and to my knowledge none are in the clinic. Still, it is worth trying: 6PGHD may be more druggable, and approaches like this are likely to provide an answer.