Fragments vs P2X1

Four years ago we highlighted a paper in which researchers performed a fragment screen against ion channels. There have been other occasional reports, but for the most part this has been a quiet area. A new open-access paper in Neuropharmacology by Andrew Thompson and collaborators at Cambridge University, University of Bern, VU University Amsterdam, and Washington State University provides another case study.

The researchers were interested in the P2X1 purinergic receptor, which allows calcium ions to pass into cells when ATP binds. An antagonist could be a safe anti-clotting agent as well as a potential male contraceptive. However, the only reported inhibitors are freakish molecules like suramin.

The paper is heavily focused on assay development and validation, in this case using cells stably transfected with P2X1. These were loaded with a voltage-sensitive fluorescent dye: when the channel opens, fluorescence increases. (Control cells not expressing P2X1 do not behave this way.) By adding potential ligands first and then adding ATP, both agonists and antagonists could be identified.

The researchers screened 1443 fragments (from IOTA) at 300 µM each. Cell-based fragment screens are rare but not unprecedented. In this case, 46 hits were obtained, and these were retested at multiple concentrations; 39 hits showed dose responses. These were both agonists and antagonists, with EC₅₀ values ranging from low micromolar to above 1 millimolar.

For confirmation, the researchers used a fluorescently labeled analog of ATP that binds to the P2X1 on transfected cells but not to cells that don’t express P2X1; the increased fluorescence of the cells could be visualized using confocal microscopy. Most of the fragment hits reduced the fluorescent signal, suggesting that they block ATP binding.

A structural analysis suggested that the hits are quite diverse, though annoyingly only a single fragment structure is provided. Still, these do look like useful assays, and the paper provides another successful example of fragment screening in a complicated cellular system.

Fragments vs ion channels

Ion channels represent an intriguing class of targets for a variety of indications, but it is difficult to develop robust high throughput assays. The gold standard is to actually measure the electrical conduction of the channels using techniques such as patch clamping, but this is time-consuming and tedious. Unfortunately, faster indirect methods are prone to high rates of false positives. Medium throughput automated patch clamp technologies are now available, and in a recent issue of Bioorg. Med. Chem. Lett. Scott Wolkenberg and colleagues at Merck decided to use one of these for a fragment screen.

The researchers screened 1280 fragments against the sodium ion channel acid-sensing ion channel 3 (ASIC3), a potential target to treat pain. Compounds were screened at 1 mM concentration, and 56 of them inhibited >50%. Interestingly, this 4.4% hit rate is comparable to other fragment screens at Merck on completely different target classes. Dose-response curves were generated for the 56 compounds, yielding 32 hits in 12 separate chemical series with ligand efficiencies > 0.3. Similarity searching led to the testing of 250 analogs, but most of these were less active than the original fragments.

In the absence of structural information, two strategies were used to try to improve the potencies. First, a series of 220 analogs were synthesized in which small (approximately 64 mass units) substituents were systematically added at various positions around the fragments. Unfortunately, most of the compounds were inactive, and those that were active were only marginally better than the starting fragment. The second strategy was to make more dramatic changes using parallel chemistry of up to a few hundred compounds. This was more successful: for example, fragment 3 led to compound 24, with 200-fold improved activity, albeit with a loss in ligand efficiency.

Unfortunately the best compounds reported still do not break the micromolar barrier, and the pharmacokinetics are not encouraging. The authors conclude that:

The present results are consistent with the analysis that advancing fragment hits to high potency (<100 nM) and/or to a preclinical development milestone in the absence of structural information is unlikely.

I think this may be overly pessimistic. While optimizing fragments in the absence of structure is certainly challenging, it is possible. Fragment screening ultimately delivered several distinct series of low micromolar compounds against ASIC3. Had these come from an HTS assay, the job of optimizing them further would not be any easier. Moreover, this report represents another example of using fragment approaches to tackle membrane proteins.

TINS and STD and SPR – oh my!

Following up on our last post on the use of the NMR technique TINS applied to a membrane protein, the same research group has now compared TINS with other techniques on a more conventional target. In addition to TINS, they conducted fragment screens using another commonly used NMR technique as well as surface plasmon resonance (SPR) and high-concentration screening; the results appear in the Journal of Biomolecular Screening.

TINS involves immobilizing a target protein onto a resin, then flowing fragments across the resin and determining whether they bind to the target as assessed by a reduction in their NMR amplitudes. A reference protein is evaluated at the same time; only fragments that bind to the target protein and not the reference are considered hits. In this case, the researchers chose the viral protein RNA-dependent RNA Polymerase (RdRP) as the target and the PH domain from the human protein Akt1 as the reference. Using a total of 4 mg of RdRP, they screened a library of 1270 commercially available fragments in pools of 3 to 5 compounds each, resulting in 74 hits.

One of the most commonly used NMR techniques for fragment screening is saturation transfer difference (STD), in which the magnetization of the protein target is saturated, and so magnetization transfers to any ligands bound to the protein. The researchers tested 133 fragments (both hits and non-hits from the TINS experiments) and found a total of 49 hits, of which 40 had also been found by TINS.

The 83 fragment hits from both TINS and STD were tested for their ability to inhibit polymerase activity at concentrations up to 2 millimolar; 70 of them showed some activity, and a few of these seemed to actually activate the enzyme.

Finally, a selected set of 62 fragments (all of which were hits in at least one of the three assays) were tested in an SPR assay at concentrations up to 0.2 millimolar. Of these, around half showed binding, and these tended to be the fragments that showed the greatest activity in the enzymatic assay.

The authors conclude that TINS picks up more hits than the other assays, though high-concentration screening comes close. This may be true, but it would have been nice if they had run the entire set of 1270 fragments through each of the different methods; it is possible that there were false negatives in the TINS experiments that could have been picked up by the other techniques. Moreover, some of the TINS hits that didn’t confirm in other assays may well have been false positives. Still, there are lots of useful data in this paper, and it demonstrates yet again the importance of using multiple, orthogonal techniques to discover and properly validate fragments.