Fragment selectivity

A constant debate in fragment-based lead discovery is whether to focus on fragments that are selective for the target of interest. Because fragments have lower complexity than larger molecules they are likely to be less specific – that is, after all, one of the main arguments for why a small set of fragments can explore more chemical space than a much larger set of lead-like molecules. But does it make sense to prioritize those fragments that are more selective? In a recent issue of J. Med. Chem. Paul Bamborough and colleagues at GlaxoSmithKline address this question experimentally.

The broad family of kinases was chosen for the investigation. Protein kinases in particular have been a rich field for drug development, including fragment-based methods. The researchers assembled a library of 1065 commercially available fragments, most of which were designed to bind to the so-called “hinge” region of protein kinases where the substrate ATP binds. Of these fragments, 936 passed quality-control and maintained stability over the course of the year-plus study.

The researchers screened these fragments at 0.4 or 0.667 mM against a panel of 30 kinases using several different assay formats: FP (fluorescence polarization), IMAP (immobilization metal affinity phosphorylation), LEADseeker (a scintillation proximity assay), and TR-FRET (time-resolved fluorescence resonance energy transfer). Various experiments suggested that FP was most susceptible to assay artifacts, though the results were still usable.

17 of the fragments screened were chosen based on common fragment motifs in the literature. One example is adenine, a fragment of ATP, which of course is used by all kinases. Despite this universality, adenine actually showed surprising specificity, inhibiting some kinases strongly and not inhibiting others at all. The same goes for other hinge-binding fragments that we’ve seen before (such as indazole). On the other hand, biaryl urea fragments designed to bind to the less-conserved adaptive pocket of kinases were quite selective, hitting just 2 kinases strongly.

Of course, especially for kinases, the trick is not getting fragment hits but in figuring out which ones to pursue. Ligand efficiency is often used to prioritize fragments, but is this necessarily a good idea? The researchers compared published high-affinity inhibitors of several kinases with fragments contained within these inhibitors and found that the fragments often would not have stood out above the pack when compared solely on the basis of ligand efficiency. Even spookier, many of the most ligand-efficient fragments appear to be assay artifacts.

What about selectivity? Are non-selective fragments bound to become non-selective leads? The authors present one example of a rather non-selective fragment that could be optimized to a highly selective molecule; PLX4032, which started life as a promiscuous azaindole, is another example.

These are just anecdotes though, so to get a broader handle on this question the authors examined a set of 577 lead-like compounds that had been screened against 203 kinases. This led to a list of 592 matched pairs of lead-like compounds and fragment substructures (most of which are likely hinge-binders) which could be analyzed for selectivity. The results recapitulate a smaller, earlier study performed with a very different data set. As Bamborough et al. put it:

It is not uncommon to find selective lead-sized compounds based upon unselective fragments. Equally, unselective leadlike compounds are frequently based upon selective fragments. It seems that the property of selectivity need not be maintained between fragments and their related lead-sized molecules.

On one level, Bamborough’s study is a bit discouraging: fragment selectivity should be used cautiously if at all in prioritizing fragments. Even ligand efficiency should not be gating; last year we discussed how a fragment with relatively modest ligand efficiency was transformed into the clinical-stage (and more ligand efficient) Hsp90 inhibitor AT13387. Other factors, such as structural novelty or how amenable a fragment will be to further elaboration, are just as if not more important for choosing fragments. All of which serves to reemphasize the fact that drug discovery is less a series of hard and fast rules than a loose system of guidelines and hunches. This lack of predictability is part of what makes the process so frustrating – and fun.