Fragments distinguish allosteric from active site binders

As discussed last year, secondary binding sites on proteins appear to be quite common. Some of these sites have no functional relevance, but others are allosteric sites, which can modulate the activity of proteins. Allosteric ligands can be useful for several reasons. First, unlike molecules that bind at the active (that is, catalytic) site of an enzyme, which usually inhibit activity, allosteric site binders can increase activity. Second, allosteric sites are usually less conserved than active sites, allowing greater selectivity. Finally, combining an allosteric inhibitor with an active site inhibitor can lead to synergy as well as lower the incidence of resistance mutations for cancer and anti-infectives. In a recent ACS Med. Chem. Lett. paper, Lukasz Skora and Wolfgang Jahnke at Novartis describe a simple NMR approach to differentiate these two classes of ligands.

The researchers used ¹⁹F NMR to screen 540 fragments containing a CF₃ group, each at 25 µM, in pools of 30 against the kinase ABL1 (at 4 µM); the BCR-ABL1 mutant form of this protein is a key driver for chronic myelogenous leukemia. Several approved drugs target the active site of ABL1, and Novartis researchers have recently launched clinical studies of a compound called ABL001, which binds to an allosteric pocket.

Fragments that bind to ABL1 showed a decreased ¹⁹F NMR signal due to line broadening. Adding ABL001 displaced fragments that bind to the allosteric site, thereby increasing their NMR signals, while adding the active-site binding drug imatinib displaced fragments that bind to the catalytic site. Follow-up experiments with individual fragments identified a selective catalytic-site binder (CAT-1) and a selective allosteric site binder (ALLO-1). Both fragments are commercially available and quite weak (K_d = 43 µM for ALLO-1 and IC₅₀ = 380 µM for CAT-1), which in this case is a feature because they can easily be displaced.

Mixing these two fluorine-containing probes with ABL1, adding test compounds, and performing ¹⁹F NMR thus provides a simple means to determine whether a ligand binds to the allosteric site, the active site, or both sites. The researchers confirmed that the approved catalytic-site binding drugs nilotinib, dasatinib, and ponatinib displace CAT-1 but not ALLO-1, while allosteric-site binders such as ABL001 displaced ALLO-1 but not CAT-1.

Interestingly, a crystal structure of imatinib with the highly related protein ABL2 shows the compound binding to both the catalytic and allosteric sites, yet although imatinib clearly displaced CAT-1 it could not displace ALLO-1. This is a useful reminder that crystal structures say nothing about affinity.

The drug crizotinib, which binds to the active site of multiple kinases, has been reported by other researchers to bind to the allosteric pocket of BCR-ABL1, but this was not borne out in the competition assays. Similarly, the drug fingolimod has also been reported as an allosteric inhibitor of ABL1. This molecule did indeed displace ALLO-1, but only at concentrations so high as to be biologically irrelevant.

This is a nice paper, and a good reminder that fragments can make useful biophysical probes in and of themselves, even without the need for optimization.