Focal molography for assessing protein-ligand interactions

The list of technologies used to find and validate fragments is long, with more than a dozen in our 2024 poll. But it’s always fun to learn about new approaches. At the Novalix conference earlier this year I learned of “focal molography,” and this is the topic of a recent paper in J. Med. Chem. by Nicholas Favalli, Andreas Frutiger, Sebastian Oehler, and colleagues at Philochem and lino Biotech.

 

The approach is described in detail in this Wikipedia article. Briefly, it is a biophysical technique based on changes in diffraction. A sensor chip contains a matrix of “molograms,” each of which is a lens constructed of micropatterned ridges and grooves. The ridges of each mologram are functionalized with unique single-stranded oligonucleotides. These capture complementary strands, each attached to a molecule to be screened. The grooves are coated with oligonucleotides without small molecules to act as a control in the subsequent readout. Proteins are then flowed over the chip. Protein binding to the small molecules of a specific mologram changes the topography of the ridge, and so the diffraction. This change can be measured in units of “coherent mass density” (pg/mm²). Because only specific binding to the ridges is detected (the grooves provide background controls for each mologram), the technique is suitable for even crude samples such as cell lysates.

 

An obvious application for focal molography is to confirm the binding of hits identified from DNA-encoded libraries (DEL), since the small molecules already have an oligonucleotide tag. This is the focus of the new paper.

 

The researchers started by assessing three known binders to the model protein carbonic anhydrase IX (CAIX). Surface plasmon resonance (SPR), the most frequently-used chip-based biophysical method, was used as a benchmark in two formats, one in which the small molecule was bound to the SPR chip, which is most comparable to focal molography, and one in which the protein was bound to the SPR chip.

 

SPR and focal molography agreed in rank order for the three compounds, but focal molography sometimes suggested tighter binding. For the highest-affinity compound, SPR gave a K_d of 28 nM for compound bound to the chip and 450-750 nM for protein bound to the chip, while focal molography gave a K_d of 0.82 nM. Agreement was better for the weaker binders, one of which was mid-nanomolar and the other low micromolar. The kinetic constants k_on and k_off also differed between focal molography and SPR by as much as two orders of magnitude.

 

Next, the researchers examined hits from a DEL screen against CAIX, some of which were selective against the related protein CAII. A total of two dozen molecules were tested by both focal molography and fluorescence polarization (FP) as an orthogonal technique. Again, the rank orders of affinities for the compounds to CAIX and CAII agreed while the absolute values varied, with focal molography usually suggesting ten-fold tighter binding. (Though the FP assay often bottomed out above the K_d values measured by focal molography.)

 

Finally, the researchers examined the binding of a known high-affinity binder to the oncology target fibroblast activation protein (FAP). In this case the measured affinities were close to the reported value.

 

This is an interesting approach, and according to the researchers “focal molography can support up to ~500 duplicate kinetic measurements per day on a single regenerable chip.” The observation that affinity measurements seem to be tighter than other methods does raise concerns. I wonder if the density of the molecules on the molograms leads to rebinding, as is sometimes seen when SPR chips are too heavily functionalized. Alternatively, since CAIX is a homodimer, perhaps avidity effects played a role. Also, the researchers suggest that the technique is best used for compounds with affinities better than 10 µM, which reduces utility for many fragment programs.

 

If you’ve used focal molography, how has it worked for you?