Differential scanning fluorimetry (DSF) for covalent ligands

Differential scanning fluorimetry (DSF), also known as a thermal shift assay, is one of the most common techniques for fragment screening according to the most recent methods poll on Practical Fragments. This popularity is in part due to the simplicity of the assay: just mix your protein with a dye such as SYPRO Orange, which binds to the hydrophobic core of unfolded proteins, heat the solution in a PCR thermocycler, and measure the change in fluorescence. Ligands that bind specifically to proteins often stabilize them, increasing the melting temperature. As we noted in 2017, some companies even use DSF to assess the ligandability of new targets.

 

Most of the focus on DSF has involved non-covalent ligands. But the approach also works for covalent ligands, according to a new open-access paper in the British Journal of Pharmacology by Nir London and colleagues at the Weizmann Institute of Science.

 

The researchers acknowledge that they are not the first to explore DSF on covalent ligands; last year we discussed a screen of 47 covalent fragments against 47 kinases, and just last month we highlighted a DSF screen that identified an unexpectedly covalent fragment hit.

 

The new paper starts by performing DSF on five drug targets (BTK, the G12C mutant of KRAS, SARS-CoV-2 MPro, Pin1, and Keap1), each with well-characterized covalent ligands (for example, ibrutinib, acalabrutinib, and evobrutinib for BTK, sotorasib and adagrasib for KRAS^G12C, etc.) For four of the proteins they also tested non-covalent or covalent reversible ligands. Almost all of the ligands increased thermal stability of the bound protein, and covalent ligands tended to have a greater effect.

 

In addition to testing specific ligands, the researchers also performed DSF experiments on the five proteins with three reactive, non-specific covalent ligands: iodoacetamide, ethyl 2-(bromomethyl)acrylate, and a chloroacetamide called RA13. Intact protein mass spectrometry confirmed that these molecules gave essentially complete modification of the five proteins, in some cases at multiple sites.

 

In contrast to the specific ligands, the reactive alkylators tended either to have marginal effects on the melting temperatures of the proteins or to actually destabilize the proteins, sometimes dramatically. That said, there were cases of stabilization. The researchers suggest that “reactive, non-specific, irreversible binders may act as destabilizers, since they form an irreversible covalent bond regardless of molecular recognition, which may result in deformation of the protein if the rest of the binder cannot be accommodated near the modified amino acid.”

 

To assess how well DSF works for covalent fragments more generally, the researchers acquired 2612 acrylamides from Enamine and screened them (each at 8 µM in pools of 5) against Keap1 for 24 hours at 4 ºC. Mass spectrometry showed that just over 100 gave at least 50% modification, which a back of the envelope calculation suggests a covalent efficiency roughly 1 M^-1s^-1. Next, 84 compounds with differing degrees of modification were tested by DSF, and, in contrast to the kinase paper we discussed last year, there was a correlation between extent of modification and either stabilization or destabilization of the protein. But consistent with the specific ligands discussed above, the more reactive fragments (as assessed by glutathione reactivity) were the only ones that caused destabilization.

 

The authors conclude by proposing “DSF as a fast and complementary follow-up approach for electrophilic fragment screening, to prioritize productive and selective covalent binders over promiscuous reactive fragments.” This seems reasonable – what do you think?