Covalent fragments writ large

We’ve written previously about irreversible covalent fragment-based lead discovery. The nice thing about irreversible inhibitors is that they have an infinite no off-rate: once they bind and react with a target, that protein is permanently out of action. A paper published today in Nature by Keriann Backus, Benjamin Cravatt, and colleagues at Scripps Research Institute takes this approach to a whole new level.

The researchers assembled a library of just over 50 fragments containing cysteine-reactive electrophiles, such as chloroacetamides and acrylamides; the average molecular weight was 284 Da. These were then screened against human cells or cell lysates using a proteomic approach called isotopic tandem orthogonal proteolysis-activity based protein profiling (isoTOP-ABPP). This technique, previously developed by the Cravatt laboratory, uses mass spectrometry to differentiate contents of treated and untreated cells and identify specific regions of proteins that are modified.

In all, 758 cysteine residues in 637 different proteins were found to be modified by at least one of the fragments. These included targets (such as BTK) with known covalent drugs as well as many proteins with no small molecule inhibitors. Even more exciting, this set included some particularly challenging classes of proteins, such as transcription factors and various adapter and scaffolding proteins. Most proteins only had a single modified cysteine, and these were not necessarily in the active site (see also here). Happily, computational docking did a good job of (retrospectively) predicting the modified cysteine residues.

The fragments themselves ranged significantly in how many cysteines they modified, from < 0.1% to > 15%, with a median of 3.8%. Interestingly, the correlation with intrinsic electrophilicity – as measured by reaction with the small molecule thiol glutathione – was fairly weak. This suggests that the fragments are modifying proteins based on other properties, such as specific interactions between fragment and protein.

The initial studies were done using cell lysates at high (500 µM) fragment concentrations. Follow-up studies in whole cells using 50-200 µM fragment gave similar results, with 64% of the cysteines from the lysate experiments reacting with the same fragments in cells, even at the lower concentrations. Interestingly though, four fragment-cysteine interactions were found only in cells and not in lysates.

One class of proteins you might expect reactive fragments to hit are cysteine proteases, such as the caspases, and indeed one chloroacetamide-containing fragment reacted with the active site cysteine of caspase-8 (CASP8). Surprisingly though, this fragment showed only marginal activity in an inhibition assay, and subsequent experiments revealed that it is selective for the inactive zymogen (or proenzyme) form of the protein, thereby preventing activation. This fragment does not react with the related caspases 2, 3, 6, or 9, though it does hit CASP10. Modest modifications led to a compound that was also selective for CASP8 over CASP10. These two molecules were used to show that both CASP8 and CASP10 appear to be essential for extrinsic apoptosis in primary human T cells, but not in the immortalized Jurkat T-cell line.

Of course, it will be essential to rigorously characterize any covalent molecules used as probes. Chloroacetamides are well-known electrophiles – so well known in fact that they are generally excluded from screening libraries, including those that helped define the original PAINS filters. A single digit percentage hit rate means that any given covalent fragment could easily hit hundreds of proteins. The researchers here do careful control experiments – such as using an inactive enantiomer and extensive proteomic analyses – but someone less careful could easily mislead themselves and others. Done rigorously, though, this is an exciting approach that may well increase the number of ligandable targets.