Screening irreversible covalent fragments, computationally and experimentally

Last week we discussed a paper that characterized commercially available irreversible fragments and screened them against ten proteins. The “warheads” used were either acrylamides or chloroacetamides. This week we’re continuing the theme of irreversible fragments with two papers, each using different warheads.

The first paper, published in Bioorg. Med. Chem. Lett. by Alexander Statsyuk and collaborators at University of Houston, Schrödinger, and Northwestern University, uses a computational approach.

Research we previously highlighted from the Statsyuk lab found that methyl vinylsulfones have a narrower range of reactivities than – for example – acrylamides. This property is important  to ensure that differences in binding to a target are caused by (specific) noncovalent interactions rather than mere differences in warhead reactivity. For the current campaign, the researchers constructed a virtual library in which 1648 commercially available carboxylic acids were coupled to H₂N-CH₂-CH=CH-SO₂-CH₃.

The researchers used the CovDock program from Schrödinger to dock the fragments. (Another computational tool for doing so is DOCKovalent, which we described back in 2014.) The specific target chosen was cathepsin L, a cysteine protease which has been implicated in a variety of diseases from cancer to osteoporosis. The virtual screen yielded 33 high-scoring compounds, five of which were synthesized based on price and diversity. Unfortunately, most of these had “solubility issues,” but compound 11 did show time-dependent inhibition of cathepsin L. The researchers also found that the racemic methyl substituent could be removed (compound 13), suggesting that compound growing might be productive. Compound 13 was also selective against three other cysteine proteases.

The second paper, by David House, Katrin Rittinger and collaborators at GlaxoSmithKline, the Francis Crick Institute, and Cellzome, was published in J. Am. Chem. Soc. The researchers were interested in protein ubiquitination, in which the small protein ubiquitin is conjugated to various other proteins to cause a variety of effects depending on the context. The biology is fiendishly complex, but the final step is done by an E3 ubiquitin ligase, of which there are more than 600 in human cells. Needless to say, selective chemical probes would be useful.

The researchers were specifically interested in LUBAC, an RBR E3 ubiquitin ligase which conjugates ubiquitin to proteins with an N-terminal methionine to modulate cellular pathways important in cancer and inflammation. The ligase itself actually consists of three protein subunits, with HOIP containing the catalytic cysteine residue. Although a couple inhibitors had been previously reported, the researchers found these to be nonspecific. Thus, they built and screened their own fragment library. For a warhead, they chose the 4-aminobut-2-enoate methyl ester, which Statsyuk had previously shown has a narrow range of reactivity and is about 10-fold less reactive than the vinylsulfones discussed above. The researchers constructed a small set of 104 fragments, grouped them into pools of 4 or 5, and screened these against 2 µM HOIP at 20 µM each using intact protein mass spectrometry. Compound 5 was one of the best hits.

Compound 5 functionally inhibited HOIP and was selective against about a dozen other cysteine-containing enzymes. The researchers obtained a crystal structure of the molecule bound to HOIP, which confirmed covalent binding to the active site cysteine. Limited SAR studies led to a slightly more potent analog (containing a six-membered ring instead of a five-membered ring), and this molecule showed pathway inhibition in a cell-based assay with EC₅₀  = 37 µM. Activity-based profiling in two cell lines revealed only 8 or 11 proteins that were significantly modified by the compound in addition to HOIP.

The molecules in both of these papers still require considerable work to become chemical probes, let alone development candidates. Nonetheless, they are useful starting points, and together demonstrate the increasing interest and utility of irreversible covalent fragments.