Characterizing and screening commercially available irreversible covalent fragments

A few years ago we highlighted the utility of irreversible fragments. Because these molecules form covalent bonds with their targets, they can be more effective than similarly sized noncovalent molecules at inhibiting proteins. However, compared with conventional fragments, the quality and quantity of commercial irreversible fragments is limited. This is changing, as described (open access!) by Nir London (Weizmann Institute of Science) and a large, multinational group of collaborators in J. Am. Chem. Soc.

The researchers assembled a collection of 993 fragments from Enamine, all of which contained a cysteine-reactive warhead, either a chloroacetamide (76%) or an acrylamide (24%). The molecules were largely rule of three compliant, even with the warhead included.

A major concern with screening irreversible fragments is that binding to the target protein can be dominated by the inherent reactivity of the warheads rather than non-covalent (and presumably target-specific) interactions from the fragment. Indeed, a previous study found that the reactivities of acrylamides ranged over more than three orders of magnitude. To assess fragments for this, the researchers developed a rapid, plate-based spectrophotometric assay based on labeling the reduced form of Ellman’s reagent. Not surprisingly, the chloroacetamides tended to be more reactive than the acrylamides, but overall the reactivity range across both classes was a relatively modest ~100-fold.

Next, the researchers screened their library against ten cysteine-containing proteins. Fragments were screened in pools of five (200 µM each) with 2 – 10 µM protein for 24 hours at 4 °C. As with Tethering, intact protein mass spectrometry was used to identify hits, which were found for seven of the ten proteins. Hit rates ranged from 0.2 to 4%.

Not surprisingly for fragments, some hits were promiscuous: they strongly labeled two or more proteins. However, these represented less than 3% of the library. Surprisingly, promiscuity did not correlate with reactivity, and in fact some of the most reactive fragments did not label any of the proteins. This suggests that non-covalent interactions are playing a role in promiscuity, and indeed many of the frequent hitters were aminothiazoles – which have previously been found to be promiscuous.

The researchers also screened their fragments (at 10 µM) against three cell lines, and here they did see a correlation with reactivity, with the most reactive fragments tending to be more toxic.

Next, the researchers began optimizing hits against two targets. The first, OTUB2, is a deubiquitinase (DUB) implicated in diverse diseases from amyotrophic lateral sclerosis to diabetes to cancer. The primary screen yielded 47 hits which labeled at least 50%, of which 37 were quite selective. Co-crystal structures were solved for 15 fragment-protein complexes, and two shared a hydrazide moiety (as in PCM-0102954) which made multiple hydrogen bonds with the protein. Two rounds of SAR-by-catalog eventually led to OTUB2-COV-1, which inhibited the enzyme with a respectable k_cat/K_I = 3.75 M^-1 s^-1. Despite containing a chloroacetamide, the molecule labeled just 26 of 2998 cysteines in proteins detected in a cell-based proteomic assay.

The researchers also found 36 fragment hits against NUDT7, a protein potentially associated with diabetes, and many of these stabilized the protein in a differential scanning fluorimetry (DSF) assay. Crystal structures were obtained for several, and compound PCM-0102716 showed an overlap with the non-covalent molecule NUDT7-REV-1 derived from a previous crystallographic fragment screen. When the researchers merged these, the resulting NUDT7-COV-1 showed low micromolar inhibition and rapid labeling (k_cat/K_I = 757 M^-1 s^-1). This is all the more impressive given that the original noncovalent hit showed no activity. NUTDT7-COV-1 also showed target engagement in a cell assay, and hit only 37 of 2025 detected cysteine residues in a proteomics screen.

This is a nice, thorough paper, though I suspect people in industry will be wary of the chloroacetamides that form the bulk of the library. Nonetheless, chemical structures and reactivity data for all the fragments are reported in the supporting information, making this a useful resource for anyone wishing to dip their toes into covalent fragment screening.