From fragment linking to covalent RAD52 inhibitor

Targeting proteins involved in DNA repair is a promising approach against cancer. However, finding effective small molecule inhibitors can be difficult because the protein-DNA interface tends to be very polar, with few classically ligandable pockets. In a new J. Am. Chem. Soc. paper, Nicolas Bocquet, Olivier Querolle, Ulrich Lücking, and collaborators at FoRx Therapeutics, WuXi AppTec, Diamond Light Source, University of Bern, Sandexis, and Weizmann Institute of Science provide a proof of concept.

 

The researchers were interested in RAD52, which mediates several DNA repair functions. The N-terminus of the protein forms an oligomeric ring structure, and each subunit contains a solvent-exposed groove that binds to single-stranded DNA. Since DNA is negatively charged, the researchers conducted a crystallographic screen against a library of 320 carboxylic acid containing fragments. This identified three related molecules, of which compound 3 had the highest occupancy. Because the compounds bind at the DNA-binding interface, the researchers developed a fluorescence polarization (FP) DNA-displacement assay and found that the fragments disrupt DNA binding at high micromolar concentrations.

 

Interestingly, the crystal structure revealed two copies of compound 3 bound to each subunit of RAD52: each positioned where a single nucleotide would normally bind, with the carboxylic acids making similar contacts as the phosphates. In other words, the fragments mimic two consecutive bases of a single-stranded oligonucleotide. Naturally, this suggested a linking approach.

Linking two close analogs of compound 3 led to compound 5, with a satisfying two orders of magnitude improvement in activity in the FP assay, which the researchers suggest is largely due to lowering the entropic penalty. Linking directly off the thiazole moieties produced similar results, and a macrocycle had slightly better activity, though not so much better as to justify the synthetic hassle. A crystal structure of compound 5 revealed a small hydrophobic cleft, and this was exploited with a phenyl moiety in compound 11, a high nanomolar inhibitor in the FP assay.

 

A crystal structure of compound 11 bound to RAD52 confirmed that the compound binds as designed. The structure also revealed a cysteine residue (C64) in close proximity to the phenyl moiety. Adding an acrylamide warhead yielded a covalent inhibitor, and further optimization ultimately led to FORX-864, which has low nanomolar activity in the FP assay after 4 hours. The researchers appropriately measured the k_inact/K_I, which came in at a respectable 230,000 M^-1s^-1, better than some covalent clinical compounds. Removing the warhead led to a dramatic loss in potency (IC₅₀ = 27 µM in the FP assay).

 

In a cellular assay to measure single-stranded annealing (SSA), FORX-864 showed roughly 1 micromolar activity, while a compound lacking the warhead was inactive. FORX-864 was also inactive in a cell line in which RAD52 had been knocked out, suggesting on-target activity. The researchers profiled the reactivity of FORX-864 in an activity-based proteomic profiling (ABPP) assay and found that it was reasonably selective. Intravenous dosing in mice showed encouraging pharmacokinetic properties, though with two carboxylic acid moieties oral dosing might be challenging.

 

Overall this is a nice example of liganding a difficult target. Given that FORX-864 mimics a dinucleotide, it may bind to other DNA- or RNA-binding proteins. However, covalent binding is required for high potency, and it is unlikely these other proteins will have cysteine residues in the appropriate position, which should help with selectivity. FORX-864 certainly appears to be the best chemical probe for RAD52 to date.