Critical Assessments of Computational Hit-finding Experiments Challenge #4: Targeting the Tyrosine Kinase Binding Domain of the E3 Ligase Casitas B-Lineage Lymphoma Proto-Oncogene B, an Immuno-Oncology Target

The critical assessments of computational hit-finding experiments (CACHE) are prospective benchmarking exercises where small molecule ligands predicted for a nominated protein of interest are procured and tested experimentally. In CACHE #4, 23 participating teams each selected up to 100 compounds designed to bind the E3 ligase CBLB, an immuno-oncology target. The organizers released the first public structure of CBLB in complex with a known ligand at the outset of the challenge to enable the discovery of chemically novel molecules. Out of the 1688 compounds collectively predicted, ten compounds from nine participants showed some sign of activity (primary hits), but only two were convincingly confirmed (validated hits) across multiple biophysical binding assays and in subsequent analogs, and only one of them was chemically novel. The apparent difficulty of finding novel CBLB ligands, previously reflected by the chemical similarity of inhibitors from the patent literature, suggests that potent CBLB hit molecules were either rare in the screened commercial libraries, or that computational methods may have struggled to retrieve presumably present but weak ligands, or that the conformational dynamics of the binding site was not sufficiently accounted for. The successful method was strikingly atypical. Selection from a chemical binding similarity classifier followed by a random forest binding affinity predictor trained on inhibitors from the patent literature was refined using a diffusion-model binding pose predictor. The road to an ML-driven breakthrough in computational hit-finding can take unexpected turns. Which of these turns reveal novel horizons and which are mere distractions will best be investigated and answered collectively. The discovery in CACHE #4 of a novel series of CBLB ligands is not a trivial achievement and may inform medicinal chemistry efforts toward clinical candidates that complement immune checkpoint inhibition to elicit antitumor immune response in cancer patients.