We describe a novel method that combines experimental biophysical data (HDX-MS) with weighted ensemble simulations (WES) to accurately predict ternary complex structures at atomic resolution. We show that the WES+HDX approach generates accurate structures (RMSD below 2.0 Å to x-ray) and can reproduce solution-state dynamic behavior of the ternary complex. We illustrate how this approach is used to predict degradation propensity of different heterobifunctional and glue molecules.

Chemo-proteomics profiling of PROTACs designed from pan-class inhibitors revealed a large difference in the degradation frequencies of the target proteins engaged by these molecules. Using protein-intrinsic features, we developed a machine learning classifier that discriminates target proteins based on their observed degradation patterns and highlights properties that dictate their degradability. Using computational structural modeling, we provide mechanistic insight into the predicted features and obtain actionable information for rational PROTAC design.

Combining the state-of-the-art molecular modeling, in heterogeneous data sources and in machine learning techniques, we are dramatically increasing the accuracy in our computational predictions. We will showcase recent successful case studies including virtual screening enrichment, ligase screening for TPD and ternary complex formation in PROTACs. Overall, the enrichment of machine learning techniques with data augmentation from molecular modeling seems to provide the necessary boost that prediction models might need.

Small molecules that induce protein degradation through ligase-mediated ubiquitination, are showing considerable promise as a new pharmacological modality. Following clinical proof of concept by Thalidomide and related IMiDs, several new molecular entities are entering clinical development. Significant progress has been made towards chemically induced targeted protein degradation using heterobifunctional small molecule ligands and exciting opportunities arise from better understanding molecular glues. We will present recent work towards a better understanding of the molecular principles that govern neo-substrate recruitment by small molecule degraders.

Three distinct characteristics of targeted protein degradation empower drug discovery; the catalytic nature that can achieve high efficacy, the ability to target previously undruggable proteins and to deliver the drug activity to selective tissues. E3 ligands hold the key to realize the full potential of TPD, but are very limited at present. Cullgen has discovered multiple novel E3 ligands for the targeted protein degradation.

Unique subcellular environment due to factors such as the location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether subcellular context of POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V (dTAG) constructs consisting of varying localisation signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localised to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome, or lysosome. Differentially localised Halo or FKBP12F36V proteins displayed varying levels of degradation using the same respective PROTACs.

Despite identification of IMiD-induced neo-substrates of E3 ligase CRL4-Cereblon in hematopoietic malignancies, the cell-type specific mechanisms of these molecules have not been extensively explored, especially in the immune context. As a result, there are a number of unknowns regarding the efficacy and safety of TPD therapeutics derived from these molecules. Here, we employed complex phenotypic assays to better understand the on-target mechanisms of this class of drugs. 

CRISPR scanning was used to broadly survey the landscape of drug resistance mutations to molecular glue degraders in neosubstrates. Integrative analysis of resistance sites revealed varying levels of sequence conservation and mutational constraint that control the emergence of different resistance mechanisms, highlighting that many regions co-opted in targeted protein degradation are inessential. Altogether, we outline a rapid and general approach to survey neosubstrate requirements necessary for effective degradation.

Clinically used as an ABL inhibitor, dasatinib promiscuously targets other tyrosine kinases, especially those in the SRC family. We developed a series of dasatinib-based PROTAC molecules, using phenyl-glutarimide as the cereblon binder. These SRC-directed PROTACs exhibited remarkable activity in LCK-activated T cell leukemia, in vitro, and in vivo. Because SRC family kinases are important therapeutic targets in cancer, we are evaluating these PROTACs systematically in ~1,000 tumors across histologic types.

Protein members of nucleosome remodeling complexes are emerging therapeutic targets. One protein of interest, the Bromodomain PHD Finger Transcription Factor, BPTF, is an essential member of the human nucleosome remodeling factor NURF. In recent years years, BPTF has become increasingly identified as a pro-tumorigenic factor, prompting investigations into the molecular mechanisms associated with BPTF function. Despite a druggable bromodomain small molecule discovery is at an early stage. Our lab has developed novel screening approaches including protein-based 19F NMR to develop both the first inhibitor of the BPTF bromodomain, and now more potent and selective chemical probes. Building on these results I will present our efforts at developing the first BPTF degraders to study the role of this protein in pediatric cancers.