Chemical probes are critical tools for measuring target engagement and elucidating the biological functions of proteins that can lead to identifying prospective therapeutic agents. Establishing a relationship between proximal (target engagement) and distal (functional inhibition) biomarkers early in preclinical studies can play an important role in better understanding an inhibitor’s mechanism of action and its potential efficacy in the clinic. A suite of chemical probes was utilized in establishing the relationship between target engagement and functional inhibition for several BTK inhibitors both in vitro and in vivo.  

We propose a novel use of α-methylene-β-lactone (MeLac) as a chemical probe warhead with broad reactivity. MeLac is reactive to thiol (Cys), hydroxyl (Ser, Thr, and Tyr), and amino (Lys) groups on multiple amino acid residues. Therefore, MeLac chemical probe can label various enzyme active sites, enable rapid large-scale activity profiling of proteins in diverse classes, and ultimately accelerate the development of efficacious medicine.

Target2035 is a recently launched initiative where a global federation of chemical biologists is joining forces to deliver chemical probes for the entire druggable human proteome by the year 2035. In a pilot project, the Structural Genomics Consortium, Toronto, is testing AI-based hit-generation technologies with a focus on WDR-containing proteins, interaction hubs often found in E3 ligase complexes. This global project, as well as one of our most recent chemical probes targeting the histone methyltransferase NSD2 will be presented.

Approach to evaluate the ability of E3-ligase components for neo-substrate degradation. Bypassing hit finding to identify specific E3-ligase binders, this approach makes use of maleimide-thiol chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). Applying COFFEE to SPSB2, a CRL5 receptor as well as to SKP1, the adaptor protein for CRL1-SCF complexes, we validate this method to define the activity of previously uncharacterized ubiquitin ligase components.

Kinases are attractive drug targets amenable to small molecule inhibition and targeted degradation. Despite this, approximately 1/4 of the kinome remains unannotated in terms of both function and disease association. In this study, we used a chemoproteomic map of the degradable kinome to identify leads for understudied kinases, and present the optimization of one of these hits into the first degraders of the understudied kinase NEK9.

PROTACs induce formation of a ternary complex involving a protein of interest and an E3 ligase that is bridged by the degrader. Gaining structural insights from ternary complexes is challenging due to the dynamic nature of the ternary complex. We present a novel approach that combines Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) with novel computational algorithms (such as protein-protein docking or weighted ensemble molecular dynamics simulations) to obtain atomic resolution models of the ternary complex.

AssayQuant® has combined chelation-enhanced fluorescence with high-throughput peptide synthesis to create optimized physiological substrate sensors to monitor the activity of protein kinases and phosphatases. The result is a simple add & read method for continuous, quantitative and direct detection of activity using recombinant enzymes or crude lysates. A modified approach allows continuous monitoring of lipid kinases (DGKs). These enabling tools and services provide a quantum improvement to accelerate nextgen drug development.

Phenotypic screens present a unique opportunity to uncover biological tools, novel biology, and discover druggable targets.  At AbbVie, a combination of phenotypic and target-based screening strategies is in place to augment our early discovery pipeline. In this presentation, I will highlight recent phenotypic screens conducted using disease relevant cellular models to identify and validate novel targets and mechanisms of action.  Using an integrated approach of cell based and target based screening, we successfully progressed these targets to the next step in small molecule drug discovery.

Development of biologic drugs including biosimilars and biobetters require rigorous bioanalytical methods for comparability, efficacy, stability, and potency testing. These often demand a complex set of cell-based assay that have proved to be quite challenging to develop. Eurofins DiscoverX addresses this challenge with off-the-shelf, ready-to-use cell-based bioassays. Here we show how these qualified, fit-for-purpose, automatable assays save at least 6-9 months of assay development time, accelerating a path to regulatory submission.

Identification of the molecular target is a key step towards the understanding of MoA of hit compounds that not only allows a more informed safety assessment but can also initiate structure-based drug designs. Here in, we provide our comprehensive chemical biology tactics involving chemical probe design, photoaffinity labeling, target identification, validation, engagement (CETSA) and identifying the binding site of the cellular target in the apolipoprotein E (apoE) phenotypic program. Our findings highlight the power of chemical biology and quantitative chemical proteomic approaches for target deconvolution of a phenotypic screening hit.

Epitranscriptomic RNA modifications play important roles in biology; however, there remains a major gap in our understanding of the scope, regulation, and function of these modifications in biological systems. Here, we present RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy to study and target RNA-modifying enzymes in their native context, and discuss its application to elucidate novel epitranscriptomic pathways for eukaryotic gene regulation.  

Recent work in chemoproteomics has enabled early drug discovery efforts on targets previously considered "undruggable." Here, we highlight these methods for target validation and the advancement of small-molecule therapeutics to generate precision oncology medicines.

Selinexor, a covalent XPO1 inhibitor, is approved in the U.S. in combination with dexamethasone for penta-refractory multiple myeloma. In this talk we will describe clickable probes based on XPO1 inhibitors selinexor and eltanexor for the labeling of XPO1 in live cells to assess target engagement and selectivity. We use mass spectrometry-based chemoproteomic workflows to profile the proteome-wide selectivity of selinexor and eltanexor and show that they are selective for XPO1.

Human cell microarray screening enables the discovery of specific primary cell surface receptors (i.e., druggable targets) as well as uncovering off-targets for a variety of biotherapeutic molecules, including antibodies, proteins, CAR T and other cell therapies. Case studies will focus on the assessment of off-target liabilities at a preclinical stage, and highlight the increasing role of the technology as a compliment to tissue cross reactivity studies prior to IND submissions.