Carbonneau, S. | Search bioRxiv

bioRxiv2026

Scalable platform for cellular and biochemical screening of combinatorial small-molecule libraries in droplets

A new pattern of hit discovery is emerging with the rise of chemocentric modalities, such as chemical induced proximity (CIP). Synthesis and functional screening of dynamic, purpose-built libraries, such as E3 ligase biased libraries for targeted protein degradation (TPD), is redefining the druggable landscape. However, this new paradigm arguably lies beyond the remit of conventional screening methods which have been optimized for static, diversity-oriented libraries. To address this gap, we developed microfluidic compound screening in droplets (MicDrop), a scalable platform where purpose-built DNA-encoded one-bead one-compound library members are individually tested at high concentrations in pico-liter sized droplets for either biochemical or cellular function. A proof-of-concept CRBN library demonstrated the robustness of the workflow through an IKZF3 degradation screen which discovered an unexpected succinimide-based IKZF3 degrader, while reproducing the relative potency ranking of reference compounds. Screening of a prospective VHL library for CDO1 recruitment and degradation demonstrated that orthogonal assays gave an informative consensus hit list and an actionable machine-learning (ML) recruitment model. These findings establish a blueprint for intentional discovery of chemical inducers of proximity, while producing reliable data to accelerate ML-based design-make-test-analyze (DMTA) cycles in pursuit of new therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/730915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@12de3cborg.highwire.dtl.DTLVardef@1c6618dorg.highwire.dtl.DTLVardef@12ea42eorg.highwire.dtl.DTLVardef@11cea77_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell Biology↗

bioRxiv2023

Reinstating targeted protein degradation with DCAF1 PROTACs in CRBN PROTAC resistant settings

Targeted protein degradation (TPD) of neo-substrates with proteolysis targeting chimeras (PROTACs) or molecular glues has emerged as a key modality in exploring new biology as well as designing new drug candidates where catalytic inhibition is neither efficacious nor an option. TPD is mediated through harnessing E3 ligases and redirecting them to ubiquitinate de novo target proteins for subsequent proteasomal degradation. Until recently, E3 ligase chemical matter available for mediating TPD has been limited to a relatively low number of ligases, considering that over 600 E3 ligases are encoded by the human genome. In addition, the most utilized ligase for TPD approaches, CRBN, has been observed to be downregulated in settings of acquired resistance to immunomodulatory inhibitory drugs (IMiDs). IMiDs are molecular glues that target IKZF transcription factors to CRBN for degradation. Resistance is potentially accelerated by non-essentiality of CRBN for cell viability. Here we investigated if the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a potent, non-covalent DCAF1 binder. We show that this binder, selective for the CRL4DCAF1 E3 ligase complex, can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments confirm specific degradation via the CRL4DCAF1 E3 ligase. We further highlight the versatility of DCAF1 for TPD by developing a DCAF1-dasatininb PROTAC targeting multiple cytosolic and membrane bound tyrosine kinases. We expand these findings towards Brutons tyrosine kinase (BTK) selective PROTACs and through extensive optimization and characterization efforts share key observations that led to a potent and selective DCAF1-BTK PROTAC (DBt-10). Finally, with this PROTAC DBt-10, we show rescue of BTK degradation in a BTK-dependent, CRBN-degradation-resistant cell line and provide a rationale for E3 ligase swap to overcome CRBN mediated resistance.

biochemistry↗