Intrinsically Disordered Regions in the Transcription Factor MYC:MAX Modulate DNA Binding via Intramolecular Interactions.

The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor (TF) MYC is in large part an intrinsically disordered oncoprotein. In complex with its obligate heterodimerization partner MAX, MYC preferentially binds E-Box DNA sequences (CANNTG). At promoters containing these sequence motifs, MYC controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. A vast network of proteins in turn regulates MYC function via intermolecular interactions. In this work, we establish another layer of MYC regulation by intramolecular interactions. We used nuclear magnetic resonance (NMR) spectroscopy to identify and map multiple binding sites for the C-terminal MYC:MAX DNA-binding domain (DBD) on the intrinsically disordered regions (IDRs) in the MYC N-terminus. We find that these binding events in trans are driven by electrostatic attraction, that they have distinct affinities, and that they are competitive with DNA binding. Thereby, we observe the strongest effects for the N-terminal MYC box 0 (Mb0), a conserved motif involved in MYC transactivation and target gene induction. We prepared recombinant full-length MYC:MAX complex and demonstrate that the interactions identified in this work are also relevant in cis, i.e., as intramolecular interactions. These findings are supported by surface plasmon resonance (SPR) experiments, which revealed that intramolecular IDR:DBD interactions in MYC decelerate the association of MYC:MAX complexes to DNA. Our work offers new insights into how bHLH-LZ TFs are regulated by intramolecular interactions, which open up new possibilities for drug discovery.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.