Discovery of Nanomolar DCAF1 Small Molecule Ligands.

DCAF1 is a substrate receptor of two distinct E3 ligases (CRL4DCAF1 and EDVP), plays a critical physiological role in protein degradation, and is considered a drug target for various cancers. Antagonists of DCAF1 could be used toward the development of therapeutics for cancers and viral treatments. We used the WDR domain of DCAF1 to screen a 114-billion-compound DNA encoded library (DEL) and identified candidate compounds using similarity search and machine learning. This led to the discovery of a compound (Z1391232269) with an SPR KD of 11 µM. Structure-guided hit optimization led to the discovery of OICR-8268 (26e) with an SPR KD of 38 nM and cellular target engagement with EC50 of 10 µM as measured by cellular thermal shift assay (CETSA). OICR-8268 is an excellent tool compound to enable the development of next-generation DCAF1 ligands toward cancer therapeutics, further investigation of DCAF1 functions in cells, and the development of DCAF1-based PROTACs.

Effects of temperature on transjunctional voltage-dependent gating kinetics in Cx45 and Cx40 gap junction channels.

Gap junctions (GJs) are intercellular channels directly linking neighbouring cells and are dodecamers of connexins. In the human heart, connexin40 (Cx40), Cx43, and Cx45 are expressed in different regions of the heart forming GJs ensuring rapid propagation of action potentials in the myocardium. Two of these connexins, Cx40 and Cx45, formed functional GJs with prominent transjunctional voltage-dependent gating (Vj-gating), which can be a mechanism to down regulate coupling conductance (Gj). It is not clear the effects of temperature on Vj-gating properties. We expressed Cx40 or Cx45 in N2A cells to study the Vj-gating extent, the kinetics of deactivation, and the recovery time course from deactivation at 22 °C, 28 °C, and 32 °C. Dynamic uncoupling between cell pairs were evaluated at different temperatures, junctional delays, and/or repeating frequencies. Cx40 or Cx45 GJs showed little changes in the extent of Vj-gating, but in both cases with a faster deactivation kinetics at high temperatures. The recovery from deactivation was faster at higher temperatures for Cx45 GJs, but not for Cx40 GJs. Cx45 GJs, but not Cx40 GJs, were dynamically uncoupled when sufficient junctional delays and/or repeating frequency in all tested temperatures. Gap junction specific dynamic uncoupling could play an important role in regulating action potential propagation speed in Cx45 enriched nodal cells in the heart.

Functional Characterization of Novel Atrial Fibrillation-Linked GJA5 (Cx40) Mutants.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Recently, four novel heterozygous Cx40 mutations-K107R, L223M, Q236H, and I257L-were identified in 4 of 310 unrelated AF patients and a followup genetic analysis of the mutant carriers' families showed that the mutants were present in all the affected members. To study possible alterations associated with these Cx40 mutants, including their cellular localization and gap junction (GJ) function, we expressed GFP-tagged and untagged mutants in connexin-deficient model cells. All four Cx40 mutants showed clustered localization at cell-cell junctions similar to that observed of wildtype Cx40. However, cell pairs expressing Cx40 Q236H, but not the other individual mutants, displayed a significantly lower GJ coupling conductance (Gj) than wildtype Cx40. Similarly, co-expression of Cx40 Q236H with Cx43 resulted in a significantly lower Gj. Transjunctional voltage-dependent gating (Vj gating) properties were also altered in the GJs formed by Q236H. Reduced GJ function and altered Vj gating may play a role in promoting the Q236H carriers to AF.