Development and Characterization of Selective FAK Inhibitors and PROTACs with In Vivo Activity.

Focal adhesion kinase (FAK) is an attractive drug target due to its overexpression in cancer. FAK functions as a non-receptor tyrosine kinase and scaffolding protein, coordinating several downstream signaling effectors and cellular processes. While drug discovery efforts have largely focused on targeting FAK kinase activity, FAK inhibitors have failed to show efficacy as single agents in clinical trials. Here, using structure-guided design, we report the development of a selective FAK inhibitor (BSJ-04-175) and degrader (BSJ-04-146) to evaluate the consequences and advantages of abolishing all FAK activity in cancer models. BSJ-04-146 achieves rapid and potent FAK degradation with high proteome-wide specificity in cancer cells and induces durable degradation in mice. Compared to kinase inhibition, targeted degradation of FAK exhibits pronounced improved activity on downstream signaling and cancer cell viability and migration. Together, BSJ-04-175 and BSJ-04-146 are valuable chemical tools to dissect the specific consequences of targeting FAK through small-molecule inhibition or degradation.

Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Structural basis for the transport and regulation mechanism of the Multidrug resistance-associated protein 2.

Multidrug resistance-associated protein 2 (MRP2) is an ATP-powered exporter important for maintaining liver homeostasis and a potential contributor to chemotherapeutic resistance. Deficiencies in MRP2 function are associated with Dubin-Johnson Syndrome and increased vulnerability to liver injury from cytotoxic drugs. Using cryogenic electron microscopy (cryo-EM), we determined the structures of human MRP2 in three conformational states: an autoinhibited state, a substrate-bound pre-translocation state, and an ATP-bound post-translocation state. These structures show that MRP2 functions through the classic alternating access model, driven by ATP binding and hydrolysis. Its cytosolic regulatory (R) domain serves as a selectivity gauge, wherein only sufficiently high concentrations of substrates can effectively compete with and disengage the R domain to initiate transport. Comparative structural analyses of MRP2 in complex with different substrates reveal how the transporter recognizes a diverse array of compounds, highlighting the transporter's role in multidrug resistance.

Exploring the target scope of KEAP1 E3 ligase-based PROTACs.

Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.

Discovery of a Pyrimidothiazolodiazepinone as a Potent and Selective Focal Adhesion Kinase (FAK) Inhibitor.

Focal adhesion kinase (FAK) is a tyrosine kinase with prominent roles in protein scaffolding, migration, angiogenesis, and anchorage-independent cell survival and is an attractive target for the development of cancer therapeutics. However, current FAK inhibitors display dual kinase inhibition and/or significant activity on several kinases. Although multitargeted activity is at times therapeutically advantageous, such behavior can also lead to toxicity and confound chemical-biology studies. We report a novel series of small molecules based on a tricyclic pyrimidothiazolodiazepinone core that displays both high potency and selectivity for FAK. Structure-activity relationship (SAR) studies explored modifications to the thiazole, diazepinone, and aniline "tail," which identified lead compound BJG-03-025. BJG-03-025 displays potent biochemical FAK inhibition (IC50 = 20 nM), excellent kinome selectivity, activity in 3D-culture breast and gastric cancer models, and favorable pharmacokinetic properties in mice. BJG-03-025 is a valuable chemical probe for evaluation of FAK-dependent biology.