Efficient Targeted Degradation via Reversible and Irreversible Covalent PROTACs.

Proteolysis targeting chimeras (PROTACs) represent an exciting inhibitory modality with many advantages, including substoichiometric degradation of targets. Their scope, though, is still limited to date by the requirement for a sufficiently potent target binder. A solution that proved useful in tackling challenging targets is the use of electrophiles to allow irreversible binding to the target. However, such binding will negate the catalytic nature of PROTACs. Reversible covalent PROTACs potentially offer the best of both worlds. They possess the potency and selectivity associated with the formation of the covalent bond, while being able to dissociate and regenerate once the protein target is degraded. Using Bruton's tyrosine kinase (BTK) as a clinically relevant model system, we show efficient degradation by noncovalent, irreversible covalent, and reversible covalent PROTACs, with <10 nM DC50's and >85% degradation. Our data suggest that part of the degradation by our irreversible covalent PROTACs is driven by reversible binding prior to covalent bond formation, while the reversible covalent PROTACs drive degradation primarily by covalent engagement. The PROTACs showed enhanced inhibition of B cell activation compared to ibrutinib and exhibit potent degradation of BTK in patient-derived primary chronic lymphocytic leukemia cells. The most potent reversible covalent PROTAC, RC-3, exhibited enhanced selectivity toward BTK compared to noncovalent and irreversible covalent PROTACs. These compounds may pave the way for the design of covalent PROTACs for a wide variety of challenging targets.

Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli.

As a large number of cancers are caused by nonsense mutations in key genes, read-through of these mutations to restore full-length protein expression is a potential therapeutic strategy. Mutations in the adenomatous polyposis coli (APC) gene initiate the majority of both sporadic and hereditary colorectal cancers (CRC) and around 30% of these mutations are nonsense mutations. Our goal was to test the feasibility and effectiveness of APC nonsense mutation read-through as a potential chemo-preventive therapy in Familial Adenomatous Polyposis (FAP), an inherited CRC syndrome patients. Ten FAP patients harboring APC nonsense mutations were treated with the read-through inducing antibiotic erythromycin for 4 months. Endoscopic assessment of the adenomas was performed at baseline, after 4 and after 12 months. Adenoma burden was documented in terms of adenoma number, maximal polyp size and cumulative polyp size per procedure. Tissue samples were collected and subjected to molecular and genetic analyses. Our results show that in the majority of patients the treatment led to a decrease in cumulative adenoma burden, median reduction in cumulative adenoma size and median reduction in adenoma number. Molecular and genetic analyses of the adenomas revealed that the treatment led to a reduced number of somatic APC mutations, reduced cellular proliferation and restoration of APC tumor-suppressing activity. Together, our findings show that induced read-through of APC nonsense mutations leads to promising clinical results and should be further investigated to establish its therapeutic potential in FAP and sporadic CRCs harboring nonsense APC mutations.

Serum starvation enhances nonsense mutation readthrough.

Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. KEY MESSAGES: Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.