Harnessing Phosphorus Fluoride Exchange (PFEx) Click Chemistry for Developing Covalent Anti-Cancer Drugs

Abstract Covalency is known to enhance both the potency and selectivity of anti-cancer drugs. Covalent drugs are equipped with a mildly reactive functional group, often referred to as a "warhead," which can form a covalent bond with the target upon protein-drug interaction. The covalent bond formation provides additional affinity beyond non-covalent interactions, leading to increased potency. Selecting the appropriate warhead is critical in the design of covalent drugs. These warheads must strike a perfect balance between stability and reactivity. They need to be stable enough during drug transportation and yet reactive enough upon drug-target interaction to generate specific covalent linkages between drug molecules and their targets. Current covalent anti-cancer drugs mainly rely on α, β-unsaturated carbonyls, which predominantly target cysteine residue. This chemical property of α, β-unsaturated carbonyls imposes strict restrictions on target selection and many protein targets lack a reactive cysteine in or near the binding pocket, greatly limiting the advance of covalent drug discovery. To address this issue, we propose to utilize Phosphorus Fluoride Exchange (PFEx), a new generation of click chemistry, to design novel privileged covalent warheads with expanded target scopes. In preliminary studies, we demonstrated that PFEx could proceed swiftly under biological conditions, and PFEx warheads, such as aryl phosphoramidofluoridate, can react with Lys, His, and Tyr to afford stable covalent bonds. Additionally, we showed aryl phosphoramidofluoridate exhibited low cytotoxicity. These results highlight the potential of PFEx in covalent drug design. In the next two years, we will: 1) synthesize PFEx warheads with different substitution groups to fine-tune the stability and reactivity of PFEx warheads; 2) compare the stability and reactivity of PFEx warheads with α, β-unsaturated carbonyls warheads; 3) test the cytotoxicity and off-target cross-linking of PFEx covalent warheads. Overall, the proposed work will identify new covalent warheads with expanded target scopes, which will open up new avenues for covalent anti-cancer drug discovery.