| dc.description.abstract |
Covalent protein kinase inhibitors have firmly established themselves as a powerful therapeutic modality, with most efforts traditionally focused on targeting reactive cysteine residues. However, many kinases lack suitably positioned cysteines, limiting the reach of current covalent strategies. Motivated by this gap, this dissertation explores covalent inhibition beyond cysteine by leveraging underexplored nucleophilic residues – specifically tyrosines and lysines.
Initial efforts applied sulfur(VI) fluoride chemistry to target tyrosines through structure-based design, using the kinases JAK3 and MK2 as model systems. Tailored analogs bearing sulfonyl fluoride and fluorosulfate warheads were designed and synthesized to engage specific tyrosines. While intact protein mass spectrometry confirmed covalent modification in some cases, functional inhibition often proved elusive. In one striking example, a fluorosulfate designed to target a tyrosine in MK2 instead modified the “catalytic” lysine, highlighting the unpredictable yet promising reactivity of this warhead class.
In light of the challenges encountered using rational design alone, a covalent-first approach was adopted. A focused electrophilic library featuring diverse hinge binders, linker architectures, and S(VI)F warheads was screened against kinases with tyrosines in privileged structural positions. This led to the identification of several novel covalent binders, most notably a fluorosulfate that modified Lys338 (HRD+2) of ALK2, a kinase involved in fibrodysplasia ossificans progressiva (FOP). This represents the first reported covalent ligand for ALK2, which lacks a ligandable cysteine in its active site and has remained inaccessible to cysteine-focused strategies.
Further structural and kinetic studies on this hit revealed key structure–activity relationships. Variations to the hinge binder notably affected inactivation rates – e.g., replacing azaindole with aminopyridine slowed down the modification – while linker changes had minor impact. As expected, a sulfonyl fluoride showed greater reactivity than fluorosulfates, which translated into faster target modification. Substituents on the aryl ring bearing the warhead also influenced the rate of target inactivation: electron-withdrawing groups enhanced it, electron-donating groups reduced it, but steric bulk could offset these effects, underscoring the balance between reactivity and spatial fit. Unexpectedly, several analogs, including the original fluorosulfate hit, exhibited an apparent ALK2 activation in biochemical assays. Though the mechanism remains unclear, these results suggest a complex structure–function relationship meriting further investigation.
Altogether, this work expands the landscape of covalent kinase inhibition by validating lysines as viable targets for S(VI)F chemistry, offering new avenues beyond cysteine and enabling covalent targeting of kinases previously considered inaccessible to covalent drug modalities. |
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