Off-target binding of the histone deacetylase inhibitor vorinostat to carbonic anhydrase II and IX

Histone deacetylase inhibitors (HDACi) are widely used in cancer therapy but often suffer from off-target effects due to their pan-inhibitory activity towards zinc-dependent enzymes. Vorinostat (SAHA), a hydroxamate-based HDACi, has been shown to lack isoform selectivity, potentially leading to unintended interactions with other metalloenzymes. Here, we report high-resolution crystal structures of SAHA bound to human carbonic anhydrase II (CA II) and a carbonic anhydrase IX (CA IX) active-site mimic. Structures determined at room temperature and 100 K revealed two distinct SAHA conformers in both CA II and the CA IX mimic, with the hydroxamate moiety displacing the zinc-bound water and adopting either a tetrahedral or pentahedral coordination to Zn²⁺. Differences in hydrophobic interactions were observed between CA II and the CA IX mimic due to the F131V amino-acid difference between the two enzymes. SwissDock modeling accurately predicted the SAHA binding orientations observed in crystallography. Thermal shift assays using nanoDSF showed minimal stabilization of either CA by SAHA, in contrast to the potent CA inhibitor acetazolamide. Binding-energy calculations suggest that SAHA may bind carbonic anhydrases with affinities comparable to its HDAC targets. These findings highlight potential off-target binding of SAHA to carbonic anhydrases, which may contribute to its clinical side effects. The results also suggest that hydroxamates may serve as a nonsulfonamide scaffold for novel CA inhibitors, although isoform selectivity remains a challenge.