Gulkis Mitchell C, Hodgkinson James T, Sele Céleste P, Knecht Wolfgang, McKenna Robert, Fisher S Zoë
Department of Biochemistry and Molecular Biology, University of Florida, 1149 Newell Drive, Gainesville, FL 32610, USA.
Leicester Institute of Structural and Chemical Biology and School of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
Acta Crystallogr F Struct Biol Commun. 2025 Sep 1;81(Pt 9):388-397. doi: 10.1107/S2053230X25007447. Epub 2025 Aug 26.
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 Zn. 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.
组蛋白去乙酰化酶抑制剂（HDACi）在癌症治疗中被广泛应用，但由于其对锌依赖性酶的泛抑制活性，常常存在脱靶效应。伏立诺他（SAHA）是一种基于异羟肟酸的HDACi，已被证明缺乏亚型选择性，这可能导致其与其他金属酶发生意外相互作用。在此，我们报告了SAHA与人碳酸酐酶II（CA II）及一种碳酸酐酶IX（CA IX）活性位点模拟物结合的高分辨率晶体结构。在室温及100 K下测定的结构显示，在CA II和CA IX模拟物中SAHA均有两种不同的构象，异羟肟酸部分取代了与锌结合的水，并与锌形成四面体或五面体配位。由于这两种酶之间存在F131V氨基酸差异，在CA II和CA IX模拟物之间观察到了疏水相互作用的差异。SwissDock建模准确预测了晶体学中观察到的SAHA结合方向。使用纳米差示扫描荧光法（nanoDSF）进行的热迁移分析表明，与强效CA抑制剂乙酰唑胺相比，SAHA对这两种CA的稳定作用极小。结合能计算表明，SAHA可能以与其HDAC靶点相当的亲和力结合碳酸酐酶。这些发现突出了SAHA与碳酸酐酶潜在的脱靶结合，这可能是其临床副作用的原因之一。结果还表明，异羟肟酸可能作为新型CA抑制剂的非磺酰胺支架，尽管亚型选择性仍然是一个挑战。
