Inhibition of Mandelate Racemase by Boron-Based Inhibitors: Different Binding Modes for Benzoxaboroles Versus Boronic Acids

Mandelate racemase (MR) catalyzes the Mg²⁺-dependent interconversion of (R)- and (S)-mandelate and has been employed as a model enzyme to demonstrate that an enzyme catalyzing the deprotonation of a carbon acid substrate may be inhibited by boronic acids. We report a detailed structure-activity-based study of the ability of various boronic acid derivatives to competitively inhibit MR. 2-Naphthylboronic acid (K_i = 0.32 ± 0.01 μM), furan-3-boronic acid (K_i = 10 ± 1 μM), and thiophene-3-boronic acid (K_i = 1.27 ± 0.06 μM) were potent inhibitors of MR, while 1-naphthylboronic acid (K_i = 28 ± 3 μM) and nitrogen-heterocycles (e.g., isoxazole, indole, 1H-indazole, pyridine, and pyrimidine) bearing boronic acid groups were generally weaker inhibitors. A chlorine substituent on the pyridine (i.e., 2-chloro-pyridine-5-boronic or 2-chloro-pyridine-4-boronic acids) or pyrimidine (i.e., 2-chloro-pyrimidine-5-boronic acid) ring enhanced the binding affinity by 3- to 27-fold. Surprisingly, benzoxaboroles, including the Antifungal agent tavaborole (i.e., 5-fluorobenzoxaborole, K_i = 1.06 ± 0.09 μM), were also potent competitive inhibitors of MR. The pH-dependence of the inhibition by benzoxaborole suggested that the species with the tetrahedral, sp³-hybridized boron atom was the more potent inhibitor. Interestingly, ¹¹B NMR spectroscopy and X-ray crystallography revealed that aryl boronic acids and benzoxaboroles interact with MR via different binding modes. Unlike phenylboronic acid, which forms an N^ε2-B bond with His 297 at the active site, the 1.8-Å resolution structure of the MR-tavaborole adduct revealed the presence of an N^ζ-B bond between the bound tavaborole and Lys 166 at the active site.