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 (<i>R</i>)- and (<i>S</i>)-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 (<i>K</i>_i = 0.32 ± 0.01 μM), furan-3-boronic acid (<i>K</i>_i = 10 ± 1 μM), and thiophene-3-boronic acid (<i>K</i>_i = 1.27 ± 0.06 μM) were potent inhibitors of MR, while 1-naphthylboronic acid (<i>K</i>_i = 28 ± 3 μM) and nitrogen-heterocycles (<i>e</i>.<i>g</i>., isoxazole, indole, 1<i>H</i>-indazole, pyridine, and pyrimidine) bearing boronic acid groups were generally weaker inhibitors. A chlorine substituent on the pyridine (<i>i</i>.<i>e</i>., 2-chloro-pyridine-5-boronic or 2-chloro-pyridine-4-boronic acids) or pyrimidine (<i>i</i>.<i>e</i>., 2-chloro-pyrimidine-5-boronic acid) ring enhanced the binding affinity by 3- to 27-fold. Surprisingly, benzoxaboroles, including the antifungal agent tavaborole (<i>i</i>.<i>e</i>., 5-fluorobenzoxaborole, <i>K</i>_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.