From lin‐Benzoguanines to lin‐Benzohypoxanthines as Ligands for Zymomonas mobilis tRNA–Guanine Transglycosylase: Replacement of Protein–Ligand Hydrogen Bonding by Importing Water Clusters

Abstract

The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of Shigella flexneri. Herein, the molecular recognition properties of a guanine binding pocket in Zymomonas mobilis TGT are investigated with a series of lin-benzohypoxanthine- and lin-benzoguanine-based inhibitors that bear substituents to occupy either the ribose-33 or the ribose-34 pocket. The three inhibitor scaffolds differ by the substituent at C(6) being H, NH(2), or NH-alkyl. These differences lead to major changes in the inhibition constants, pK(a) values, and binding modes. Compared to the lin-benzoguanines, with an exocyclic NH(2) at C(6), the lin-benzohypoxanthines without an exocyclic NH(2) group have a weaker affinity as several ionic protein-ligand hydrogen bonds are lost. X-ray cocrystal structure analysis reveals that a new water cluster is imported into the space vacated by the lacking NH(2) group and by a conformational shift of the side chain of catalytic Asp102. In the presence of an N-alkyl group at C(6) in lin-benzoguanine ligands, this water cluster is largely maintained but replacement of one of the water molecules in the cluster leads to a substantial loss in binding affinity. This study provides new insight into the role of water clusters at enzyme active sites and their challenging substitution by ligand parts, a topic of general interest in contemporary structure-based drug design.

Chasing Protons: How Isothermal Titration Calorimetry, Mutagenesis, and pKa Calculations Trace the Locus of Charge in Ligand Binding to a tRNA-Binding Enzyme

“…A hydrogen bond is formed to a nearby water molecule (2.8 Å) and the backbone NH of Thr71 (2.7 Å). 20 As expected, the binding affinity of 2 determined by ITC measurements drops for both mutant variants as favorable interactions are lost in both cases. For the Asp156Asn mutant, a reduction by a factor of nearly four (wild type, 49 ± 5 nM; Surprisingly, 2 does not show any significant buffer dependency upon binding to the two mutants (Table 1).…”

“…A newly published crystal structure of Z. mobilis TGT in complex with 2-amino-lin-benzohypoxanthine-type inhibitors shows that Asp102 adopts a significantly different orientation in the complexes compared to the corresponding lin-benzoguanine complexes. 20 This residue is rotated away from the ligand toward the ribose-34 subpocket as similarly found in the apo enyzme. 20,25 In the new position, Asp102 forms an interaction with the carboxamide group of Asn70 and the backbone NH of Thr71.…”

“…Both the lin-benzoguanines and the lin-benzohypoxanthines show unsatisfactory membrane permeation in PAMPA measurements. 20 This study demonstrates the importance to analyze the protonation properties of structurally closely related ligands. ITC titrations, performed in different buffer conditions, allow elucidating the net protonation inventory and stoichiometry.…”

“…20,24 Unfortunately, no values are available for the morpholinoethyl substituent in 1; we therefore assume a value similar to that of parent morpholinoethyl. The value for the latter has been reported to be pK a = 7.7.…”

Nexus Between Protein–Ligand Affinity Rank-Ordering, Biophysical Approaches, and Drug Discovery

“…8 Recent experimental studies, employing highresolution X-ray crystallography, on a series of complexes of tRNA-guanine transglycosylase with lin-benzoguanines and linbenzohypoxanthines revealed the presence of a water cluster in the active site, whose specific structure was strongly tied to the observed inhibition constants for this series. 9 A similar idiosyncratic importation of water clusters has now been documented for glutamate racemase, where experimental and computational work indicated a wide range of ligand-specific water-mediated contacts in a buried polar active site. 10 MD-based sampling and experimental biophysical methods are being employed in increasingly synergistic ways to benefit SBDD, but the full potential will be only be realized when and if these methods are more routinely integrated into drug discovery work flows.…”