Effectiveness of Tetrahedral Adducts as Transition-State Analogs and Inhibitors of the Class C β-Lactamase of Enterobacter cloacae P99

Abstract

Previous studies have shown that β-lactamases are inhibited by boronates and phosphonates, both of which form covalent tetrahedral adducts with the active-site serine residue. These have been interpreted, as have similar complexes formed with serine proteinases, as transition-state analog structures. Not all molecules capable of forming such tetrahedral adducts are good inhibitors of serine β-lactamases, however. In this paper, a series of molecules potentially capable of forming anionic tetrahedral adducts at the active site [PhCH2CONHCH2M(XY)-OSer] have been assessed as sources of transition-state analogs and as inhibitors of the class C β-lactamase of Enterobacter cloacae P99. It was found by experiment that the aldehyde, the silanetriol, and the α-keto acid (and its methyl ester) of the series were significantly poorer inhibitors than the structurally analogous boronate. This result was explored computationally. From the starting point of the crystal structure of a phosphonate adduct (Lobkovsky et al. Biochemistry 1994, 33, 6762), the various inhibitors were introduced into the active site and the complete structure relaxed by energy minimization in a force field. Tetrahedral structures derived from analogous substrates were similarly treated, and the final structures were analyzed both structurally and energetically. From the point of view of energy, it was found that the boronate (M = B, X = Y = OH), phosphonate (M = P, X = Y = O), and carbon substrate-derived analog (M = C, X = O, Y = OH) interacted comparably strongly in a noncovalent sense with the active-site residues, while the aldehyde (M = C, X = O, Y = H), silicate (M = Si, X = O, Y = OH), and α-keto acid derivatives (M = C, X = O, Y = COR) interacted more weakly. The order of energies of interaction between the tetrahedral ligands and the active site was shown to best correlate with the electrostatic interactions of the MXY- moiety with the two conserved lysine residues of the β-lactamase active site, here Lys 67 and Lys 315. There appeared to be no positive correlation between the interaction energy of X- with the oxyanion hole and the total interaction energy; the oxyanion hole therefore appears to contribute uniformly to the ligand binding but not to discrimination between ligands. There was, however, a correlation between the active site interaction energies and the interaction energy between MXY- and the H2(α2) helix dipole. The H2 helix may therefore contribute selectively toward catalysis and inhibition. The structures were interpreted in terms of the mechanism of Oefner et al. (Oefner et al. Nature (London) 1990, 343, 284). The relationship of the calculations to the measured inhibitory properties of the parent molecules is discussed as well as projections to further inhibitor design.

The Oxyanion Hole in Serine β-Lactamase Catalysis: Interactions of Thiono Substrates with the Active Site

“…This conformation will be henceforward referred to as Structure A (Scheme 2) and is represented below and shown for 5o in detail in Fig. 2 of the previous paper (36). A structure apparently very similar to A was also attained by 5s, the thiono analog of 5o (Table 2).…”

“…The final derivative of Although it is probable that longer range interactions might influence the absolute value of calculated interaction energies and conceivably even their relative values, the major aim of the present work was to examine the effects of the local environment and in particular the residues involved in catalysis. Previous results with the P99 ␤-lactamase indicated that long-range interactions did not discriminate between tetrahedral adducts (36). The Builder module of INSIGHT II was also used to construct substrate complexes 15 and 16 along a Bürgi-Dunitz trajectory (42,43).…”

“…In particular, it seemed that a neutral Tyr 150 hydroxyl group was hydrogen-bonded to the Ser 64 O ␥ oxygen (distance C 1 ) and to the Lys 67 ammonium ion (distance B). Energy minimization in the CV force field led, as described previously (36), to the movement of Tyr 150 to a position closer to a phosphonyl oxygen than Table 2 are indicated by letter-labeled dashed lines.…”

“…Recently we have used computational methods to judge the ability of tetrahedral anionic adducts to interact with the active site of the class C ␤-lactamase of Enterobacter cloacae P99 (36). As a logical extension of that work we present here a comparison between the series of oxo and thiono adducts 1-5 in order to obtain insight into any differences that there might be between the interactions of oxo and thiono species with this active site at the tetrahedral intermediate stage and thus further address the SCHEME 1 question of the molecular basis of the S test.…”

Matrix Metallo‐Proteinase (MMP‐2) Organoboronate Inhibitors

“…compounds containing both boronate and phosphonate groups, can serve as MMP-2 inhibitors, Pergament et al [13]. In addition, X-ray crystallography could show that boronates attach zinc to certain aminopeptidases [14]. This encouraged us to synthesize new organoborane derivatives and test them for possible synergistic * M. S. a and R. R. b are affiliated with the Bloom Center for Pharmacy at the Hebrew University.…”