Protein Electric Fields Enable Faster and Longer-Lasting Covalent Inhibition of β-Lactamases

Abstract

The widespread design of covalent drugs has focused on crafting reactive groups of proper electrophilicity and positioning toward targeted amino-acid nucleophiles. We found that environmental electric fields projected onto a reactive chemical bond, an overlooked design element, play essential roles in the covalent inhibition of TEM-1 β-lactamase by avibactam. Using the vibrational Stark effect, the magnitudes of the electric fields that are exerted by TEM active sites onto avibactam's reactive C═O were measured and demonstrate an electrostatic gating effect that promotes bond formation yet relatively suppresses the reverse dissociation. These results suggest new principles of covalent drug design and off-target site prediction. Unlike shape and electrostatic complementary which address binding constants, electrostatic catalysis drives reaction rates, essential for covalent inhibition, and deepens our understanding of chemical reactivity, selectivity, and stability in complex systems.

Figure 1.

Electrostatic catalysis applied to covalent inhibition. (a) Common considerations for covalent drug design, using a carbonyl warhead as an example. This work presents a new design principle: the electric fields that are produced by protein dipoles and charged groups and act on chemical bonds undergoing reactions. (b) Shape and electrostatic complementarity in contrast to electrostatic catalysis. The former considers all of the protein−ligand interactions and thus largely determines K_M or K_i; the latter focuses on the electric fields experienced by the reactive bond and therefore contributes to k_cat or k_on, as presented in this work.

Figure 2.

Reactions and key electrostatics in the active site of TEM β-lactamases. (a) Mechanism of acylation by PenG. The hydroxy residue of S70 acts as a nucleophile to attack PenG’s β-lactam C=O, forming an oxyanion intermediate before turning into an acyl-enzyme complex, which is subject to hydrolysis (not shown). (b) Mechanism of covalent inhibition by AVB. A similar nucleophilic attack of AVB’s urea C=O by S70 traps the enzyme in the carbamate complex without turnover. Recyclization, the reverse reaction of carbamylation, regenerates AVB, making it a reversible covalent inhibitor. Proton movement is omitted in the electron-pushing mechanism. (c) Model of electrostatic catalysis. The conversion of a C=O to an oxyanion intermediate passes through a transition state where charges are more separated between C and O atoms, generating a reaction difference dipole ($\Delta {\overrightarrow{\mu }}_{\text{rxn}}$), which interacts with the electric field in the enzyme active site $\overrightarrow{F}$ to reduce the free energy barrier by ΔΔG^‡. (d) H-bond donated by the A237 backbone amide, a key contributor to the enzyme electric field in β-lactamases (and many other enzymes), can be perturbed by the A237Y mutation and even removed by replacing the amide with an ester in A237Y^e.

Figure 3.

Electrostatic perturbation by A237 mutations measured using isotope-edited infrared spectroscopy. (a) Key active site residues in the WT and A237Y crystal structures of TEM−AVB (Figure S5). The two key H-bonds between the carbamate C=O and the backbone amides are highlighted as red dashed lines. The electron density maps of AVB (2mF_o − DF_c, 1.5σ) are depicted. (b) Substitution of a backbone amide to an ester by incorporation of p-hydroxy-l-phenyllactic acid (HPLA), the α-hydroxy acid counterpart of tyrosine, and the high-resolution MS of A237Y, A237Y^e, and the two protein fragments of hydrolyzed A237Y^e (Table S3). (c) ¹³C-Labeled AVB and PenG. (d, e) ¹²C−¹³C difference infrared absorption spectra for TEM·AVB (trapped by the S70G mutation) (d) and TEM−AVB (e). The experimental curve (black) is fitted to a sum (gray) of ¹²C (positive) and ¹³C (negative) peaks (curve fitting details in Tables S16 and S17). Peaks belonging to the same positive−negative pair are filled with the same color. (f−h) Fitted ¹²C peaks in infrared absorption spectra for WT and A237 mutants of TEM·AVB (f), TEM−AVB (g), and TEM·PenG (h). The top electric field axes are mapped from the bottom frequency axes based on calibration results as detailed in the Supporting Information.

Figure 4.

Role of electric fields in covalent inhibition and drug design. (a) Plot of free energy barrier (ΔG^‡) against the magnitude of electric fields (F) projected on the reactive C=O for AVB carbamylation, recyclization, and PenG acylation. Expressing ΔG^‡ in kcal/mol and F in units of kcal/mol/D (top axis), the linear regression lines are ΔG^‡ = 1.40F + 25.0 (AVB carbamylation), ΔG^‡ = 1.42F + 28.6 (AVB recyclization), and ΔG^‡ = 1.42F + 24.0 (PenG acylation). (b) Contribution of electrostatic catalysis, $\Delta \Delta G_{\Delta F}^{‡}$, to the ΔG^‡ gap between WT AVB carbamylation and recyclization. (c) Conceptual illustration of using two handles together—bond electrophilicity and environmental electric field—to tune the rate of covalent inhibition (illustrated by the shade of orange). Ideally, only the high-field target reaches high reactivity (filled in orange) while the lowfield, off-target sites remain under-reactive (unfilled).