Rational design of a beta-lactamase inhibitor achieved via stabilization of the trans-enamine intermediate: 1.28 A crystal structure of wt SHV-1 complex with a penam sulfone

Abstract

beta-Lactamases are one of the major causes of antibiotic resistance in Gram negative bacteria. The continuing evolution of beta-lactamases that are capable of hydrolyzing our most potent beta-lactams presents a vexing clinical problem, in particular since a number of them are resistant to inhibitors. The efficient inhibition of these enzymes is therefore of great clinical importance. Building upon our previous structural studies that examined tazobactam trapped as a trans-enamine intermediate in a deacylation deficient SHV variant, we designed a novel penam sulfone derivative that forms a more stable trans-enamine intermediate. We report here the 1.28 A resolution crystal structure of wt SHV-1 in complex with a rationally designed penam sulfone, SA2-13. The compound is covalently bound to the active site of wt SHV-1 similar to tazobactam yet forms an additional salt-bridge with K234 and hydrogen bonds with S130 and T235 to stabilize the trans-enamine intermediate. Kinetic measurements show that SA2-13, once reacted with SHV-1 beta-lactamase, is about 10-fold slower at being released from the enzyme compared to tazobactam. Stabilizing the trans-enamine intermediate represents a novel strategy for the rational design of mechanism-based class A beta-lactamase inhibitors.

Figure 1

a. Proposed reaction scheme for inhibitors with class A β-lactamases. The transient inactivation of SHV-1 involves the trans-enamine intermediate which energetically is more favorable than the cis-enamine intermediate. To escape the transient inhibition, the reaction needs to proceed back to the imine intermediate before it can continue and result in either the reactivation of the active SHV-1 enzyme or irreversible inactivation. The k_react measured corresponds to: $$\frac{(k_{\mathrm{inact}}+k_{\mathrm{deacyl}})(k_{\mathrm{c}\to \mathrm{i}})(k_{\mathrm{t}\to \mathrm{c}})}{k_{\mathrm{c}\to \mathrm{t}}{k}_{\mathrm{i}\to \mathrm{c}}+(k_{\mathrm{c}\to \mathrm{t}}+k_{\mathrm{c}\to \mathrm{i}})(k_{\mathrm{inact}}+k_{\mathrm{deacyl}})}$$ b. Schematic diagram of SA2-13 and tazobactam.

Figure 1

a. Proposed reaction scheme for inhibitors with class A β-lactamases. The transient inactivation of SHV-1 involves the trans-enamine intermediate which energetically is more favorable than the cis-enamine intermediate. To escape the transient inhibition, the reaction needs to proceed back to the imine intermediate before it can continue and result in either the reactivation of the active SHV-1 enzyme or irreversible inactivation. The k_react measured corresponds to: $$\frac{(k_{\mathrm{inact}}+k_{\mathrm{deacyl}})(k_{\mathrm{c}\to \mathrm{i}})(k_{\mathrm{t}\to \mathrm{c}})}{k_{\mathrm{c}\to \mathrm{t}}{k}_{\mathrm{i}\to \mathrm{c}}+(k_{\mathrm{c}\to \mathrm{t}}+k_{\mathrm{c}\to \mathrm{i}})(k_{\mathrm{inact}}+k_{\mathrm{deacyl}})}$$ b. Schematic diagram of SA2-13 and tazobactam.

Figure 2

Electron density of the SA2-13 compound bound in the active site of wt SHV-1 β-lactamase. Side-by-side stereo view of 1.28 Å resolution |Fo|-|Fc| omit electron density contoured at 2.5 σ is depicted in green. Atom types are color coded accordingly: oxygen (red), nitrogen (blue), sulfur (yellow), and carbon (light and dark grey for the protein and SA2-13, respectively). Waters labeled W1-4 are waters numbered 339A, 339B, 154, and 203, respectively, in the deposited coordinate set.

Figure 3

Stereo diagram depictuing the interactions of SA2-13 within the active site of SHV-1 β-lactamase. Hydrogen bonds are depicted as black dashed lines. Water molecule are highlighted as red spheres. Residue N170 has two alternative conformations (shown as #1 and #2). This causes the catalytic deacylation water, in close proximity to N170 and E166, to also have two alternate positions that pair with each of the N170 conformations (indicated as W1 and W2, respectively).

Figure 4

Schematic diagram depicting the interactions of SA2-13 when bound to wt SHV-1 β-lactamase. Hydrogen bonds are depicted as dashed lines (distances are shown in Å). Van der Waals interactions are shown as a curved line with perpendicular stripes. The double bond between atoms C5 and C6 is in the trans configuration which, together with the intramolecular hydrogen bond involving the N4 atom, identify the trans-enamine intermediate for the covalently bound SA2-13. Interactions and active site changes that might contribute to enhanced stabilization of the trans-enamine intermediate of SA2-13 are indicated by i-iv and are discussed in the text.

Figure 5

Overlay of SA2-13 and HEPES when bound to SHV-1. The active sites of the HEPES bound SHV-2 structure and that of our SA2-13 wt SHV-1 structure were superimposed. The figure shows the active site of wt SHV-1 depicting both SA2-13 (blue stick) and HEPES (yellow stick) illustrating the similar position of the sulfone+linker moiety of HEPES and that of the carboxyl+linker moiety of SA2-13.

Figure 6

Superposition of the SA2-13 and tazobactam bound structures. Shown are SA2-13 (blue) when bound to wt SHV-1 and tazobactam (magenta) when bound to the deacylation deficient E166A variant of SHV-1. Only the protein atoms of the wt SHV-1:SA2-13 complex are shown. The water molecule involved in forming a hydrogen bond (yellow dashed line) with the triazolyl moiety of tazobactam when complexed to the E166A SHV-1 variant is depicted as a red sphere.