Non-classical transpeptidases yield insight into new antibacterials

Abstract

Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the β-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of β-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for β-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.

Figure 1. Interactions of β-lactams with mycobacterial l,d-transpeptidases and activity of biapenem and faropenem in the mouse model of TB

(a) Binding of faropenem to M. tuberculosis enzymes Ldt_Mt1 and Ldt_Mt2, and M. abscessus enzymes Ldt_Mab1 and Ldt_Mab2. In each panel, the top plot displays the titration of faropenem to enzyme, and the bottom panel displays the non-linear fit of heat exchange across increasing ligand:enzyme molar ratios. (b) Nitrocefin hydrolyzing activity of Ldt_Mt2 following incubation with various β-lactams. (c) Log₁₀ colony forming units of M. tuberculosis in the lungs of BALB/c mice before, during and after treatment with biapenem and faropenem alone or in combination with rifampin and control regimens. Data represent the mean (n = 5 mice per group per time point) and error bars represent the standard deviation. (d–k) Gross pathology of lungs of M. tuberculosis-infected mice at the end of treatment. One representative lung from each treatment group is shown: (d) no treatment, (e) isoniazid, (f) rifampin, (g) isoniazid + rifampin, (h) biapenem, (i) biapenem + rifampin, (j) faropenem, (k) faropenem + rifampin. Scale bar, 1 cm.

Figure 2. Molecular details of interactions of M. tuberculosis l,d-transpeptidases with (carba)penems

(a) Crystal structures of Ldt_Mt2 bound by faropenem at 2.17 Å, (b) Ldt_Mt1 bound by faropenem at 2.25 Å, and (c) Ldt_Mt2 bound by doripenem at 2.18 Å. Residues that make significant interactions are shown in green and adducts are shown in cyan. The 2Fo-2Fc difference fourier map (gray) is contoured at 1.0 σ. Distances are in Å. (d) Kinetics of inhibition of Ldt_Mt2 by doripenem, biapenem, faropenem and tebipenem. Kinetic constants k_inact and K_app were determined spectrophotometrically. Data represent the mean of three independent experiments and error bars represent standard deviation.

Figure 3. Design and activity of evolved carbapenems

(a) Model of carbapenem bound to the catalytic core of Ldt_Mt2. While the carbapenem core ring is tightly bound to the catalytic core (gray) of Ldt_Mt2 by covalent bonding to cysteine 354, its R1 group protrudes and makes extensive contacts with either the inner cavity (pocket colored purple, ligand colored beige) or the outer cavity (pocket colored yellow, ligand colored green), depending on the ligand binding mode. (b) Chemical structures of evolved carbapenems T123, T206, T208 and T210. (c) Kinetics of acylation of Ldt_Mt2 by T208 and T210. Kinetic constants k_inact and K_app were determined spectrophotometrically. Data represent the mean of three independent experiments and error bars represent standard deviation.

Figure 4. Crystal structures of Ldt_Mt2 with evolved carbapenems

a–c) Structure with T206 at the catalytic site of Ldt_Mt2a) Conformation A1, b) conformation A2 and c) conformation B showing interactions of T206. Structure of adducts (cyan) of (d) T208, (e) T210 and (f) T224 at the catalytic site of Ldt_Mt2. For each panel, the 2Fo-2Fc difference fourier map (gray) is contoured at 1.0 σ. Distances are in Å.

Figure 5. Proposed mechanism of acylation of l,d-transpeptidases by (carba)penems

(a) Reaction of faropenem with Ldt_Mt1 or Ldt_Mt2, and reaction of Ldt_Mt2 with (b) doripenem and (c) T208.