In vitro validation of acetyltransferase activity of GlmU as an antibacterial target in Haemophilus influenzae

Abstract

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.

FIGURE 1.

A, the steady-state ordered bisubstrate kinetic mechanism in which acetyl-CoA is the first substrate, both substrates cause substrate inhibition, and the inhibitor competes with acetyl-CoA is one plausible kinetic mechanism for the E. coli GlmU acetyltransferase activity based on the data herein. B, comparison of the experimental data to the best-fit non-linear least squares regression for the mechanism in A.

FIGURE 2.

Isothermal titration calorimetry studies of compound 2 binding to E. coli GlmU (K_d = 1.9 μm). Observed heats evolved over time are shown in the top, and the corresponding fitted one-site binding curve is shown at the bottom.

FIGURE 3.

One-dimensional WaterLOGSY spectral comparison of 200 μm acetyl-CoA binding to 10 μmH. influenzae GlmU with (bottom) and without (top) the addition of 200 μm compound 4 (left) or 5 (right). Signals of proton a, b, and c from acetyl-CoA are labeled and compared in the WaterLOGSY spectra. The signals in the dashed boxes are from the compounds. The NMR samples were prepared in 50 mm HEPES (pH 7.5), 5 mm MgCl₂, 5 mm DTT, 0.1 mm EDTA, and 5% D₂O. The spectra were acquired at 298 K.

FIGURE 4.

A, a view of the inhibitor binding pocket in the E. coli GlmU trimer. B, electron density (2F_o − F_c map at 1.2σ) of compound 3 in complex with GlmU. C, binding interactions of compound 3 and acetyl-CoA. Compound 3 has been depicted in thick yellow sticks, and acetyl-CoA is shown in thin gray lines. The three monomers are colored pink, gray, and cyan. The residues mutated in the resistant strains are highlighted in darker shades of the respective monomer color. Hydrogen bonds are depicted as dotted lines.

FIGURE 5.

Compound 5 preferentially inhibits N-acetylglucosamine and acetic acid incorporation into macromolecules of H. influenzae acrB::cat. Inhibition of synthesis of protein (leucine and valine incorporation; not shown), RNA (uridine incorporation; circles), fatty acids (acetate incorporation; triangles), peptidoglycan (N-acetylglucosamine incorporation; squares), and DNA (thymidine incorporation; not shown) was measured as described under “Experimental Procedures.” The incorporation rates of added precursors into uninhibited cells were 21, 65, 1100, 7600, 3.1, and 35 μmol/h/A₆₀₀, respectively. Inhibition of leucine, valine, thymidine, and uridine all showed similar modest inhibition and stayed below 50% even at the highest compound concentration tested. For clarity, only uridine is shown as a representative of these four. Data are averages of two independent measurements; S.D. are ∼10%.

FIGURE 6.

Amino acid sequences of the carboxyl-terminal end of four GlmU isozymes, numbered according to the sequence of the E. coli isozyme. Thr-420 and Trp-449, which when mutated to Ile and Gly respectively led to resistance of H. influenzae acrB::cat against compound 5, are highlighted.