Université catholique de Louvain (UCL-Bruxelles)
Louvain Drug Research Institute > Cellular and Molecular Pharmacology
Novel antibiotic targets and drug design

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World-wide increase of bacterial resistance to antibiotics makes essential the discovery of new agents directed against unexploited bacterial targets. 

In this context, we analyse natural mutants with impaired growing capabilities to detect genes products having a character of essentiality.   We then undertake the design or proactive selection of inhibitors of the function of these proteins. 

These inhibitors could represent new classes of antibiotics.

These research programs are also linked to those exploring drug-membrane interactions and the chemotherapy of intracellular infection.




Main research programs

Inhibitors of D-Ala-D-Ala ligases

The D-alanine:D-alanine (D-Ala:D-Ala) ligase, which catalyses dimerization of D-alanine before its incorporation in late peptidoglycan precursors, is essential for bacteria since natural mutants having lost activity are incapable of growing unless rescued by the addition of D-Ala:D-Ala in their culture medium.  The reaction catalysed by the D-Ala:D-Ala ligase involves the formation of D-alanylphosphate as a tetrahedral intermediate. 

The enzyme is strongly inhibited by D-cycloserine and methylphosphinophosphate but none of these inhibitors fulfil the requirements for becoming useful drugs. 

In collaboration with the 'Université libre de Bruxelles', we have characterized the active site of the enzyme by 3-D computer-aided modelling (Figure 1).  The model successfully explained the inactivity of four spontaneous mutants, including those which could not be explained by inspection of the published X-ray structures of this enzyme.

Figure 1: 
3D-model ( tube diagram) of the D-Ala:D-Ala ligase of E. faecalis using the X-ray structure of the Escherichia coli enzyme complexed with ADP and the methylphosphinophosphate inhibitor as a template.  The image highlights the residues (depicted as balls) found in contact with ADP and phosphinophosphate. 

Color code of residues: 

  • green, hydrophobic; 
  • pink, aromatic; 
  • dark blue, basic; 
  • light blue, polar; 
  • red, acidic. 
From Prevost et al., 2000

Inhibitors with drugable properties are now looked for using (i) rational, target-based approaches, (ii) empirical pharmacochemical, structure-based approaches starting from known inhibitors, (iii) screening of natural sources. 

These novel compounds are tested for their capacity to inhibit the enzyme (using a purified protein) as well as the bacterial growth (using clinical isolates of important human pathogens).

A first successful example is represented by benzoxazoles, which were designed de novo starting from computational modeling (Figure 2).

Figure 2:

Left: Docked pose of the most active benzoxazole (IT16) in the E. coli D-Ala-D-Ala ligase enzymatic pocket. IT16 is depicted as ball-and-sticks and interacting residues and ATP are represented as thick lines. The two magnesium ions are depicted as pink spheres. Oxygens, nitrogens, carbons, phosphorus and hydrogens are colored in red, blue, green, magenta and white respectively

Right: chemical structure, inhibitory activity towards E. faecalis D-Ala-D-Ala ligase, scoring in computational analysis, and antimicrobial activity of benzoxazole compounds.

From Tytgat et al., 2008

Selected References
on D-Ala-D-Ala ligases and inhibitors thereof (by reverse chronological order; for a full reference list, see our publication list)

Other targets

Other targets are being actively explored. Three orientations are being followed, namely

Figure 3A

Figure 3 B

Figure 3:

Left: Comparative activity of the glycopeptide vancomycin and of the lipopglycopeptide telavancin. S. aureus was exposed to increasing concentrations of each of these drugs during 3 h. The graph shows the change in bacterial counts from the initial inoculum. telavancin is bactericidal (3 log decrease in colony forming units [CFU]) as soon as its concentration reaches 10 x its Minimal Inhibitory Concentration (MIC), while vancomycin never reaches a bactericidal effect. The mode of action of lipoglycopeptides is further discussed in the section on drug-membrane interactions.

From Barcia-Macay et al, 2006

Right: Delocalization of PBP2 in MRSA as determined by fluorescence microscopy in bacteria grown in broth containing oxacillin or oxacillin and epicathecin gallate (ECg). This delocalization (shown by the group of P. Taylor) is thought to impair the interaction of PBP2 (acting as transglycosylase) with PBP2a (acting as transpetptidase), which is essential for maintenance of growth of MRSA in the presence of beta-lactams. This may explain why ECg sensitizes MRSA to beta-lactams although it does not interact with PBP2a (shown by our group). These features of ECg-induced phenotype can be explained by changes in the fluid dynamics of the membrane.

From Bernal et al., 2010

Selected References other targets (by reverse chronological order; for a full reference list, see our publication list)


Additional information:  <tulkens@facm.ucl.ac.be>
Last significant update: December 2010