Ben Bardsley

Die Vancomycin‐Antibiotica und der Kampf gegen resistente Bakterien

Die letzte Verteidigungslinie gegen „Superbakterien” bilden die Antibiotica der Vancomycin-Gruppe. Wie aus der umfassenden Aufklarung des Wirkungsmechanismus der Antibiotica und eines Resistenzmechanismus gegen diese Wirkstoffe hevorgeht, kann diese Form der Resistenz uberwunden werden, ohne die Bindungsstelle des Antibioticums fur die Zellwandvorstufen pathogener Bakterien direkt zu modifizieren.

Estimating binding constants – The hydrophobic effect and cooperativity

The reliable estimation of binding constants by computational means for drugs binding to receptors represents one of the major challenges in molecular recognition. Many approaches to this problem have relied on the partitioning of binding free energy according to each of the interactions which are made or broken on binding, e.g., hydrogen bonds, salt bridges, π-π interactions, or the hydrophobic effect. With particular reference to the hydrophobic effect, we illustrate how such partitioning ignores one of the fundamental properties of systems of non-covalent interactions, namely that of cooperativity. This leads to estimates for the free energy benefit of the hydrophobic effect which vary according to the method of determination used, and whether the system in which the measurements are taken exhibits cooperativity. The context dependence of the magnitude of the hydrophobic effect illustrates the problem associated with considering weak interactions in isolation from the system in which they occur. The approach of partitioning binding affinities can be very useful, but until the problem of cooperativity can be better addressed by computational chemists, there remains the possibility that attempts to estimate at least some binding constants will unfortunately be seriously flawed.

Cooperativity in ligand binding expressed at a model cell membrane by the vancomycin group antibiotics

Dimerisation or the use of a membrane anchor enhances the binding of the glycopeptide antibiotics at the surface of a model cell membrane.

Measurement of the different affinities of the two halves of glycopeptide dimers for acetate

The titration of sodium acetate into solutions of the vancomycin group antibiotics eremomycin and chloroeremomycin has been studied by observing the downfield movement of the 1 H NMR signals due to amide protons in the antibiotic binding pocket; this change in chemical shift has been used to determine the different affinities of the two halves of the dimers for acetate ligand.

AN ILLUSTRATION OF THE EXPRESSION OF COOPERATIVE BINDING ENERGY IN ARRAYS OF NON-COVALENT INTERACTIONS

An analysis of a thermodynamic cycle for the formation of ligand-bound dimers gives a simple illustration as to how a cooperative binding energy (ΔG°coop) can be expressed over a range of interfaces, rather than at just one of the interfaces within the array.

Use of model cell membranes to demonstrate templated binding of vancomycin group antibiotics

In this paper we demonstrate the importance of binding geometry and dimerisation at the surface of model cell membranes in the mode of action of the clinically important glycopeptide antibiotics. This has been achieved through the use of model cell membranes (micelles and vesicles) to which cell wall analogues are anchored via a hydrophobic decanoyl chain. A number of –D-Ala-terminating cell wall analogues, ranging from two to six residues in length, have been used. Dipeptide, pentapeptide and hexapeptide display enhanced binding to the antibiotic at the model cell surface, but tripeptide and tetrapeptide do not. The possible implications of the observed binding geometries for bacterial systems are discussed.

A limitation of two-state analysis for transitions between disordered and weakly ordered states

BACKGROUND The stability of the secondary structure of particular peptide regions is often used to investigate the involvement of the region in protein folding. When analysing the relatively small populations of associated states that are formed by weak interactions (i.e. those interactions that are comparable to thermal energies), it is common practice to characterise the associated state by a parameter that is measured when this state is highly occupied. The accuracy of this method, however, has not yet been determined. RESULTS Using as a model the vancomycin group of antibiotics, either forming dimers or binding to cell wall precursors, we have investigated the dependence of the limiting (i.e. fully associated) chemical shifts of two protons on the equilibrium constants for the formation of the fully associated states. The chemical shift shows a large variation with the equilibrium constant for the formation of the fully associated state. CONCLUSIONS The results demonstrate, in two systems, that a parameter representing a fully associated state (chemical shift) varies greatly with the equilibrium constant for the formation of that associated state. The results have implications for two-state analyses of populations of protein fragments in which a parameter representing the fully associated state is taken to be independent of the equilibrium constant for its formation. Using two-state analysis to determine the population of associated states of protein fragments could result in an underestimation of the population of these associated states.

The increasing tightness of fully associated states as a function of their increasing stability. The dimerisation of carboxylic acids

Literature data describing the dilution of carboxylic acids in cyclohexane are reinterpreted according to a monomer–dimer equilibrium (higher order oligomers had previously been postulated in order to explain the data). Apparent dimerisation constants for each of the acids were calculated by incorporating a factor to take into account the varying relative permittivity of the solution during the titration. Dilution titrations of propionic acid using diluents of varying relative permittivities were also carried out and apparent dimerisation constants calculated in a similar manner. The dimerisation constants were found to vary according to the relative permittivity of the diluent, and the tightness of the dimers formed (as evidenced by the chemical shift of the carboxy proton) also varied according to the diluent relative permittivity. The results demonstrate that complex stability is directly related to the strength of the hydrogen bonds which go to form that complex. They also clarify a long-standing debate regarding the origin of an anomalous downfield shift of the carboxylic acid proton seen on dilution of simple aliphatic carboxylic acids with cyclohexane.

Cooperativity between ligand binding and dimerisation in a derivative of ristocetin A

The dimerisation constant of the vancomycin group antibiotic ristocetin A has previously been shown to be lower when it is fully bound by ligand (analogues of bacterial cell wall precursors terminating in –Lys-D-Ala-D-Ala) than in its absence, i.e. dimerisation is anticooperative with ligand binding. A derivative of ristocetin A, desrhamno-ristocetin, has now been produced by enzymatic degradation, and the dimerisation constant of this derivative has been measured in the absence and presence of the bacterial cell wall precursor analogue N-acetyl-D-Ala-D-Ala. The dimerisation constant is shown to be greater in the presence of the ligand than in its absence, i.e. dimerisation is cooperative with ligand binding. This change in behaviour from anticooperativity to cooperativity is postulated to be associated with the partial equalisation of the binding affinities of the two sides of the dimer for ligand. It is therefore energetically more favourable for two ligand molecules to bind to the two halves of a desrhamno-ristocetin dimer than to two monomers.

Aggregation, binding, and dimerisation studies of a teicoplanin aglycone analogue (LY154989)

LY154989 is a vancomycin group antibiotic closely related in structure to teicoplanin aglycone. In view of the clinical importance of teicoplanin, the dimerisation, aggregation, and binding of bacterial cell wall analogues by LY154989 are of interest. These properties have been studied by proton NMR spectroscopy. LY154989 has been shown to form concentration-dependent aggregates in aqueous solution, similar to those of teicoplanin, even though it does not possess a C11 acyl chain, which was hitherto thought to be the cause of this aggregation. The aggregation can be disrupted by the addition of bacterial cell wall precursor analogues such as Ac2-KDADA, Ac-DADA or Ac-DA. Thus, growing bacteria may disrupt aggregates of teicoplanin and LY154989. LY154989 dimerises weakly in aqueous solution and the dimerisation is weakly cooperative with ligand binding.