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A recent study explored the antibacterial effects of modified forms of vancomycin on antibiotic-resistant bacteria. The results suggest C1-aminomethylene vancomycin may be an effective agent against methicillin-resistant Staphylococcus aureus (MRSA) and other antibiotic-resistant bacteria.
The ability of bacterial strains to acquire resistance to antibiotics faster than antibacterial therapies can be developed is of growing concern. One strategy which has shown some efficacy in overcoming antibiotic resistance is the modification of existing antibiotics less prone to the development of resistance, altering them to target mechanisms that make resistance possible. Further modifications to structural elements of antibiotic substances not directly related to resistance are being explored, hoping to further decrease likelihood of resistance and develop more effective antibiotics.
Though vancomycin – typically used as a last line of defense against methicillin-resistant Staphylococcus aureus (MRSA) – is from a class of resistance-delaying antibiotics, vancomycin-resistant bacterial strains have emerged. Vancomycin binds to certain protein precursors that permit the formation of bacterial cell walls. The binding pocket of vancomycin targets the tail ends of these precursors, which end in double alanine (Ala-Ala) residues. Targeting these precursors prevents bacterial cell walls from maturing, hampering bacterial function and contributing to bacterial death. Vancomycin-resistant bacteria, however, can sense vancomycin and change the Ala-Ala ends to alanine-lactate (Ala-Lac), preventing vancomycin from binding. Pocket-modified forms of vancomycin, capable of binding to both Ala-Ala and Ala-Lac ends and therefore enabling antibacterial activity against both vancomycin-susceptible and resistant bacteria, are in development. Further, adding 4-chlorobiphenyl-methyl (CBP) to a region of vancomycin outside of the binding pocket has been shown increase its efficacy.
In a recent study published in the Proceedings of the National Academy of Sciences, researchers investigated the effects of modifications to vancomycin on its antibacterial properties. Eighteen forms of vancomycin were evaluated: unaltered vancomycin, with an oxygen molecule in its binding pocket (vancomycin–O); pocket-modified vancomycin, for which the oxygen molecule has been replaced with a sulphur molecule (vancomycin–S), an imine group (vancomycin–NH), or 2 hydrogen atoms (vancomycin–H2); unaltered or pocket-modified vancomycin with a CBP molecule attached to a region outside the binding pocket (CBP–O, CBP–S, CBP–NH, and CBP–H2, respectively); vancomycin–O or vancomycin–H2 with an amine group attached to 1 of 4 other sites outside the binding pocket (RNH–O1, RNH–O2, RNH–O3, RNH–O4, and RNH–H2, respectively); and vancomycin–O or vancomycin–H2 with both CBP and RNH modifications (CBP-RNH–O1, CBP-RNH–O2, CBP-RNH–O3, CBP-RNH–O4, and CBP-RNH–H2, respectively). To test antibacterial activity, the smallest doses required to visibly limit growth (minimum inhibitory concentration, MIC) in MRSA, vancomycin-susceptible S. aureus (VSSA), and Enterococcus strains resistant to vancomycin A or B were recorded; cell wall maturity was assessed with respect to the prevalence of the cell wall protein precursor UDP-N-acetylmuramyl-depsipentapeptide (UAMDPP), greater amounts of which would suggest little incorporation into the cell wall and therefore less maturation; and cell wall integrity was assessed with respect to the amount of vancomycin detected inside VanA-resistant Enterococci.
The MIC for vancomycin–O-treated VSSA was 0.5 μg/mL. Comparatively, vancomycin–S had an MIC of 32 μg/mL, 0.03 μg/mL for CBP–O and CBP–NH, 2 μg/mL for CBP–S, and 0.5 μg/mL for CBP–H2. The MIC for vancomycin–O-treated MRSA was 0.5 μg/mL, compared to 32 μg/mL for vancomycin–S, 0.03 μg/mL for CBP–O, 2 μg/mL for CBP–S, 0.06 μg/mL for CBP–NH, and 0.25 for CBP–H2. The MIC for vancomycin–O-treated VanA-resistant E. faecalis and E. faecium were both 250 μg/mL, compared to 32 μg/mL for vancomycin–S, 0.5 μg/mL for vancomycin–NH, and 31 μg/mL for vancomycin–H2. For CBP–O, the MIC were both 2.5 μg/mL, compared to 4 μg/mL for CBP–S, 0.005 μg/mL for CBP–NH, and 0.13 and 0.06 for CBP–H2. For RNH–O1, the MIC were both 500 μg/mL, compared to 63 and 126 μg/mL for RNH–O2, 4 and 2 μg/mL for RNH–O3, 2 μg/mL for RNH–O4, and 0.16 μg/mL for RNH–H2. For CBP-RNH–O1, the MIC were both 5 μg/mL, compared to 0.25 μg/mL for CBP-RNH–O2, 2 μg/mL for CBP-RNH–O3 and CBP-RNH–O4, and 0.01 and 0.005 for CBP-RNH–H2. The MIC for vancomycin–O-treated VanB-resistant E. faecalis was 8 μg/mL, compared to 32 μg/mL for vancomycin–S, 0.03 μg/mL for CBP–O, 2 μg/mL for CBP–S, 0.06 μg/mL for CBP–NH, and 0.5 μg/mL for CBP–H2.
UAMDPP levels were around 10 μg in vancomycin-free VanA-resistant E. faecium and E. faecalis. In both strains, vancomycin–O did not significantly increase levels of UAMDPP. Vancomycin–H2 and RNH–H2 resulted in UAMDPP levels around 75 μg in E. faecalis and 45 μg in E. faecium. CBP–S, CBP-RNH–O3, and CBP-RNH–O4 resulted in levels around 100 μg and 80 μg. CBP–O and CBP–H2 resulted in levels around 130-140 μg and 110-115 μg. UAMDPP levels were around 120 μg for CBP-RNH–O1 in E. faecalis. The highest levels were observed around 160-170 μg and 120-130 μg with CBP-RNH–O2 and CBP-RNH–H2, respectively.
Compared to vancomycin–O, RNH–H2 and CBP-RNH–H2 were abundant inside VanA-resistant E. faecium. Levels of CBP-RNH–O2, RNH–O3 and RNH–O4 were also significantly increased and slight increases were observed with CBP-RNH–O1, CBP-RNH–O3, and CBP-RNH–O4.
Resistance was slow to develop among the vancomycin–H2 derivatives. After 50 exposures, the MIC increased only fourfold for CBP-RNH–H2, eightfold for CPB–H2, and sixteen-fold for RNH–H2 in E. faecium.
Overall, the results suggest that C1-aminomethylene vancomycin (CBP-RNH–H2), which can bind to either Ala-Ala or Ala-Lac residues, inhibits cell wall maturation and reduces cell wall integrity, making it 25,000-50,000 times more potent than unaltered vancomycin, and indicating that it may serve as an effective antibiotic against MRSA and vancomycin-resistant strains. The findings also suggest that the CBP and RNH modifications contribute to its antibacterial activity through Ala-Ala/Ala-Lac-independent inhibition of cell wall maturation and the reduction of cell wall integrity, respectively. Further research into vancomycin analogues with different binding pockets and peripheral alterations may be beneficial.
Written By: Raishard Haynes, MBS