Tuesday, 6 June 2017

Fighting back! Vancomycin (plus) my molecule for June

This month I decided to wait for the release of a paper that the press announced as an example of magic! Since all magic can be explained by science, and therefore science is not magic; I thought it would be appropriate to set the record straight. Dale Boger's group at The Scripps Institute in California published a series of chemical modifications to the "last resort" antibiotic vancomycin that impact not only on its potency, but also look to have made significant inroads to reducing the emergence of "resistance". The excellent final figure in their recent publication in Proceedings of the National Academy of Scienceis shown below. It reveals the complexity of the molecule and it identifies the chemical groups associated with its antibiotic properties. But first let me provide some background to vancomycin. 

Vancomycin was discovered in the same year that Watson and Crick discovered the double helical nature of DNA (1953), around 24 years after Fleming published the discovery of Penicillin. By 1953, resistance to penicillin treatment had become a real clinical issue, particularly in Staphylococcus aureus (recall MRSA). With the discovery of vancomycin (the vanquisher!), it looked like an alternative treatment for resistant strains was now in sight. In fact the drug was fast-tracked into hospitals and was in use just five years after its discovery.

Vancomycin is a naturally occurring heptapeptide, originally isolated from the organism Amycolatopsis orientalis, which was originally identified by the Harvard trained organic chemist, Edmund Kornfeld at Eli Lilly, working with soil samples collected from the jungles of Borneo by missionary workers! We now know that this seven amino acid peptide is synthesised by non-ribosomal protein synthesis (NRPS), after which it is chemically modified in a a complex series of secondary metabolism steps. the aromatic rings are a combination of modified phenyl-glycine and tyrosine which are chlorinated and glycosylated. The sequence of amino acids is 

(1) Leucine (2) Tyrosine (modified by hydroxylation) (3) Asparagine (4) Glycine (modified by phenyl-hydroxylation) (5) Glycine (modified by phenyl-hydroxylation) (6) Tyrosine (modified by hydroxylation) (7) Glycine (modified by addition of dihyxdroxylated benzene)

starting from the bottom RH corner and proceeding clockwise in the structural diagram at the top.

As you might imagine, this is too short a sequence to be synthesised as heptameric units on the ribosome (what is the shortest polypeptide to be synthesised in a mature form via the ribosome? and what are the constraints on chain length?). The role of vancomycin, a complex secondary metabolite in the physiology of Amycolatopsis orientalis, as with other antibiotics is presumably to serve as a defence against bacterial threats to its survival, but it also reveals that complex carbohydrates etc can be introduced into microbial polypeptide chains in a way that we usually associate with proteins in much more elevated species. The 7 modules of vancomycin (centred on each amino acid) are generated and "finalised" for function by a set enzymes that utilise ATP to provide the necessary energy through an adenylate intermediate. You can read more here about these enzymes and their genes. 

In my mind there are always three main questions that need addressing when trying to rationalise the mode of action of antibiotics. The first is pretty well understood and relates to the target (or targets). The second is less clear, and that is an understanding of the mechanism of killing versus growth arrest. The third question relates to the likelihood and mechanism of resistance. In the case of vancomycin and its synthetic derivatives, it would appear that there are three targets. The biosynthesis of the bacterial cell wall is essential for the normal growth of most bacteria. Vancomycin is a bivalent inhibitor of this process, interfering with two distinct enzymatic steps (shown in blue at 6 0'clock and 12 o'clock in the diagram). In addition, the positively charged moiety (bottom LHS) is thought to disrupt the cell membrane. The combination of these three weapons not only reduces the minimal inhibitory concentration (MIC) of vancomycin, but it also massively reduces the emergence of resistance in the target organism. So the Boger group have made significant progress in taking a natural product and improving it, in the great traditions of natural product chemistry and contemporary organic synthesis. It is now up to the molecular biologists to provide the explanations relating to cell death and ultimately the mechanisms of resistance, which will be when and not if!

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