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Synthesis and evaluation of microcin C analogs as aminoacyl-tRNA synthetase inhibitors.
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Summary</>
</>
This dissertation developed
from an initial interest in extending the McC concept to a series of potent
antimicrobial compounds that could target a variety of aaRSs (Ch.2). Along the
way, several new side projects wereinvestigated that evolved in response to
further optimize drug-likeness (Ch. 3) and selectivity for the bacterial enzyme
(Ch. 5). We alsoundertook several attempts to prevent possible resistance
mechanismsto potential antibiotics that might result from this work (Ch. 4). </>
Increasing resistance to antibiotics is a major
problem worldwide and provides the stimulus for development of new bacterial
inhibitors with preferably different modes of action. In search for new leads,
several new bacterial targets are being exploited beside the use oftraditional
screening methods. Hereto, inhibition of bacterial protein synthesis is a
long-standing validated target. Aminoacyl-tRNA synthetases (aaRSs) play an
indispensable role in protein synthesis and their structures proved quite
conserved in prokaryotes and eukaryotes.However, some divergence has occurred
allowing the development of selective aaRS inhibitors. Following an outline on
the action mechanismof aaRSs, an overview has been given of already existing
aaRS inhibitors, which are largely based on mimics of the aminoacyl-adenylates,
the natural reaction intermediates. This is followed by a discussion on more
recent developments in the field and the bioavailability problemand the
so-called Trojan horse inhibitors, as based on microcin C (McC) and the
sideromycins.</>
Microcin C (21</>)
is a potent antibacterial compound produced by some E. coli</> strains. McC functions through a Trojan horse mechanism: it
is actively taken up inside a sensitive cell through the function of the
YejABEF transporter and then processed by cellular aminopeptidases. Processed
McC (22</>) is a non-hydrolyzable
aspartyl-adenylate analogue that inhibits aspartyl-tRNA synthetase (AspRS). A
new synthesis is described that allows for the production of a wide variety of
McC analogues in acceptable amounts. Using this synthesis a number of diverse
compounds was synthesized with altered target specificity. It was shown that
these compounds exhibit potent whole-cell activity. In addition, further
characteristics of the YejABEF transporter were determined using these
compounds, showing a preference for the more polar aminoacyl residues at the C</>-terminal position.</>
McC analogues with variable length of the peptide
moiety were synthesized and evaluated in order to characterize substrate
preferences of the YejABEF transporter. It was shown that a minimal peptide-chain
length of six amino acids and the presence for N</>-terminal
formyl-methionyl-arginyl sequence are required for transport.</>
N</></>-methylated
aaSAs were synthesized to investigate their potential as aaRS inhibitors and to
establish if these would escape acetylation of the alpha amine (with
concomitant inactivation of the inhibitor), which is a self-protective
mechanism in the bacterial cell. It was shown however that these compounds are
not able anymore to effectively inhibit their respective aaRSs. In addition we
showed that (</>D</>)D-SA
(i.e.</> Asp-SA with a </>D</>-configuration of the Asp), is a potent inhibitor of
AspRS. However, we also showed that the inhibitory effect of (</>D</>)D-SA is relatively short-lasting. This was shown to
be attributable to a large extent to acetylation by RimL, although it was also
shown that other unknown factors also play a role in the inactivation of this
compound.</>
McC analogues with </>D</>-amino acids at positions two to six were shown to be
resistant against metabolization by the different peptidases and could
therefore not liberate the active moiety. This observation could not be
reversed by the incorporation of L-amino acids at position six, showing that none
of the peptidases could exhibit endopeptidase activity.</>
The substitution of the adenine by aryl-tetrazole
moieties linked via</> a two-carbon
spacer in aaSA proved unsuccessful. Although all synthesized compounds, both in
McC as in SDC form, showed nice potential when evaluated in in vitro</> aminoacylation experiments, the whole-cell activity was
virtually abolished. Onlythe activity of compound 7</> (CB432) against S. aureus </>could
be confirmed. It was shown that lack of activity in whole-cell assays was due
to an inability of these compounds to pass the cell membrane. Nevertheless, it
was also shown that the synthesized SDCs could readily be metabolized by the
peptidases PepA, PepB and PepN.</>
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