Introduction
b-Lactam antibiotics have been in clinical use for more than 65 years
and have proven to be safe and efficient. They include penicillins,
cephalosporins, monobactams and carbapenems. The market for b-lactam antibiotics has been valued at US$ billion 8.1 in 2002,
representing 52% of the total antibiotic market. b-Lactam agents form the cornerstone of the antibiotic arsenal and it
is therefore not surprising that the most commonly acquired mechanism of
antibiotic resistance is the ability of bacteria to express enzymes
(b-lactamases) inactivating these agents. Extended-spectrum
b-lactamases (ESBLs) in particular are capable of inactivating
virtually every type of b-lactam antibiotics and b-lactamase inhibitors. The expression of ESBL enzymes is conserved in
multi-drug resistant (MDR) Enterobacteriaceae, which have the
capacity to resist several antibiotics from different classes, including agents
often used as the last line of effective antibiotic treatment. These bacteria
have been listed among the top 6 organisms to which new antibiotics are urgently
needed. However there are virtually no novel-mechanism antibacterials against Enterobacteriaceae strains in clinical
development currently.
Host defence peptides (HDPs) are multifunctional
molecular effectors of innate immunity, the first line of defence against
infection in multicellular organisms. They have potent, rapid and broad-spectrum
antibacterial activities. Their multiple functions in host defences, associated
with their low susceptibility to classical mechanisms of drug resistance and low
propensity to select resistant mutants, support the rationale of developing
novel peptide-based therapeutics harnessing the effector mechanisms of innate
immunity. However, to date, HDPs have not been clinically successful as single
agents.
Technology
b-Lactam Host Defence Conjugates
The objective of this technology is to:
generate hybrid antibiotics of a b-lactam (cephalosporin) and a host defence peptide, joined by a
cleavable linker exploit the mechanism of action of the cephalosporin and/or the
main mechanism of bacterial resistance against b-lactam agents to selectively disconnect these 2 antibiotic
agents.
Approach:
Conjugation of the peptide at the 3’-position of the cephalosporin
(figure below) allows its release, triggered by the mechanism of action of the
cephalosporin (interaction with the penicillin-binding proteins, PBPs), and/or
the main mechanism of bacterial resistance against b-lactam
agents. In the latter case, the release of the conjugated peptide occurring in a
catalytic mode, the hybrid molecule can constitute a b-lactamase-dependent prodrug where a cephalosporin promoiety
transiently masks a functional group (N terminus) of the peptide.
Synthetic method:
Linking a peptide to a b-lactam involves the development of a synthetic approach which meets the
(divergent) requirements of both components. Two approaches have been developed
for this conjugation, one of them based on ‘click-chemistry’
(copper(I)-catalysed azide-triazole cyclo-addition) which allows the efficient,
reliable and versatile attachment of a peptide, without restriction on the
sequence content, to the cephalosporin. A candidate based on cephalothin and an
8-mer, proteolytically stable (all-D) sequence derived from the bactenecin
peptide has been prepared and evaluated.
Results
Chemical and enzymatic hydrolysis assays performed with a purified
b-lactamase indicate that the cephalothin-bactenecin conjugate can release its
peptide component upon cleavage of the b-lactam
ring, while no background hydrolysis is observed in buffer alone.
Representative strains of Gram-positive (Staphylococcus aureus, including MRSA strains) and Gram-negative (Escherichia coli, including clinical MDR strains) organisms were used to assess the antimicrobial activities
of the conjugate. Minimum Inhibitory Concentrations (MICs) in the 1-8
mM
range were
obtained against S.
aureus. Higher MICs were achieved against E. coli (3-16 mM), but
it is expected that these results are associated with a reduced penetration of
the bacterial outer membrane which could be overcome by modifying the side-chain
of the cephalosporin.
Advantages
Hybrid antibiotics of a b-lactam (cephalosporin) and a host defence peptide
represent:
1.dual-acting antibiotic candidates with a predicted polypharmacology
profile; the ability of an antibiotic agent to address more than one bacterial
target increases its efficacy and delays bacterial resistance
development
2.novel-mechanism (b-lactam-based) antibacterials with restored activities against
b-lactam-resistant pathogens
3.b-lactamase-dependent prodrug candidates of host defence peptides for
sequences requiring a free N-terminus; a prodrug technology can address some
clinical shortcomings of HDPs in systemic therapies and can be exploited to selectively target MDR Enterobacteriaceae producing ESBLs (niche indication expected to represent the second most attractive
antibiotic market opportunity).
Contact:
Dr Aoife
Gallagher, RCSI Technology Transfer, 123 St Stephen’s Green, Dublin 2, Ireland.
Email: aoifegallagher1@rcsi.ie. Tel: +353 1
4022394