Herein, we present the synthesis of two novel adamantane appended supramolecular self-associating amphiphiles. The antimicrobial efficacy of these compounds is determined against both clinically ...relevant Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Escherichia coli. We also explore the self-associative properties of these amphiphiles in both polar organic DMSO-d
6
0.5% H
2
O and H
2
O (D
2
O)/EtOH 19:1 solutions, confirming aggregate stability through the determination of zeta potential values, aggregate size through a combination of
1
H NMR DOSY and dynamic light scattering studies as appropriate, and critical aggregate concentration through the derivation of concentration-dependent surface tension values. We also perform a variety of
1
H NMR dilution studies and in-silico modelling to further explore the roles of intermolecular hydrogen bonding and lipophilicity within aggregate formation and antimicrobial efficacy. Finally, we perform haemolysis and Galleria mellonella toxicity assays to establish the potential of these compounds to undergo further development as antibiotic agents.
SSAs are a class of supramolecular self-associating amphiphilic salt, the anionic component of which contains a covalently bound hydrogen bond donor-acceptor motif. This results in a monomeric unit ...which can adopt multiple hydrogen bonding modes simultaneously. Previous investigations have shown examples of SSAs to act as antimicrobial agents against clinically relevant methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report an intrinsically fluorescent SSA which can self-associate producing dimers, spherical aggregates and hydrogels dependent on solvent environment, while retaining antimicrobial activity against both model Gram-positive (MRSA) and Gram-negative (Escherichia coli) bacteria. Finally, we demonstrate the SSA supramolecular hydrogel to tolerate the inclusion of the antibiotic ampicillin, leading to the enhanced inhibition of growth with both model bacteria, and derive initial molecular structure-physicochemical property-antimicrobial activity relationships.
Reliable antimicrobial susceptibility testing is essential in informing both clinical antibiotic therapy decisions and the development of new antibiotics. Mammalian cell culture media have been ...proposed as an alternative to bacteriological media, potentially representing some critical aspects of the infection environment more accurately. Here, we use a combination of NMR metabolomics and electron microscopy to investigate the response of Escherichia coli and Pseudomonas aeruginosa to growth in differing rich media to determine whether and how this determines metabolic strategies, the composition of the cell wall, and consequently susceptibility to membrane active antimicrobials including colistin and tobramycin. The NMR metabolomic approach is first validated by characterizing the expected E. coli acid stress response to fermentation and the accompanying changes in the cell wall composition, when cultured in glucose rich mammalian cell culture media. Glucose is not a major carbon source for P. aeruginosa but is associated with a response to osmotic stress and a modest increase in colistin tolerance. Growth of P. aeruginosa in a range of bacteriological media is supported by consumption of formate, an important electron donor in anaerobic respiration. In mammalian cell culture media, however, the overall metabolic strategy of P. aeruginosa is instead dependent on consumption of glutamine and lactate. Formate doping of mammalian cell culture media does not alter the overall metabolic strategy but is associated with polyamine catabolism, remodelling of both inner and outer membranes, and a modest sensitization of P. aeruginosa PAO1 to colistin. Further, in a panel of P. aeruginosa isolates an increase between 2- and 3-fold in sensitivity to tobramycin is achieved through doping with other organic acids, notably propionate which also similarly enhances the activity of colistin. Organic acids are therefore capable of nonspecifically influencing the potency of membrane active antimicrobials.
Chronic myeloid leukemia is the first disease in which the potential of molecular targeted therapy with tyrosine kinase inhibitors (TKIs) was realized. Despite this success, a proportion of patients, ...particularly with advanced disease, are, or become, resistant to this treatment. Overcoming resistance and uncovering the underlying mechanisms is vital for further improvement of clinical outcomes. Here we report the identification, development, and characterization of a novel chronic myeloid leukemia cell line carrying the additional chromosomal aberration t(3;12)(q26;p13) resulting in expression of the TEL/MDS1/EVI1 fusion protein, which is resistant to TKIs. Resistance to TKIs was overcome by the co-administration of the BH3-mimetic, ABT-737. In addition, application of EVI1-specific small interfering RNA decreased expression of the TEL/MDS1/EVI1 fusion, reduced resistance to imatinib, and increased sensitivity to ABT-737. Subsequent studies revealed a role for the BH3-only protein BAD, probably via a phosphoinositide 3-kinase/AKT-dependent pathway, as pharmacological inhibition of AKT could also resensitize cells to death from TKIs. These findings indicate a novel pathway of TKI resistance regulated by EVI1 proteins and provide a promising means for overcoming resistance in chronic myeloid leukemia and other hematological malignancies displaying EVI1 overexpression.
Antimicrobial resistance has become a major global concern. Development of novel antimicrobial agents for the treatment of infections caused by multidrug resistant (MDR) pathogens is an urgent ...priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents initially discovered and isolated from natural sources. Recently, C8-linked PBD biaryl conjugates have been shown to be active against some MDR Gram-positive strains. To explore the role of building block orientations on antibacterial activity and obtain structure activity relationship (SAR) information, four novel structures were synthesized in which the building blocks of previously reported compounds were inverted, and their antibacterial activity was studied. The compounds showed minimum inhibitory concentrations (MICs) in the range of 0.125–32 μg/mL against MDR Gram-positive strains with a bactericidal mode of action. The results showed that a single inversion of amide bonds reduces the activity while the double inversion restores the activity against MDR pathogens. All inverted compounds did not stabilize DNA and lacked eukaryotic toxicity. The compounds inhibit DNA gyrase in vitro, and the most potent compound was equally active against both wild-type and mutant DNA gyrase in a biochemical assay. The observed activity of the compounds against methicillin resistant S. aureus (MRSA) strains with equivalent gyrase mutations is consistent with gyrase inhibition being the mechanism of action in vivo, although this has not been definitively confirmed in whole cells. This conclusion is supported by a molecular modeling study showing interaction of the compounds with wild-type and mutant gyrases. This study provides important SAR information about this new class of antibacterial agents.
Some antimicrobial peptides (AMPs) have potent bactericidal activity and are being considered as potential alternatives to classical antibiotics. In response to an infection, such AMPs are often ...produced in animals alongside other peptides with low or no perceivable antimicrobial activity, whose role is unclear. Here we show that six AMPs from the Winter Flounder (WF) act in synergy against a range of bacterial pathogens and provide mechanistic insights into how this increases the cooperativity of the dose-dependent bactericidal activity and potency that enable therapy. Only two WF AMPs have potent antimicrobial activity when used alone but we find a series of two-way combinations, involving peptides which otherwise have low or no activity, yield potent antimicrobial activity. Weakly active WF AMPs modulate the membrane interactions of the more potent WF AMPs and enable therapy in a model of
burn wound infection. The observed synergy and emergent behaviour may explain the evolutionary benefits of producing a family of related peptides and are attractive properties to consider when developing AMPs towards clinical applications.
Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant
(MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we ...identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism,
. Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.
The
fluoroquinolone class of antibiotics has a well-established
structure–activity relationship (SAR) and a long history in
the clinic, but the effect of electron-rich benzofused substituents
at the ...N1 position remains poorly explored. Because groups at this
position are part of the topoisomerase–DNA binding complex
and form a hydrophobic interaction with the major groove of DNA, it
was hypothesized that an electron-rich benzofused N1 substituent could
enhance this interaction. Molecular modeling techniques were employed
to evaluate the binding of certain N1-modified fluoroquinolones to
DNA gyrase targets from both
Staphylococcus aureus
and
Klebsiella pneumoniae
species
compared with ciprofloxacin and norfloxacin. Seven N1-modified fluoroquinolones
were subsequently synthesized and tested against a panel of Gram-negative
pathogens to determine minimum inhibitory concentration (MIC) values.
Gram-negative outer membrane penetration was investigated using the
membrane permeabilizer polymyxin B nonapeptide and compound efflux
via
resistance–nodulation–division-family
efflux transporters was evaluated using the known efflux pump inhibitor
phenylalanine–arginine β-naphthylamide. Additionally,
the target inhibitory activity of representative compound
6e
was determined in a cell-free environment. A correlation between
N1 substituent hydrophobicity and activity was observed across the
MIC panel, with compound activity decreasing with increased hydrophobicity.
Those compounds with highest hydrophobicity were inactive because
of poor solubility profiles whereas compounds with intermediate hydrophobicity
were inactive because of impaired outer membrane penetration, and
reduced inhibition of topoisomerase targets, the latter in contrast
to modeling predictions. This study adds new information to the fluoroquinolone
SAR and suggests limited utility of large hydrophobic substituents
at the N1 position of fluoroquinolones.
Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency ...of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs.
The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular ...bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.
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