Antibiotic resistance is a major public health challenge, and Gram-negative multidrug-resistant bacteria are particularly dangerous. The threat of running out of active molecules is accelerated by the extensive use of antibiotics in the context of the COVID-19 pandemic, and new antibiotics are urgently needed. Colistin and polymyxin B are natural antibiotics considered as last resort drugs for multi-resistant infections, but their use is limited because of neuro- and nephrotoxicity. We previously reported a series of synthetic analogues inspired in natural polymyxins with a flexible scaffold that allows multiple modifications to improve activity and reduce toxicity. In this work, we focus on modifications in the hydrophobic domains, describing analogues that broaden or narrow the spectrum of activity including both Gram-positive and Gram-negative bacteria, with MICs in the low µM range and low hemolytic activity. Using biophysical methods, we explore the interaction of the new molecules with model membranes that mimic the bacterial inner and outer membranes, finding a selective effect on anionic membranes and a mechanism of action based on the alteration of membrane function. Transmission electron microscopy observation confirms that polymyxin analogues kill microbial cells primarily by damaging membrane integrity. Redistribution of the hydrophobicity within the polymyxin molecule seems a plausible approach for the design and development of safer and more selective antibiotics.
The efficient preparation of novel bioactive peptide drugs requires the availability of reliable and accessible chemical methodologies together with suitable analytical techniques for the full characterisation of the synthesised compounds. Herein, we describe a novel acidolytic method with application to the synthesis of cyclic and linear peptides involving benzyl-type protection. The process consists of the in situ generation of anhydrous hydrogen bromide and a trialkylsilyl bromide that acts as protic and Lewis acid reagents. This method proved to be useful to effectively remove benzyl-type protecting groups and cleave Fmoc/tBu assembled peptides directly attached to 4-methylbenzhydrylamine (MBHA) resins with no need for using mild trifluoroacetic acid labile linkers. The novel methodology was successful in synthesising three antimicrobial peptides, including the cyclic compound polymyxin B3, dusquetide, and RR4 heptapeptide. Furthermore, electrospray mass spectrometry (ESI-MS) is successfully used for the full characterisation of both the molecular and ionic composition of the synthetic peptides.
Antibiotic resistance is a daunting challenge for public health systems worldwide. A major goal to fight resistant bacteria involves the design, discovery and development of new antibiotics, particularly against multi-drug-resistant strains. Currently, there is renewed interest in polymyxins, an old class of antimicrobial cyclic lipopeptides, highly potent against therapeutically relevant Gramnegative bacteria. Polymyxins are now used as last resort antibiotics in hospitals because of their nephrotoxicity and neurotoxicity that requires careful monitoring of the patient. Our group has embarked on a project to design and develop new polymyxins devoid of toxicity problems using a versatile and chemically accessible scaffold structure [1,2]. Compounds show excellent activity against Gram-negative bacteria. Synergistic and antibiofilm activities have also been recently described in combination with imipenem [3]. Herein, the latest results of our recently designed polymyxin analogs will be presented.
Acknowledgments:The research was supported by the University of Barcelona, Fundació Bosch i Gimpera, Xarxa de Referència en Biotecnologia, 2016LLAVO0018 grant (Generalitat de Catalunya) and the European Institute of Innovation and Technology (EIT Health). The authors are members of the ENABLE (European Gramnegative Antibacterial Engine) consortium (IMI-ND4BB, http://www.imi.europa.eu/projects-results/projectfactsheets/enable).
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