The antimicrobial peptide SET-M33. Strategies to improve the manufacturing procedures and production of back-up molecules as novel antibiotics

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. In this thesis are repoted strategies to improve the biopharmaceutical development and manufacturing process of this peptide. First, the secondary structure of SET-M33 was investigated by NMR to fully characterize the product in the framework of preclinical studies. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, It is also reported the studies on a new salt formulation, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient to decrease the manufacturing peptide cost and did not increase the toxicity of the antimicrobial drug. In addition, to identify back-up molecules, a panel of modified versions of SET-M33 was tested in order to produce new molecules with better performance in terms of pharmaceutical profile and manufacturing costs. Amongst them, the opportunity of using SET-M33D-L-Ile and SET-M33D-Leu/Ile will allow to decrease the costs in the synthesis process and SET-M33-Gly/Ala, to eliminate the degradation site for bacterial proteases, without altering the strong antimicrobial activity of the original peptide. Finally cloning strategies, expression systems, purifications and structural characterizations of various proteins of mammalian inflammasomes performed at Boston Children's Hospital, affiliated with Harvard Medical School, are described.