molecules Article NMR Study of the Secondary Structure and Biopharmaceutical Formulation of an Active Branched Antimicrobial Peptide Francesca Castiglia 1, Fabrizia Zevolini 1, Giulia Riolo 1, Jlenia Brunetti 1 , Alessandra De Lazzari 2, Alberto Moretto 2, Giulia Manetto 2, Marco Fragai 3, Jenny Algotsson 4, Johan Evenäs 4, Luisa Bracci 1,5, Alessandro Pini 1,5 and Chiara Falciani 1,5,* 1 Department of Medical Biotechnology, University of Siena, via Aldo Moro 2, 53100 Siena, Italy; [email protected] (F.C.); [email protected] (F.Z.); [email protected] (G.R.); [email protected] (J.B.); [email protected] (L.B.); [email protected] (A.P.) 2 Zambon S.p.a, Via della Chimica, 9. 36100 Vicenza, Italy; [email protected] (A.D.L.); [email protected] (A.M.); [email protected] (G.M.) 3 Magnetic Resonance Center (CERM), University of Florence, via Luigi Sacconi 6, 50019 Sesto Fiorentino (Firenze), Italy; [email protected]fi.it 4 Red Glead Discovery AB, Medicon Village, 223 81 Lund, Sweden; [email protected] (J.A.); [email protected] (J.E.) 5 SetLance srl, via Aldo Moro 2, 53100 Siena, Italy * Correspondence: [email protected]; Tel.: +0039-577-232022 Academic Editor: Hirokazu Tamamura Received: 15 October 2019; Accepted: 20 November 2019; Published: 25 November 2019 Abstract: 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. The secondary structure of this 40 amino acid peptide was investigated by NMR to fully characterize the product in the framework of preclinical studies. The possible presence of helixes or β-sheets in the structure had to be explored to predict the behavior of the branched peptide in solution, with a view to designing a formulation for parenteral administration. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, we also report the studies on a new salt form, 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 from a process development point of view and did not increase the toxicity of the antimicrobial drug. Keywords: antimicrobial peptides; branched peptides; NMR structure; counter-ion 1. Introduction The synthetic antimicrobial peptide SET-M33 is being developed as a possible new candidate for the development of a new antibacterial drug [1]. Synthesized in branched form with four copies of the same peptide sequence (KKIRVRLSA) on a three-lysine core (Figure1), it is more resistant to proteases and more suitable for clinical applications than its linear analogues [2–5]. SET-M33 shows strong antimicrobial activity against several multi-resistant Gram-negative bacteria, including clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and some Molecules 2019, 24, 4290; doi:10.3390/molecules24234290 www.mdpi.com/journal/molecules Molecules 2019, 24, x 2 of 13 Molecules 2019, 24, 4290 2 of 13 clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and some enterobacteriaceaeenterobacteriaceae [[66,,77].]. ItIt also also shows shows anti-inflammatory anti-inflammatory properties properties thanks thanks to its capacity to its capacity to neutralize to neutralizeLPS-induced LPS cytokine-induced release cytokinein vitroreleaseand inin vitro vivo andand in to vivo prevent and septic to prevent shock septic in animals shock infected in animals with infectedbacteria with of clinical bacteria interest of clinical [8]. interest [8]. Figure 1. Tetra-branched structure of the antimicrobial peptide SET-M33. Figure 1. Tetra-branched structure of the antimicrobial peptide SET-M33. The upstream and downstream processes of SET-M33 are currently being developed together withThe full upstream characterization and downstream of the product. processes Like mostof SET peptides,-M33 are SET-M33 currently is being routinely developed characterized together by withHPLC full and characterization mass spectrometry of the for product. its purity Like and most chemical peptides, structure. SET-M33 Here is we routinely also report characterized its 1H/13C/ 15byN 1 13 15 HPLCNMR and spectroscopy mass spectrometry characterization for its purity for full and atom-specific chemical structure assignment. Here and we its also primary report and its H/ secondaryC/ N NMRpeptide spectroscopy structure based characterization on observed for chemical full atom shifts.-specific Assessment assignment of SET-M33and its primary secondary and structuresecondary in peptidewater allows structure predicting based on aggregation observed chemical potential, shifts. which Assessment can affect drug of SET product-M33 designsecondary and structure formulation in waterstrategy. allows Aqueous predicting environments aggregation are potential, involved which in most can typical affect peptide drug product drug manufacturing design and formulation processes, strategy.that can A bequeous crucial environment for successfuls are development, involved in such most as typical the lyophilization peptide drug process manufacturing and the preparation processes, thatof injectable can be crucial pharmaceutical for successful forms. development, such as the lyophilization process and the preparation of injectableSalt formation pharmaceutical is important forms. in biopharmaceutical drug development as it modulates drug solubility,Salt formation stability andis important bioavailability. in biopharmaceutical Like almost all synthetic drug development peptides, SET-M33 as it modulates is obtained drug as a solubilitrifluoroacetatety, stability salt and (TFA), bioavailability. due to the presence Like almost of trifluoroacetic all synthetic acidpeptides, in the SET cleavage-M33 is and obtained purification as a trifluoroacetatesettings. Preliminary salt (TF studiesA), due on to thethe epresencefficacy and of trifluoroacetic toxicity of SET-M33 acid inrevealed the cleavage that and SET-M33 purification acetate settings.is less toxic Preliminary to human studies cells andon the animals efficacy than and SET-M33 toxicity TFAof SET [9-].M33 The revealed SET-M33 that peptide SET-M33 is therefore acetate isconverted less toxic to to the human acetate cells form and at animals the end ofthan synthesis. SET-M33 The TF counter-ionA [9]. The SET exchange-M33 peptide requires is the therefore use of a convertedquaternary to ammoniumthe acetate form salt resinat the and end is of unsuitable synthesis. forThe industrial counter-ion scaling exchange [10]. Inrequires order tothe avoid use of this a quaternarystep, hydrochloric ammonium acid wassalt resin chosen and as is a strongerunsuitable acid for than industrial TFA, so scaling that the [10]. chloride In order counter-ion to avoid could this step,replace hydrochloric trifluoroacetate acid was [11– chosen13]. Complete as a stronger replacement acid than of TFA, TFA withso that chloride the chloride and stability counter of-ion SET-M33 could replaceto HCl trifluoroacetate treatment were [11 assessed.–13]. Complete Finally, thereplacement antimicrobial of TF activityA with chloride and toxicity and stability of SET-M33 of SET with-M33 the tothree HCl di treatmentfferent counter-ions, were assessed. TFA, Finally, acetate the and antimicrobial chloride, were activity assessed and in toxicity mammalian of SET cells-M33 and with in mice. the three different counter-ions, TFA, acetate and chloride, were assessed in mammalian cells and in mice.2. Results and Discussion 2.2.1. Results NMR and Characterization Discussion of SET-M33 Peptide The 40 amino acid long peptide SET-M33 was characterized using 1H-/13C-/15N-NMR spectroscopy 2.1. NMR Characterization of SET-M33 Peptide to perform complete atom-specific assignment, to verify the primary peptide structure and to evaluate possibleThe secondary40 amino or acid other long ordered peptide structures SET- basedM33 onwas observed characterized chemical using shift values1H-/13C and-/15N possible-NMR spectroscopymedium- and to long-range perform complete NOEs. atom-specific assignment, to verify the primary peptide structure and toTo evaluate allow a fasterpossible NMR secondary data collection, or other aordered 12.5 mg structure/mL peptides based solution on observed was used. chemical The pH shift was valuesadjusted and to possible pH 4.9 by medium a small addition- and long of-range TFA-d NOEs.in order to minimize the rate of proton exchange between amideTo groupsallow a of faster the peptide NMR data and watercollection, molecules a 12.5 resulting mg/mL inpeptide clear observation solution was of theused. backbone The pH amide was adjusted to pH 4.9 by a small addition of TFA-d in order to minimize the rate of proton exchange MoleculesMoleculesMolecules2019 2019 2019,,24 ,24 24,, 4290,x x 333 of of 1313 13 betweenbetween amideamide groupsgroups ofof thethe peptidepeptide andand waterwater moleculesmolecules resultingresulting inin clearclear observationobservation