Investigation of the N-Terminus Amino Function of Arg10-Teixobactin

molecules Communication Investigation of the N-Terminus Amino Function of Arg10-Teixobactin Shimaa A. H. Abdel Monaim 1, Sikabwe Noki 1, Estelle J. Ramchuran 1, Ayman El-Faham 2,3 ID , Fernando Albericio 1,2,4,5,6,* ID and Beatriz G. de la Torre 1,7,* 1 Catalysis and Peptide Research Unit, School of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa; [email protected] (S.A.H.A.M.); [email protected] (S.N.); [email protected] (E.J.R.) 2 Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] 3 Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 12321, Egypt 4 School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa 5 Department of Organic Chemistry, University of Barcelona, Barcelona 08028, Spain 6 CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Barcelona 08028, Spain 7 KRISP, College of Health Sciences, University of KwaZulu-Natal, Durban 4001, South Africa * Correspondence: [email protected] (F.A.); [email protected] (B.G.d.l.T.); Tel.: +27-614-047-528 (F.A.); +27-614-009-144 (B.G.d.l.T.) Received: 28 August 2017; Accepted: 25 September 2017; Published: 28 September 2017 Abstract: Teixobactin is a recently described antimicrobial peptide that shows high activity against gram-positive bacteria as well as mycobacterium tuberculosis. Due to both its structure as a head-to-side chain cyclodepsipeptide and its activity, it has attracted the attention of several research groups. In this regard, a large number of analogs with substitutions in both the cycle and the tail has been described. Here, we report the contribution of the N-terminus residue, N-Me-D-Phe, to the activity of Arg10-teixobactin. On the basis of our findings, we conclude that the N-terminus accepts minimum changes but not the presence of long alkyl chains. The presence of a positive charge is a requirement for the activity of the peptide. Furthermore, acylation of the N-terminus leads to total loss of activity. Keywords: antimicrobial peptides; teixobactin; lipophilicity; solid-phase peptide synthesis; cyclic depsipeptides 1. Introduction The isolation of a new anti-microbial peptide (AMP) called teixobactin in 2015 by Ling et al. raised great expectations because it is one of the few new antibiotics to have been reported in recent years [1,2]. This antibiotic shows high activity against gram-positive bacteria as well as Mycobacterium tuberculosis. Teixobactin (Figure1) is a head-to-side chain cyclodepsipeptide that contains hydrophilic and hydrophobic residues [3,4]. Thus, the tail of the natural peptide contains four polar amino acids, L-allo-enduracididine, two Ser, and D-Gln, and four non-polar Ile residues, one Ala and one N-Me-D-Phe residue at the N-terminus. Finally, a D-Thr is the branching unit to form the cyclic moiety. Molecules 2017, 22, 1632; doi:10.3390/molecules22101632 www.mdpi.com/journal/molecules Molecules 2017, 22, 1632 2 of 9 Molecules 2017, 22, 1632 2 of 8 Figure 1. Chemical structure of teixobactin. Taking Arg10-teixobactin (1) as a reference, in which the unusual L-allo-Enduracididne is replaced by Taking Arg -teixobactin (1) as a reference, in which the unusual L-allo-Enduracididne is replaced L-Arg [2,5], Nowick’s10 and our group have independently confirmed the importance of maintaining a by L-Arg [2,5], Nowick’s and our group have independently confirmed the importance of maintaining balance between the hydrophobic and hydrophilic residues for keeping the activity [3,4]. The main a balance between the hydrophobic and hydrophilic residues for keeping the activity [3,4]. The main conclusions drawn from those studies are that the replacement of polar or neutral amino acids by Lys conclusions drawn from those studies are that the replacement of polar or neutral amino acids by Lys retains or improves the activity of the molecule, while replacing the non-polar amino acids by Lys retainsleads to or total improves loss of the activity. activity Only of the the molecule, replacemen whilet of replacing non-polar the Ile non-polar by cyclohexylglycine amino acids by (Chg) Lys leads has tobeen total reported loss of to activity. retain Onlyactivity the [4]. replacement In a furthe ofr step non-polar towards Ile understanding by cyclohexylglycine the contribution (Chg) has of been the reportedcationic residues to retain activityto the activity [4]. In a furtherof teixobactin, step towards our group understanding has prepared the contribution a library ofof themore cationic than residues25 teixobactin to the analogs activity in of which teixobactin, extra basic our group residues has (Lys, prepared Orn, aDap) library were of introduced more than 25into teixobactin positions analogsoccupied in by which polar/neutral extra basic amino residues acids, (Lys, without Orn, Dap)affecting were the introduced N-terminus into residue positions [6]. occupied That study by N- polar/neutralconcluded that amino the activity acids, withoutof a cationic affecting teixobac the tinterminus analog residuerequires [ 6a]. ma Thatximum study of concluded three or four that thepositive activity charges. of a cationicThus, the teixobactin introduction analog of an requires extra charge a maximum leads to oftotal three loss or of four activity positive [6]. charges. Thus,However, the introduction little attention of an extra has charge been paid leads to to the total role loss of of the activity N-terminus [6]. residue for teixobactin, However, little attention has been paid to the role of the N-terminus residue for teixobactin, N-Me-D-Phe. We and Madder-Taylor-Singh’s group have demonstrated the importance of the D- N-Me-D-Phe. We and Madder-Taylor-Singh’s group have demonstrated the importance of the D- configuration, and reported that the Me group cannot be switched for an acetyl (N-Ac-D-Phe-Arg10- configuration,teixobactin (9)) and [5,7]. reported Furthermore, that the in Me a grouptotally cannotdifferent be switchedapproach, for Nowick an acetyl et al (N. -Ac-preparedD-Phe-Arg a short10- teixobactinversion of teixobactin, (9)) [5,7]. Furthermore,namely lipobactin, in a totallywhich differentretains most approach, of its activity Nowick and et in al. which prepared the last a short five version of teixobactin, namely lipobactin, which retains most of its activity and in which the last residues (two Ile (L, and D-allo), Ser, D-Gln, and N-Me-D-Phe) are replaced by a dodecanoyl moiety [4,8]. five residuesWith the (twoaim to Ile further (L, and exploreD-allo), the Ser, contributionD-Gln, and of Nthe-Me- N-Me-D-PheD-Phe) are to replaced the activity by of a dodecanoylteixobactin, moiety [4,8]. here we synthesized a small library of Arg10-teixobactin analogs. As we previously demonstrated that With the aim to further explore the contribution of the N-Me-D-Phe to the activity of teixobactin, the replacement of N-Me-D-Phe by N-Me-D-Lys leads to total loss of activity [6], here we concentrated hereon increasing we synthesized the hydrophobicity a small library ofof Argthe10 N-teixobactin-terminus analogs.by introducing As we previouslyextra tails demonstratedto this terminus that. theLipophilicity replacement plays of N a-Me- crucialD-Phe role by Nin-Me- the Dactivity-Lys leads of all to totalpeptides, loss of as activity well as [6 ],in here their we transport concentrated and ondistribution increasing within the hydrophobicitybiological systems of [9–11]. the N-terminus However, byfor introducingAMPs, increasing extra hydrophobicity tails to this terminus. can be Lipophilicitya way to promote plays binding a crucial to bacterial role in the membranes, activity of thereby all peptides, causing as greater well as disruption. in their transport For instance, and distributionsome species within such biologicalas S. aurus systems show a [9 limited–11]. However, electrostatic for AMPs, surface increasing potential hydrophobicity with the hydrophilic can be aAMPs, way to causing promote the binding AMPs to to bind bacterial to the membranes, bacterial me therebymbrane causingin a different greater way disruption. [12]. Furthermore, For instance, the somemembrane species binding such asof suchS. aurus peptidesshow is a salt limited sensitive, electrostatic leading to surface limited potential potency withat physiological the hydrophilic ionic AMPs,strength; causing however, the increasing AMPs to bind their to hydrophobicity the bacterial membrane means reducing in a different their salt way sensitivity, [12]. Furthermore, as well as theincreasing membrane their binding bactericidal of such potency. peptides As is an salt example, sensitive, increasing leading to the limited hydrophobicity potency at physiological of the AMP ionicCNY21 strength; (CNYITELeRRQHARASHLGLAR) however, increasing their hydrophobicity improved pept meanside reducing adsorption, their led salt to sensitivity, peptide asliposome well as increasingleakage, and their enhanced bactericidal activity potency. against As Escherichia an example, coli increasing and Pseudomonas the hydrophobicity aeruginosa of[12]. the Furthermore, AMP CNY21 (CNYITELeRRQHARASHLGLAR)it has been demonstrated that the replacement improved of peptide the aliphatic adsorption, acyl moiety led to of peptide polymyxin liposome B or colistin leakage, by andaromatic enhanced or fatty activity acid substituents against Escherichia has a great coli andimpactPseudomonas on the antimicrobial aeruginosa activity[12]. Furthermore, of the analogs it has [13–17]. been demonstrated that the replacement of the aliphatic acyl moiety of polymyxin B or colistin by aromatic or fatty acid substituents has a great impact on the antimicrobial activity of the analogs [13–17]. Molecules 2017, 22, 1632 3 of 9 Molecules 2017, 22, 1632 3 of 8 2. Results Results and and Discussion Discussion 2.1. Synthetic Synthetic Strategy Strategy N 2 6 8 10 The N-terminus-terminus Arg Arg10-teixobactin-teixobactin analogs analogs (Figure (Figure 22,, 2––6,, 8––10)) were were synthesized synthesized using using strategies strategies developed by our group for the previous analogs [[2,3,7].2,3,7].

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