Characterization of Tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties

In e na ional Jou nal o Molecula Sciences A icle Cha ac e iza ion o Tachyplesin Pep ides and Thei Cyclized Analogues o Imp o e An imic obial and An icance P ope ies Felici as Ve nen 1, Pe a J. Ha ey 1, Susana A. Dias 2, Ana SaloméVeiga 2, Yen-Hua Huang 1, Da id J. C aik 1, Nicole Law ence 1and Sónia T oei a Hen iques 1,3,* 1Ins i u e o Molecula Bioscience, The Uni e si y o Queensland, B isbane, Queensland 4072, Aus alia 2Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, 1649-028 Lisboa, Po ugal 3 School o Biomedical Sciences, Facul y o Heal h, Ins i u e o Heal h & Biomedical Inno a ion, Queensland Uni e si y o Technology, T ansla ional Resea ch Ins i u e, B isbane, Queensland 4102, Aus alia *Co espondence: [email p o ec ed]; Tel.: +61-7-34437342 Recei ed: 26 July 2019; Accep ed: 21 Augus 2019; Published: 26 Augus 2019   Abs ac : Tachyplesin I, II and III a e hos de ense pep ides om ho seshoe c ab species wi h an imic obial and an icance ac i i ies. They ha e an amphipa hic β -hai pin s uc u e, a e highly posi i ely-cha ged and di e by only one o wo amino acid esidues. In his s udy, we compa ed he s uc u e and ac i i y o he h ee achyplesin pep ides alongside hei backbone cyclized analogues. We assessed he pep ide s uc u es using nuclea magne ic esonance (NMR) spec oscopy, hen compa ed he ac i i y agains bac e ia (bo h in he plank onic and bio ilm o ms) and a panel o cance ous cells. The impo ance o pep ide-lipid in e ac ions was examined using su ace plasmon esonance and luo escence spec oscopy me hodologies. Ou s udies showed ha achyplesin pep ides and hei cyclic analogues we e mos po en agains G am-nega i e bac e ia and melanoma cell lines, and showed a p e e ence o binding o nega i ely-cha ged lipid memb anes. Backbone cycliza ion did no imp o e po ency, bu imp o ed pep ide s abili y in human se um and educed oxici y owa dhuman edbloodcells. Pep ide-lipidbindinga ini y, o ien a ion wi hin hememb ane, and abili y o dis up lipid bilaye s di e ed be ween he cyclized pep ide and he pa en coun e pa . We show ha achyplesin pep ides and cyclized analogues ha e simila ly po en an imic obial and an icance p ope ies, bu ha backbone cycliza ion imp o es hei s abili y and he apeu ic po en ial. Keywo ds: achyplesin; hos de ense pep ide; an icance ; an imic obial; an ibio ilm; pep ide-memb ane in e ac ion; s uc u e-ac i i y; model memb anes; nuclea magne ic esonance solu ion s uc u e 1. In oduc ion The hos de ense pep ides (HDPs) achyplesin I, II and III (TI, TII and TIII) a e ac i e agains a b oad ange o G am-nega i e and G am-posi i e bac e ia and ungi [ 1 – 4 ] and possess an icance p ope ies [ 5 – 15 ]. Each analogue was isola ed om a di e en species o ho seshoe c ab, bu hey sha e high sequence homology (Table 1). TI, TII and TIII possess 17 amino acid esidues, wo disul ide bonds, a C- e minal α -amida ion, and hei s uc u e is o ganized in a β -hai pin [ 2 , 3 , 16 ]. Like o he HDPs [ 17 ], TI, TII and TIII possess an amphipa hic seconda y s uc u e (i.e., posi i ely cha ged and hyd ophobic amino acids seg ega e in o dis inc clus e s), hough o be essen ial o hei an imic obial ac i i y. Ca ionic amphipa hic HDPs selec i ely a ge he anionic su aces o mic obes, a he han he neu al su ace o hos cells, and kill hem by a mechanism ha in ol es binding o and inse ion in o cell memb anes. The ini ial binding is media ed by elec os a ic a ac ions be ween he posi i ely-cha ged esidues o HDPs and he anionic mic obial su ace [ 18 ], and is ollowed by he In . J. Mol. Sci. 2019,20, 4184; doi:10.3390/ijms20174184 www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2019,20, 4184 2 o 25 inse ion o hyd ophobic esidues in o lipid memb anes in a p ocess ha in ol es an-de -Waal’s in e ac ions wi h he phospholipids [18,19]. Simila o bac e ial cells, he su ace o cance cells is nega i ely-cha ged due o he inc eased exp ession and exposu e o phospholipids con aining he anionic phospha idylse ine (PS) headg oup [ 20 – 23 ]. In con as , cell memb anes o heal hy mammalian cells a e asymme ic and phospholipids con aining PS-headg oups a e ound exclusi ely in he inne lea le . Va ia ions in he o e all cell su ace cha ge [ 24 ] in phospholipids wi h exposed [ 25 , 26 ] memb ane luidi y and cu a u e [ 18 , 27 ], ha e been shown o modula e he selec i e oxici y o HDPs owa ds cance cells. These di e ences egula e he a ini y o pep ides o cell memb anes, he e ec i e pep ide- o-lipid a io and he abili y o HDP o kill cance ous cells o pa hogens a he han heal hy cells [28–30]. Se e al s udies ha e in es iga ed he ac i i y, s uc u e and mechanism o he ac ion o TI, bu ew ha e examined he ac i i y o TII and TIII. Ea ly s udies sugges ed ha TI kills bac e ial cells by a mechanismin ol inginne memb anepe meabiliza ionand he apide luxo K + [ 31 – 33 ]. La e , TI was shown o ansloca e ac oss lipid bilaye s, cause a phospholipid lip- lop and o m o oidal po es [ 33 , 34 ]. In cance ous cells, TI was epo ed o induce cell dis up ion and la e apop osis/nec osis [ 12 , 35 ]. Pa edes-Game o e al. p oposed ha he cance cell dea h mechanism was dependen on he pep ide dose: a high concen a ions, he pep ide-induced di ec cell memb ane dis up ion, and a lowe concen a ions, i ac i a ed in acellula cell dea h mechanisms [12]. Because o he exp ession o TI, TII and TIII in dis inc species o ho seshoe c ab, we we e speci ically in e es ed in compa ing hei s uc u e, ac i i y and mode-o -ac ion. As hese pep ides ha e a po en ial applica ion as an icance and/o an imic obial agen s, we in es iga ed whe he backbone cycliza ion would inc ease he s abili y and main ain ac i i y. Ou s udies show ha TI, TII and TIII, and hei cyclic analogues cTI, cTII and cTIII, ha e simila s uc u es and ac i i ies agains bac e ia and cance ous cells. Backbone cycliza ion educed he hemoly ic ac i i y and inc eased pep ide s abili y while main aining po en an icance and an imic obial ac i i ies. cTI and cTIII especially showed po en ial o be conside ed o he de elopmen o an icance pep ide-based d ugs. 2. Resul s 2.1. P ope ies o Tachyplesin I–III and Thei Cyclic Analogues The amino acid sequences o TI, TII and TIII di e in posi ions 1 o 15, which can be a lysine o an a ginine esidue (Table 1). So a , TI is he mos s udied and he only analogue wi h epo ed s uc u e calcula ions [ 36 – 38 ]. We we e in e es ed in compa ing TI, TII and TIII and hei backbone-cyclized analogues (cTI–III) o iden i y simila i ies and di e ences in hei h ee-dimensional s uc u e and s abili y; and o de e mine whe he hese cha ac e is ics a ec he memb ane in e ac ions and biological ac i i y o he pep ides. All pep ides we e syn hesized using solid-phase pep ide syn hesis, oxidized and co ec ly olded, as sugges ed by he obse ed masses using elec osp ay ioniza ion mass spec oscopy (ESI-MS; Supplemen a y Figu e S1 and Table 1) and con i med h ough clea ly dispe sed peaks in he amide egion o hei espec i e One-dimensional (1D) 1 H NMR spec a [ 39 ]. The pep ides we e pu i ied o >95%, as con i med by analy ical e e se-phase high-pe o mance liquid ch oma og aphy (RP-HPLC; see ch oma og ams o pu e pep ides in Supplemen a y Figu e S1a). Despi e mino di e ences, hei o e all hyd ophobici y ollows he end cTI >cTII >cTIII >TI > TII >TIII (Table 1), as indica ed by hei e en ion ime (RT) on analy ical RP-HPLC (Supplemen a y Figu e S1). The cyclic analogues appea o be o e all mo e hyd ophobic (less pola ) han he pa en pep ides, which is consis en wi h he loss o he N- e minal cha ge and he C- e minal amida ion esul ing om cycliza ion. In . J. Mol. Sci. 2019,20, 4184 3 o 25 Table 1. The sequence and physicochemical p ope ies o achyplesin I–III (TI-TIII) and hei cyclic analogues (cTI-cTIII). Pep ide Sequence 1Mass (Da) 2RT (min) 3Cha ge 4 Calc. Obs. TI KWCFRVCYRGICYRRCR * 2263.8 2263.5 18.04 +7 TII RWCFRVCYRGICYRKCR * 2263.8 2263.5 17.79 +7 TIII KWCFRVCYRGICYRKCR * 2235.8 2235.6 17.68 +7 cTI KWCFRVCYRGICYRRCRG2303.8 2303.7 18.69 +6 cTII RWCFRVCYRGICYRKCRG 2303.8 2303.7 18.53 +6 cTIII KWCFRVCYRGICYRKCRG 2275.8 2275.5 18.44 +6 1 Pep ide sequences and amino acid esidues di e ing om TI a e in bold. * deno es C- e minal amida ion. 2 A e age mass calcula ed (Calc.) om he amino acid sequence and expe imen ally obse ed (Obs.) using syn he ic pep ide and de e mined om m/z 3+in ESI-MS. 3 Re en ion ime (RT) o pep ides on an analy ical RP-HPLC; ch oma og ams shown in Supplemen a y Figu e S1a we e ob ained wi h a 2%/min g adien o 0–40% sol en B (90% ace oni ile; 0.05% i luo oace ic acid (TFA) ( / )) in sol en A (H 2 O, 0.05% TFA ( / )) a a low a e o 0.3 mL/min. 4 Cha ge o he pep ides a pH 7.4. 2.2. S uc u e o Tachyplesins and Backbone-Cyclised Analogues The h ee-dimensional (3D) s uc u es o TII and TIII, and o he backbone-cyclized analogues cTI–cTIII we e de e mined wi h solu ion NMR spec oscopy. All backbone esonances we e ully assigned apa om he N- e minal amides o he wo linea pep ides, and he R1/G18 amides o cTII, e lec i e o some deg ee o lexibili y in hese egions. The seconda y α H chemical shi s o he na i e pep ides and he cyclic analogues we e highly simila , indica ing a negligible change in he backbone s uc u e and he β-s ands (W2-Y8; I11-R17) (Figu e 1a,b). The 3D solu ion s uc u es o each pep ide we e calcula ed om dis ance es ain s, anging om 154 o he linea pep ides, o 172–284 o he cyclic analogues, along wi h dihed al angle es ain s o aling 34 o 36. The inal amily o s uc u es o each o he pep ides has good s uc u al and ene gy s a is ics, as indica ed by an o e all MolP obi y sco e o less han 1.6, shown in Table S1. Analysis o he s uc u es by PROMOTIF [ 40 ] de ines an ipa allel β -s ands being o med by esidues W2-Y8 and I11-R17 in all bu one o he pep ides. The excep ion is TIII which has sligh ly sho e s ands o med be ween esidues C3-Y8 and I11-C16. All disul ide bonds a e de ined by PROMOTIF as adop ing he sho igh -hand hook con igu a ion. S uc u es o he achyplesin pep ides and he cyclized analogues di e ed p ima ily in he lexibili y o he N- and C- e mini o he pa en pep ides (Figu e 1b–d). The educ ion o he amino acid side-chain lexibili y in he e minal egions due o backbone cycliza ion is emphasized in Figu e 1d wi h he side chain o he esidues K/R1 and K/R15, which di e be ween TI/cTI, TII/cTII and TIII/cTIII; he side chain o W2 is also shown, as his esidue is used o moni o he pep ide pa i ioning in o lipid bilaye s (see Sec ion 2.5.2). No signi ican ca ion- π in e ac ions be ween he R/K1 and W2 we e no ed o any o he achyplesin pep ides. Such an in e ac ion migh be e ealed by NMR in he o m o subs an ial de ia ions o he chemical shi s o a ginine/lysine esidues bu none we e obse ed (see Figu e 1a). The lack o ca ion- π in e ac ions is also suppo ed by he NMR solu ion s uc u es ha e eal a high deg ee o lexibili y in he e mini o he linea pep ides, and by he di e en o ien a ions ha hese esidues acqui e in he cyclic pep ides, as shown in Figu e 1d. The pep ide s uc u es we e deposi ed wi h he P o ein Da a Bank (PDB) and he Biological Magne ic ResonanceDa a Bank (BMRB):TII—PDB ID:6PI2, BMRB ID:30617; TIII—PDB ID:6PI3, BMRB ID: 30618; cTI—PDB ID: 6PIN; BMRB ID: 30619; cTII—PDB ID: 6PIO, BMRB ID: 30620; cTIII—PDB ID: 6PIP, BMRB ID: 30621. In . J. Mol. Sci. 2019,20, 4184 4 o 25 In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 o 25 Figu e 1. The NMR s uc u es o TI and cTI ( ed), TII and cTII (cyan), TIII and cTIII (magen a). (a) Seconda y αH chemical shi a 298 K de e mined om 1 H NMR spec a. Shi s we e calcula ed by sub ac ion o andom coil 1 H NMR shi s [41] om he expe imen al alues. Fo achyplesin I (TI), he shi s o he pep ide syn hesized in ou lab we e compa ed o shi s ob ained om da a banks: TI—PDB ID 2RTV [37] , and TI—BMRB ID 1135 [38] . Posi i e shi s g ea e han 0.1 ppm sugges β- s ands, indica ed by g ey a ows. (b) O e lay o he cyclic analogues (colo ed backbone) wi h hei espec i e pa en pep ide (g ey backbone). Hyd ogen bonds be ween β-s ands a e indica ed in blue. The s uc u e o TI was ob ained om he PDB (ID 1WO0). (c) O e lay o TI–TIII and cTI–cTIII. (d) Mobili y o he esidues (K o R) a posi ion 1 and 15, which di e be ween he pep ides, and o W2 o he na i e sequences and cyclic analogues, espec i ely. 2.3. Imp o ed S abili y and Reduced Hemoly ic Ac i i y o Cyclized Tachyplesin Pep ides Cycliza ion has been shown o inc ease s abili y [42,43] and educe he hemoly ic ac i i y o some pep ides [44,45]. Compa ison o esis ance o human p o eases showed ha he cTI analogue did no deg ade a e ea men o 24 h in 25% ( / ) human se um, whe eas only 25% o TI emained in he solu ion (see analy ical RP-HPLC ch oma og ams in supplemen a y Figu e S2). A pep ide (linea and wi hou disul ide bonds) used as a con ol was ully deg aded unde he same condi ions Figu e 1. The NMR s uc u es o TI and cTI ( ed), TII and cTII (cyan), TIII and cTIII (magen a). ( a ) Seconda y α H chemical shi a 298 K de e mined om 1 H NMR spec a. Shi s we e calcula ed by sub ac ion o andom coil 1 H NMR shi s [ 41 ] om he expe imen al alues. Fo achyplesin I (TI), he shi s o he pep ide syn hesized in ou lab we e compa ed o shi s ob ained om da a banks: TI—PDB ID 2RTV [ 37 ], and TI—BMRB ID 1135 [ 38 ]. Posi i e shi s g ea e han 0.1 ppm sugges β -s ands, indica ed by g ey a ows. ( b ) O e lay o he cyclic analogues (colo ed backbone) wi h hei espec i e pa en pep ide (g ey backbone). Hyd ogen bonds be ween β-s ands a e indica ed in blue. The s uc u e o TI was ob ained om he PDB (ID 1WO0). ( c ) O e lay o TI–TIII and cTI–cTIII. ( d ) Mobili y o he esidues (K o R) a posi ion 1 and 15, which di e be ween he pep ides, and o W2 o he na i e sequences and cyclic analogues, espec i ely. 2.3. Imp o ed S abili y and Reduced Hemoly ic Ac i i y o Cyclized Tachyplesin Pep ides Cycliza ion has been shown o inc ease s abili y [ 42 , 43 ] and educe he hemoly ic ac i i y o some pep ides [ 44 , 45 ]. Compa ison o esis ance o human p o eases showed ha he cTI analogue did no deg ade a e ea men o 24 h in 25% ( / ) human se um, whe eas only 25% o TI emained in he solu ion (see analy ical RP-HPLC ch oma og ams in Supplemen a y Figu e S2). A pep ide (linea and In . J. Mol. Sci. 2019,20, 4184 5 o 25 wi hou disul ide bonds) used as a con ol was ully deg aded unde he same condi ions (Figu e 2a). TI has p e iously been shown o be comple ely s able o 2 h in mouse o human se um [46]. The pe cen age o hemolysis o human ed blood cells (RBCs) ollowed he end TI >TII > TIII >cTII >cTI >cTIII when compa ed a 128 µ M, he highes concen a ion o pep ide es ed. TI was he mos hemoly ic o he na i e sequences. Backbone cycliza ion educed he hemoly ic ac i i y o he cTI and cTIII compa ed o hei pa en coun e pa s, bu led o no clea imp o emen o cTII. The C-amida ed pep ides TI–TIII lysed 66%, 56% and 41% o RBCs a 64 µ M espec i ely (Figu e 2b and Table 2). A simila end bu lowe hemoly ic ac i i y had been epo ed o TI, TII, and TIII lacking C- e minal amida ion a highe pep ide concen a ions [ 47 ], which is known o impac pep ide ac i i y [48,49]. The posi i e con ol meli in, a hemoly ic pep ide om honeybee enom, induces 100% hemolysis in human RBCs a concen a ions abo e 2 µ M. By compa ison, a his concen a ion, all achyplesin pep ides ha e a low hemoly ic ac i i y o a ound 10%. In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 o 25 (Figu e 2a). TI has p e iously been shown o be comple ely s able o 2 h in mouse o human se um [46]. The pe cen age o hemolysis o human ed blood cells (RBCs) ollowed he end TI > TII > TIII > cTII > cTI > cTIII when compa ed a 128 µM, he highes concen a ion o pep ide es ed. TI was he mos hemoly ic o he na i e sequences. Backbone cycliza ion educed he hemoly ic ac i i y o he cTI and cTIII compa ed o hei pa en coun e pa s, bu led o no clea imp o emen o cTII. The C-amida ed pep ides TI–TIII lysed 66%, 56% and 41% o RBCs a 64 µM espec i ely (Figu e 2b and Table 2). A simila end bu lowe hemoly ic ac i i y had been epo ed o TI, TII, and TIII lacking C- e minal amida ion a highe pep ide concen a ions [47], which is known o impac pep ide ac i i y [48,49]. The posi i e con ol meli in, a hemoly ic pep ide om honeybee enom, induces 100% hemolysis in human RBCs a concen a ions abo e 2 µM. By compa ison, a his concen a ion, all achyplesin pep ides ha e a low hemoly ic ac i i y o a ound 10%. Figu e 2. The pep ide s abili y in human se um and ac i i y agains human ed blood cells (RBCs). (a) S abili y o TI and cyclic analogue cTI in 25% ( / ) human se um o 24 h. The samples we e analyzed by analy ical RP-HPLC and he pe cen age o pep ide emaining was de e mined om he a ea unde he pep ide peak in he ch oma og am in compa ison o peak a ea a ime ze o. A linea 18 amino acid pep ide (KGGGGSGQLIDSMANSFV) was included as he posi i e con ol. (b) Hemoly ic ac i i y o achyplesins and hei cyclic analogues we e es ed up o 128 µM agains human RBCs (0.25% ( / ), 1 h incuba ion a 37 °C). Meli in, a highly hemoly ic pep ide, was included as he posi i e con ol. Table 2. Concen a ion o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) equi ed o induce 50% lysis in RBCs (HC50). a. Pep ide HC50 (µM) TI 34.9 ± 2.8 TII 55.4 ± 6.6 TIII 86.4 ± 12.2 cTI 106.9 ± 21.0 cTII 64.1 ± 9.4 cTIII > 128 a Values we e de e mined om a minimum o h ee independen eplica es and depic ed as mean ± SEM. 2.4. Biological Ac i i y o Tachyplesin I–III and o Thei Cyclic Analogues 2.4.1. Ac i i y agains Bac e ia The G am-nega i e Esche ichia coli s ains ATCC 25922 and DC2 CGSC 7139 and he G am- posi i e S aphylococcus au eus s ains ATCC 25923 and ATCC 6538 we e used o de e mine he an imic obial ac i i y o he pa en achyplesin pep ides and hei cyclic analogues agains bac e ia wi h di e ences in he physical and chemical p ope ies o hei cell wall. E. coli ATCC 25922 Figu e 2. The pep ide s abili y in human se um and ac i i y agains human ed blood cells (RBCs). ( a ) S abili y o TI and cyclic analogue cTI in 25% ( / ) human se um o 24 h. The samples we e analyzed by analy ical RP-HPLC and he pe cen age o pep ide emaining was de e mined om he a ea unde he pep ide peak in he ch oma og am in compa ison o peak a ea a ime ze o. A linea 18 amino acid pep ide (KGGGGSGQLIDSMANSFV) was included as he posi i e con ol. ( b ) Hemoly ic ac i i y o achyplesins and hei cyclic analogues we e es ed up o 128 µ M agains human RBCs (0.25% ( / ), 1 h incuba ion a 37 ◦C). Meli in, a highly hemoly ic pep ide, was included as he posi i e con ol. Table 2. Concen a ion o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) equi ed o induce 50% lysis in RBCs (HC50). a. Pep ide HC50 (µM) TI 34.9 ±2.8 TII 55.4 ±6.6 TIII 86.4 ±12.2 cTI 106.9 ±21.0 cTII 64.1 ±9.4 cTIII >128 aValues we e de e mined om a minimum o h ee independen eplica es and depic ed as mean ±SEM. 2.4. Biological Ac i i y o Tachyplesin I–III and o Thei Cyclic Analogues 2.4.1. Ac i i y agains Bac e ia The G am-nega i e Esche ichia coli s ains ATCC 25922 and DC2 CGSC 7139 and he G am-posi i e S aphylococcus au eus s ains ATCC 25923 and ATCC 6538 we e used o de e mine he an imic obial In . J. Mol. Sci. 2019,20, 4184 6 o 25 ac i i y o he pa en achyplesin pep ides and hei cyclic analogues agains bac e ia wi h di e ences in he physical and chemical p ope ies o hei cell wall. E. coli ATCC 25922 (“smoo h” LPS) and S. au eus ATCC 25923 a e common con ol s ains o an imic obial suscep ibili y es ing [ 50 , 51 ]. E. coli DC2 CGSC 7139 is hype sensi i e o an ibac e ial agen s and mo e pe meable o dyes [ 52 ], while S. au eus ATCC 6538 is known o o m bio ilms [ 53 ]. Tachyplesin pep ides and hei cyclic analogues we e es ed agains plank onic cul u es o all s ains and agains S. au eus ATCC 6538 in he bio ilm o m o di ec compa ison o hei an imic obial ac i i y. Tachyplesin I–III we e wo- o- ou imes mo e po en han hei cyclic analogues agains all bac e ial s ains in hei plank onic g ow h o m (Table 3). Gene ally, he G am-nega i e s ains we e mo e suscep ible han he G am-posi i e s ains. Simila MICs ha e been epo ed p e iously o TI agains E. coli ATCC 25922, [ 1 ] and TI and TII agains S. au eus ATCC 25923 [ 2 , 3 ]. Tachyplesin pep ides we e mos ac i e agains E. coli DC2 CGSC 7139 and E. coli ATCC 25922. The ac i i y agains hese bac e ial s ains was educed when he pep ides we e cyclized. The pep ides we e leas ac i e agains S. au eus ATCC 6538 in he plank onic o m, and no di e ence in ac i i y was obse ed be ween achyplesin pep ides and cyclic analogues (Table 3). Table 3. The minimal inhibi o y concen a ions (MICs) o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) agains plank onic bac e ia. MIC (µM) Pep ide E. coli DC2 CGSC 7139 E. coli ATCC 25922 S. au eus ATCC 25923 S. au eus ATCC 6538 TI 0.5–1 0.0625–0.5 1–4 4–8 TII 0.5–1 0.125–0.5 1–4 4–8 TIII 0.25–0.5 0.0625–0.5 1–4 4–8 cTI 4 1 2–8 8 cTII 4–8 1–2 2–8 8 cTIII 4–8 1–2 2–8 8 To de e mine whe he he achyplesin pep ides could ac agains bac e ial bio ilms, he cells me abolic ac i i y was measu ed o bio ilms o med by S. au eus ATCC 6538 (Figu e 3and Table 4). All pep ides exhibi ed simila ac i i y agains he bac e ia in he bio ilm o m, wi h a 50% loss o cell me abolic ac i i y obse ed a ~20 µ M. Howe e , app oxima ely 40% o he bio ilm was me abolically ac i e a 32 µ M, he highes concen a ion es ed, sugges ing a educed ac i i y agains bio ilm compa ed o he plank onic S. au eus ATCC 6538 bac e ia. In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 25 (“smoo h” LPS) and S. au eus ATCC 25923 a e common con ol s ains o an imic obial suscep ibili y es ing [50,51]. E. coli DC2 CGSC 7139 is hype sensi i e o an ibac e ial agen s and mo e pe meable o dyes [52], while S. au eus ATCC 6538 is known o o m bio ilms [53]. Tachyplesin pep ides and hei cyclic analogues we e es ed agains plank onic cul u es o all s ains and agains S. au eus ATCC 6538 in he bio ilm o m o di ec compa ison o hei an imic obial ac i i y. Tachyplesin I–III we e wo- o- ou imes mo e po en han hei cyclic analogues agains all bac e ial s ains in hei plank onic g ow h o m (Table 3). Gene ally, he G am-nega i e s ains we e mo e suscep ible han he G am-posi i e s ains. Simila MICs ha e been epo ed p e iously o TI agains E. coli ATCC 25922, [1] and TI and TII agains S. au eus ATCC 25923 [2,3]. Tachyplesin pep ides we e mos ac i e agains E. coli DC2 CGSC 7139 and E. coli ATCC 25922. The ac i i y agains hese bac e ial s ains was educed when he pep ides we e cyclized. The pep ides we e leas ac i e agains S. au eus ATCC 6538 in he plank onic o m, and no di e ence in ac i i y was obse ed be ween achyplesin pep ides and cyclic analogues (Table 3). Table 3. The minimal inhibi o y concen a ions (MICs) o achyplesin I–III (TI-TIII) and o hei cyclic analogues (cTI-cTIII) agains plank onic bac e ia. MIC (µM) Pep ide E. coli DC2 CGSC 7139 E. coli ATCC 25922 S. au eus ATCC 25923 S. au eus ATCC 6538 TI 0.5–1 0.0625–0.5 1–4 4–8 TII 0.5–1 0.125–0.5 1–4 4–8 TIII 0.25–0.5 0.0625–0.5 1–4 4–8 cTI 4 1 2–8 8 cTII 4–8 1–2 2–8 8 cTIII 4–8 1–2 2–8 8 To de e mine whe he he achyplesin pep ides could ac agains bac e ial bio ilms, he cells me abolic ac i i y was measu ed o bio ilms o med by S. au eus ATCC 6538 (Figu e 3 and Table 4). All pep ides exhibi ed simila ac i i y agains he bac e ia in he bio ilm o m, wi h a 50% loss o cell me abolic ac i i y obse ed a ~20 µM. Howe e , app oxima ely 40% o he bio ilm was me abolically ac i e a 32 µM, he highes concen a ion es ed, sugges ing a educed ac i i y agains bio ilm compa ed o he plank onic S. au eus ATCC 6538 bac e ia. Figu e 3. The ac i i y o achyplesin pep ides and hei cyclic analogues agains an S. au eus ATCC 6538 bio ilm. The esponse cu e o bio ilm ea ed wi h inc easing he concen a ions o pep ide (n = 4, ± SEM). Figu e 3. The ac i i y o achyplesin pep ides and hei cyclic analogues agains an S. au eus ATCC 6538 bio ilm. The esponse cu e o bio ilm ea ed wi h inc easing he concen a ions o pep ide (n=4, ±SEM). In . J. Mol. Sci. 2019,20, 4184 7 o 25 Table 4. Ac i i y o achyplesin I–III (TI–TIII) and cyclic analogues (cTI–cTIII) agains S. au eus ATCC 6538 in he bio ilm g ow h o m. Pep ide CC50 (µM) 1Me abolically Ac i e Cells (%) 2 TI 21.5 ±2.2 37.7 ±4.3 TII 23.1 ±2.9 39.9 ±2.9 TIII 24.2 ±4.1 43.3 ±4.1 cTI 17.8 ±3.6 31.9 ±6.0 cTII 19.4 ±1.5 34.6 ±7.1 cTIII 18.0 ±1.4 37.5 ±7.2 1 Pep ide concen a ion equi ed o induce he educ ion o 50% o he me abolically ac i e cell popula ion (CC50) de e mined using one-si e speci ic binding wi h he Hill slope. 2 Pe cen age o cells emaining me abolically ac i e a 32 µM, he highes pep ide concen a ion es ed. 2.4.2. Ac i i y agains Cance ous Cells TI, TII, TIII, and hei cyclic analogues we e es ed agains h ee melanoma (MM96L, HT144 and WM164) and one ce ical cance (HeLa) cell line (Supplemen a y Tables S2 and S3) o de e mine whe he he pep ides exhibi di e en cy o oxic ac i i ies. The aneuploid immo al ke a inocy e cell line HaCaT was included as a non-cance ous con ol. Cy o oxici y owa d cance cell lines was compa ed by de e mining pep ide concen a ions equi ed o achie e 50% o cell dea h (CC50) om dose- esponse cu es (Table 5). Table 5. The cy o oxici y o achyplesin I–III (TI-TIII) and cyclic analogues (cTI-cTIII) agains cul u ed cells. CC50 (µM) 1Melanoma Selec i i y 5 Pep ide MM96L 2HT144 2WM164 2HeLa 3HaCaT 4 TI 1.5 ±0.1 1.7 ±0.2 2.5 ±0.1 13.1 ±1.2 11.6 ±1.6 2–21 TII 1.6 ±0.1 2.0 ±0.1 1.6 ±0.1 18.0 ±3.9 3.7 ±0.2 3–35 TIII 1.8 ±0.1 2.0 ±0.1 1.7 ±0.1 21.7 ±1.1 7.3 ±0.5 5–48 cTI 1.3 ±0.1 1.4 ±0.1 2.7 ±0.1 6.7 ±0.6 7.9 ±0.5 2–76 cTII 1.1 ±0.1 0.8 ±0.04 2.4 ±0.3 7.2 ±0.4 2.4 ±0.3 3–58 cTIII 1.7 ±0.1 0.9 ±0.03 1.3 ±0.1 9.3 ±0.4 7.5 ±0.3 3–98 1 The concen a ion necessa y o kill 50% o cells was calcula ed om dose- esponse cu es (n ≥ 3, ± SEM). 2 Melanoma cell lines: MM96L, HT144, WM164, 3 ce ical cance cell line: HeLa, 4 heal hy epi helial con ol cell line: HaCaT (aneuploid immo al ke a inocy e). Desc ip ion and e i ica ion o each cell line a e de ailed in Supplemen a y Tables S2 and S3). 5 selec i i y o melanoma cell lines was es ima ed h ough he ac i i y- oxici y index (ATI). ATI =MHC/MCC50 (modi ied om Re e ence [ 46 ]), wi h MHC being he minimal concen a ion necessa y o induce 10% (lowe alue) o 50% (highe alue) cell dea h in human RBCs and MCC50 being he median o CC50 alues o all melanoma cell lines. Values abo e 1 indica e a highe selec i i y o he cance ous cells o e RBCs. The cy o oxic ac i i ies o he achyplesin pep ides and hei cyclic analogues a e dependen on he cell lines (Table 5). All pep ides we e mos e ec i e agains he melanoma cell lines MM96L, HT144 and WM164 (cy o oxic ac i i ies anged be ween CC50 0.8–2.7 µ M). Compa ed o he con ol cell line HaCaT, he cy o oxic ac i i ies o he pep ides agains melanoma we e signi ican ly di e en (p<0.05) wi h he excep ion o cTII agains WM164. The ce ical cance cell line HeLa was mo e esis an owa ds he achyplesin pep ides compa ed o he melanoma cell lines and highe pep ide concen a ions we e necessa y o each 50% cell dea h. Di e ences in cy o oxic ac i i y agains HeLa, compa ed o he con ol cell line HaCaT, can be obse ed o all pep ides excep TI and cTI. The cyclic analogues cTI–cTIII we e app oxima ely 2x mo e po en agains HeLa han he pa en pep ides TI–TIII (p<0.05). The pep ides ha e a simila selec i i y o he melanoma cell lines a lowe pep ide concen a ions which would induce ≤10% hemolysis in RBCs. A concen a ions inducing ≤50% hemolysis in RBCs, In . J. Mol. Sci. 2019,20, 4184 8 o 25 he cyclic analogues cTI–cTIII we e mo e selec i e. O e all, he mos p omising he apeu ic ange was obse ed o TIII, cTI and cTIII (Table 5). 2.5. Mechanis ic S udies Pep ides wi h an amphipa hic a angemen o cha ged and hyd ophobic esidues a e known o ac agains bac e ial and cance cells ia selec i e memb ane a ge ing, pene a ion and/o lysis. To cha ac e ize how pep ide-memb ane in e ac ions a ec biological ac i i y, we unde ook de ailed pep ide-memb ane binding s udies ha compa e pa en and cyclic achyplesin pep ides. 2.5.1. Pep ide Binding o Model Memb anes The abili y o TI o bind o model memb anes has been p e iously shown and a p e e ence o nega i ely-cha ged memb anes was ound [ 31 , 46 ]. We we e in e es ed in compa ing he memb ane-binding p ope ies o he pa en achyplesins e sus he cyclic analogues o de e mine whe he di e ences in memb ane binding could explain he ela i e biological ac i i ies (see Table 5). Gi en he simila po encies among he h ee achyplesins, and among he h ee cyclic analogues, we compa ed he in e ac ion o TI and cTI, as ep esen a i e o pa en and cyclic achyplesin pep ides, espec i ely, wi h model memb anes using su ace plasmon esonance (SPR). Phospholipids con aining PC-headg oups a e he mos common in he ou e lea le o he mammalian plasma memb ane [ 54 , 55 ]; hus, we p epa ed model memb anes composed o he zwi e ionic POPC (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine), which o ms luid bilaye s a 25 ◦ C and mimics he o e all luidi y and neu al su ace o heal hy euka yo ic cells [ 56 ]. Phospholipids con aining he nega i ely-cha ged PS-headg oups a e no mally es ic ed o he inne lea le in euka yo ic cell memb anes bu a e exposed a he cell su ace o cance ous cells [ 20 , 22 , 23 ]. The e o e, we used model memb anes wi h 20% o POPS (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine), POPC/POPS (4:1 mola a io), o ep esen he nega i ely-cha ged su ace o cance cells. Bo h TI and cTI bound wi h highe a ini y o nega i ely-cha ged POPC/POPS (4:1) han o zwi e ionic POPC lipid bilaye s. cTI had s onge a ini y o bo h lipid sys ems han he pa en pep ide, as shown by a highe pep ide- o-lipid (P/L) a io du ing associa ion, a slowe dissocia ion a e (k o ) om he lipids, and a highe amoun o pep ide emaining associa ed o he memb ane a he end o dissocia ion (P/L o ) (Figu e 4a,b and Table 6). Addi ionally, TI and cTI dis up ed la ge unilamella esicles (LUVs) o POPC/POPS (4:1) wi h highe e icacy han LUVs o POPC, which ag ees wi h hei p e e ence o nega i ely-cha ged o e neu al memb anes. Howe e , TI dis up ed LUVs o POPC and o POPC/POPS (4:1) mo e e icien ly han he cyclic analogue cTI (Figu e 4c, Table 6). Thus, he highe binding sa u a ion and a ini y o cTI (see P/L max and kine ic pa ame e s in Table 6) o he memb anes did no co ela e wi h i s abili y o dis up memb anes. The highe e icacy o TI in dis up ing memb anes, compa ed o he cyclic analogue, migh explain he highe ac i i y o he pa en TI–TIII o plank onic bac e ial cells (see Table 3), compa ed o hei cyclic analogues. In . J. Mol. Sci. 2019,20, 4184 9 o 25 In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 o 25 hemolysis in RBCs, he cyclic analogues cTI–cTIII we e mo e selec i e. O e all, he mos p omising he apeu ic ange was obse ed o TIII, cTI and cTIII (Table 5). 2.5. Mechanis ic S udies Pep ides wi h an amphipa hic a angemen o cha ged and hyd ophobic esidues a e known o ac agains bac e ial and cance cells ia selec i e memb ane a ge ing, pene a ion and/o lysis. To cha ac e ize how pep ide-memb ane in e ac ions a ec biological ac i i y, we unde ook de ailed pep ide-memb ane binding s udies ha compa e pa en and cyclic achyplesin pep ides. 2.5.1. Pep ide Binding o Model Memb anes The abili y o TI o bind o model memb anes has been p e iously shown and a p e e ence o nega i ely-cha ged memb anes was ound [31,46]. We we e in e es ed in compa ing he memb ane- binding p ope ies o he pa en achyplesins e sus he cyclic analogues o de e mine whe he di e ences in memb ane binding could explain he ela i e biological ac i i ies (see Table 5). Gi en he simila po encies among he h ee achyplesins, and among he h ee cyclic analogues, we compa ed he in e ac ion o TI and cTI, as ep esen a i e o pa en and cyclic achyplesin pep ides, espec i ely, wi h model memb anes using su ace plasmon esonance (SPR). Phospholipids con aining PC-headg oups a e he mos common in he ou e lea le o he mammalian plasma memb ane [54,55]; hus, we p epa ed model memb anes composed o he zwi e ionic POPC (1- palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine), which o ms luid bilaye s a 25 °C and mimics he o e all luidi y and neu al su ace o heal hy euka yo ic cells [56]. Phospholipids con aining he nega i ely-cha ged PS-headg oups a e no mally es ic ed o he inne lea le in euka yo ic cell memb anes bu a e exposed a he cell su ace o cance ous cells [20,22,23]. The e o e, we used model memb anes wi h 20% o POPS (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine), POPC/POPS (4:1 mola a io), o ep esen he nega i ely-cha ged su ace o cance cells. Bo h TI and cTI bound wi h highe a ini y o nega i ely-cha ged POPC/POPS (4:1) han o zwi e ionic POPC lipid bilaye s. cTI had s onge a ini y o bo h lipid sys ems han he pa en pep ide, as shown by a highe pep ide- o-lipid (P/L) a io du ing associa ion, a slowe dissocia ion a e (k o ) om he lipids, and a highe amoun o pep ide emaining associa ed o he memb ane a he end o dissocia ion (P/L o ) (Figu e 4a,b and Table 6). Addi ionally, TI and cTI dis up ed la ge unilamella esicles (LUVs) o POPC/POPS (4:1) wi h highe e icacy han LUVs o POPC, which ag ees wi h hei p e e ence o nega i ely-cha ged o e neu al memb anes. Howe e , TI dis up ed LUVs o POPC and o POPC/POPS (4:1) mo e e icien ly han he cyclic analogue cTI (Figu e 4c, Table 6). Thus, he highe binding sa u a ion and a ini y o cTI (see P/L max and kine ic pa ame e s in Table 6) o he memb anes did no co ela e wi h i s abili y o dis up memb anes. The highe e icacy o TI in dis up ing memb anes, compa ed o he cyclic analogue, migh explain he highe ac i i y o he pa en TI–TIII o plank onic bac e ial cells (see Table 3), compa ed o hei cyclic analogues. Figu e 4. Memb ane binding and dis up ion induced by achyplesin I (TI) and cyclic achyplesin I (cTI). Model memb anes composed o 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine (POPC) and POPC/1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine (POPS) (4:1) we e compa ed. (a) Su ace plasmon esonance senso g ams ob ained wi h 32 µM pep ide injec ed o e lipid bilaye s deposi ed Figu e 4. Memb ane binding and dis up ion induced by achyplesin I (TI) and cyclic achyplesin I (cTI). Model memb anes composed o 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine (POPC) and POPC/1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine (POPS) (4:1) we e compa ed. ( a ) Su ace plasmon esonance senso g ams ob ained wi h 32 µ M pep ide injec ed o e lipid bilaye s deposi ed on an L1 chip su ace o 180 s (associa ion); dissocia ion was moni o ed o 600 s. Response uni s (RU) we e con e ed in o a pep ide- o-lipid a io (P/L (mol/mol)) o ake in o conside a ion he di e ences in lipid packing esul ing in di e en amoun s being deposi ed o co e he chip su ace. (b) The dose- esponse cu es show P/L ob ained a he end o he associa ion phase ( =170 s) and plo ed as a unc ion o pep ide concen a ion injec ed. ( c ) Pe cen age o esicle leakage de e mined by luo escence emission in ensi y o 5-ca boxy luo escein ( λex =490 nm, λem =513 nm) leaking om LUVs a inc easing concen a ions o pep ide. Lipid concen a ion used was 5 µ M, and he pep ide was es ed up o 10 µ M. Dose- esponse cu es we e i ed wi h one-si e speci ic binding wi h Hill slope equa ion in G aphPad P ism. Table 6. The kine ic and a ini y pa ame e s om su ace plasmon esonance analysis o he in e ac ion o 32 µ M achyplesin I (TI) and cyclic achyplesin I (cTI) wi h neu al (POPC) and nega i ely-cha ged model memb anes (POPC/POPS (4:1)) and leakage induced by he same pep ides and lipid sys ems. Pep ide Lipid Sys em P/Lmax (mol/mol) 1KD (µM) 1ko (x 10−2s−1)2P/Lo (mol/mol) 2LCmax (%) 3 TI POPC 0.26 ±0.06 22.4 ±10.9 1.50 ±0.11 0.046 ±0.001 39.5 ±4.6 cTI 0.33 ±0.07 16.7 ±8.4 0.91 ±0.03 0.065 ±0.001 32.9 ±9.4 TI POPC/POPS (4:1) 0.37 ±0.04 11.8 ±2.4 2.75 ±0.22 0.096 ±0.001 76.0 ±2.8 cTI 0.48 ±0.08 9.2 ±3.4 0.70 ±0.03 0.137 ±0.002 58.5 ±3.6 1 P/L max and K D we e calcula ed om he dose- esponse cu es (one-si e speci ic binding wi h Hill slope equa ion, G aphPad P ism) in Figu e 4b. The P/L max alue ep esen s he pep ide- o-lipid a io (mol/mol) when pep ide-lipid binding eaches sa u a ion, K D is he pep ide concen a ion necessa y o each he hal -maximal binding esponse. 2 k o is he dissocia ion cons an and P/L o is he pep ide-lipid a io a he end o associa ion phase calcula ed om he senso g ams ob ained wi h 32 µ M pep ide in Figu e 4a. k o and P/L o we e i ed in G aphPad P ism, assuming a Langmui kine ic. 3 Pe cen age o leakage achie ed when incuba ing 10 µ M pep ide wi h 5 µ M LUVs. POPC is 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphocholine; POPS is 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphose ine. To examine he abili y o cTI o bind model memb anes ha mimic bac e ial cell memb anes, we p epa ed esicles wi h an E. coli pola lipid ex ac composed o zwi e ionic phospha idyle hanolamine (PE)-phospholipids, nega i ely-cha ged phospha idylglyce ol (PG)-phospholipids, and ca diolipin (CA) in he p opo ion 67:23.2:9.8 (w /w %). The SPR senso g am and dose- esponse cu es (Supplemen a y Figu e S3) show ha cTI has a high a ini y o E. coli lipids. Compa ison o he dose- esponse cu es and i ed pa ame e s show ha he maximum amoun o cTI bound o E. coli lipids (P/L max , Supplemen a y Table S4) is no as high as o he o he es ed nega i ely-cha ged memb anes o o he zwi e ionic POPC, bu he P/L o is highe han om he o he es ed memb anes (see Supplemen a y Table S4), sugges ing ha a la ge amoun o pep ide emains bound o he bilaye s ha mimic bac e ial memb anes. To in es iga e whe he cTI dis inguishes he nega i ely-cha ged headg oups p esen in bac e ia (i.e., PG) om hose in cance cells (i.e., PS), we compa ed he binding o cTI o POPC/POPG (1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphoglyce ol; In . J. Mol. Sci. 2019,20, 4184 16 o 25 0.05% ( / ) TFA) un il he desi ed pu i y o >95%. The co ec pep ide mass was con i med wi h ESI-MS, while na i e disul ide connec i i y was in e ed om he dispe sion o peaks in he 1D NMR spec a using a B uke A ance 600 MHz spec ome e (Bille ica, USA). The pep ide concen a ion was de e mined om he abso bance a 280 nm ( E280 =8730 M −1 .cm −1 as es ima ed ex inc ion coe icien based on he con ibu ion o Ty and T p esidues, and disul ide bonds). 4.2. NMR Spec oscopy Fo he s uc u al analysis o TI–III and cTI–III, pep ide (1 mg/mL) was dissol ed in H 2 O/D 2 O (10:1, / ) and he pH adjus ed o pH 4–5. 1D 1 H spec a, wo-dimensional o al co ela ed spec oscopy (TOCSY) and nuclea O e hause e ec spec oscopy (NOESY) we e acqui ed wi h a B uke A ance 600 MHz NMR spec ome e (Bille ica, USA) a a empe a u e o 298 K. Addi ional spec a o 1 H- 13 C HSQC and 1 H- 15 N HSQC in H 2 O/D 2 O (10:1, / ) and exclusi e co ela ion spec a (E.COSY) in D 2 O we e acqui ed. Spec a we e e e enced o an in e nal s anda d 2,2-dime hyl-2-silapen one-5-sul ona e (DSS) a 0 ppm. CYANA 3.97 was used o au oma ically calcula e and e ine s uc u es based on dis ance es ain s de i ed om he NOESY spec a [ 72 ], and o sion angles ( φ and ϕ ) gene a ed using TALOS-N and H α , C α , C β , HN chemical shi s de i ed om NOESY, 1 H- 13 C HSQC and 1 H- 15 N HSQC spec a [ 73 ]. Se e al χ 1 side-chain angle es ain s we e added based on E.COSY and NOESY da a. A inal se o s uc u es was gene a ed wi h CNS [ 74 ] using o sion angle dynamics, e inemen and ene gy minimiza ion in explici sol en . Final s uc u es we e assessed o s e eochemical quali y using MolP obi y [75]. 4.3. Se um S abili y The se um s abili y assay was ca ied ou as p e iously desc ibed [ 42 ] wi h some modi ica ions. B ie ly, achyplesin a ian s we e incuba ed in 25% ( / ) human se um dilu ed in phospha e-bu e ed saline (PBS) a a inal concen a ion o 50 µ M a 37 ◦ C. T iplica es we e collec ed a ime 0 h and 24 h and he se um p o eins we e p ecipi a ed wi h ace oni ile (1:3 a io) supplemen ed wi h 3% TFA. Samples we e kep on ice o 10 min be o e cen i uga ion a 17,000 × g o 10 min a 4 ◦ C. The pep ide con aining supe na an o each sample was ha es ed and quan i ied using RP-HPLC (10 o 45% sol en B, 1%/min g adien ). The pe cen age o pep ide s abili y in human se um was calcula ed by compa ing he a ea o he pep ide peak ob ained a 24 h o ha a ime 0 h. A linea pep ide con aining 18 amino acid esidues (KGGGGSGQLIDSMANSFV) was included as a con ol suscep ible o p o eoly ic deg ada ion. 4.4. Hemoly ic S udies A small amoun o blood was collec ed om h ee heal hy human dono s. The blood was immedia ely dilu ed in PBS and cen i uged 4–5 imes o 1 min a 4000 pm o wash and sepa a e he human ed blood cells (RBCs). RBCs suspension (0.25% ( / ) in PBS) was incuba ed wi h pep ides wi h wo- old se ial dilu ions o he pep ide (highes concen a ion es ed was 128 µ M, and he lowes was 0.25 µ M) in a 96-well pla e. Meli in, a memb ane dis up i e pep ide, was used as con ol. The pla es we e incuba ed o 1 h a 37 ◦ C. A e incuba ion, he pla es we e cen i uged o 5 min a 1000 pm o pelle any non-lysed RBCs. A o al o 100 µ L o he supe na an we e ans e ed o a new 96-well pla e [ 76 ]. The hemoglobin eleased in o he supe na an om lysed cells was measu ed by abso bance a 415 nm using he Tecan in ini e M1000P o mul ipla e eade (Männedo , Swi ze land). 4.5. Cell Cul u e Cells we e g own in cell cul u e lasks and incuba ed in a humidi ied a mosphe e (5% CO 2 , 37 ◦ C). The cance cell lines HeLa and he con ol cell line HaCaT we e g own in a DMEM medium supplemen ed wi h 1% ( / ) penicillin/s ep omycin and 10% ( / ) e al bo ine se um (FBS). The cance cell lines MM96L, HT144 and WM164 we e g own in a RPMI medium supplemen ed wi h 1% ( / ) penicillin/s ep a idin, 10% ( / ) FBS, 20 mM l-glu amine, and 10 mM sodium py u a e. Cell cul u es In . J. Mol. Sci. 2019,20, 4184 17 o 25 we e main ained by dilu ion upon eaching con luence, each 48–72 h. Mo e in o ma ion abou he cell lines [77] is a ailable in Tables S2 and S3. 4.6. Cy o oxici y Assays Cells we e seeded in o 96-well la -bo om pla es a 5 × 10 3 cells/well and incuba ed o e nigh . The medium was emo ed and eplaced wi h 90 µ L se um- ee medium, 10 µ L o 10x concen a ed pep ide solu ions in PBS we e added. PBS was added as blank and 0.1% ( / ) T i on X-100 was used o es ablish 100% o cell dea h. A e 2 h incuba ion a 37 ◦ C, 10 µ L o il e ed 0.05% (w/ ) esazu in solu ion was added o each well [ 70 ]. Resazu in is con e ed o he pink and luo escen compound eso u in by iable cells [ 78 ]. A e incuba ion o e nigh , he luo escence in ensi y ( λex =565 nm and λem =584 nm ) was measu ed wi h he Tecan in ini e M1000P o mul ipla e eade (Männedo , Swi ze land). The selec i i y was calcula ed h ough he ac i i y- oxici y index (ATI) [ 46 ]. ATI =MHC/MCC50, wi h MHC being he minimal concen a ion necessa y o induce 10% o 50% cell dea h in human ed blood cells and MCC50 he median o cy o oxic concen a ions (CC50) o all es ed melanoma cell lines. 4.7. An imic obial S udies S. au eus ATCC 25923, S. au eus ATCC 6538, E. coli ATCC 25922, and E. coli DC2 CGSC 7139 we e g own in Muelle Hin on B o h (Sigma Ald ich, S . Luis, USA). Bac e ial cul u es in he exponen ial g ow h phase we e dilu ed o an OD600nm o 0.001 and seeded in o 96-well pla es. Pep ides a di e en concen a ions, s a ing a 64 µ M and wi h wo- old se ial dilu ions (i.e., 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625 µ M), we e incuba ed wi h cells [ 43 ]; 0.05% ( / ) esazu in was added o he cul u es he nex day and he con e sion o eso u in was measu ed a e 1 h o incuba ion a 37 ◦ C using a 565 nm exci a ion and 584 nm emission wa eleng h, as abo e. 4.8. Bio ilm S udies S. au eus ATCC 6538 (1 × 10 6 c u/mL) was cul u ed in T yp ic Soy B o h, con aining 0.25% (w/ ) glucoseand incuba ed in96-well mic o i e la -bo omed polys y enepla es o 24ha 37 ◦ C.P e o med bio ilms we e hen washed wi h Muelle Hin on B o h o emo e non-adhe en cells. Two- old se ial dilu ions o each pep ide (highes concen a ion es ed was 32 µ M and lowes was 0.25 µ M) we e added o he bio ilms o 4 h. Un ea ed 24 h p e o med bio ilms we e used as a con ol. The me abolic ac i i y o bio ilm-embedded cells was de e mined using a esazu in educ ion luo ome ic assay as p e iously desc ibed [53]. 4.9. Lipid Vesicle P epa a ion Mix u es wi h syn he ic lipids (POPC, POPS, POPG, A an i Pola Lipids) o E. coli lipid ex ac (A an i Pola Lipids, Alabas e . USA) we e ex uded in HEPES bu e (10 mM HEPES, 150 mM NaCl, pH 7.4) o p oduce lipid esicles, as p e iously desc ibed [ 43 , 79 ]. LUVs (Ø ≤ 100 nm) we e used in luo escence spec oscopy assays and small unilamella esicles (SUVs, Ø ≤50 nm) o SPR. 4.10. Fluo escence Spec oscopy Assays Fluo escence emission spec a (300–400 nm, exci a ion a 280 nm, sli s 3/3 mm) o 12.5 µ M pep ide and l-T p in HEPES bu e (in qua z cu e es, pa h leng h o 0.5 cm) we e scanned upon i a ion wi h LUVs composed o a ious lipid composi ions (up o 3 mM POPC, and up o 1.5 mM POPC/POPS (4:1) o POPC/POPG (4:1)) [ 80 ] using a Fluo oMax-4 spec o luo ome e (Ho iba, Kyo o, Japan). In eg a ed a eas o he luo escence emission spec a we e co ec ed o luo opho e dilu ion and ligh dispe sion due o i a ion wi h LUVs suspension; he blank was discoun ed. In . J. Mol. Sci. 2019,20, 4184 18 o 25 4.11. Quenching o T yp ophan Fluo escence The memb ane in-dep h loca ion o he T p esidue wi hin he pep ides was ollowed using Ac ylamide (Sigma Ald ich, S . Luis, USA) and 5- and 16-DS (Sigma Ald ich, S . Luis, USA). Quenching induced by ac ylamide was moni o ed wi h 12.5 µ M pep ide in a HEPES bu e , in he p esence o 1 mM o POPC, 0.1 mM POPC/POPS (4:1), 1 mM POPC/POPS (4:1), o 0.1 mM POPC/POPG (4:1) i a ed wi h inc easing concen a ion o ac ylamide [ 81 ]. The luo escence emission spec a we e de e mined wi h an exci a ion wa eleng h o 290 nm o educe he quenche / luo opho e ligh abso p ion a io. The luo escence emission spec a a ea was co ec ed o he inne il e e ec [ 82 ] due o inc eased abso bance o ac ylamide. Da a poin s we e analyzed using he S e n-Volme ep esen a ion (Equa ion (1)), he KSV is de e mined om he slope. I0 I=1+KSV[Q](1) A o al o 12.5 µ M o TI, o o cTI, in HEPES bu e and 1 mM POPC/POPS (4:1) we e i a ed wi h inc easing concen a ion o 5DS, o o 16DS. The luo escence emission spec a a ea was co ec ed o he inne il e e ec [ 82 ] due o inc eased abso bance o 5-,16DS. The e ec i e concen a ion o 5DS and 16DS in he lipid bilaye was calcula ed: pa i ion coe icien s o 5DS and 16DS in o luid memb anes a e 89,000 and 9730, espec i ely [ 59 , 81 ]. The da a poin s had a nega i e de ia ion o linea i y and he KSV was de e mined by i ing he da a wi h he Leh e equa ion (Equa ion (2)) [59,81]. I0 I=1+KSV[Q] (1+KSV[Q])(1− B)+ B (2) I0= luo escen in ensi y in bu e I= luo escen in ensi y in p esence o quenche KSV =S e n-Volme cons an [Q]=quenche concen a ion B= ac ion o ligh accessible o he quenche =I0,B I0 I0,B = luo escen in ensi y o he accessible popula ion o he quenche when [Q] =0 The a e age dis ance o he T p esidue om he bilaye cen e (Å) was de e mined using he pa allax me hod [62] ollowing he equa ion, z1F=1 −πCln F1 F2−L2 21 2L21 (3) zcF =z1F+Lc1(4) in which he z 1F is he dis ance be ween he T p esidue and he quenche moie y in 5DS, L 21 is he dis ance be ween he quenche g oups in 5 and 16DS (5 Å), F 1 is he luo escence o 12.5 µ M o TI, o o cTI, in he p esence o 1 mM POPC/POPS (4:1) and 0.4 mM 5DS, F 2 is he luo escence o 12.5 µ M o TI, o o cTI, in he p esence o 1 mM POPC/POPS (4:1) and 0.4 mM 16DS. C is he mola ac ion o quenche wi hin he o al lipid concen a ion pe uni a ea (assuming he su ace o lipid o be 70 Å2) [62]. z cF is he dis ance be ween he T p esidue and he cen e o he bilaye , L c1 is he dis ance be ween he cen e o he bilaye s and he quenche g oup o 5DS (15 Å). 4.12. Su ace Plasmon Resonance (SPR) SPR was used o in es iga e he a ini y and binding kine ics o pep ides o memb anes o di e en composi ions. The expe imen s we e conduc ed wi h an L1 biosenso chip (GE Heal hca e) a 25 ◦ C using a BIAco e 3000 ins umen (GE Heal hca e, Chicago, USA) [ 83 , 84 ]. The HEPES bu e was used o sample p epa a ion and as a unning bu e . Lipid bilaye s we e immobilized on o L1 chip by In . J. Mol. Sci. 2019,20, 4184 19 o 25 injec ion o SUVs a a low a e o 2 µ L/min. Pep ide samples wi h wo- old se ial dilu ions ( he highes concen a ion es ed was 64 µ M and he lowes was 1 µ M) we e injec ed o e he lipid bilaye a a low a e o 5 µ L/min. Associa ion o he pep ides on o he lipid bilaye was ollowed o 180 s and he dissocia ion om he lipid o 600 s. The BIAe al so wa e was used o analyze he senso g ams. The esponse uni s (RU) we e no malized o he pep ide- o-lipid a io (P/L); he P/L ob ained a a ixed ime poin a he end o he associa ion cu e (a 170 s), a which he esponse has eached a pla eau and he binding is close o equilib ium, was used o compa e he a ini y o he pep ides o he di e en lipid sys ems [84]. 4.13. Vesicle Leakage Assay LUVs (Ø ≤ 100 nm) we e p epa ed wi h he HEPES bu e con aining 40 mM o he luo escen 5-ca boxy luo escein (CF, Sigma Ald ich, S . Luis, USA). LUVs illed wi h CF a sel -quenching concen a ions we e sepa a ed om he non-encapsula ed dye on a Sephadex G-50 column equilib a ed wi h HEPES bu e [ 85 ]. The concen a ion o CF-LUVs was de e mined h ough a calib a ion cu e p epa ed om he o iginal lipid mix u e using S ewa ’s assay (abso bance a 485 nm) [ 86 ]. Pep ides we e incuba ed (25 ◦ C, 20 min) a wo- old dilu ions (s a ing a 10 µ M) wi h LUVs (5 µ M) in he HEPES bu e in a 96-well la -bo om black op ipla es (Pe kin Elme , Wal ham, USA). Vesicles and pep ides we e incuba ed o 20 min in da k and he elease o CF was measu ed in a Tecan in ini e M1000P o mul ipla e eade using 490 nm as he exci a ion and 513 nm as emission wa eleng hs. 4.14. S a is ical Analysis Values (mean o i ± SEM) we e analyzed in G aphPad P ism 7 o es o signi ican di e ences in he cy o oxic ac i i y o he pep ides. The mul iple - es unc ion wi h he Holm–Sidak me hod was applied when indica ed. P alues below 0.05 we e conside ed o be signi ican . Supplemen a y Ma e ials: Supplemen a y ma e ials can be ound a h p://www.mdpi.com/1422-0067/20/17/ 4184/s1. Au ho Con ibu ions: F.V. concep ualized his s udy wi h supe ision and inpu om S.T.H. and N.L. F.V., S.A.D., and P.J.H. conduc ed expe imen al wo k: F.V. olded and pu i ied he pep ides, conduc ed oxici y assays, model memb anes, su ace plasmon esonance and luo escence spec oscopy assays; S.A.D. conduc ed he bio ilm assay and helped F.V. wi h he an imic obial assays; P.J.H. pe o med NMR spec oscopy and s uc u al analysis o he pep ides; Y.-H.H. es ablished he se um s abili y assay. F.V. analyzed and in e p e ed da a wi h assis ance om S.T.H. and expe con ibu ions om all he co-au ho s. S.T.H., N.L., P.J.H., D.J.C., Y.-H.H. and A.S.V. e ised and edi ed he manusc ip . S.T.H. and D.J.C. p o ided he esou ces and acqui ed he unding. Funding: This p ojec was unded by a Na ional Heal h Medical Resea ch Council (NHMRC) p ojec g an (APP1084965). F.V. was suppo ed by he UQ Resea ch Schola ship, S.T.H. is an Aus alian Resea ch Council (ARC) Fu u eFellow(FT150100398), D.J.C. isanARC Aus alianLau ea e Fellow(FL150100146). Ma ie Skłodowska-Cu ie Resea ch and Inno a ion S a Exchange g an (RISE; call: H2020-MSCA-RISE-2014, g an ag eemen 644167) unded secondmen s o S.A.D. and o A.S.V. o he Uni e si y o Queensland. The T ansla ional Resea ch Ins i u e is suppo ed by a g an om he Aus alian Go e nmen . Acknowledgmen s: Many hanks o QUEDDI, he Ma Coope lab and he Helmu Schaide lab o he cell lines HaCaT, HT144 and WM164 espec i ely. The au ho s hank Oli ie Chene al and Joachim Weidmann (IMB, UQ) o hei assis ance wi h pep ide syn hesis. Con lic s o In e es : The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip , o in he decision o publish he esul s. Abb e ia ions 1D One-dimensional 2-CTC 2-chlo o i yl esin 3D Th ee-dimensional ACN Ace oni ile ATI Ac i i y/ oxici y index ATTC Ame ican Type Cul u e Collec ion In . J. Mol. Sci. 2019,20, 4184 20 o 25 BMRB Biological magne ic esonance da a bank CC Cy o oxic concen a ion CF 5-ca boxy luo escein cTI Cyclic Tachyplesin I cTII Cyclic Tachyplesin II cTIII Cyclic Tachyplesin III cTI–cTIII Cyclic Tachyplesin I, II and III DS Doxyl-s ea ic acid E.COSY Exclusi e co ela ion spec a ESI-MS Elec osp ay ioniza ion mass spec oscopy FBS Fe al bo ine se um Fmoc 9- luo enylme hoxyca bonyl HC50 Hemoly ic concen a ion necessa y o 50% lysis o RBCs HDP Hos de ense pep ide KSV S e n – Volme cons an LPS Lipopolysaccha ide LUV La ge unilamella esicle MCC Minimal cy o oxic concen a ion MHC Minimal hemoly ic concen a ion MIC Minimal inhibi o y concen a ion NMR Nuclea magne ic esonance NOESY Nuclea O e hause e ec spec oscopy PBS Phospha e bu e ed saline PDB P o ein da a bank PC Phospha idylcholine PS Phospha idylse ine PG Phospha idylglyce ol P/L Pep ide- o-lipid a io POPC 1-palmi oyl-2-oleoyl-glyce o-3-phosphocholine POPS 1-palmi oyl-2-oleoyl-sn-glyce o-3-phospho-L-se ine POPG 1-palmi oyl-2-oleoyl-sn-glyce o-3-phospho-(10- ac-glyce ol) RBCs Red blood cells RP-HPLC Re e se-phase high-pe o mance liquid ch oma og aphy RT Re en ion ime RU Response uni SEM S anda d e o o mean SPPS Solid phase pep ide syn hesis SPR Su ace plasmon esonance SUV Small unilamella esicle TI Tachyplesin I TII Tachyplesin II TIII Tachyplesin III TI–TIII Tachyplesin I, II and III TFA T i luo oace ic acid TOCSY To al co ela ed spec oscopy Re e ences 1. Edwa ds, I.A.; Ellio , A.G.; Ka anagh, A.M.; Zuegg, J.; Blasko ich, M.A.T.; Coope , M.A. Con ibu ion o Amphipa hici y and Hyd ophobici y o he An imic obial Ac i i y and Cy o oxici y o β-Hai pin Pep ides. ACS In ec . Dis. 2016,2, 442–450. [C ossRe ] [PubMed] 2. Miya a, T.; Tokunaga, F.; Yoneya, T.; Yoshikawa, K.; Iwanaga, S.; Niwa, M.; Takao, T.; Shimonishi, Y. An imic obial pep ides, isola ed om ho seshoe c ab hemocy es, achyplesin II, and polyphemusins I and II: chemical s uc u es and biological ac i i y. J. Biochem. 1989,106, 663–668. [C ossRe ] [PubMed] In . J. Mol. Sci. 2019,20, 4184 21 o 25 3. Nakamu a,T.; Fu unaka, H.; Miya a,T.; Tokunaga, F.; Mu a, T.; Iwanaga, S.; Niwa,M.; Takao, T.; Shimonishi, Y. Tachyplesin,aclasso an imic obialpep ide om hehemocy eso heho seshoec ab(Tachypleus iden a us). Isola ion and chemical s uc u e. J. Biol. Chem. 1988,263, 16709–16713. [PubMed] 4. Oh a, M.; I o, H.; Masuda, K.; Tanaka, S.; A akawa, Y.; Wacha o ayankun, R.; Ka o, N. Mechanisms o an ibac e ial ac ion o achyplesins and polyphemusins, a g oup o an imic obial pep ides isola ed om ho seshoe c ab hemocy es. An imic ob. Agen s Chemo he . 1992,36, 1460–1465. [C ossRe ] [PubMed] 5. Bu i, M.V.; To qua o, H.F.V.; Ba os, C.C.; Ide, J.S.; Mi anda, A.; Pa edes-Game o, E.J. Compa ison o Cy o oxic Ac i i y in Leukemic Lineages Re eals Impo an Fea u es o be a-Hai pin An imic obial Pep ides. J. Cell. Biochem. 2017,118, 1764–1773. [C ossRe ] [PubMed] 6. Chen, J.; Xu, X.M.; Unde hill, C.B.; Yang, S.; Wang, L.; Chen, Y.; Hong, S.; C eswell, K.; Zhang, L. Tachyplesin ac i a es he classic complemen pa hway o kill umo cells. Cance Res. 2005 ,65, 4614–4622. [C ossRe ] [PubMed] 7. Ding, H.; Jin, G.; Zhang, L.; Dai, J.; Dang, J.; Han, Y. E ec s o achyplesin I on human U251 glioma s em cells. Mol. Med. Rep. 2015,11, 2953–2958. [C ossRe ] [PubMed] 8. Kuzmin, D.V.; Emel’yano a, A.A.; Kalashniko a, M.B.; Pan elee , P.V.; O chinniko a, T.V. In Vi o S udy o An i umo E ec o An imic obial Pep ide Tachyplesin I in Combina ion wi h Cispla in. Bull. Exp. Biol. Med. 2018,165, 220–224. [C ossRe ] 9. Li, Q.F.; Ou Yang, G.L.; Li, C.Y.; Hong, S.G. E ec s o achyplesin on he mo phology and ul as uc u e o human gas ic ca cinoma cell line BGC-823. Wo ld J. Gas oen e ol. 2000,6, 676–680. [C ossRe ] 10. Li, X.; Dai, J.; Tang, Y.; Li, L.; Jin, G. Quan i a i e P o eomic P o iling o Tachyplesin I Ta ge s in U251 Gliomasphe es. Ma . D ugs 2017,15, 20. [C ossRe ] 11. Ouyang, G.L.; Li, Q.F.; Peng, X.X.; Liu, Q.R.; Hong, S.G. E ec s o achyplesin on p oli e a ion and di e en ia ion o human hepa ocellula ca cinoma SMMC-7721 cells. Wo ld J. Gas oen e ol. 2002,8, 1053–1058. [C ossRe ] 12. Pa edes-Game o, E.J.; Ma ins, M.N.; Cappabianco, F.A.; Ide, J.S.; Mi anda, A. Cha ac e iza ion o dual e ec s induced by an imic obial pep ides: egula ed cell dea h o memb ane dis up ion. Biochim. Biophys. Ac a 2012,1820, 1062–1072. [C ossRe ] 13. Ro han, H.A.; Ambikabo hy, J.; Ramasamy, T.S.; Rashid, N.N.; Yuso , R. A P elimina y S udy in Sea ch o Po en ial Pep ide Candida es o a Combina ional The apy wi h Cance Chemo he apy D ug. In . J. Pep . Res. The . 2017,25, 115–122. [C ossRe ] 14. Shi, S.L.; Wang, Y.Y.; Liang, Y.; Li, Q.F. E ec s o achyplesin and n-sodium bu y a e on p oli e a ion and gene exp ession o human gas ic adenoca cinoma cell line BGC-823. Wo ld J. Gas oen e ol. 2006 ,12, 1694–1698. [C ossRe ] 15. Zhang, H.T.; Wu, J.; Zhang, H.F.; Zhu, Q.F. E lux o po assium ion is an impo an eason o HL-60 cells apop osis induced by achyplesin. Ac a Pha macol. Sin. 2006,27, 1367–1374. [C ossRe ] 16. Mu a, T.; Fujimo o, T.; Nakajima, H.; Iwanaga, S. Tachyplesins Isola ed om Hemocy es o Sou heas Asian Ho seshoe C abs (Ca cinosco pius o undicauda and Tachypleus gigas): Iden i ica ion o a New Tachyplesin, Tachyplesin III, and a P ocessing In e media e o I s P ecu so 1. J. Biochem. 1990,108, 261–266. [C ossRe ] 17. Zaslo , M. An imic obial pep ides o mul icellula o ganisms. Na u e 2002,415, 389–395. [C ossRe ] 18. Teixei a, V.; Feio, M.J.; Bas os, M. Role o lipids in he in e ac ion o an imic obial pep ides wi h memb anes. P og. Lipid Res. 2012,51, 149–177. [C ossRe ] 19. Yeaman, M.R.; Youn , N.Y. Mechanisms o An imic obial Pep ide Ac ion and Resis ance. Pha macol. Re . 2003,55, 27–55. [C ossRe ] 20. U sugi, T.; Sch oi , A.J.; Conno , J.; Bucana, C.D.; Fidle , I.J. Ele a ed exp ession o phospha idylse ine in he ou e memb ane lea le o human umo cells and ecogni ion by ac i a ed human blood monocy es. Cance Res. 1991,51, 3062–3066. 21. Ran, S.; Downes, A.; Tho pe, P.E. Inc eased Exposu e o Anionic Phospholipids on he Su ace o Tumo Blood Vessels. Cance Res. 2002,62, 6132. 22. Zwaal, R.F.; Com u ius, P.; Be e s, E.M. Su ace exposu e o phospha idylse ine in pa hological cells. Cell. Mol. Li e Sci. 2005,62, 971–988. [C ossRe ] 23. Riedl, S.; Rinne , B.; Asslabe , M.; Schaide , H.; Walze , S.; No ak, A.; Lohne , K.; Zwey ick, D. In sea ch o a no el a ge - phospha idylse ine exposed by non-apop o ic umo cells and me as ases o malignancies wi h poo ea men e icacy. Biochim. Biophys. Ac a 2011,1808, 2638–2645. [C ossRe ] In . J. Mol. Sci. 2019,20, 4184 22 o 25 24. Iwasaki, T.; Ishibashi, J.; Tanaka, H.; Sa o, M.; Asaoka, A.; Taylo , D.; Yamakawa, M. Selec i e cance cell cy o oxici y o enan iome ic 9-me pep ides de i ed om bee le de ensins depends on nega i ely cha ged phospha idylse ine on he cell su ace. Pep ides 2009,30, 660–668. [C ossRe ] 25. Lei e Na á lia, B.; Au de ho s -Robe s, A.; Palma Ma io, S.; Connell Simon, D.; Ne o Jo ã o, R.; Beales Paul, A. PE and PS Lipids Syne gis ically Enhance Memb ane Po a ion by a Pep ide wi h An icance P ope ies. Biophys. J. 2015,109, 936–947. [C ossRe ] 26. Ma suzaki, K.; Sugishi a, K.; Fujii, N.; Miyajima, K. Molecula Basis o Memb ane Selec i i y o an An imic obial Pep ide, Magainin 2. Biochemis y 1995,34, 3423–3429. [C ossRe ] 27. Hoskin, D.W.; Ramamoo hy, A. S udies on an icance ac i i ies o an imic obial pep ides. Biochim. Biophys. Ac a 2008,1778, 357–375. [C ossRe ] 28. Ha is, F.; Dennison, S.R.; Singh, J.; Phoenix, D.A. On he selec i i y and e icacy o de ense pep ides wi h espec o cance cells. Med. Res. Re . 2013,33, 190–234. [C ossRe ] 29. Gaspa , D.; Veiga, A.S.; Cas anho, M.A. F om an imic obial o an icance pep ides. A e iew. F on . Mic obiol. 2013,4, 294. [C ossRe ] 30. Ma suzaki, K. Why and how a e pep ide–lipid in e ac ions u ilized o sel -de ense? Magainins and achyplesins as a che ypes. Biochim. Biophys. Ac a 1999,1462, 1–10. [C ossRe ] 31. Ma suzaki, K.; Fukui, M.; Fujii, N.; Miyajima, K. In e ac ions o an an imic obial pep ide, achyplesin I, wi h lipid memb anes. Biochim. Biophys. Ac a 1991,1070, 259–264. [C ossRe ] 32. Iwanaga, S.; Mu a, T.; Shigenaga, T.; Seki, N.; Kawano, K.; Ka su, T.; Kawaba a, S. S uc u e- unc ion ela ionships o achyplesins and hei analogues. Ciba Found. Symp. 1994,186, 160–174. 33. Imu a, Y.; Nishida, M.; Ogawa, Y.; Takaku a, Y.; Ma suzaki, K. Ac ion mechanism o achyplesin I and e ec s o PEGyla ion. Biochim. Biophys. Ac a 2007,1768, 1160–1169. [C ossRe ] 34. Ma suzaki, K.; Yoneyama, S.; Fujii, N.; Miyajima, K.; Yamada, K.; Ki ino, Y.; Anzai, K. Memb ane pe meabiliza ion mechanisms o a cyclic an imic obial pep ide, achyplesin I, and i s linea analog. Biochemis y 1997,36, 9799–9806. [C ossRe ] 35. Kuzmin, D.V.; Emeliano a, A.A.; Kalashniko a, M.B.; Pan elee , P.V.; Balandin, S.V.; Se eb o skaya, E.O.; Belogu o a-O chinniko a, O.Y.; O chinniko a, T.V. Compa a i e in i o s udy on cy o oxici y o ecombinan be a-hai pin pep ides. Chem. Biol. D ug Des. 2017,91, 294–303. [C ossRe ] 36. Laede ach, A.; And eo i, A.H.; Ful on, D.B. Solu ion and micelle-bound s uc u es o achyplesin I and i s ac i e a oma ic linea de i a i es. Biochemis y 2002,41, 12359–12368. [C ossRe ] 37. Kushibiki, T.; Kamiya, M.; Aizawa, T.; Kumaki, Y.; Kikukawa, T.; Mizuguchi, M.; Demu a, M.; Kawaba a, S.; Kawano, K. In e ac ion be ween achyplesin I, an an imic obial pep ide de i ed om ho seshoe c ab, and lipopolysaccha ide. Biochim. Biophys. Ac a 2014,1844, 527–534. [C ossRe ] 38. Kawano, K.; Yoneya, T.; Miya a, T.; Yoshikawa, K.; Tokunaga, F.; Te ada, Y.; Iwanaga, S. An imic obial pep ide, achyplesin I, isola ed om hemocy es o he ho seshoe c ab (Tachypleus iden a us). NMR de e mina ion o he be a-shee s uc u e. J. Biol. Chem. 1990,265, 15365–15367. 39. Mielke, S.P.; K ishnan, V.V. Cha ac e iza ion o p o ein seconda y s uc u e om NMR chemical shi s. P og. Nucl. Magn. Reson. Spec osc. 2009,54, 141–165. [C ossRe ] 40. Hu chinson, E.G.; Tho n on, J.M. PROMOTIF–a p og am o iden i y and analyze s uc u al mo i s in p o eins. P o ein Sci. 1996,5, 212–220. [C ossRe ] 41. Wisha , D.S.; Bigam, C.G.; Yao, J.; Abildgaa d, F.; Dyson, H.J.; Old ield, E.; Ma kley, J.L.; Sykes, B.D. 1H, 13C and 15N chemical shi e e encing in biomolecula NMR. J. Biomol. NMR 1995,6, 135–140. [C ossRe ] 42. Chan, L.Y.; Zhang, V.M.; Huang, Y.H.; Wa e s, N.C.; Bansal, P.S.; C aik, D.J.; Daly, N.L. Cycliza ion o he an imic obial pep ide gomesin wi h na i e chemical liga ion: in luences on s abili y and bioac i i y. Chembiochem. 2013,14, 617–624. [C ossRe ] 43. T oei a Hen iques, S.; Law ence, N.; Chaousis, S.; Ra ipa i, A.S.; Chene al, O.; Ben ield, A.H.; Ellio , A.G.; Ka anagh, A.M.; Coope , M.A.; Chan, L.Y.; e al. Redesigned Spide Pep ide wi h Imp o ed An imic obial and An icance P ope ies. ACS Chem. Biol. 2017,12, 2324–2334. [C ossRe ] 44. Kondejewski, L.H.; Fa me , S.W.; Wisha , D.S.; Kay, C.M.; Hancock, R.E.; Hodges, R.S. Modula ion o s uc u e and an ibac e ial and hemoly ic ac i i y by ing size in cyclic g amicidin S analogs. J. Biol. Chem. 1996,271, 25261–25268. [C ossRe ] In . J. Mol. Sci. 2019,20, 4184 23 o 25 45. Tam, J.P.; Lu, Y.A.; Yang, J.L. Ma ked inc ease in memb anoly ic selec i i y o no el cyclic achyplesins cons ained wi h an an ipa allel wo-be a s and cys ine kno amewo k. Biochem. Biophys. Res. Commun. 2000,267, 783–790. [C ossRe ] 46. Edwa ds, I.A.; Ellio , A.G.; Ka anagh, A.M.; Blasko ich, M.A.T.; Coope , M.A. S uc u e-Ac i i y and -Toxici y Rela ionships o he An imic obial Pep ide Tachyplesin-1. ACS In ec . Dis. 2017 ,3, 917–926. [C ossRe ] 47. Ma gg a , M.B.; Pan elee , P.V.; Emeliano a, A.A.; So okin, M.I.; Boloso , I.A.; Buzdin, A.A.; Kuzmin, D.V.; O chinniko a, T.V. Cy o oxic Po en ial o he No el Ho seshoe C ab Pep ide Polyphemusin III. Ma . D ugs 2018,16, 466. [C ossRe ] 48. Hilchie, A.L.; Hoskin, D.W.; Powe Coombs, M.R. An icance Ac i i ies o Na u al and Syn he ic Pep ides. In An imic obial Pep ides: Basics o Clinical Applica ion; Ma suzaki, K., Ed.; Sp inge Singapo e: Singapo e, 2019; pp. 131–147. 49. Kuzmin, D.V.; Emeliano a, A.A.; Kalashniko a, M.B.; Pan elee , P.V.; O chinniko a, T.V. E ec o N- and C-Te minal Modi ica ions on Cy o oxic P ope ies o An imic obial Pep ide Tachyplesin I. Bull. Exp. Biol. Med. 2017,162, 754–757. [C ossRe ] 50. Ri e a, M.; Be asso, A.; McCa ey, C.; Geo gopapadakou, N.H. Po ins and lipopolysaccha ide o Esche ichia coli ATCC 25922 and isogenic ough mu an s. FEMS Mic obiol. Le . 1993,108, 183–187. [C ossRe ] 51. T eangen, T.J.; Maybank, R.A.; Enke, S.; F iss, M.B.; Di iak, L.F.; Ka aolis, D.K.; Ko en, S.; Ondo , B.; Phillippy, A.M.; Be gman, N.H.; e al. Comple e Genome Sequence o he Quali y Con ol S ain S aphylococcus au eus subsp. au eus ATCC 25923. Genome Announc. 2014,2, e01110-14. [C ossRe ] 52. Cla k, D. No el an ibio ic hype sensi i e mu an s o Esche ichia coli gene ic mapping and chemical cha ac e iza ion. FEMS Mic obiol. Le . 1984,21, 189–195. [C ossRe ] 53. Pin o,S.N.; Dias,S.A.; C uz,A.F.; Mil-Homens, D.; Fe nandes,F.; Valle, J.; And eu, D.; P ie o,M.; Cas anho,M.; Cou inho, A.; e al. The mechanism o ac ion o pepR, a i al-de i ed pep ide, agains S aphylococcus au eus bio ilms. J. An imic ob. Chemo he . 2019, dkz223. [C ossRe ] 54. Wa son, H. Biological memb anes. Essays Biochem. 2015,59, 43–69. [C ossRe ] 55. Ingol sson, H.I.; Melo, M.N.; an Ee den, F.J.; A na ez, C.; Lopez, C.A.; Wassenaa , T.A.; Pe iole, X.; de V ies, A.H.; Tieleman, D.P.; Ma ink, S.J. Lipid o ganiza ion o he plasma memb ane. J. Am. Chem. Soc. 2014 , 136, 14554–14559. [C ossRe ] 56. Van Mee , G.; Voelke , D.R.; Feigenson, G.W. Memb ane lipids: whe e hey a e and how hey beha e. Na . Re . Mol. Cell Biol. 2008,9, 112–124. [C ossRe ] 57. E ink, M.R. Fluo escence Quenching Reac ions. In Biophysical and Biochemical Aspec s o Fluo escence Spec oscopy; Dewey, T.G., Ed.; Sp inge US: Bos on, MA, USA, 1991; pp. 1–41. 58. Ghisaidoobe, A.B.; Chung, S.J. In insic yp ophan luo escence in he de ec ion and analysis o p o eins: a ocus on Fo s e esonance ene gy ans e echniques. In . J. Mol. Sci. 2014,15, 22518–22538. [C ossRe ] 59. San os, N.C.; P ie o, M.; Cas anho, M.A.R.B. Quan i ying molecula pa i ion in o model sys ems o biomemb anes: an emphasis on op ical spec oscopic me hods. Biochim. Biophys. Ac a 2003 ,1612, 123–135. [C ossRe ] 60. Fe nandes, M.X.; Ga c í a de la To e, J.; Cas anho, M.A.R.B. Join de e mina ion by B ownian dynamics and luo escence quenching o he in-dep h loca ion p o ile o biomolecules in memb anes. Anal. Biochem. 2002 , 307, 1–12. [C ossRe ] 61. Lakowicz, J.R. Topics in Fluo escence Spec oscopy P inciples; Sp inge US: Bos on, MA, USA, 2002. 62. Cha opadhyay, A.; London, E. Pa allax me hod o di ec measu emen o memb ane pene a ion dep h u ilizing luo escence quenching by spin-labeled phospholipids. Biochemis y 1987,26, 39–45. [C ossRe ] 63. Ky ychenko, A.; Ladokhin, A.S. Molecula Dynamics Simula ions o Dep h Dis ibu ion o Spin-Labeled Phospholipids wi hin Lipid Bilaye . J. Phys. Chem. B 2013,117, 5875–5885. [C ossRe ] 64. Ba oni, G.; Maise a, G.; Esin, S. An imic obial pep ides and hei in e ac ion wi h bio ilms o medically ele an bac e ia. Biochim. Biophys. Ac a 2016,1858, 1044–1060. [C ossRe ] 65. G assi, L.; Maise a, G.; Esin, S.; Ba oni, G. Combina ion S a egies o Enhance he E icacy o An imic obial Pep ides agains Bac e ial Bio ilms. F on . Mic obiol. 2017,8, 2409. [C ossRe ] In . J. Mol. Sci. 2019,20, 4184 24 o 25 66. Mina di, D.; Ghiselli, R.; Ci ioni, O.; Giacome i, A.; Kamysz, W.; O lando, F.; Sil es i, C.; Pa i, G.; Kamysz, E.; Scalise, G.; e al. The an imic obial pep ide Tachyplesin III coa ed alone and in combina ion wi h in ape i oneal pipe acillin- azobac am p e en s u e e al s en Pseudomonas in ec ion in a a subcu aneous pouch model. Pep ides 2007,28, 2293–2298. [C ossRe ] 67. Yu, K.; Lo, J.C.; Yan, M.; Yang, X.; B ooks, D.E.; Hancock, R.E.; Lange, D.; Kizhakkeda hu, J.N. An i-adhesi e an imic obial pep ide coa ing p e en s ca he e associa ed in ec ion in a mouse u ina y in ec ion model. Bioma e ials 2017,116, 69–81. [C ossRe ] 68. Oishi, O.; Yamashi a, S.; Nishimo o, E.; Lee, S.; Sugiha a, G.; Ohno, M. Con o ma ions and o ien a ions o a oma ic amino acid esidues o achyplesin I in phospholipid memb anes. Biochemis y 1997 ,36, 4352–4359. [C ossRe ] 69. Dohe y, T.; Wa ing, A.J.; Hong, M. Dynamic s uc u e o disul ide- emo ed linea analogs o achyplesin-I in he lipid bilaye om solid-s a e NMR. Biochemis y 2008,47, 1105–1116. [C ossRe ] 70. To ca o, I.M.; Huang, Y.H.; F anquelim, H.G.; Gaspa , D.; C aik, D.J.; Cas anho, M.A.; T oei a Hen iques, S. Design and cha ac e iza ion o no el an imic obial pep ides, R-BP100 and RW-BP100, wi h ac i i y agains G am-nega i e and G am-posi i e bac e ia. Biochim. Biophys. Ac a 2013,1828, 944–955. [C ossRe ] 71. Chene al, O.; Sch oede , C.I.; Du ek, T.; Walsh, P.; Huang, Y.H.; Li as, S.; P ice, D.A.; C aik, D.J. Fmoc-based syn hesis o disul ide- ich cyclic pep ides. J. O g. Chem. 2014,79, 5538–5544. [C ossRe ] 72. Gün e , P. Au oma ed NMR S uc u e Calcula ion wi h CYANA. In P o ein NMR Techniques; Downing, A.K., Ed.; Humana P ess: To owa, NJ, USA, 2004; pp. 353–378. 73. Shen, Y.; Bax, A. P o ein backbone and sidechain o sion angles p edic ed om NMR chemical shi s using a i icial neu al ne wo ks. J. Biomol. NMR 2013,56, 227–241. [C ossRe ] 74. B unge , A.T. Ve sion 1.2 o he C ys allog aphy and NMR sys em. Na . P o oc. 2007,2, 2728. [C ossRe ] 75. Chen, V.B.; A endall, W.B., 3 d; Headd, J.J.; Keedy, D.A.; Immo mino, R.M.; Kap al, G.J.; Mu ay, L.W.; Richa dson, J.S.; Richa dson, D.C. MolP obi y: all-a om s uc u e alida ion o mac omolecula c ys allog aphy. Ac a C ys allog . D Biol. C ys allog . 2010,66, 12–21. [C ossRe ] 76. Huang, Y.H.; Colg a e, M.L.; Cla k, R.J.; Ko ze, A.C.; C aik, D.J. Lysine-scanning mu agenesis e eals an amendable ace o he cyclo ide kala a B1 o he op imiza ion o nema ocidal ac i i y. J. Biol. Chem. 2010 , 285, 10797–10805. [C ossRe ] 77. Huang, Y.; Liu, Y.; Zheng, C.; Shen, C. In es iga ion o C oss-Con amina ion and Misiden i ica ion o 278 Widely Used Tumo Cell Lines. PLoS ONE 2017,12, e0170384. [C ossRe ] 78. Riss, T.L.; Mo a ec, R.A.; Niles, A.L.; Duellman, S.; Benink, H.A.; Wo zella, T.J.; Mino , L. Cell Viabili y Assays. In Assay Guidance Manual; Si ampalam, G.S., Coussens, N.P., B imacombe, K., G ossman, A., A kin, M., Auld, D., Aus in, C., Baell, J., Bejcek, B., Chung, T.D.Y., e al., Eds.; Eli Lilly & Company and he Na ional Cen e o Ad ancing T ansla ional Sciences: Be hesda, MD, USA, 2016. 79. Maye , L.D.; Hope, M.J.; Cullis, P.R. Vesicles o a iable sizes p oduced by a apid ex usion p ocedu e. BBA-Biomemb anes 1986,858, 161–168. [C ossRe ] 80. Hen iques, S.T.; Pa enden, L.K.; Aguila , M.-I.; Cas anho, M.A.R.B. The Toxici y o P ion P o ein F agmen P P(106 − 126) is No Media ed by Memb ane Pe meabiliza ion as Shown by a M112W Subs i u ion. Biochemis y 2009,48, 4198–4208. [C ossRe ] 81. Hen iques, S.T.; Cas anho, M.A.R.B. En i onmen al ac o s ha enhance he ac ion o he cell pene a ing pep ide pep-1: A spec oscopic s udy using lipidic esicles. Biochim. Biophys. Ac a 2005 ,1669, 75–86. [C ossRe ] 82. Capu o, G.A.; London, E. Using a no el dual luo escence quenching assay o measu emen o yp ophan dep h wi hin lipid bilaye s o de e mine hyd ophobic alpha-helix loca ions wi hin memb anes. Biochemis y 2003,42, 3265–3274. [C ossRe ] 83. Hen iques, S.T.; Huang, Y.H.; Cas anho, M.A.; Baga olli, L.A.; Sonza, S.; Tachedjian, G.; Daly, N.L.; C aik, D.J. Phospha idyle hanolamine binding is a conse ed ea u e o cyclo ide-memb ane in e ac ions. J. Biol. Chem. 2012,287, 33629–33643. [C ossRe ] 84. Hen iques, S.T.; Huang, Y.-H.; Roseng en, K.J.; F anquelim, H.G.; Ca alho, F.A.; Johnson, A.; Sonza, S.; Tachedjian, G.; Cas anho, M.A.R.B.; Daly, N.L.; e al. Decoding he memb ane ac i i y o he cyclo ide kala a B1: he impo ance o phospha idyle hanolamine phospholipids and lipid o ganiza ion on hemoly ic and an i- HIV ac i i ies. J. Biol. Chem. 2011,286, 24231. [C ossRe ] In . J. Mol. Sci. 2019,20, 4184 25 o 25 85. Huang, Y.-H.; Colg a e, M.L.; Daly, N.L.; Keleshian, A.; Ma inac, B.; C aik, D.J. Biological Ac i i y o he P o o ypic Cyclo ide Kala a B1 Is Modula ed by he Fo ma ion o Mul ime ic Po es. J. Biol. Chem. 2009 ,284, 20699–20707. [C ossRe ] 86. S ewa , J.C. Colo ime ic de e mina ion o phospholipids wi h ammonium e o hiocyana e. Anal. Biochem. 1980,104, 10–14. [C ossRe ] © 2019 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).