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. 2020 Jul 10;11(9):1041–1047. doi: 10.1039/d0md00172d

Synthesis and biological evaluation of S-lipidated lipopeptides of a connexin 43 channel inhibitory peptide†

Sung-Hyun Yang a,b,‡, Connor A Clemett c,‡, Margaret A Brimble a,b,d, Simon J O'Carroll c,✉, Paul W R Harris a,b,d,✉
PMCID: PMC7513673  PMID: 33479696

graphic file with name d0md00172d-ga.jpgLipidated Peptide5 analogues are able to mediate hemichannel openings leading to inhibition of chemical messengers to the extracellular matrix.

Abstract

The synthesis and biological activity of 42 novel S-lipidated analogues of a connexin 43 channel inhibitory Peptide5 is described. Unmodified Peptide5 moderates hemichannels and gap junctions that are both implicated in the progression of neurological disease. Peptide5 was site-specifically modified with a cysteine residue, which then underwent thiol–ene mediated S-lipidation to afford S-lipidated Peptide5 analogues containing straight-chain, branched, or aromatic lipids. The modified peptides were assessed for their effect on hemichannel opening and the most promising candidates were evaluated in serum stability studies.

Introduction

Biologically active peptides attract much attention as potential drug candidates due to their intrinsic nature of selective high binding affinity for their receptors with high potency and low toxicity.1 However, the use of peptides as therapeutics is limited by poor chemical and physical stability such as proteolytic degradation.2 A plethora of chemical methodologies are available for the modification of native peptides to generate peptide mimetics with improved pharmacokinetic and pharmacodynamic properties. Common methods include substitution with unnatural (d)-amino acid3/non-proteinogenic amino acid,4 beta-amino acid,5 cyclisation,6N-methylation of the amide bond,7 PEGylation,8 glycosylation,9 phosphorylation10 and lipidation.11 Lipidation of native peptides is an effective strategy to improve pharmacokinetic and pharmacodynamic properties of native peptides by increasing proteolytic stability, receptor selectivity and potency. This strategy has been verified by the FDA-registered lipopeptide drugs including the anti-diabetic agents semaglutide (Ozempic®),12 liraglutide (Victoza®)13 and insulin detemir (Levemir®).14

Connexin (Cx)43 is a protein widely expressed within the central nervous system where it acts to provide syncytial formation and a high degree of intercellular communication, particularly in endothelial and glial cells.15 Membrane-bound connexin family members assemble into both homo- and heteromeric hexamers referred to as connexons; like a pore, these are dynamic in their ability to regulate the opening of a central channel (Fig. 1). Two connexons of neighbouring cells appose one another, forming a gap junction to link the respective intracellular spaces.16 Perturbation of this system can occur through tissue injury, metabolic failure, or inflammatory activation. This leads to disruption of existing gap junctions and the opening of unopposed connexons, referred to as hemichannels, thereby creating a continuation of cytoplasm with the extracellular space.17 Pathological opening of hemichannels can lead to cell swelling,18 excitotoxic cell death,19 and vascular haemorrhage.20 Opening of hemichannels leads to the release of molecules into the extracellular space. An example of this is adenosine triphosphate (ATP), which when released from Cx43 hemichannels activates purinergic signalling21 and the inflammasome pathway, ultimately leading to the secretion of proinflammatory cytokines.22 Cx43 hemichannels are therefore a potential drug target for treatment of neurological conditions.23

Fig. 1. Multimerisation of membrane anchored Cx43 protein. Six monomers form a hemichannel connexon which then dock with a neighbouring connexon to form a gap junction.

Fig. 1

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Small peptide mimetics to each of the Cx43 extracellular loops (EL1 and EL2) have been a source of interest in targeting the pharmacological closure of hemichannels in cases of tissue injury as they act to mimic the EL–EL interaction of an opposing connexon prompting channel closure.24 A promising candidate, Peptide5 (1), (VDCFLSRPTEKT), derived from EL2 in Cx43 has been demonstrated to provide therapeutic benefit in models of inflammatory nervous tissue and vascular injury (Fig. 2).25 Peptide5 was found to restrict hemichannel leakage of ATP into the extracellular space following ischaemic injury in vitro26 providing a mechanism for its protective effects. Existing gap junction blockers such as carbenoxolone (CBX) have been shown to be protective in a number of disease models27 and in limiting ATP leakage in vitro,26 but also interfere with beneficial connexin gap-junctions. Importantly, Peptide5 (1) induces hemichannel closure with reduced gap junction disruption,28 which makes it a better candidate for the specific targeting of Cx43 hemichannels. However, further work is required to maximise both the efficacy and serum stability of Peptide5-based candidates to demonstrate their potential as agents to treat human disease.

Fig. 2. Origin of Peptide5 derived from the extracellular loop 1 (EL1) of the trans membrane domains of Cx43. CL1 = cytoplasmic loop.

Fig. 2

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Our previous alanine scanning studies have identified key amino acids required for the observed biological activity of Peptide5 (1) and provided insight into potential sites for further modification.26 In the current work, we report the preparation of a library of S-lipidated analogues of Peptide5 (1), informed by our alanine scanning work, and enabled by our ‘CLipPA’ (cysteine lipidation of peptides or amino acids) lipidation technology. Six amino acids in the native Peptide5 (1) sequence were substituted sequentially with the thiol-containing amino acid cysteine or a thiol acid which then underwent S-lipidation with a range of lipidated vinyl esters creating a unique 42-member library of novel lipopeptides. It was found that employing high molecular weight lipids, particularly in the SRPTEKT region within Peptide5 impaired the ability of Peptide5 to induce hemichannel closure in an endothelial ischaemic injury model. Pleasingly, some substitutions that conserved the approximate mass and polarity of the native side chains demonstrated improved functional efficacy.

Results and discussion

Synthesis of peptide scaffolds (2)–(8)

We have developed a facile process to directly install lipids onto a thiol-containing peptide, coined CLipPA.29,30,35 CLipPA involves a conventional thiol–ene coupling initiated by a radical reaction between a sulphur atom and sp2–sp2 unsaturated carbon bond bearing a lipid chain (Scheme 1). CLipPA displays high levels of chemoselectivity with a broad range of functional group tolerance and results in exclusively anti-Markovnikov regioselectivity. The scope of the direct S-lipidation of peptides using the CLipPA technique has been established by the synthesis of lipidated peptide hormones,30 rapid preparation of antimicrobial peptides,29b cyclic lipopeptides,29d synthesis of TLR-2 agonists for cancer vaccines29c,e and novel stapled peptides.29f

Scheme 1. S-Lipidation of fully unprotected peptides by CLipPA reaction employing vinyl ester containing lipids under UV light. All amino acid side chains are unprotected and remain unmodified.

Scheme 1

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We hypothesised that adding a lipid to Peptide5 (1)31 may help to anchor the resultant lipopeptide to hydrophobic cellular membranes thereby improving its gap junction function. Lipidation may also serve to increase the half-life of the unmodified Peptide5 (1), which has a short half-life, thereby limiting its clinical usefulness. Based on our previous alanine scanning study of Peptide5 (1), we synthesised seven scaffold peptides bearing a thiol-containing amino acid or a thiol alkanoic acid (Table 1). Six analogues, 2–7 possessed a single mutation in place of the native amino acid residues (1Val, 4Phe, 6Ser, 9Thr, 10Glu or 12Thr) employing cysteine as a convenient thiol handle. For peptide 8 the N-terminus of the peptide sequence was readily capped with 3-mercaptopropionic acid. Peptide5 (1) contains a cysteine residue at the 3 position that needs to be preserved. To avoid S-lipidation at this site, a temporary acetamidomethyl (Acm) protecting group was introduced, which was removed following S-lipidation on all thiol-containing peptide scaffolds.

Table 1. Structures of the 42-member library of S-lipopeptide analogues of Peptide5 (1) with assorted lipid tails at the mutation sites of 1Val, 4Phe, 6Ser, 9Thr, 10Glu, 12Thr and N-terminus.

1Val-2Asp-3Cys-4Phe-5Leu-6Ser-7Arg-8Pro-9Thr-10Glu-11Lys-12Thr Peptide5 1 graphic file with name d0md00172d-u1.jpg graphic file with name d0md00172d-u2.jpg graphic file with name d0md00172d-u3.jpg graphic file with name d0md00172d-u4.jpg graphic file with name d0md00172d-u5.jpg graphic file with name d0md00172d-u6.jpg graphic file with name d0md00172d-u7.jpg
graphic file with name d0md00172d-u8.jpg 2 (62) 9 (62) 16 (64) 23 (55) 30 (53) 37 (55) 44 (58)
graphic file with name d0md00172d-u9.jpg 3 (65) 10 (65) 17 (63) 24 (62) 31 (45) 38 (64) 45 (48)
graphic file with name d0md00172d-u10.jpg 4 (52) 11 (52) 18 (58) 25 (60) 32 (55) 39 (49) 46 (55)
graphic file with name d0md00172d-u11.jpg 5 (55) 12 (55) 19 (51) 26 (55) 33 (62) 40 (53) 47 (61)
graphic file with name d0md00172d-u12.jpg 6 (62) 13 (58) 20 (52) 27 (51) 34 (51) 41 (55) 48 (51)
graphic file with name d0md00172d-u13.jpg 7 (65) 14 (60) 21 (57) 28 (51) 35 (57) 42 (58) 49 (53)
graphic file with name d0md00172d-u14.jpg 8 (52) 15 (52) 22 (55) 29 (52) 36 (51) 43 (51) 50 (55)
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aParentheses indicate isolated yields of crude peptide following the resin cleavage.

bParentheses indicate isolated yields following lipidation, Acm removal and RP-HPLC purification.

Peptide scaffolds 2–8 were synthesized by Fmoc solid phase peptide synthesis (SPPS) on aminomethyl polystyrene resin32 using an automated peptide synthesizer with the reagents depicted in Scheme 2. Commercially available Fmoc-Thr(tBu)-HMPP (hydroxylmethylphenoxypropionic acid) was used to immobilise the first amino acid-linker for the synthesis of peptides 2–6 and 8, while 2-chlorotrityl resin used to anchor the racemisation-prone cysteine residue using non-activating conditions for peptide 7. Elongation of the peptide chain on both loaded resins was then achieved employing 20% (v/v) piperidine/DMF for Nα-Fmoc protecting group removal and HATU/N,N′-diisopropylethylamine as the coupling reagent/base for the condensation of the incoming Fmoc-amino acid. Racemisation of Na-protected cysteine is a well-known side reaction during chain assembly, hence racemisation suppression conditions were employed for the coupling of Fmoc-Cys(Trt)-OH and Fmoc-Cys(Acm)-OH by using HATU/HOAt, and 2,4,6-trimethylpyridine in DCM/DMF (1 : 1, v/v).33 A representative synthesis of peptide scaffold 2 is depicted (Scheme 2).

Scheme 2. The representative synthesis of peptide scaffold 2 and S-lipopeptide 9. Conditions and reagents. (a) Fmoc-Thr(tBu)-HMPP (2 equiv.), DIC (2 equiv.), DCM : DMF (9 : 1, v/v), r.t., overnight.; (b) iterative Fmoc-SPPS using a Tribute® automated peptide synthesizer i) 20% piperidine/DMF (v/v), 7 min × 2, r.t.; ii) Fmoc-AA-OH (5 equiv.), HATU (4.75 equiv.), DIPEA (10 equiv.), DMF, 50 min; for either Fmoc-Cys(Trt)-OH or Fmoc-Cys(Acm)-OH coupling; (5 equiv.), HATU (4.75 equiv.), HOAt (4.75 equiv.), 2,4,6-trimethylcollidine (8 equiv.), DCM : DMF (1 : 1, v/v), 50 min × 2;. iii) 20% Ac2O/DMF (v/v), 1 min, r.t.; (c) TFA : H2O : EDT : TIPS (91.5 : 5 : 2.5 : 1, v/v/v/v), r.t. 2 h; (d) vinyl butyrate (70 equiv.), DMPA (3 equiv.), tnonylmercaptan (80 equiv.), TIPS (80 equiv.), 5% TFA (v/v), NMP, 365 nm UV irradiation, r.t. 1 h; (e) i) AgOAc (50 equiv.), MeCN : H2O (+0.1% TFA, 1 : 1 v/v), overnight; ii) DTT (300 equiv.), 6.0 M Gn·HCl, r.t., 1 h.

Scheme 2

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Peptides (2–8) were then released from the resin with concomitant removal of side chain protecting groups by treatment of the peptidyl-resin with the cleavage cocktail, TFA : H2O : EDT : TIPS (91.5 : 5 : 2.5 : 1, v/v/v/v). All peptides were obtained with high purity which did not require any further purification prior to lipidation. With peptide scaffolds 2–8 easily accessible, the S-lipidation of the free thiol was next examined.

S-Lipidation of peptide scaffolds using CLipPA chemistry

Readily available diverse vinyl esters were chosen as the lipid component for the CLipPA reaction. Vinyl esters were selected containing lipids that included straight chain alkyl groups (propyl, nonyl, pentadecyl, heptadecyl), a branched alkyl group (tert-butyl) and an aryl group (4-tert-butylphenyl). These were selected as it has been reported that conjugation of bioactive peptides with lipids affects the activity and stability of the resultant lipopeptide that is dependent on the type (length, bulkiness) and composition (polarity) of the lipid.34 Our optimised reaction conditions employed the vinyl ester (70 equiv.), triisopropylsilane (80 equiv.), 5% (v/v) TFA in N-methylpyrrolidinone (NMP), 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photo-induced radical initiator, under UV light irradiation at 365 nm and an exogenous thiol as a radical quencher. Recently, we replaced the malodorous tert-butylmercaptan thiol quencher by the more agreeable tert-nonylmercaptan with no effect on the reaction and this modified procedure was also adopted in the present work.35 Pleasingly, S-lipidation of all peptide scaffolds 2–8 proceeded smoothly and lipidated peptides could be obtained directly following precipitation from diethyl ether requiring no further purification.35 The Acm protecting group on 3Cys was then removed by treatment with AgOAc (ref. 36) and the library of forty-two S-lipopeptides 9–50 were obtained in moderate to high yield after a single RP-HPLC purification step (Table 1).

Biological evaluation of S-lipopeptide analogues of Peptide5 (1)

Limiting injury associated with Cx43 hemichannel opening is a key mechanism for the anti-inflammatory behaviour of Peptide5 (1) in both in vivo, and simple in vitro models of cell or tissue slice culture.23 To test the effect of the peptide modifications on Peptide5 activity, we employed a model of ischemic insult on a cell culture model of the brain microvasculature to replicate tissue injury and damage responses, including that of Cx43 hemichannel opening and ATP release.26,28 This model is also applicable in the case of a systemically delivered therapy as it is the inflammatorily activated vasculature that likely sees the highest levels of administered drug. The leakage of intracellular ATP into media is a readily quantifiable means of describing the extent of injury-induced hemichannel opening.15b To detect this, immortalized human cerebral microvascular endothelial cells (Applied Biological Materials, Canada; cat # T0259) were incubated in Hypoxic-Acidic Ion-shifted Ringers (HAIR) solution, designed to replicate the disrupted pH and ion-balance in damaged vasculature. As ATP leaks via hemichannels into this solution, it is easily sampled and quantified fluorometrically by the firefly luciferase/d-luciferin reaction (see ESI,† method 9). Consistent with previous observations, Peptide5 (1) treatment provided a significant reduction of ATP release over untreated controls (53.8 ± 3.1%) as did carbenoxolone (CBX; 23.3 ± 3.4%), an inhibitor of both Cx43 hemichannels and gap junctions (Fig. 3A).

Fig. 3. Modification of peptide5 side chains modulates functional behaviour in a mass- and position-dependent manner. (A) ATP release assays in hCMVEC cultures exposed to model ischaemic injury (HAIR) alone or in combination with pan-Cx43 inhibitor CBX or Peptide5 (1) (both 10 μM). ATP release assay in hCMVEC cultures treated with HAIR + Peptide5 mimetics 9–50 (10 μM) modified with (B) propyl, (C) nonyl, (D) pentadecyl, (E) heptadecyl, (F) tert-butyl, (G) 4-tert-butylphenyl side-chains. A line at 53.75% on each figure indicates the level of Peptide5 (1) control. All values represent mean ± SEM% relative to untreated injury (HAIR). n = 3 (S-lipopeptides), n = 16 (HAIR, NR, HAIR + CBX, HAIR + 1. (H) Linear regression of lipid MW at AA sites 4, 10, and 12 within Peptide5 vs. the degree of ATP release when applied to injured cerebral endothelium). One-way ANOVA with multiple comparisons vs. P5(1), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0001. * indicates a change above P5, # indicates a change below Peptide5.

Fig. 3

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Of the 42 S-lipopeptide analogues (9–50) of Peptide5 (1) tested, the presence of a butyl lipid chain at each amino acid position on Peptide5 (1) did not interfere with its bioactivity in preventing ATP leakage compared to unmodified Peptide5 (1) (Fig. 3B). Peptide 13 (32.0 ± 5.1%, p = 0.0139), and 15 (28.8 ± 4.2%, p = 0.0029) demonstrated improved bioactivity by further limiting ATP release compared to Peptide5 (1) (53.8 ± 3.1%). By way of contrast, it was observed that the substitution of high molecular weight, longer/straight chain aliphatic lipids (nonyl, pentadecyl, and heptadecyl) did not improve the bioactivity of Peptide5 (1) and lipidation at positions 9 (analogues 26, 33), 10 (analogues 20, 27, 34), and 12 (analogues 28, 35) reduced the efficacy of hemichannel closure below that of the native Peptide5 (1) sequence as demonstrated by an increase in ATP release (Fig. 3C–E, Table 2). Given that the shorter butyl chain is also non-polar although lesser so, but did not demonstrate such functionality this may be suggestive of steric hindrance by longer lipid chains occurring at the peptide–Cx43 interface. Indeed, ATP release following treatment with modified Peptide5 mimetics correlates positively to the similarity of the molecular mass of the newly introduced side chains at amino acid positions Phe-4, Glu-10, and Thr-12 (Fig. 3H). Indeed, our previous alanine substitution studies identified that amino acids within the SRPTEKT sequence (positioned at 6Ser–12Thr) within Peptide5 (1) were key to elicit closure of Cx43 hemichannels.26

Table 2. Addition of high MW straight aliphatic lipid chains to the Peptide5 (1) at AA-positions 9, 10, and 12 increases ATP release in ischaemic conditions.

S-Lipopeptide analogues Side chain modification Mutation site ATP (% HAIR) p-Value vs. P5 (1) p-Value vs. HAIR
Peptide 5 (1) n/a — 53.8 ± 3.1 — <0.0001
26 Pentadecyl Thr-9 102.0 ± 21.0 <0.0001 0.9998
33 Heptadecyl Thr-9 102.2 ± 8.1 <0.0001 0.9997
20 Nonyl Glu-10 96.0 ± 7.4 <0.0001 0.9994
27 Pentadecyl Glu-10 114.3 ± 22.1 <0.0001 0.8012
34 Heptadecyl Glu-10 83.7 ± 13.2 0.0066 0.4192
28 Pentadecyl Thr-12 131.4 ± 13.0 <0.0001 0.0091
35 Heptadecyl Thr-12 129.2 ± 21.8 <0.0001 0.0294
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Thus, it appears that the presence of the native amino acid side chains is required to elicit hemichannel closure in some instances. This is consistent with the above findings wherein it was found that altering Peptide5 (1) at Thr-9, Glu-10, or Thr-12 with lipids other than propyl, disrupts its mode of action. The addition of long aliphatic chains to Peptide5 (1) at these sites constitutes a significant change to the overall hydrophobicity of the molecule and, likely too, its ability to bind with an opposing Cx43 EL domain. At each of the tested positions, substitution with the aromatic 4-tert-butylphenyl, a linear butyl, or the branched tert-butyl lipid chain did not significantly impair Peptide5 (1) hemichannel blockade (Fig. 3B, F and G). However, the addition of the tert-butyl lipid chain significantly improved peptide-induced hemichannel closure when introduced at Phe-4 (analogue 38, 23.4 ± 2.7%, p = 0.0006) (Fig. 3F). This substitution maintains the polarity and approximate mass of the native amino acid at this position. These data demonstrate that chemical modification of the Peptide5 (1) structure alters its functional behaviour in a modification- and amino acid sequence specific-manner. The work reported herein suggests that alteration of sites within the core Peptide5 (1) structure to gain functional improvement can be achieved but due care must be taken to avoid significantly disrupting key binding interactions between it and the target Cx43 domains.

Serum stability assay

Proteolytic degradation of peptides by proteases leads to low tissue accumulation,37 and this is one of the inherent limiting factors posed by peptide-based drug candidates. To overcome this, many strategies have been employed to improve the proteolytic stability while maintaining activity. Peptide lipidation is of interest as the resulting lipopeptide shows a tendency to exhibit an extended half-life time by exploiting the long half-life of the most abundant serum protein, human serum albumin (HSA, ∼19 days), by binding via the lipid to fatty acid binding sites that are present on serum albumin.38 In order to understand the effect of S-lipidation on the proteolysis rate, the proteolytic stability of the native Peptide5 (1) and representative lipopeptide analogues was carried out in vitro by measuring the half-life of the subjected peptides at 37 °C (pH 7.4) in rat serum.39 The half-life was estimated by analysis of reverse-phase high performance liquid chromatography (RP-HPLC) data by taking of aliquots at different time points. Synthetic Pepetide-5 (1) was used as a positive control while lipopeptide analogues 15, 17, 25, 34, 38 and 45 were chosen from each representative lipid group that displayed the highest activity from the luminescence assay.

As a positive control, the half-life of Peptide5 (1) was determined, which rapidly degraded in rat serum, with a (t1/2) of just 5 min. Unfortunately, the half-life of all the elected Pepitde5 lipopeptides did not improve, and similar half-life values approaching the native peptide sequence, Peptide5 (1) were obtained. The current proteolytic degradation results may suggest that peptide degradation relates to the peptide primary sequence irrespective of the type and position of the lipid chain.

Conclusions

In conclusion, a library of S-lipopeptide analogues of Peptide5 (1) were synthesized using efficient “in-house” CLipPA S-lipidation chemistry based on Fmoc SPPS. The effect of varying the nature of the lipid chain substituents on Cx43 hemichannel opening and ATP release was examined using luminescence assay in vitro and proteolytic stability was assessed in a rat serum stability assay. Generally, analogues possessing shorter lipid chains displayed greatly enhanced functional efficacy activity, while half-lives were not dramatically improved relative to the non-lipidated Peptide5 (1). The tools developed in this study will be used to further design lipidated analogues of Peptide5 (1) to improve proteolytic stability and maintain gap junction function.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

Click here for additional data file. (5.9MB, pdf)

Acknowledgments

We thank the Auckland Medical Research Foundation (AMRF) for generous financial support of this research (Grant Ref. 1117016). Mr Clemett was funded by a doctoral scholarship generously provided by The CatWalk Trust.

Footnotes

†Electronic supplementary information (ESI) available: Experimental details for the synthesis of all peptides, analytical LC and MS, bioluminescence assay. See DOI: 10.1039/d0md00172d

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