Article pubs.acs.org/jmc
Glycosylated Enfuvirtide: A Long-Lasting Glycopeptide with Potent Anti-HIV Activity Shuihong Cheng,†,⊥ Xuesong Chang,† Yan Wang,‡ George F. Gao,†,§ Yiming Shao,‡ Liying Ma,‡ and Xuebing Li*,†,⊥,§ †
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Chaoyang District, Beijing 100101, China ⊥ National Engineering Research Center for Carbohydrate Synthesis, Jiangxi Normal University, Nanchang 330022, China ‡ State Key Laboratory for Infection Disease Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Changping District, Beijing 102206, China § Center for Influenza Research and Early-warning, Chinese Academy of Sciences (CASCIRE), Chaoyang District, Beijing 100101, China S Supporting Information *
ABSTRACT: Many peptide-based therapeutics have short circulatory half-lives. We report here that the pharmacokinetics of an antiHIV peptide drug enfuvirtide (ENF) can be dramatically improved by a chemical glycosylation approach. A set of glycosylated ENFs with varying glycosylation sites and glycan structures were synthesized. Among these, a sialic acid-introduced peptide (SL-ENF) demonstrated a 15-fold extended half-life in rats relative to ENF (T1/2: 23.1 vs 1.5 h), and its antiviral potency was comparable to that of ENF (EC50: 2 vs 3 nM). SL-ENF bound to a functional fragment of the HIV fusogenic protein gp41 and formed complexes with high affinity and α-helicity, revealing the mechanism behind its potent antiviral activity. Because it is widely accepted in biology that glycosylation protects proteins from denaturation and proteases, our approach may be useful for the development of novel protein and peptide drugs with enhanced pharmaceutical properties.
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tion.10 As an alternative, chemical or chemoenzymatic glycosylation was highlighted11,12 and explored for pharmacokinetic modulation of peptide-based drugs.13−17 This route is attractive because it may give access to structurally well-defined glycoproteins or glycopeptides and offer additional therapeutic benefits such as tissue targeting,18 beyond the conventional approaches such as PEG conjugation. The usefulness of chemical glycosylation is illustrated here with a glycosylated peptide that shows a dramatically prolonged half-life without a reduction in the inherent anti-HIV potency. We employed a 36-residue synthetic peptide drug, enfuvirtide (ENF),19 as a model to demonstrate our strategy. ENF is a potent inhibitor of HIV-1 infection that binds to the viral envelope fusogenic protein gp41 and blocks the gp41-mediated membrane fusion process between the virus and host cell. Despite its successful use in the clinical treatment of AIDS, ENF can be rapidly cleared from the circulation principally by degradation in the liver and kidneys, thus requiring frequent subcutaneous injections (twice daily) that often result in
INTRODUCTION Protein glycosylation is ubiquitous in organisms and fundamental to many biological processes such as cellular recognition, immune response, and infection. A well-established function of glycosylation is to increase the in vivo stability of proteins by protecting them from proteolytic enzymes.1,2 In particular, glycans with a terminal sialic acid residue contribute to the extended circulatory half-life of glycoproteins such as erythropoietin, because the sialic acid moiety confers additional resistance to liver-mediated clearance.3−6 In the discovery of protein and peptide therapeutics, a major challenge is the pharmacokinetic control of exogenous biomolecules that tend to be rapidly removed by the metabolic and immune systems. A number of strategies have been developed to address this issue and include conjugation of the therapeutics with polyethylene glycol (PEG)7 or other molecules.8 Similar to these approaches, glycosylation has also been established as a potential tool to improve pharmacokinetics by modulating the biophysical and physiological properties.2,9 However, current methods to produce therapeutic proteins and peptides rely heavily on recombinant expression systems that have limited control over glycosyla© 2015 American Chemical Society
Received: October 28, 2014 Published: January 16, 2015 1372
DOI: 10.1021/jm5016582 J. Med. Chem. 2015, 58, 1372−1379
Journal of Medicinal Chemistry
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adverse drug reactions.20,21 Moreover, the negligible solubility of ENF under physiological conditions also complicates treatment regimens.22 The shortcomings of ENF have motivated considerable efforts to develop more persistent or soluble alternatives that can function in a similar fashion.23−34 For example, conjugation of C34 (1, another peptide inhibitor of HIV fusion that overlaps with a part of ENF sequence) with cholesterol results in marked improvements in both half-life and antiviral potency.24 Glycosylation of ENF or peptide 1 with α-galactose- or mannose-containing oligosaccharides is described31−34 and, in one case, the manno-oligosaccharidepeptide conjugate displays greatly enhanced proteolytic resistance and fair antiviral activity.34 In this study, we synthesized four glycopeptide mimics containing two types of glycans, lactose (Lac) or sialyllactose (SL), attached to the ENF peptide backbone at various positions (Figure 1), to
Article
RESULTS AND DISCUSSION The structural design of glycosylated ENFs (glyENFs) was based on the following essential considerations. First, either the N- or C-terminus of ENF was chosen as the site for modification to minimize the impairment of glycosylation on the peptide structure, which is required for antiviral activity. To this end, a cysteine residue was chemically introduced into either terminus to allow for the attachment of the glycans through an easy thiol-maleimide coupling reaction.35 Second, Lac and SL can be used as elements for pharmacokinetic controls, because these simple structures represent the terminal sugar residues of many mammalian glycoproteins or glycolipids. Their hydrophilic features could also facilitate the solubility and stability of glyENFs. Finally, an oligoethylene glycol spacer, bearing a terminal maleimide group, was used to tether the glycans to the peptide. This would endow flexibility between the two moieties and reduce nonspecific interactions with undesirable biological components. On the basis of the design elements mentioned above, we synthesized glyENFs using a modular strategy31,32 (Scheme 1). Scheme 1. Synthesis of GlyENFsa
a Reaction conditions: (a) CMP-Neu5Ac, α2,6-sialyltransferase, TrisHCl buffer (pH 8.0), 37 °C, 85%; (b) N-methoxycarbonyl maleimide, saturated aq solution of NaHCO3, 0 °C, 84%; (c) sodium ascorbate, CuSO4·5H2O, THF/H2O mixture, 35 °C, 75% for 6 and 70% for 7; (d) phosphate buffer (pH 7.2), rt, 5 min, 80% for Lac-ENF, 85% for ENF-Lac, 78% for SL-ENF, and 80% for ENF-SL (overall yields from compound 2: 50% for Lac-ENF, 54% for ENF-Lac, 46% for SL-ENF, and 47% for ENF-SL).
Figure 1. Peptide 1, ENF, cysteine-incorporated ENFs (cENF and ENFc), and glyENFs (Lac-ENF, SL-ENF, ENF-Lac, and ENF-SL). All peptides were protected by N-terminal acetylation and C-terminal amidation.
Enzymatic sialylation of 2′-azidoethyl lactoside 236 with α2,6sialyltransferase and sialyl donor CMP-Neu5Ac afforded sialylated trisaccharide 337 in 85% yield. Using a simple Cu(I)-catalyzed Huisgen cycloaddition reaction,38 azides 2 and 3 were coupled with an alkyne-functionalized oligoethylene glycol linker 5, which was synthesized from the known primary amine 439 by treatment with N-methoxycarbonyl maleimide, generating corresponding glycosides 6 and 7, in 75% and 70%
demonstrate the feasibility of our approach to improve the pharmacological profile of therapeutic peptides and proteins. It was anticipated that the modification with the sialic acidcontaining SL would result in long-lasting therapeutics, because of the imparted resistance to hepatic degradation. 1373
DOI: 10.1021/jm5016582 J. Med. Chem. 2015, 58, 1372−1379
Article
Journal of Medicinal Chemistry
XTT cytotoxicity assay.41 The monocysteine-incorporated peptides (cENF and ENFc) revealed antiviral activity with EC50 values comparable to that of ENF, whereas their biscysteine counterpart (cENFc) showed considerably lower potency. The N-terminally glycosylated peptides (Lac-ENF and SL-ENF) inhibited viral infection as well as ENF, whereas the C-terminal glycosylations (ENF-Lac and ENF-SL) resulted in obvious reductions in the antiviral efficacy. The C-terminal residues of ENF (WNWF) are essential for its inhibitory activity, as this lipophilic segment can facilitate ENF targeting to the cell lipid membrane where fusion occurs.42 Replacement of the WNWF motif with ANAA completely inactivated the peptide.43 Conjugation of cholesterol24 or albumin25 to the Cterminus induced a significant decrease of the activity. In contrast, modification of the N-terminus with albumin25 or octyl44 did not heavily impact on the antiviral potency. Thus, our results correlate well with previous findings and the potent anti-HIV glyENFs, especially Lac-ENF and SL-ENF, which were designed for the development of long-lasting therapeutics, warranted further investigation. We next verified that the antiviral activity of glyENFs was attributed to their interactions with the viral gp41 protein. This was analyzed by surface plasmon resonance (SPR) experiments using a 46-residue peptide fragment of gp41, designated N46 (8, for the sequence, see the Experimental Section),42 which represents the N-heptad repeat (NHR) domain of gp41 and the binding target of ENF and peptide 1. Both Lac-ENF and SLENF strongly bound to peptide 8, with affinities of 452 nM and 250 nM, respectively, which are of the same order of magnitude as observed for ENF binding (182 nM) (Figure 3). This result
yields, respectively. The maleimide functionalities of 6 and 7 enabled rapid modification (96%, in 46−54% overall yields from compound 2) and characterized by mass spectroscopy (Figure 2). The glyENFs retained the intact sequence required for the bioactivity and showed a significant enhancement in their aqueous solubility (≥1.0 M, pH 7.2) when compared with ENF.
Figure 2. ESIMS analysis and HPLC purification (inset) of glyENFs. Shown are the data of SL-ENF. Other glyENFs have similar characteristics. HPLC chromatographic conditions: Phenomenex Synergi Hydro-RP C18 column (4 μm, 10 × 250 mm), 40 → 70% CH3CN/H2O linear gradient over 11 min at 2.5 mL/min, UV detection at 220 nm.
The antiviral activity of glyENFs were examined in a singlecycle infectivity assay using TZM-bl cells, which express high levels of HIV-1 entry receptors (CD4, CCR5, and CXCR4) and contain an integrated luciferase reporter gene under the control of HIV-1 long terminal repeat promoter.40,41 The cells were infected with a laboratory adapted HIV-1 strain (SF33) in the presence or absence of the test peptides and the inhibitory activities were determined by luciferase expression on day 2 postinfection. All the peptides potently inhibited HIV-1 infection at nanomolar concentrations (Table 1). None of the peptides affected cell viability at concentrations below the micromolar range (
