Conjugation of a Nonspecific Antiviral Sapogenin with a Specific HIV

Article pubs.acs.org/jmc

Conjugation of a Nonspecific Antiviral Sapogenin with a Specific HIV Fusion Inhibitor: A Promising Strategy for Discovering New Antiviral Therapeutics Chao Wang,† Lu Lu,‡ Heya Na,† Xiangpeng Li,† Qian Wang,‡ Xifeng Jiang,† Xiaoyu Xu,† Fei Yu,§ Tianhong Zhang,† Jinglai Li,† Zhenqing Zhang,† Baohua Zheng,† Guodong Liang,† Lifeng Cai,† Shibo Jiang,*,‡,§ and Keliang Liu*,† †

Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai 200032, China § Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10065, United States ‡

S Supporting Information *

ABSTRACT: Triterpene saponins are a major group of active components in natural products with nonspecific antiviral activities, while T20 peptide (enfuvirtide), which contains a helix zone-binding domain (HBD), is a gp41-specific HIV-1 fusion inhibitor. In this paper, we report the design, synthesis, and structure−activity relationship (SAR) of a group of hybrid molecules in which bioactive triterpene sapogenins were covalently attached to the HBD-containing peptides via click chemistry. We found that either the triterpenes or peptide part alone showed weak activity against HIV-1 Env-mediated cell−cell fusion, while the hybrids generated a strong cooperative effect. Among them, P26−BApc exhibited anti-HIV-1 activity against both T20-sensitive and -resistant HIV-1 strains and improved pharmacokinetic properties. These results suggest that this scaffold design is a promising strategy for developing new HIV-1 fusion inhibitors and possibly novel antiviral therapeutics against other viruses with class I fusion proteins.

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INTRODUCTION

energy for virus−cell membrane fusion and entry into host cells.12 The synthetic peptides derived from CHR sequences can bind to their counterpart, NHR, to prevent intramolecular fusogenic 6-HB formation, thereby inhibiting membrane fusion and viral infection.13,14 T20 (generic name, enfuvirtide; brand name, Fuzeon) was approved by the U.S. Food and Drug Administration in 2003 as the only HIV-1 fusion inhibitor for the treatment of HIV-1 infections.15 Another peptide, C34, which has a sequence that partially overlaps with that of T20, exhibits promising potency in inhibiting HIV-1-mediated membrane fusion.2 However, due to its poor solubility under physiological conditions, C34 has only been used as a laboratory tool to study the structure and function of HIV-1 gp41. Previous studies have shown that T20 contains a lipidbinding domain (LBD), whereas C34 consists of a pocketbinding domain (PBD), and both share a helix zone-binding domain (HBD) (Figure 1A).16 The HBD exhibits only marginal anti-HIV-1 activity. However, the addition of a PBD to the N-terminus of the HBD or a LBD to the C-terminus of the HBD makes the peptides much more potent than peptides

Antiviral agents can be designed by targeting different steps of the enveloped virus replication cycle.1 Among the various life cycle events, the entry of enveloped viruses into host cells by fusion of the viral membrane with the cellular membrane is the first step.2 This process has been considered as an attractive target and has received wide attention.3,4 The human immunodeficiency virus type 1 (HIV-1) features a common mechanism to catalyze membrane fusion as other enveloped viruses with class I fusion proteins.5 The envelope glycoproteins (Env) of HIV-1, a class I membrane fusion protein, play important roles in the early stage of HIV-1 entry.6,7 HIV-1 Env is composed of a complex of noncovalently associated glycoproteins surface subunit gp120 and transmembrane subunit gp41.8 Its surface subunit gp120 engages in receptor binding, and gp41 mediates fusion of the virus with the target cell.9 With typical sequence motifs homologous to those identified in class I enveloped viruses, gp41 contains an extracellular domain, a transmembrane domain, and a cytoplasmic tail.10,11 It is well-known that the formation of a general six-helix bundle (6-HB) between three C-terminal heptad repeats (CHRs) and an N-terminal heptad repeat (NHR) trimer of the gp41 extracellular domain provides the © XXXX American Chemical Society

Received: May 16, 2014

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dx.doi.org/10.1021/jm500763m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Schematic representation of HIV-1 gp41 and the designed sapogenin−peptide conjugates. (A) HIV-1 gp41 contains a fusion peptide (FP), an N-terminal heptad repeat (NHR), a C-terminal heptad repeat (CHR), a transmembrane domain (TM), and a cytoplasm domain (CP). The key conformational change in gp41 can be blocked by CHR-derived fusion inhibitors T20 and C34. Both of them contain a partial helix zone-binding domain (HBD), which is crucial for these CHR-based fusion inhibitors to interact with the helix zone in the NHR. (B) Chikusetsusaponin IVa extracted from Alternanthera philoxeroides (Mart.) Griseb. (C) The designed sapogenin−peptide conjugates.

containing the HBD alone.17 Furthermore, some peptides corresponded to the C-proximal region of α1-antitrypsin, designated virus-inhibitory peptide (VIRIP),18,19 or derived from the E2 envelope protein of GB virus C (GBV-C),20 can interact with the HIV-1 gp41 fusion peptide and effectively prevent the virus from entering into the host cell. Saponins are a group of bioactive natural compounds mainly produced by plants to counteract pathogens and herbivores; in addition, they are a resource of lead compounds in drug discovery due to their tremendous range of pharmacological properties.21 Several pentacyclic triterpene saponins, such as chikusetsusaponin IVa and glycyrrhizin, show wide antiviral activities, and their administration to virus-infected patients resulted in delayed progression of infection symptoms (Figure 1B).22,23 Although saponins have immense structural diversity, they all share similar structural components: a hydrophobic isoprenoid-derived sapogenin covalently linked to a hydrophilic single saccharide or oligosaccharide moiety.24 Previous reports have indicated that the overall conformation of saponins harmoniously constructed with both hydrophobic and hydrophilic segments is essential for their biological effects.25,26 Inspired by the strong cooperative effect of the aglycone and sugar chain units generated in saponins, we hypothesized that the triterpene sapogenin may serve as the PBD or LBD and that its addition to the N- or C-terminus of a peptide containing only the HBD may result in a significant increase of the anti-HIV-1 activity of the peptides. Herein, we designed novel HIV-1 fusion inhibitors that consisted of the HBD-containing peptide P26 linked with a sapogenin derivative at either the N- or C-terminus of P26 (Figure 1C). The triterpene sapogenin−peptide conjugates were tested as

inhibitors of HIV-1 Env-mediated cell−cell fusion and laboratory-adapted HIV-1IIIB (subtype B, X4) or HIV-1BaL (subtype B, R5) replication as well as against both T20sensitive and resistant HIV-1 strains. One of these conjugates, P26−BApc, which exhibited promising anti-HIV-1 activity, was further subjected to in vitro and in vivo pharmacokinetic studies. The results of this study will provide a promising strategy for the rational design of novel antiviral peptides against HIV-1 and other enveloped viruses with class I fusion proteins.

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DESIGN Because the interaction between NHR and CHR of HIV-1 gp41 plays a critical role in virus−cell membrane fusion, it is conceivable that interfering with this interaction might prevent fusogenic gp41 core formation and thus block HIV-1 infection.27 Indeed, both C34 and T20 peptides derived from the CHR region exhibit anti-HIV-1 activities in the nanomolar range.9 In our prior studies, we have shown that C34 and T20 contain a common 4−3 heptad repeat (HR) sequence, which acts as a structure domain and is responsible for its interaction with the helix zone in the gp41 NHR region.16,28 A synthetic peptide CHR-3, which contains the entire 4−3 HR sequence shared by C34 and T20 but lacks the PBD or LBD sequences, exhibits only weak antiviral activity.17 These results suggest that although the 4−3 HR sequence is essential for these C-peptides to interact with the helix-zone domain in the viral gp41 NHR region, the functional domains, e.g., the PBD in C34 and the LBD in T20, are also critical for the interactions with their corresponding targets. In this study, the HBD-derived peptide B



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Figure 2. Chemical structures of triterpene derivatives.

Scheme 1. Synthesis of BA Derivativesa

(i) TBDPSCl, imidazole, DMAP, DMF, 80 °C; (ii) 4-pentynoic acid, DIC, DMAP, DCM, rt; (iii) TBAF, THF, rt; (iv) propargyl bromide, K2CO3, DMF, rt; (v) allyl bromide, K2CO3, DMF, rt; (vi) 4-pentynoic acid, DIC, DMAP, DCM, rt; (vii) acetic anhydride, pyridine, DMAP, DCM, rt; (viii) propargyl bromide, K2CO3, DMF, rt.

a

the β-Ala spacer was inserted between the triterpene and the target-specific peptide P26 to allow for flexibility. Except for the hydrophobic aglycone precursor of triterpene saponins, pentacyclic triterpenes exist in nature as free acids and also possess a wide range of pharmacological profiles.21 Betulinic acid (BA) is a member of the triterpene family that is present in a variety of plants.30 In an independent line of investigation, Lee, Fujioka, and colleagues isolated BA from Syzigium claviflorum and demonstrated its inhibitory potency against HIV-1 replication in H9 lymphocytes.31 Subsequently, Lee’s group designed a panel of BA derivatives with

P26 was used as the lead compound and fusion inhibitors with novel scaffolds were synthesized by covalently attaching bioactive triterpene sapogenins to the N- or C-terminus of P26. We previously reported a set of conjugates in which the small-molecule fusion inhibitors targeting the hydrophobic cavity in the gp41 NHR-trimer can act as a substitute for the pocket-binding domain (PBD) of the C34 peptide.29 We also studied different linkers between a small molecule and a peptide, in which a flexible linker, β-alanine (β-Ala), could simply and nicely maintain the proper binding orientation and conformation of the small molecules. Therefore, in this study, C



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Scheme 2. Synthesis of OA and UA Derivativesa

(i) TBDPSCl, imidazole, DMAP, DMF, 80 °C; (ii) 4-pentynoic acid, DIC, DMAP, DCM, rt; (iii) TBAF, THF, rt; (iv) propargyl bromide, K2CO3, DMF, rt; (v) allyl bromide, K2CO3, DMF, rt; (vi) 4-pentynoic acid, DIC, DMAP, DCM, rt.

a

resin with TFA. Unfortunately, this route was unsuccessful as a result of the transformation of the olefinic bond of the pentacyclic triterpenes in a TFA medium, which was consistent with previous reports.38 Therefore, the triterpene derivatives were covalently linked to the N- or C-terminus of the P26 peptide, respectively, via click chemistry. The synthetic route to the triterpene derivatives is outlined in Schemes 1 and 2. In general, to synthesize the triterpene aglycone derivatives modified at the C3 position, the C28 carboxylic acid group was first protected by reacting it with tertbutylchlorodiphenylsilane (TBDPSCl) to yield the silyl ester. Next, the 4-pentynyl group was introduced at the C3 position as a carboxylic ester. Finally, the TBDPS protecting group was removed with tetrabutylammonium fluoride in tetrahydrofuran (TBAF in THF) to afford the C3-modified series. On the other hand, a propargyl group was directly introduced to the C28 carboxylic acid group to provide compounds in the C28modified series. As shown in Scheme 1, BApc and BApo were also synthesized such that the C28 carboxylic acid group or the C3 hydroxyl group on the scaffold of BA was permanently protected by an allyl group or an acetyl group, respectively. As shown in Scheme 2, UApc and OApc were also synthesized such that the C28 carboxylic acid group on the scaffolds of UA and OA was permanently protected by an allyl group, respectively. The azidoacetic acid was prepared in good yields by substitution of sodium azide on the corresponding bromoacetic acid in water. To synthesize sapogenin−peptide conjugates in which a triterpene aglycone was attached to the N-terminus of the P26 peptide, P26 peptide plus βAla as the linker was initially synthesized on solid support using Rink Amide resin following the standard Fmoc strategy synthesis procedure. This βAla−P26 peptide was then functionalized on the N-terminus

modifications at its C3 and C28 positions, respectively, and identified lead compounds with enhanced anti-HIV-1 potency.32,33 Structure−activity relationship (SAR) analysis34 has shown that BA derivatives containing modifications to the carboxylic acid group at C28, e.g., RPR103611 and IC9564 reported by Lee’s group, act at the entry step of infection. In addition to the inhibitory effects on HIV-1 entry, BA derivatives containing alterations to the hydroxyl group at the C3 position, such as Bevirimat, have been demonstrated to inhibit viral maturation specifically.35 Some other pentacyclic triterpenes and their derivatives identified by Lee’s group, e.g., oleanolic acid (OA) and ursolic acid (UA), which have a six-membered E ring rather than the five-membered E ring found in BA, have also exhibited antifusion or antimaturation activity.36,37 From the previous SAR studies described above, the functional groups at the C28 and C3 positions of these triterpene scaffolds are critical for their bioactivities. Therefore, we designed and synthesized four parallel series (series A−D) of triterpene derivatives in which an alkyne group was introduced into the parent structures of these sapogenins as the intermediate for conjugation with the HBD-derived peptide P26 via a triazole linkage by click chemistry to generate the sapogenin−peptide conjugates (Figure 2).

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CHEMISTRY Because the unmodifed sapogenins have a carboxyl group functionality, it would have been more straightforward to couple the sapogenin derivatives to the amino group of the peptides on the resin as the final step of solid-phase synthesis, while the hydroxyl group of the sapogenins had been protected as a tert-butyl ether during the reaction. All peptide protecting groups, including the tert-butyl group on the nonpeptide moiety, could be removed during peptide cleavage from the D



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Figure 3. Strategy for the preparation of (A) BAc−P26 and (B) P26−BApc conjugates. Click chemistry provides an orthogonal and productive way to link two large molecules.

Table 1. Inhibitory Activities of Sapogenin−Peptide Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion compda

sequenceb

EC50 (nM)c

BAo−P26 UAo−P26 OAo−P26 BAc−P26 UAc−P26 OAc−P26 BApc−P26 BApo−P26 P26−BAo P26−BAc P26−BApc P26−BApo P26−UApc P26−OApc BA UA OA P26 Ptrz−P26 P26−Ptrz P26 + BAo T20

BAo-a-NNYTSLIHSLIEESQNQQEKNEQELL UAo-a-NNYTSLIHSLIEESQNQQEKNEQELL OAo-a-NNYTSLIHSLIEESQNQQEKNEQELL BAc-a-NNYTSLIHSLIEESQNQQEKNEQELL UAc-a-NNYTSLIHSLIEESQNQQEKNEQELL OAc-a-NNYTSLIHSLIEESQNQQEKNEQELL BApc-a-NNYTSLIHSLIEESQNQQEKNEQELL BApo-a-NNYTSLIHSLIEESQNQQEKNEQELL NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BAo) NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BAc) NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BApc) NNYTSLIHSLIEESQNQQEKNEQELL-a-K(BApo) NNYTSLIHSLIEESQNQQEKNEQELL-a-K(UApc) NNYTSLIHSLIEESQNQQEKNEQELL-a-K(OApc) betulinic acid ursolic acid oleanolic acid NNYTSLIHSLIEESQNQQEKNEQELL Ptrz-a-NNYTSLIHSLIEESQNQQEKNEQELL NNYTSLIHSLIEESQNQQEKNEQELL-a-K(Ptrz) NNYTSLIHSLIEESQNQQEKNEQELL + BAo YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

89.4 ± 2.4 145 ± 17 176 ± 45 15.1 ± 2.5 51.5 ± 25 28.6 ± 5.1 197 ± 55 327 ± 21 19.6 ± 5.0 44.2 ± 10 3.94 ± 0.3 7.94 ± 1.5 3.35 ± 1.1 3.31 ± 1.0 >100000 >100000 >100000 3240 ± 560 3580 ± 156 2183 ± 786 2390 ± 612 10.1 ± 1.4

a

When a nonpeptide moiety is conjugated to the N-terminus of P26, the hybrid has carboxyamide at the C-terminus. When a nonpeptide moiety is attached to the C-terminus of P26, the conjugate has an acetyl group at the N-terminus and carboxyamide at the C-terminus. P26 and T20 have an acetyl group at the N-terminus and carboxyamide at the C-terminus. ba, β-Alanine; Ptrz, 4-propyl-1H-1,2,3-triazol. cCompounds were tested in triplicate, and the data are presented as the mean ± standard deviation.

reaction was applied after routine TFA-mediated cleavage and purification (Figure 3B). Click chemistry provides an orthogonal and productive way for linking two large molecules. The reaction can be catalyzed by cuprous ion from in situ reduction of copper(II) sulfate pentahydrate through ascorbate.39 Although the formation of byproducts resulting from Cu(II)-mediated oxidative side reactions, e.g., Glaser-type alkyne coupling processes, is suppressed as any dissolved dioxygen is reduced by ascorbate, the ascorbate−Cu(II) system induced reactive oxygen species have been shown to lead to histidine oxidation or produce nondisulfide oxidative cross-linking. 4 0 , 4 1 The tris(triazolylmethyl)amine ligands, e.g., N,N,N-Tris[(1-benzyl-1H1,2,3-triazol-4-yl)methyl]amine (TBTA), have been described as a sacrificial reductant and a powerful stabilizing ligand for

with an azido moiety by acylation with azidoacetic acid. Peptide cleavage from the resin was carried out successfully, and the crude azido-precursors were purified by reverse-phase HPLC. Thereafter, sapogenin derivatives were attached to the azidoprecursors via click chemistry, which was achieved in the presence of CuSO4·5H2O and sodium ascorbate in a mixture of H2O and tert-butyl alcohol at room temperature (Figure 3A). To synthesize conjugates in which a pentacyclic triterpene was “capped” to the C-terminus of the P26 peptide, a lysine (Lys) with allyloxycarbonyl (Alloc) group-modified side chains was initially introduced to the C-terminus of the P26 peptide through a βAla spacer. After routine synthesis, the Alloc protecting group was selectively removed on-resin by palladium-mediated deprotection. Next, an azido group was introduced to the side chain of the Lys on-resin, and a click E



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2, a good agreement between fusion inhibition and inhibition of HIV-1IIIB or HIV-1BaL infection was observed. P26−BApc,

copper(I), intercepting hydroxyl radicals formation and protecting Cu(I) from oxidation and disproportionation.40 Fortunately, neither histidine oxidation nor oxidative connections was observed after 60 min in our experimental condition (Supporting Information, Figure S1A). To analyze the effect of TBTA on the copper-catalyzed azide−alkyne cycloaddition (CuAAC), we further performed a cycloaddition reaction between BAc and azido-precursor with addition of 5 equiv of TBTA relative to Cu. There were also no oxidized byproducts observed. However, the reaction rate was reduced, which was consistent with previous reports (Supporting Information, Figure S1B).

Table 2. Anti-HIV-1 Activities and Cytotoxicities of the Conjugatesa EC50 (nM) for inhibiting compd BAo−P26 UAo−P26 OAo−P26 BAc−P26 UAc−P26 OAc−P26 BApc−P26 BApo−P26 P26−BAo P26−BAc P26−BApc P26−BApo P26−UApc P26−OApc T20

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RESULTS AND DISCUSSION Inhibitory Activities of the Sapogenin−Peptide Conjugates on HIV-1 Env-Mediated Cell−Cell Fusion. First, we used an HIV-1 Env-mediated cell−cell fusion assay to measure the inhibitory activities of these conjugates. The activities of the compounds are shown in Table 1. In our assay, BA, UA, and OA all exhibited no inhibitory activity in the cell− cell fusion assay. P26 displayed moderate inhibitory activity, with a 50% effective concentration (EC50) value of 3240 ± 560 nM. Attaching BAo, UAo, or OAo to the N-terminus of P26 dramatically increased the activity, and this series of conjugates had EC50 values of 89.4 ± 2.4, 145 ± 17, and 176 ± 45 nM, respectively, which were 18−40-fold greater than that of P26 alone. The inhibitory activity was further increased when the compounds in series A were conjugated to P26, with EC50 values of 15.1 ± 2.5, 51.5 ± 25, and 28.6 ± 5.1 nM, respectively, providing compounds with approximately 60-fold more potency than that of P26 alone and reaching the potency of T20. After the C28 carboxylic acid group and the C3 hydroxyl group were blocked with allyl and acetyl groups, respectively, BApc−P26 exhibited 13-fold less potency than BAc−P26, whereas BApo−P26 was only 3.6-fold less potent than BAo−P26. These findings suggest that the C28 carboxylic acid group has a more critical role than the C3 hydroxyl group in inhibiting HIV-1 Env-mediated cell−cell fusion. In another group of conjugates, the triterpene derivatives were linked to the C-terminus of the P26 peptide. When the C28 or C3 group was protected, the inhibitory potency was further increased. However, the C28-protected hybrids (e.g., P26−BApc) exhibited stronger inhibitory activities than the C3-protected hybrids (e.g., P26−BApo). Compared with P26−BApc, P26− UApc and P26−OApc showed similar inhibitory activities. It is noteworthy that the triazole linkage makes limited contact with the viral proteins at either the N- or C-terminus of P26. To clarify whether P26 and sapogenin in a mixture state also resulted in significant synergistic effect as in a covalently linked state, the inhibitory activity of a mixture of P26 and BAo (1:1 ratio, unlinked) was tested as the control. Although P26−BAo exhibited promising inhibitory activity, the synergy was lost when simply mixing P26 with BAo in 1:1 ratio, which displayed similar potency as P26 alone (EC50 2390 ± 612 nM). Moreover, P26−BApc and P26−OApc showed solubility values of 18.2 mg mL−1 and 12.4 mg mL−1, respectively, in pure water, suggesting their potential as drug candidates (Supporting Information, Table S1). Inhibitory Activities of the Conjugated Peptides on Viral Replication and Their Cytotoxicities. Next, we tested the inhibitory activity of these compounds on the replication of laboratory-adapted HIV-1 strains, IIIB (subtype B, X4) and BaL (subtype B, R5), as described previously.42 As shown in Table

HIV-1IIIB replication 475 565 369 133 387 94.0 242 501 154 61.6 4.28 41.4 2.18 3.11 10.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

87 130 2.0 53 252 15 11 122 9.0 16 0.7 1.0 0.8 0.1 0.4

HIV-1BaL replication

CC50 (μM)

SIb

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

>25 >25 >25 >25 >25 >25 >25 >25 >25 >25 14.3 ± 1.0 >25 7.92 ± 1.3 8.22 ± 2.4 >25

>52 >44 >68 >187 >64 >266 >103 >52 >50 >406 3348 >603 3633 2643 >2293

456 288 498 98.0 113 150 519 99.0 135 83.1 6.90 20.8 2.51 0.271 2.88

72 32 87 26 55 21 98 12 15 8.3 0.1 5.5 0.5 0.01 0.1

a

Compounds were tested in triplicate, and the data are presented as the mean ± standard deviation. bSI (selectivity index) = CC50/EC50 for inhibiting HIV-1IIIB infection.

P26−UApc, and P26−OApc, which exhibited promising inhibitory activities in the cell−cell fusion assay, also strongly inhibited HIV-1IIIB infection with EC50 values of 4.28 ± 0.7, 2.18 ± 0.8, and 3.11 ± 0.1 nM, respectively, reaching the antiviral potency of T20. Furthermore, we also tested the cytotoxicities of these compounds on MT-2 cells using an XTT cytotoxicity assay (XTT: sodium 3′-[1-(phenylamino)-carbonyl]-3,4-tetrazolium-bis(4-methoxy-6-nitro)bezenesulfonic acid hydrate). The low or absent cytotoxicity of conjugates on MT-2 cells suggest that the sapogenin−peptide conjugates are suitable for future study as drug candidates (Table 2). The Function of BA Modification As Determined by a Prime/Wash Assay in Cell−Cell Membrane Fusion. Numerous biological effects of saponins have been ascribed to their membrane perturbation.43 Previous reports have indicated that saponins as well as sapogenins can incorporate into lipid bilayers to alter the fluidity of the plasma membrane, thus showing broad antiviral potency.44 To illustrate membrane binding affinity, a prime/wash assay was performed. Conjugates BApc−P26 and P26−BApc, as well as T20 and C34 as controls, were preincubated with target cells (TZM-b1 cells) at 37 °C for 1 h, followed by washing to remove unbound peptide before addition of the HL2/3 cells to initiate fusion (Figure 4). We compared the EC50 values of peptide with or without washing the cells to evaluate the binding ability of conjugates to target cell membrane.45,46 We found that the antiviral activities of C34 and BApc−P26 were dramatically decreased (>769-fold and

Figure 4. Strategy for the prime/wash assay of cell−cell membrane fusion. F



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concomitant increase in fluorescence according to their potencies, could be detected in this assay. The positive control, C34, strongly inhibited the 6HB formation, with IC50 values of 2.84 μM. As shown in Figure 5A, Ptrz−P26 alone exhibited no competitive inhibition with env2.0, whereas triterpene−P26 hybrids exhibited inhibitory activities, with IC50 values ranging from 8.31 to 15.5 μM (Supporting Information, Table S2). P26−BAc exhibited much lower potency than BAc−P26, although it could also block the 6HB formation, suggesting a different mechanism of action. These results suggest that triterpene located at the N-terminus of P26 may serve as a PBD to bind to the pocket on the gp41 NHR region, resulting in the increased inhibition of 6HB formation and HIV-1 Envmediated cell−cell fusion. The specific interactions between BAc−P26 conjugates and HIV-1 gp41 NHR were further confirmed by size-exclusion HPLC (SE-HPLC). Compared with CHR−peptides alone, the complex formed between C34 or BAc−P26 and the N36 peptide eluted at the earlier retention time. However, no new peak corresponding to the mixture of P26−BAc peptide and N36 at the corresponding retention time was observed (Figure 5B−D). These results indicate that BAc− P26, similar to C34, is able to interact with N36 to form complexes in PBS. Sapogenin−Peptide Conjugates Inhibit HIV-1 Entry by Time-of-Addition Assay. To determine whether P26− BApc acquires another mechanism of action, a time-of-addition assay was carried out to evaluate the inhibitory activity of P26− BApc against HIV-1 replication when it was added to cells at different intervals postinfection. As shown in Figure 6, P26− BApc was highly effective in inhibiting HIV-1 replication when it was added to the cells with virus together but exhibited significantly decreased inhibitory activity when it was added 1 h or longer after virus was added to the cells. However, the nucleoside reverse transcriptase inhibitor zidovudine continued to exert its full effect even when it was added 6 h postinfection. These results suggest that P26−BApc exhibit anti-HIV-1 potency at an early stage of viral entry with a window of approximately 1 h.

>85-fold, respectively) as a result of their insufficient interaction with the cell membrane. Similar to T20, which interacts with the target cell membrane via its LBD, P26−BApc only showed an 11-fold decrease in potency, indicating a strong binding affinity with the cell membrane (Table 3). These results suggest that BApc located at the C-terminus, rather than the Nterminus, of P26 may serve as a LBD. Table 3. Interaction of Conjugates with Lipid Bilayers HIV-1 Env-mediated cell−cell fusion EC50 (nM) compd

a

BApc−P26 P26−BApc T20 C34

without washing

with washing

± ± ± ±

>10000 (>85) 139 ± 21 (11) 542 ± 13 (39) >1000 (>769)

117 13 14 1.3

24 5.9 5.5 0.7

a

Compounds were tested in triplicate, and the data are presented as the mean ± standard deviation; the values in parentheses indicate relative changes (n-fold) in the EC50 compared with the EC50 without washing.

Sapogenin−Peptide Conjugates Interact with HIV-1 gp41 NHR and Inhibit 6HB Formation of the gp41 NHR and CHR. Another interesting feature of triterpene derivatives is that the modified position (position C3 or C28 of the triterpene scaffold) can orchestrate their mechanism of action.22 Thus, a previously described fluorescence resonance energy transfer (FRET) assay29 was used to test the inhibitory activity of the triterpene−P26 conjugates on gp41 6HB formation. Briefly, the ligand 2,2′-bipyridine-5′-carboxylate (bpy) was linked to the N-terminus of the N-peptide containing the hydrophobic pocket of gp41. The bpy−Npeptide formed a trimeric coiled coil, namely env2.0, by chelation with Fe(II), and acted as a fluorescence quencher of the probe CP2-LY (MTWBEWDREIBNYTSLIC-LY), which was modified with Lucifer yellow dye at its C-terminus and specifically bound to env2.0. Competitive inhibition of env2.0− probe interaction by sapogenin−peptide conjugates, with a

Figure 5. (A) Inhibitory activities of conjugates on 6HB formation and determination of specific interactions between (B) C34, (C) BAc−P26, (D) P26−BAc and N36 using SE-HPLC analysis. G



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and determined the in vitro and in vivo half-lives of P26−BApc and T20, respectively. As shown in Figure 7, P26−BApc exhibited a longer in vitro half-life than T20 in the liver (0.815 vs 0.516 h) and kidney homogenates (0.598 vs 0.281 h), as well as with proteinase K (a broad-spectrum serine proteinase) digestion (0.905 vs 0.324 h). Meanwhile, P26−BApc also exhibited a longer in vivo half-life than T20 (3.13 vs 2.18 h) (Table 5). T20 is the first peptidic HIV-1 fusion inhibitor approved for the treatment of HIV-1 infections. It offers several advantages, e.g., high activity, high specificity, and low toxicity, which are similar to other peptide and protein drugs.48 Like insulin, T20 cannot be administered orally as it is rapidly inactivated by gastrointestinal enzymes. Furthermore, due to its degradation by proteolytic enzymes in the blood, T20 also suffer from short half-life in vivo. These weaknesses render T20 to be sc injected twice daily at high dosage (90 mg), resulting in high cost to patients and serious local injection reactions. Strikingly, our designed sapogenin−peptide conjugates showed enhanced pharmacokinetic properties over T20, prolonging the existence of hybrids in the circulatory system that may lead to a lower frequency of injections or lower dose. Currently, oral delivery of macromolecular drugs is still a challenge because of the enzymatic degradation and poor penetration across the intestinal membrane.49 In addition to pharmaceutical approaches, chemical modifications also have shown promising results for enhancing oral bioavailability of peptides and proteins. Lipidization of peptides (e.g., palmitoylation of insulin and development of hexyl-insulin) has shown exciting results, primarily due to enhanced enzymatic stability and improved intestinal permeability.50 Similarly, our sapogenin−peptide conjugates also exhibited prolonged in vitro and in vivo halflives due to the attachment of the strong hydrophobic triterpenes to P26 peptide. We speculated that, despite meeting with limited success to improve oral bioavailability, our strategy to design the peptide conjugates opens a new avenue to develop peptide fusion inhibitors with improved pharmacological properties.

Figure 6. Time-of-addition assay. P26−BApc (100 nM) was added at different intervals postinfection by HIV-1IIIB in MT-2 cells. Zidovudine (200 nM) was used as a control. The assay was done in triplicate, and the experiment was repeated at least twice. The data are presented in mean ± SEM (standard error of mean) in one representative experiment out of three.

Sapogenin−Peptide Conjugates Are Highly Potent against T20-Sensitive and T20-Resistant HIV-1 Strains. Although the first peptidic anti-HIV drug T20 has shown promise in treating HIV/AIDS patients who have failed to respond to the current reverse transcriptase inhibitors and protease inhibitors, the successful clinical use of T20 has stimulated efforts to develop potent peptides against T20resistant strains.47 Therefore, we tested our designed conjugates against infection by T20-sensitive and resistant HIV-1 strains. A panel of HIV-1NL4−3 mutants, including one T20-sensitive and five T20-resistant strains, was used in our experiments. As shown in Table 4, T20 was much less effective against T20Table 4. Activity of Conjugates against T20-Sensitive and -Resistant Strains NL4-3 mutanta

T20 (EC50, nM)

D36G (36G)V38A (36G)V38A/N42D (36G)N42T/N43K (36G)V38E/N42S (36G)V38A/N42T

40.6 ± 7.8 >2000 (>50) >2000 (>50) >2000 (>50) >2000 (>50) >2000 (>50)

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P26−BApc (EC50, nM) 29.2 135 34.4 192 322 308

± ± ± ± ± ±

CONCLUSION In conclusion, we designed peptide conjugates by covalently attaching a bioactive triterpene sapogenin to HBD-containing peptide P26 derived from the middle portion of the HIV-1 gp41 CHR domain. In this newly designed scaffold, the sapogenin unit may serve as PBD or LBD in the hybrid molecule to interact with the HIV-1 gp41 pocket or the target cell membrane, respectively. These conjugates showed a strong cooperative effect between the sapogenin moiety and the P26 peptide, therefore, exhibited potent antiviral activity against both HIV-1 X4 and R5 viruses as well as T20-resistant HIV-1 strains. This study thus provides an efficient strategy to design new anti-HIV-1 compounds by combining natural products with target-specific peptides. From a medicinal chemistry perspective, triterpene saponins are a major group of active components in natural products with universal antiviral activities. Thus, exploiting their potential in anti-HIV-1 compound design will open new avenues for developing viral fusion inhibitors against other viruses with class I membrane fusion proteins.

1.3 29 (4.6) 3.7 (1.2) 10 (6.5) 52 (11) 5.5 (10)

a

Compounds were tested in triplicate, and the data are presented as the mean ± standard deviation; the values in parentheses indicate relative changes (n-fold) in the EC50 compared with the EC50 in the presence of the D36G substitution; HIV-1NL4−3(D36G) is a T20-sensitive strain and the others are T20-resistant strains.

resistant strains, even at a concentration as high as 2000 nM. However, P26−BApc was highly effective against both T20sensitive and resistant strains, with EC50 values ranging from 29.2 to 322 nM, indicating that these conjugates have the potential to treat patients for whom T20 therapy is ineffective. Pharmacokinetic Studies. One of the major limitations of using synthetic peptide T20 for the treatment of HIV/AIDS patients is its high sensitivity to proteolytic enzymes in the blood, leading to its poor pharmacokinetics in vivo.2 We speculated that our designed sapogenin−peptide conjugates would be more resistant than T20 to proteolytic enzymes. Therefore, we investigated the pharmacokinetic profile of P26− BApc in rats after intravenous administration of a single dose

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EXPERIMENTAL SECTION

Chemistry. Melting points were measured with a RY-1 melting point apparatus without correction. 1H (400 MHz) and 13C (100 MHz) NMR spectra were measured on a JNM-ECA-400 spectrometer

H



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Figure 7. Metabolic stability of P26−BApc and T20 (8 μg/mL) in (A) liver homogenate, (B) kidney homogenate, and (C) a proteinase K digestion assay. (D) Pharmacokinetic profile of P26−BApc and T20 in plasma following the administration of a single intravenous dose (4 mg/kg) to rats.

Table 5. Pharmacokinetic Parameters of P26−BApc and T20 in Rats Following a Single Dose iv Administration Derived from Model-Independent Analysis compd

AUC (0−t) (μg/(ml·h))

MRT (0−t) (h)

t1/2 (h)

Vd (L/kg)

Cmax (mg/L)

P26−BApc T20

101 ± 24 26.9 ± 2.6

3.18 ± 0.19 2.10 ± 0.68

3.13 ± 0.45 2.18 ± 0.86

0.17 ± 0.05 0.44 ± 0.14

40.4 ± 18 25.5 ± 2.7

using TMS as the internal standard. The solvent used was CDCl3 unless otherwise indicated. Mass spectra (MS) were measured on an API-150 mass spectrometer with an electrospray ionization source from ABI Inc. TLC was performed on silica gel GF254 plates. Silica gel (200−300 mesh) from Qingdao Haiyang Chemical Company was used for column chromatography. All chemicals were obtained from Beijing Chemical Works or Sigma-Aldrich, Inc., and solvents were distilled and dried before use according to literature procedures. For tested compounds, purity was determined by analytical RP-HPLC using an Agilent 1200 series and a Phenomenex Jupiter C4 column (150 mm × 4.6 mm, 5 μm) using two different solvent systems (methods A and B in the Supporting Information) and a flow rate of 1 mL/min with detection wavelength at 210 nm. All tested compounds were purified, which resulted in ≥95% purity for assay testing. Synthesis of 2 and 10a−b. Under enclosed conditions flushed with N2, TBDPSCl (1.2 equiv, 0.23 mL, 0.79 mmol) was added to a stirred solution of BA, OA, or UA (1.0 equiv, 0.30 g, 0.66 mmol), imidazole (2.5 equiv, 0.11 g, 1.65 mmol), and DMAP (0.1 equiv, 8.0 mg, 0.06 mmol) in N,N-dimethylformamide (DMF) (2 mL). The mixture was continuously stirred, heated to reflux overnight, and monitored by TLC. After the reaction was finished, DCM (25 mL) was added. The organic phase was washed with 1 M HCl, and the aqueous phase was extracted with DCM three times (20 mL × 3). The organic phase was pooled, washed with brine, and dried over anhydrous Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by silica gel column chromatography

using petroleum ether/ethyl acetate (30:1) as the eluent to afford 2 and 10a−b as a white solid. tert-Butyldiphenylsilyl 3β-Hydroxylup-20(29)-en-28-oate (2). Yield 0.35 g (78%); mp 139−141 °C. MS (m/z): calcd for C46H66O3Si, 694. LC-MS (m/z (rel intens)): 695 (M + H, 14). 1H NMR (CDCl3): δ 7.69 (m, 4H), 7.36 (m, 6H), 4.66 (s, 1H), 4.55 (s, 1H), 3.17 (dd, J = 11.2, 4.76 Hz, 1H), 2.40 (m, 1H), 2.23 (m, 1H), 2.04 (m, 1H), 1.80 (m, 1H), 1.13−1.65 (m, 32H), 0.95 (m, 9H), 0.75 (m, 8H). 13C NMR (CDCl3): δ 175.21, 150.66, 135.40, 135.25, 132.10, 129.95, 127.66, 109.50, 78.96, 77.32, 77.00, 76.69, 57.73, 55.33, 50.60, 48.93, 46.27, 42.51, 40.69, 38.83, 37.76, 37.15, 37.02, 34.37, 32.38, 30.40, 29.88, 27.96, 27.39, 27.02, 25.54, 20.82, 19.36, 19.30, 18.28, 16.20, 15.85, 15.34, 14.61. tert-Butyldiphenylsilyl 3β-Hydroxyolean-12-en-28-oate (10a). Yield 0.33 g (73%); mp 114−116 °C. MS (m/z): calcd for C46H66O3Si, 694. LC-MS (m/z (rel intens)): 695 (M + H, 18). 1H NMR (CDCl3): δ 7.67 (m, 4H), 7.34 (m, 6H), 5.25 (t, J = 3.36 Hz, 1H), 3.18 (dd, J = 10.6, 5.04 Hz, 1H), 2.87 (dd, J = 12.8, 3.92 Hz, 1H), 2.00 (m, 1H), 1.80 (m, 3H), 1.11−1.64 (m, 36H), 0.90−0.97 (m, 15H). 13C NMR (CDCl3): δ 176.59, 143.57, 135.77, 135.73, 132.05, 132.05, 131.93, 129.88, 127.51, 127.47, 122.37, 79.00, 77.31, 77.00, 76.68, 55.14, 47.89, 47.53, 46.27, 41.86, 41.61, 39.18, 38.71, 38.44, 36.94, 33.07, 32.38, 30.72, 28.08, 27.16, 27.08, 25.66, 23.51, 23.34, 19.33, 18.26, 16.97, 15.58, 15.32. tert-Butyldiphenylsilyl 3β-Hydroxyurs-12-en-28-oate (10b). Yield 0.31 g (68%); mp 134−136 °C. MS (m/z): calcd for C46H66O3Si, 694. LC-MS (m/z (rel intens)): 695 (M + H, 21). 1H NMR (CDCl3): δ I



Article

7.67 (m, 4H), 7.36 (m, 6H), 5.21 (t, 1H), 3.20 (m, 1H), 2.23 (d, 1H), 0.81−1.92 (m, 56H). 13C NMR (CDCl3): δ 176.24, 137.79, 135.85, 135.80, 131.99, 129.86, 127.48, 127.45, 125.62, 79.01, 77.31, 77.00, 76.69, 55.17, 53.27, 49.35, 47.53, 42.26, 39.41, 39.36, 38.95, 38.70, 38.65, 36.88, 36.66, 33.21, 30.83, 28.13, 27.89, 27.21, 27.11, 24.72, 23.17, 21.21, 19.33, 18.25, 17.25, 17.05, 15.63, 15.50. Synthesis of 3 and 11a−b. DIC (5.0 equiv, 0.23 g, 1.8 mmol) dissolved in dry DCM (4 mL) was added dropwise over 2 min to a stirred solution of 2 or 10a−b (1.0 equiv, 0.25 g, 0.36 mmol), DMAP (1.0 equiv, 0.04 g, 0.36 mmol), and pentinoic acid (3.0 equiv, 0.11 g, 1.08 mmol) in dry DCM (6 mL). The mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, 25 mL of DCM was added, and the organic phase was washed with 10% citric acid, saturated sodium bicarbonate solution, and brine and dried over Na2SO4. After the mixture was filtered and the filtrate was evaporated under reduced pressure, the residue was purified by silica gel column chromatography using petroleum ether/ethyl ether (30:1) as the eluent to yield 3 and 11a−b as a white solid. tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28oate (3). Yield 0.21 g (78%); mp 172−174 °C. MS (m/z): calcd for C51H70O4Si, 774. LC-MS (m/z (rel intens)): 797 (M + Na, 7). 1H NMR (CDCl3): δ 7.68 (m, 4H), 7.36 (m, 6H), 4.66 (s, 1H), 4.55 (s, 1H), 4.49 (m, 2H), 2.52 (m, 4H), 2.23 (m, 1H), 2.12 (m, 1H), 1.96 (m, 1H), 1.79 (m, 1H), 1.11−1.65 (m, 37H), 0.73−0.94 (m, 19H). 13 C NMR (CDCl3): δ 175.21, 171.50, 150.62, 135.39, 135.24, 132.08, 129.93, 127.65, 109.54, 82.64, 81.34, 77.31, 77.00, 76.68, 68.99, 57.71, 55.39, 50.48, 48.90, 46.29, 42.50, 40.69, 38.33, 37.80, 37.72, 37.04, 34.28, 33.81, 32.35, 30.38, 29.85, 27.93, 27.01, 25.48, 23.66, 19.33, 19.29, 18.13, 16.50, 16.23, 15.84, 14.57, 14.51. tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)olean-12-en-28-oate (11a). Yield 0.22 g (80%); mp 103−105 °C. MS (m/z): calcd for C51H70O4Si, 774. LC-MS (m/z (rel intens)): 775 (M + H, 25). 1H NMR (CDCl3): δ 7.66 (m, 4H), 7.34 (m, 6H), 5.25 (t, J = 3.08 Hz, 1H), 4.51 (t, J = 9.24 Hz, 1H), 2.87 (d, J = 17.6 Hz, 1H), 2.53 (m, 4H), 0.85−1.97 (m, 67H). 13C NMR (CDCl3): δ176.59, 171.49, 143.59, 135.77, 135.73, 129.88, 127.51, 127.47, 122.25, 82.63, 81.32, 77.31, 77.00, 76.69, 69.00, 55.22, 47.88, 47.44, 46.24, 41.86, 41.60, 39.19, 38.08, 37.69, 36.81, 33.81, 33.06, 30.72, 28.04, 27.08, 25.62, 23.51, 23.34, 19.33, 16.95, 16.73, 15.37, 14.52. tert-Butyldiphenylsilyl 3β-(Pent-4′-ynoyloxy)urs-12-en-28-oate (11b). Yield 0.18 g (68%); mp 98−99 °C. MS (m/z): calcd for C51H70O4Si, 774. LC-MS (m/z (rel intens)): 775 (M + H, 57). 1H NMR (CDCl3): δ 7.66 (m, 4H), 7.34 (m, 6H), 5.20 (t, J = 3.64 Hz, 1H), 2.53 (m, 4H), 2.25 (d, J = 10.9 Hz, 1H), 1.96 (m, 2H), 1.76 (m, 5H), 0.83−1.56 (m, 47H). 13C NMR (CDCl3): δ 176.25, 171.48, 137.83, 135.85, 135.80, 132.00, 129.86, 127.48, 127.45, 125.48, 82.63, 81.33, 77.31, 77.00, 76.68, 69.00, 55.24, 53.25, 49.34, 47.44, 42.25, 39.41, 39.35, 38.94, 38.28, 37.68, 36.76, 33.81, 30.82, 28.08, 27.87, 27.11, 24.71, 23.54, 23.20, 23.14, 21.21, 19.33, 17.23, 17.08, 15.79, 15.54, 14.52. Synthesis of BAc, OAc, and UAc. Under enclosed conditions flushed with N2, tetrabutylammonium fluoride (TBAF) (1.5 equiv, 0.14 g, 0.52 mmol) was added to a solution of 3 (1.0 equiv, 0.27 g, 0.34 mmol) in 5 mL of THF. The mixture was stirred for 3 h at room temperature with monitoring by TLC. After the reaction was complete, 15 mL of DCM was added. The organic phase was washed with 1 M HCl and brine and dried over Na2SO4. After the mixture was filtered and the filtrate was evaporated under reduced pressure, the residue was purified by silica gel column chromatography using petroleum ether/ethyl acetate (8:1) as the eluent to yield BAc as a white solid. The same procedure described above was used to obtain OAc and UAc from 11a−b. 3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28-oic Acid (BAc). Yield 0.16 g (90%); mp 253−255 °C. MS (m/z): calcd for C35H52O4, 535. LCMS (m/z (rel intens)): 536 (M+, 100). 1H NMR (CDCl3): δ 4.73 (s, 1H), 4.61 (s, 1H), 4.50 (m, 1H), 2.53 (m, 4H), 2.25 (m, 2H), 1.96 (m, 2H), 0.83−1.69 (m, 41H). 13C NMR (CDCl3): δ 181.75, 181.62, 171.55, 150.38, 109.73, 82.63, 81.33, 69.01, 56.31, 55.34, 50.30, 49.17, 46.90, 42.36, 40.62, 38.33, 38.24, 37.80, 37.05, 36.99, 34.15, 33.79,

32.09, 30.49, 29.63, 27.92, 25.36, 23.65, 20.80, 19.30, 18.10, 16.48, 16.13, 15.97, 14.63, 14.51. 3β-(Pent-4′-ynoyloxy)olean-12-en-28-oic Acid (OAc). Yield 0.17 g (95%); mp 199−202 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS (m/z (rel intens)): 536 (M+, 100). 1H NMR (CDCl3): δ 5.27 (t, J = 3.36 Hz, 1H), 4.53 (t, J = 8.12 Hz, 1H), 2.80 (dd, J = 13.7, 4.2 Hz, 1H), 2.54 (m, 4H), 0.58−1.97 (m, 46H). 13C NMR (CDCl3): δ 183.64, 171.49, 159.63, 145.66, 143.57, 122.51, 82.61, 81.30, 69.01, 56.83, 55.25, 47.49, 46.50, 46.20, 41.52, 40.21, 39.23, 37.92, 37.71, 36.94, 33.82, 33.04, 32.47, 30.64, 28.03, 27.98, 25.89, 23.54, 23.32, 22.89, 18.08, 17.12, 16.68, 15.33, 14.52. 3β-(Pent-4′-ynoyloxy)urs-12-en-28-oic Acid (UAc). Yield 0.16 g (92%); mp 242−245 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS (m/z (rel intens)): 536 (M+, 100). 1H NMR (CDCl3): δ 5.24 (t, J = 3.36 Hz, 1H), 4.53 (t, J = 8.16 Hz, 1H), 2.54 (m, 4H), 2.21 (d, J = 7.84 Hz, 1H), 0.77−1.97 (m, 45H). 13C NMR (CDCl3): δ 176.84, 143.36, 122.57, 78.98, 74.36, 55.16, 51.61, 47.57, 46.73, 45.80, 43.85, 41.66, 41.24, 39.33, 38.72, 38.40, 36.99, 33.78, 33.06, 32.70, 32.14, 30.66, 28.06, 27.62, 27.15, 25.81, 23.58, 23.37, 22.97, 18.30, 17.06, 15.55, 15.31. Synthesis of BAo, OAo, and UAo. To a solution of BA (1.0 equiv, 0.5 g, 1.1 mmol) in 10 mL of DMF were added potassium carbonate (3.0 equiv, 0.46 g, 3.3 mmol) and propargyl bromide (2.0 equiv, 171 μL, 2.2 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. After the reaction was finished, the remaining potassium carbonate and generated potassium bromide were removed by filtration, and the solvent was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using ethyl acetate/ petroleum ether (10:1) as the eluent to afford BAo as a white solid. The above method was used to synthesize compounds OAo and UAo by using OA and UA instead of BA. Prop-2′-ynyl 3β-Hydroxylup-20(29)-en-28-oate (BAo). Yield 0.42 g (78%); mp 185−187 °C. MS (m/z): calcd for C33H50O3, 494. LCMS (m/z (rel intens)): 495 (M + H, 28). 1H NMR (CDCl3): δ 4.65 (m, 4H), 3.17 (dd, J = 5.04, 11.52 Hz, 1H), 3.00 (m, 1H), 2.42 (t, J = 2.52 Hz, 1H), 2.19 (m, 2H), 1.90 (m, 2H), 0.73−1.67 (m, 40H). 13C NMR (CDCl3): δ 175.23, 150.43, 109.64, 78.93, 74.32, 56.54, 55.28, 51.31, 50.50, 49.41, 46.80, 42.33, 40.73, 38.82, 38.65, 38.20, 37.13, 36.76, 34.26, 31.89, 30.44, 29.59, 27.94, 27.34, 25.47, 20.81, 19.33, 18.23, 16.11, 15.96, 15.33, 14.68. Prop-2′-ynyl 3β-Hydroxyolean-12-en-28-oate (OAo). Yield 0.44 g (82%); mp 158−160 °C. MS (m/z): calcd for C33H50O3, 494. LC-MS (m/z (rel intens)): 495 (M + H, 25). 1H NMR (CDCl3): δ 5.30 (t, J = 3.64 Hz, 1H), 4.60 (qd, 2H), 3.22 (m, 1H), 2.85 (m, 1H), 2.41 (t, J = 2.24 Hz, 1H), 1.85−1.98 (m, 3H), 0.74−1.64 (m, 42H). 13C NMR (CDCl3): δ 176.85, 143.36, 122.57, 78.98, 78.09, 74.37, 55.14, 51.61, 47.55, 46.73, 45.78, 41.65, 41.22, 39.33, 38.72, 38.39, 36.98, 33.77, 33.05, 32.69, 32.14, 30.64, 28.06, 27.62, 27.15, 25.80, 23.57, 23.38, 22.96, 18.29, 17.04, 15.55, 15.32. Prop-2′-ynyl 3β-Hydroxyurs-12-en-28-oate (UAo). Yield 0.39 g (80%); mp 145−147 °C. MS (m/z): calcd for C33H50O3, 494. LC-MS (m/z (rel intens)): 495 (M + H, 18). 1H NMR (CDCl3): δ 5.27 (t, J = 3.64 Hz, 1H), 4.60 (qd, 2H), 3.20 (dd, J = 5.04, 10.64 Hz, 1H), 2.42 (t, J = 2.52 Hz, 1H), 2.25 (d, J = 10.9 Hz, 1H), 2.03 (m, 1H), 1.89 (m, 2H), 1.31−1.69 (m, 18H), 0.76−1.08 (m, 25H). 13C NMR (CDCl3): δ 176.66, 137.70, 125.82, 78.99, 78.06, 74.39, 55.14, 52.76, 51.57, 48.13, 47.49, 42.00, 39.54, 39.02, 38.73, 38.58, 36.91, 36.38, 32.99, 30.58, 28.09, 27.97, 27.18, 24.14, 23.45, 23.25, 21.15, 18.26, 17.15, 16.97, 15.61, 15.45. Synthesis of BApc, OApc, and UApc. To a solution of BA, OA, or UA (1.0 equiv, 1.0 g, 2.0 mmol) in 10 mL of DMF were added potassium carbonate (2.0 equiv, 0.61 g, 4.0 mmol) and allyl bromide (1.6 equiv, 284 μL, 3.3 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. After the reaction was finished, the remaining potassium carbonate and generated potassium bromide were removed by filtration, and the solvent was concentrated under reduced pressure. The product was directly used for the next step without further purification. In the next step, DIC (6.0 equiv, 0.23 g, 1.8 mmol) dissolved in dry DCM (4 mL) J



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used. Coupling of the amino acids was achieved using O-benzotriazol1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU, GL Biochem, Shanghai, China) and diisopropylethylamine (DIEA, Acrose) as an activator and an active base, respectively, in N,Ndimethylformamide (DMF) solution. The Fmoc protection group was removed using 20% piperidine/DMF. Between every coupling or Fmoc removal, the resin was washed five times with DMF and three times with dichloromethane (DCM). The carboxyl termini were amidated upon cleavage from the resin, and the amino termini were capped with acetic acid anhydride. The peptides were cleaved from the Rink Amide resin and deprotected with Reagent K, which contained 82.5% trifluoroacetic acid (TFA), 5% thioanisole, 5% m-cresol, 5% water, and 2.5% ethanedithiol. The crude peptide products were precipitated with cold diethyl ether, lyophilized, and purified by preparative reverse-phase high-performance liquid chromatography (HPLC) using a Waters preparative HPLC system (PrepLC 4000): gradient elution of 30−50% solvent B in solvent A (0.1% TFA in H2O, solvent A; 0.1% TFA in 70% CH3CN/H2O, solvent B) over 60 min at 16 mL/min on a Waters X-bridge C8, 10 μm, 19.5 mm × 250 mm column. Analytical RP-HPLC was performed on a RP-C8 column (Zorbax Eclipse XDB-C8, 5 μm, 4.6 mm × 150 mm) with gradient elution of 5−100% solvent B in solvent A over 25 min at a flow rate of 1 mL/min. Compounds were detected by UV absorption at 210 nm with a Shimadzu SPD-10A detector. All peptides were purified to >95% purity. The molecular weight of the peptides was confirmed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS; Autoflex III, Bruker Daltonics). Conjugates Synthesis (Click Chemistry). In brief, purified azido-peptide precursor (1.0 equiv, 0.005 mmol) was dissolved in 1 mL of H2O, to which triterpene derivatives (1.2 equiv, 0.006 mmol) dissolved in 1 mL of tert-butyl alcohol was added. Then CuSO4·5H2O (1.0 equiv, 0.005 mmol) and sodium ascorbate (5.0 equiv, 0.025 mmol) were added to the mixture, which was left stirring at room temperature for 4 h. The reaction was monitored by analytical RPHPLC with a Phenomenex Jupiter C4 column (150 mm × 4.6 mm, 5 μm) using 0.1% TFA in water (A) and 0.1% TFA in 90% CH3CN/ H2O (B) as the eluents, and the linear gradients as follows: 50−80% (B) in 5 min to 100% (B) in 3 min, washing step at 100% (B) for 20 min, flow 1 mL/min. After 4 h incubation, the reaction was complete and the resulting conjugated peptide was purified to >95% purity as described above (65−80 yields) and confirmed by MALDI-TOF-MS. Cell−Cell Fusion Assay. Cell−cell fusion assays were performed as previously described.51 HL2/3 cells, which stably express HIV Gag, Env, Tat, Rev, and Nef proteins, and TZM-bl cells, which stably express large amounts of CD4 and CCR5, were obtained from the NIH AIDS Reference and Reagent Program (contributed by Drs. Barbara Felber and George Pavlakis or Drs. John C. Kappes and Xiaoyun Wu, respectively). TZM-bl cells (2.5 × 104/well) and HL2/3 cells (7.5 × 104/well) were coincubated in 96-well plates (Corning Costar) at 37 °C in 5% CO2 in the presence of different concentrations of inhibitors. After incubation for 6−8 h, the medium was aspirated, the cells were washed and lysed, and luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). HIV-1 Infection Assay. For measuring the inhibitory activity of the peptides on infection of HIV-1IIIB, HIV-1BaL, and T20-resistant HIV-1 strains, 1 × 104 MT-2 cells were infected with 100 TCID50 of a virus in the presence or absence of the peptides at graded concentrations. On the fourth day postinfection, the culture supernatants were collected for detection of p24 antigen using an enzyme-linked immunosorbent assay (ELISA). The percent inhibition by the peptides and 50% effective concentration (EC50) values were calculated using Calcusyn software.52,53 Cytotoxicity Assay. The in vitro cytotoxicity of conjugated peptides to virus target cells (MT-2) was measured by the XTT assay. Briefly, 100 μL of conjugates at graded concentrations were added to equal volumes of cells (5 × 105/mL) in wells of 96-well plates. After incubation at 37 °C for 4 days, 50 μL of XTT solution (1 mg/mL) containing 0.02 μM phenazine methosulfate (PMS) were added. After

was added dropwise over 2 min to a stirred solution of 6 or 14a−b (1.0 equiv, 0.15 g, 0.3 mmol), DMAP (1.0 equiv, 0.04 g, 0.3 mmol), and pentinoic acid (3.0 equiv, 0.09 g, 0.9 mmol) in dry DCM (6 mL). The mixture was stirred at room temperature and monitored by TLC. After the reaction was complete, 25 mL of DCM was added, and the organic phase was washed with 10% citric acid, saturated sodium bicarbonate solution, and brine and dried over Na2SO4. After the mixture was filtered and the filtrate was evaporated under reduced pressure, the residue was purified by silica gel column chromatography using ethyl acetate/petroleum ether (20:1) as the eluent to yield BApc, OApc, and UApc as a white solid. Allyl 3β-(Pent-4′-ynoyloxy)lup-20(29)-en-28-oate (BApc). Yield 0.78 g (68%); mp 120−122 °C. MS (m/z): calcd for C38H56O4, 576. LC-MS (m/z (rel intens)): 577 (M + H, 10). 1H NMR (CDCl3): δ 5.92 (m, 1H), 5.24 (q, 2H), 4.73 (s, 1H), 4.57 (m, 4H), 3.00 (m, 1H), 2.53 (m, 4H), 2.25 (m, 2H), 1.96 (m, 3H), 0.83−1.68 (m, 38H). 13C NMR (CDCl3): δ 175.52, 171.50, 150.53, 132.54, 118.08, 109.61, 82.63, 81.35, 69.00, 64.56, 56.53, 55.40, 50.42, 49.41, 46.93, 42.17, 40.71, 38.21, 38.17, 37.21, 37.07, 36.98, 34.20, 33.80, 32.09, 30.32, 29.60, 27.92, 24.21, 23.11, 20.87, 19.32, 18.13, 16.51, 16.14, 15.93, 14.65, 14.51. Allyl 3β-(Pent-4′-ynoyloxy)olean-12-en-28-oate (OApc). Yield 0.66 g (58%); mp 140−142 °C. MS (m/z): calcd for C38H56O4, 576. LC-MS (m/z (rel intens)): 577 (M + H, 7). 1H NMR (CDCl3): δ 5.89 (m, 1H), 5.29 (m, 3H), 4.52 (m, 3H), 2.86 (dd, J = 13.5, 3.84 Hz, 1H), 2.53 (m, 4H), 0.72−1.67 (m, 44H). 13C NMR (CDCl3): δ 177.33, 171.47, 143.69, 132.53, 122.28, 117.67, 82.60, 81.31, 69.01, 64.77, 55.26, 47.92, 46.98, 45.81, 41.65, 41.30, 39.02, 38.06, 37.78, 36.88, 33.81, 33.07, 32.61, 32.52, 30.09, 28.03, 27.62, 25.83, 23.60, 23.50, 23.37, 23.00, 18.18, 16.92, 16.82, 15.33, 14.53. Allyl 3β-(Pent-4′-ynoyloxy)urs-12-en-28-oate (UApc). Yield 0.80 g (70%); mp 150−151 °C. MS (m/z): calcd for C38H56O4, 576. LC-MS (m/z (rel intens)): 577 (M + H, 21). 1H NMR (CDCl3): δ 5.87 (m, 1H), 5.21 (m, 3H), 4.50 (m, 3H), 2.53 (m, 4H), 2.24 (d, J = 11.2 Hz, 1H), 1.90 (m, 4H), 0.72−1.67 (m, 40H). 13C NMR (CDCl3): δ 177.10, 171.43, 138.06, 132.51, 125.46, 117.71, 82.58, 81.28, 68.99, 64.77, 55.24, 52.83, 48.06, 47.40, 41.98, 39.52, 39.01, 38.99, 37.89, 37.67, 35.88, 35.82, 33.79, 33.00, 30.61, 28.03, 27.93, 24.16, 23.48, 23.23, 21.15, 18.13, 17.00, 16.73, 15.45, 14.48. Synthesis of BApo. A mixture of BA (1.0 equiv, 4.0 g, 8.76 mmol), pyridine (25 mL), and acetic anhydride (2.0 equiv, 3.5 mL) was stirred at room temperature overnight until it became homogeneous. The mixture was then poured into ice-cold water and stirred for 20 min. The intermediate 8 was filtered off and directly used for the next step without further purification. In the next step, to a solution of 8 (1.0 equiv, 1.0 g, 2.0 mmol) in 10 mL of DMF were added potassium carbonate (3.0 equiv, 0.91 g, 6.0 mmol) and propargyl bromide (2.0 equiv, 342 μL, 4.0 mmol). The mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. After the reaction was finished, the remaining potassium carbonate and generated potassium bromide were removed by filtration, and the solvent was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using ethyl acetate/petroleum ether (10:1) as the eluent to afford 0.8 g of BApo as a white solid in 78% yield. Prop-2′-ynyl 3β-Acetoxylup-20(29)-en-28-oate (BApo). Melting point 180−182 °C. MS (m/z): calcd for C35H52O4, 536. LC-MS (m/z (rel intens)): 537 (M + H, 17). 1H NMR (CDCl3): δ 4.64 (q, 4H), 4.45 (m, 1H), 3.00 (m, 1H), 2.43 (m, 1H), 2.24 (m, 2H), 2.04 (s, 3H), 1.90 (m, 2H), 0.82−1.68 (m, 40H) . 13C NMR (CDCl3): δ 175.16, 171.00, 150.41, 109.69, 80.92, 78.12, 74.28, 56.57, 55.40, 51.30, 50.45, 49.45, 46.84, 42.36, 40.17, 38.38, 38.22, 37.77, 37.08, 36.77, 34.21, 31.91, 30.46, 29.31, 27.91, 25.47, 23.67, 21.31, 20.87, 19.33, 18.14, 16.46, 16.18, 15.99, 14.73. Peptide Synthesis. Peptides were synthesized using a Liberty automated microwave peptide synthesizer (CEM Co., Matthews, NC) with a standard solid-phase N-(9-fluorenyl)methoxycarbonyl (Fmoc) chemistry protocol. All protected amino acids used were purchased from GL Biochem Ltd. (Shanghai, China). Rink Amide resin (0.38− 0.45 mmol/g, Nankai Hecheng S&T Co. Ltd., Tianjin, China) was K



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4 h, the absorbance at 450 nm (A450) was measured with an ELISA reader. The 50% cytotoxicity concentrations (CC50) were calculated using the CalcuSyn software. The Solubility of Peptide−Sapogenin Conjugates. We estimated the solubility of the peptide−sapogenin conjugates as previously described.12 Approximately 1 mg of each peptide was weighed, and 10 μL of dd-H2O or phosphate-buffered saline (PBS) were added to dissolve the samples. The solutions were centrifuged, and the supernatants were recovered. The concentration of each tyrosine-containing hybrid was calculated using a NanoDrop-2000c UV spectrophotometer (Thermo Scientific, USA) by measuring the UV absorbance of the supernatants at 280 nm. A Prime/Wash Assay. To illustrate the key mechanism of action of the peptide−sapogenin conjugates, a prime/wash assay of cell−cell membrane fusion was performed. First, BAc−P26, P26−BApc, T20, and C34 were incubated with TZM-b1 cells at 37 °C for 1 h, followed by washing. Then, HL2/3 cells were added to initiate infection. After incubation for 48 h, the EC50 values of the peptide with or without washing were calculated to evaluate the ability of the residual peptides to inhibit cell−cell fusion. Fluorescence Resonance Energy Transfer (FRET) Assay. Briefly, the ligand 2,2′-bipyridine-5′-carboxylate (bpy) was linked to the N-terminus of the N-peptide containing the hydrophobic pocket of gp41 (a 31-mer NHR peptide: Bpy-GQAVEAQQHLLQLTVWGIKQLQARILAVEKK-NH2). The bpy−N-peptide formed a trimeric coiled coil, namely env2.0, by chelation with Fe(II), and acted as a fluorescence quencher of the probe CP2-LY (MTWBEWDREIBNYTSLIC-LY), which was modified with Lucifer yellow dye at its Cterminus and specifically bound to env2.0. Competitive inhibition of env2.0-probe interaction by sapogenin−peptide conjugates, with a concomitant increase in fluorescence according to their potencies, could be detected in this assay. Typically, 7 μM binding sites (three per receptor trimer) and 1 μM CP2-LY were used in the assay. Binding Assays by Size-Exclusion Chromatography. N36 was mixed with conjugated peptides (final concentration = 0.20 mM) at a molar ratio of 1:1 in 50 mM sodium phosphate/150 mM NaCl (pH 7.2) and incubated at 37 °C for 30 min. The mixture or peptide (20 μL) was applied to the Phenomenex BioSep-SEC-s2000, 300 mm × 7.80 mm HPLC column equilibrated with H2O and eluted at 0.8 mL/ min, and fractions were monitored at 210 nm. Effect on HIV-1 Entry by Time-of-Addition Assay. For HIV1IIIB strain, 100 nM P26−BApc, or 200 nM Zidovudine were added to MT-2 cells, respectively, at different intervals postinfection (0, 0.5, 1, 3, and 6 h). At 3 or 4 days postinfection, p24 antigen was measured as described previously.54

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81072581 and 81373456) and a National Science and Technology Major Project of China grant (2012ZX09301003).

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ABBREVIATIONS USED CHR, C-terminal heptad repeat; NHR, N-terminal heptad repeat; 6HB, six-helix bundle; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine

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ASSOCIATED CONTENT

S Supporting Information *

Analytical HPLC methods and characterization data of key compounds, HPLC analysis of BAc−P26 conjugate synthesis, the solubility of peptide−sapogenin conjugates, inhibitory activity of peptide conjugates on 6HB formation, and pharmacokinetic experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Pan, C.; Liu, S.; Jiang, S. HIV-1 gp41 fusion intermediate: a target for HIV therapeutics. J. Formosan Med. Assoc. 2010, 109 (2), 94−105. (2) Cai, L.; Jiang, S. Development of peptide and small-molecule HIV-1 fusion inhibitors that target gp41. ChemMedChem 2010, 5 (11), 1813−1824. (3) Harrison, S. C. Viral membrane fusion. Nature Struct. Mol. Biol. 2008, 15, 690−698. (4) Naider, F.; Anglister, J. Peptides in the treatment of AIDS. Curr. Opin. Struct. Biol. 2009, 19 (4), 473−482. (5) Porotto, M.; Yokoyama, C. C.; Palermo, L. M.; Mungall, B.; Aljofan, M.; Cortese, R.; Pessi, A.; Moscona, A. Viral entry inhibitors targeted to the membrane site of action. J. Virol. 2010, 84 (13), 6760− 6768. (6) Berkhout, B.; Eggink, D.; Sanders, R. W. Is there a future for antiviral fusion inhibitors? Curr. Opin. Virol. 2012, 2, 50−59. (7) Naito, T.; Izumi, K.; Kodama, E.; Sakagami, Y.; Kajiwara, K.; Nishikawa, H.; Watanabe, K.; Sarafianos, S. G.; Oishi, S.; Fujii, N.; Matsuoka, M. SC29EK, a peptide fusion inhibitor with enhanced αhelicity, inhibits replication of human immunodeficiency virus type 1 mutants resistant to enfuvirtide. Antimicrob. Agents Chemother. 2009, 53 (3), 1013−1018. (8) Chan, D. C.; Fass, D.; Berger, J. M.; Kim, P. S. Core structure of gp41 from the HIV envelope glycoprotein. Cell 1997, 89, 263−273. (9) Chan, D. C.; Kim, P. S. HIV entry and its inhibition. Cell 1998, 93, 681−684. (10) Gallo, S. A.; Puri, A.; Blumenthal, R. HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process. Biochemistry (Moscow) 2001, 40, 12231−12236. (11) Ashkenazi, A.; Shai, Y. Insights into the mechanism of HIV-1 envelope induced membrane fusion as revealed by its inhibitory peptides. Eur. Biophys. J. 2011, 40 (4), 349−357. (12) Shi, W.; Cai, L.; Lu, L.; Wang, C.; Wang, K.; Xu, L.; Zhang, S.; Han, H.; Jiang, X.; Zheng, B.; Jiang, S.; Liu, K. Design of highly potent HIV fusion inhibitors based on artificial peptide sequences. Chem. Commun. 2012, 48, 11579−11581. (13) Jiang, S.; Zhao, Q.; Debnath, A. K. Peptide and non-peptide HIV fusion inhibitors. Curr. Pharm. Des. 2002, 8, 563−580. (14) Wild, C. T.; Shugars, D. C.; Greenwell, T. K.; McDanal, C. B.; Matthews, T. J. Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 9770−9774. (15) Ferrer, M.; Kapoor, T. M.; Strassmaier, T.; Weissenhorn, W.; Skehel, J. J.; Oprian, D.; Schreiber, S. L.; Wiley, D. C.; Harrison, S. C. Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements. Nature Struct. Biol. 1999, 6 (10), 953−960. (16) Qi, Z.; Shi, W.; Xue, N.; Pan, C.; Jing, W.; Liu, K.; Jiang, S. Rationally designed anti-HIV peptides containing multifunctional domains as molecule probes for studying the mechanisms of action of the first and second generation HIV fusion inhibitors. J. Biol. Chem. 2008, 283 (44), 30376−30384. (17) Liu, S.; Jing, W.; Cheung, B.; Lu, H.; Sun, J.; Yan, X.; Niu, J.; Farmar, J.; Wu, S.; Jiang, S. HIV gp41 C-terminal heptad repeat contains multifunctional domains. Relation to mechanisims of action of anti-HIV peptides. J. Biol. Chem. 2007, 282 (13), 9612−9620. (18) Münch, J.; Ständker, L.; Adermann, K.; Schulz, A.; Schindler, M.; Chinnadurai, R.; Pohlmann, S.; Chaipan, C.; Biet, T.; Peters, T.;

AUTHOR INFORMATION

Corresponding Authors

*For K.L.: phone, 86-10-6816-9363; fax, 86-10-6821-1656; Email, [email protected]. *For S.J.: phone, 86-21-54237673; fax, 86-21-54237465; Email, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported, in part, by grants from the National Science Foundation of China (81373266, 81273434, L



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