EHOOPrAHH^ECKAa XHMH3, 2013, moM 39, № 5, c. 547-551
SYNTHESIS AND ACTIVITY OF A CYCLO-HEPTAPEPTIDE CONTAINING LYS-GLY-ASP-SEQUENCE AS A NOVEL ANTI-PLATELET AGENT
© 2013 Jian Jing
Department of Bioorganic and Biological Chemistry, Beijing Key Lab, Beijng Normal University, Beijing, 100875 P.R. China
Received March 20, 2013; in final form, April 12, 2013
Linear hepta-peptide Cys-Lys-Gly-Asp-Trp-Asp-Cys was synthesized first and then disulfide bond was formed between the Cysl and Cys7 to develop cyclo-heptapeptide containing Lys-Gly-Asp-sequence. Structural simulation showed that Lys-Gly-Asp-motif (KDG) displayed functional conformation. The cyclo-hep-tapeptide exhibited potent anti-platelet aggregation activity based on specific recognition and interaction with the GPIIb-IIIa receptor on platelet cell membrane. The specific and potent anti-platelet activity makes the KGD-containing cyclo-heptapeptide a possible therapeutic agent.
Keywords: KGD-containing cyclo-heptapeptide, platelet aggregation inhibitory activity, integrin binding selectivity
DOI: 10.7868/S0132342313050060
INTRODUCTION
Platelet aggregation is a vital process during the thrombosis-related cardio- or cebrelvascular dis-ease[1]. aIIbp3 is the most abundant adhesive receptor on the platelet surface with the number of 80 000 on each platelet [2]; it mediates platelet aggregation in hemostasis and thrombosis [3]. Disintegrins are the largest family of naturally occurring antagonists of integrin present in the venom of viperidare snakes [4, 5] that inhibit platelet aggregation via the occupancy of fibrinogen-integrin aIIbp3 receptor [6—9]. Disinte-grin structures has been well characterized by NMR and molecular modeling [10]. They all present an exposed loop with the integrin binding motif in its apex. Barbourin, isolated from viper venom, which is a 73-amino acid, disulfide-rich peptide that is highly homologous to other members of the disintegrin family, is an antagonist with specificity for integrin aIIbp3, known as glycoprotein(GP) IIb-IIIa, and also is unique in that it contains the KGD-functional motif [11]. Lys-for-Arg substitution is the sole structural feature responsible for this integrin receptor recognition and binding specificity [11]. In this study, we found a small, conformationally constrained peptide containing the KGD sequence which was derived from functional KGD-motif of barbourin and shown to be a potent and specific inhibitor of platelet GPIIb-IIIa versus other closely related integrins, such as avp3.
# Corresponding author (phone: +86 (010) 5880-2065; fax: +86 (010) 5880-7720; e-mail: jjing@bnu.edu.cn).
RESULTS AND DISCUSSION
The simulated structure of (CKGDWDC)-cyclo polypeptide is shown in Fig 1. Two cysteines located at N- and C-terminus of peptide are connected through disulfide-bond linkage to develop cyclo-peptide. And also, another role of this disulfide bond linkage is to provide some conformational constrain to the KGD-motif which plays an important role in interaction with the corresponding integrin receptor GPIIb-IIIa on surface of platelet cells.
The MALDI-TOF-MS analysis resutls are shown in Fig. 2. The molecular weight of KGD-cyclo-hep-
Fig. 1. Modelling of KGD-cyclo-heptapeptide.
Absorbance at 280 nm
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Fig. 2. RP-HPLC and mass spectrometry assay of synthesized and purified cyclo- or linear-heptapeptide. RP-HPLC was conducted utilizing a Vydac CjS column (5 ^m, 4.6 x 150 mm) which was eluted at a flow rate of 0.6 mL min-1 with a linear gradient of acetonitrile (5-60%) containing 0.1% trifluoroacetic acid. RP-HPLC profiles are displayed on the left, ass spectrum are shown on the right. (A) linear-heptapeptide; (B) cyclo-heptapeptide, respectively.
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Fig. 3. ADP stimulated platelet aggregation inhibition. (A) Evaluation of platelet aggregation inhibition. (B) The rate curve of ADP-stimulated platelet aggregation.
tapeptide determined was 819.3 dalton which is very close to the theoretical value of 820.9 dalton as calculated according to amino acids composition of the peptide. The result of mass spectrometry also showed a high homogeneity of KGD-cyclo-heptapeptide sample with single and clear peaks (Fig. 2).
DTNB, known as Ellman agent, which can react with the free-thiol group of side chain of cysteine residues of protein or peptide samples to produce yellow products as a dose-dependent relationship, was adopted to detect whether disulfide bond linkage has been developed among KGD-cyclo-heptapeptide. No color effect in the solution occurrs if no free thiol-group among protein or peptide samples. DTNB test showed that there is no any color phenomenon which suggested that no free thiol group existed in the KGD-cyclo-heptapeptide-containing solution. This assay suggested that all cysteines of the synthesized KGD-cyclo-heptapeptide have formed disulfide-bond linkage.
The next experiment displayed that KGD-cyclo-heptapeptide can inhibit efficiently ADP-stimulated platelet aggregation in a concentration-dependent manner as shown in Fig. 3A, in contrast with 0.9% NaCl solution. Cyclic KGD peptide was found to be a potent inhibitor of platelet aggregation with a half-maximum inhibition occurring at about 620 x 10-9 mol L-1.
SYNTHESIS AND ACTIVITY OF A CYCLO-HEPTAPEPTIDE
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Light transmission spectrum recorded the platelet aggregation process. The slope of initial stage in the light transmission spectrum was found different which suggested different aggregation rate during ADP-stim-ulated platelet aggregation process. The rates of platelet aggregation at different KGD-cyclo-heptapeptide concentrations were calculated as shown in Fig. 3B. The result shows that platelet aggregation rate gradually decreases KGD-cyclo-heptapeptide concentration at solution increases. This phenomenon suggests that with increase of concentration of KGD-cyclo-heptapeptide, the sensitivity of platelets to stimulus ADP displays a down effect and interaction of platelets with each other becomes more and more difficult.
We synthesized a linear Lys-Gly-Asp-containing peptide (linear-heptapeptide, C-K-G-D-W-D-C-NH2) as well as the corresponding cyclic peptide (cyclo- hep-tapeptide, cyclo(S,S)-C-K-G-D-W-D-C-NH2) with a disulfide bond connecting the amino- and carboxyl-terminal cysteine residues of the peptide. Evaluation of the synthesized peptide in the integrin binding assay showed a significant enhancement of binding potency to GPIIb-IIIa integrin for the cyclic Lys-Gly-Asp-con-taining analog 2 which means that incorporation of KGD sequence into cyclic peptide structures could increase the affinity of this peptide for IIb-IIIa integrin receptor (Fig. 4A). To ascertain their integrin receptor binding selectivity, the ability of both the linear and cyclic forms of these peptides to inhibit vitronectin binding to integrin avp3 was also examined. While cycliza-tion of KGD-containing peptides increased their affinity for GPIIb-IIIa receptor, both the linear and cyclic KGD analogs remained unreactive with avp3 receptor at concentrations up to 25 x 10-6 mol L-1 (Fig. 4B). The KGD peptide has specific affinity to IIb-IIIa receptor, but cyclization of KGD sequence could increase this affinity and at the same time not affect its integrin receptor selectivity. Thus, the cyclic KGD heptapetide is a potent and selective GPIIb-IIIa integrin receptor antagonist.
Platelet aggregation is a key event in thrombus formation and is dependent upon the binding of adhesive proteins to the GPIIb-IIIa complex on the platelet surface [12]. Based on molecular dynamics simulation and known relationship between conformation and function, it is possible to design short peptide containing KGD-sequence to interact with GPIIb-IIIa receptor specifically and efficiently, which would make it an anti-platelet therapeutic agent candidates [13]. Formation of disulfide bond linkage between N- and C-terminus of peptide reduced the probability of irregular conformation and increased conformational constrain which is beneficial to produce stable and regular structure to interact with GPIIb-IIIa. The molecular structure simulations also show that the existence of the structural constrains in the formation of hydrogen bonds between tryptophan and lysine residues and possible presence of hydrogen bond is advan-
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Fig. 4. Inhibition of receptor-ligands binding assay. (A) Fibrinogen as ligand binding to GPIIb-IIIa receptor. (B) Vitronectin as ligand binding to avß3 receptor, known as vitronectin receptor, respectively. Various doses of sample peptides compete for binding by corresponding receptors ofbiotinylated fibrinogen and vitronectin. Bound peptide ligands were quantified by enzyme-linked immunosorbent method. (■) linear-heptapeptide; (•) cyclo-KGD-containing analog, respectively.
tageous to further maintain the stability of the spatial structure of the polypeptide. The DTNB test showed that free thiol group does not exist in this annular polypeptide, all of the cysteine residues connected each other existed with disulfide bonds. Moreover, further analysis of biological activity seems to confirm that disulfide bond formation favored to present active conformation to recognize and bind with the corresponding GP receptor. The KGD annular polypeptide showed high activity of inhibiting p
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