Research paperDesign, synthesis of novel tryptophan derivatives for antiplatelet aggregation activity based on tripeptide pENW (pGlu-Asn-Trp)
Graphical abstract
Introduction
Aggregation of platelets is the leading cause of arterial thrombosis and hemostatis. Severe cardiovascular disorders, such as transient ischemic attack, myocardial infarction, and unstable angina pectoris have been reported to be related to uncontrolled platelet aggregation and platelet adhesion to the subendothelium of damaged blood vessels [1].
Several mechanisms have been found in the induction of the aggregation of platelets, including the stimulation by ADP, thrombin, collagen, TXA2 or sheer stress [2], [3], [4]. In response to these stimulations, GPIIb/IIIa, a heterodimeric membrane protein expressed on the surface of platelets, undergoes a substantial conformational change which enhances its binding affinity to fibrinogen, a multi-valent plasma protein. Binding of multiple GPIIb/IIIa molecules to a single molecule of fibrinogen is the key step for crosslinking of the platelets and thrombus formation (Fig. 1) [5], [6], [7], [8], [9], [10], therefore, development of small molecular compounds to inhibit the binding of fibrinogen to GPIIb/IIIa has been considered as an attractive strategy for suppressing aggregation of platelets and thrombus formation.
Three GPIIb/IIIa inhibitors have been approved to use in clinic, among them Abciximab is an antibody, while Eptifibatide and Tirofiban are peptidic mimetics mimicking tri-peptide Arg-Gly-Asp (RGD), the minimum binding epitope of fibrinogen to the GPIIb/IIIa receptor [11], [12], [13]. Currently, many RGD mimetics are in different stage of clinical trials as antithrombotic agents, however, most of them either fail to demonstrate sufficient efficacy or show severe side effects, such as thrombocytopenia and bleeding risk. Therefore, development of new antithrombotic drugs with improved efficacy and reduced side effects is urgently needed [14], [15], [16], [17], [18], [19], [20].
Recently, we found that pENW (Fig. 2), a three mer peptide derived from Agkistrodon acutus Guenther venom can inhibit platelet aggregation and thrombus formation in both arterial and venous thrombosis models [21]. Moreover, it could prolong the coagulation time in mice without extending the haemorrhage time. Because pENW inhibits platelet aggregation induced by all of the four agonists, we hypothesize that it may function by disturbing the interaction of GPIIb/IIIa with fibrinogen. Therefore, we tested its inhibition to GPIIb/IIIa receptor using fibrinogen/GPIIb/IIIa enzyme-linked immunosorbent assay (ELISA). In this assay, pENW shows an IC50 value of 12.5 μM, similar to a known GPIIb/IIIa receptor inhibitor, RGDS (IC50 = 5.1 μM) [22], [23], thus representing a promising lead compound. However, pENW has very short half-life, and its activity in anti-platelet aggregation is not very potent, therefore, in our present study, we performed extensive modifications to pENW with the purpose to improve the activity in anti-platelet aggregation and to reduce the bleeding risk.
Section snippets
Design and hit identification
Our previous study indicated that GPIIb/IIIa receptor is one of targets of pENW. Based upon the crystal structure of Tirofiban in complex with GPIIb/IIIa (PDB code: 2VDM), we have performed the docking study for pENW in binding to GPIIb/IIIa receptor using CDOCKER and the result is shown in Fig. 3. In the crystal structure, the 4-piperidyl group in Tirofiban has salt bridge interaction with the carboxyl group of the Asp224 residue in GPIIb subunit. The carboxyl group forms several hydrogen
Conclusion
In our previous work [22], [23], we have identified a three mer peptide pENW from Agkistrodon acutus Guenther venom as an effective antagonist of GPIIb/IIIa receptor. By comparing the binding model of pENW with GPIIb/IIIa receptor with the crystal structure of Tirofiban in complex with GPIIb/IIIa receptor, we found that the Trp moiety in pENW is the major portion for interacting with the GPIIb/IIIa receptor. Based upon this moiety, we designed and synthesized a series of Trp derivatives by
Chemistry
All reagents were from commercial sources. With tetramethylsilane (TMS) as internal standard, the 1H NMR and 13C NMR were recorded on Bruker AV-300 apparatus by using deuterated solvents. HR-MS was collected on Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments. Every targeted compound was purified via silica gel (60 Å, 70–230 mesh) column chromatography. Melting points were measured by XT-4 melting point apparatus.
Acknowledgments
This work was supported by the project 81273375 of National Natural Science Foundation of China, China Pharmaceutical University. The authors declare no other conflicts of interest.
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