Synthesis of cyclotides with potential Anti-HIV Activity
Introduction
Cyclotides are small globular microproteins(ranging from 28 to 37 amino acids)with a unique head-to-tail cyclized backbone, which is stabilized by disulfide bonds forming a cystine-knot motif. This cyclic cystine-knot framework provides a rigid molecular platform with exceptional stability towards physical, chemical and biological degradation. These microproteins can be considered natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot(1). Furthermore, naturally occurring cyclotides have been shown to possess various pharmacologically relevant activities and have been reported to cross the cell membranes(3). Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes.
The CXCR4 receptor is one of the 19 chemokine receptors known so far. This receptor is activated exclusively by the cytokine CXCL12, also known as stromal cell-derived factor-1alpha; (SDF1alpha;). Activation of CXCR4 promotes chemotaxis in leukocyte progenitor cell migration and embryonic development of the cardiovascular, hemaotopoietic, and central nervous systems. CXCR4 has also been associated with multiple types of cancers where its over expression/ activation promotes metastasis, angiogenesis, and tumor growth and/or survival. Furthermore, CXCR4 is involved in HIV replication, as it is a co-receptor for viral entry into host cells. Altogether, these features make CXCR4 a very attractive target for drug discovery. Several small disulfide cyclic peptides derived from the horseshoe crab peptides polyphemusin-I/II have recently been reported to be efficient CXCR4 antagonists and effective as anti-HIV-1 and antimetastatic agents. Some of these peptides, however, have shown limited stability and/or poor bioavailability. As a result, we will use MCoTI-I (Figure A) as a molecular scaffold to produce a novel cyclotide with CXCR4 antagonistic activity.
MCoTI cyclotides have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of the cucurbitaceae family, and are potent trypsin inhibitors (Ki asymp;20minus;30 pM)(4). MCoTI-cyclotides show very low toxicity in human cells(5) and represent a desirable molecular scaffold for engineering new compounds with unique biological properties. Hence, we are going to design novel peptides with potential anti-HIV activity by grafting topologically modified CVX15 based peptides onto the loop 6 of the cyclotide MCoTI-I. According to the X-Ray crystal structure of CVX15 bound to CXCR4, the N- and C- termini of the CVX15 peptide are deeply buried into the CXCR4 binding pocket. Therefore, a circularly permuted version of the CVX15 peptide is grafted into the loop 6 of the cyclotide MCo-TI-I in order to preserve the biological activity of the grafted peptide. The CVX15 sequence is designed by linking the original N- and C- termini directly, removing residues D-Pro8 and Pro9 and leaving the new N- and C- terminal groups on residues Tyr10 and Lys7 respectively. Residues Gln6 is also replaced by citruline, which has been shown to increase the affinity of CVX15 to CXCR4.
The synthesis of cyclotides is accomplished by solid phase peptide synthesis (SPPS), which is based on sequential addition of amino acids ( side-chain protected) to an insoluble polymeric support. Base-labile Fmoc-group and acid-labile Boc-group are used for N-terminal protection(2). After removal of this protecting group, the next protected amino acid is added using coupling reagents such as PyBop, DIEA or HBTU. The resulting peptide is attached to a resin through C-termini and can be cleaved to thioester by Ethyl thioglycolate catalyzed by sodium thiophenolate(2). Side-chain protecting groups can be removed by trifluoroacetic acid (TFA) and scavengers, such as Triisopropylsilane (Tis) and water, either simultaneously with or after the detachment of the peptide from the resin. The general procedures of SPPS are shown in Scheme 1.
Scheme 1
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