Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo

The G-protein coupled chemokine receptor 4 (CXCR4) plays an important role in several human diseases, including cancer, autoimmune disease, and HIV infection [1]. CXCR4 and its ligand CXCL12, also known as SDF1α, are extremely important during development and in normal physiology by promoting directional migration of cells [2,3], and are required for homing of immune cells [3,4]. The CXCL12-mediated activation of CXCR4 promotes cell proliferation and survival and chemotaxis through activation of the Akt and mitogen-activated protein kinase (MAPK) pathways [5], and its inhibition has been shown to reduce metastasis and sensitize tumors for radiation, immune, and chemotherapy [[6], [7], [8]]. Furthermore, CXCR4 is also key for HIV replication and plays an important role as a co-viral receptor for viral entry in host cells [9,10].

These features make CXCR4 a very attractive target for drug and imaging agent discovery [[11], [12], [13]]. Consequently, several small peptides/proteins, monoclonal antibodies, and small molecules have been developed to antagonize CXCR4 [[14], [15], [16], [17]] for anticancer and anti-HIV activity [13,18,19] (see reference [20] for a recent review on targeting CXCR4 for drug development). We have recently developed a cyclotide-based potent antagonist of CXCR4, MCo-CVX-5c [18]. This cyclotide inhibited CXCL12-activation of CXCR4 and HIV infection of Jurkat cells with IC50 values 0.3 nM and 15 nM, respectively [18,21].

Cyclotides are globular microproteins (ranging from 28 to 37 amino acids) stabilized by three disulfide bonds forming a cystine-knot motif and backbone cyclized (Fig. 1) [22,23]. This unique cyclic cystine-knot (CCK) motif confers cyclotides with a rigid molecular framework that is resistant to physical, chemical, and biological degradation [22,23]. Cyclotides can also be produced chemically or using standard heterologous expression systems [[24], [25], [26], [27]]. In addition, cyclotides contain up to 5 hypervariable loops that are amenable to substantial sequence variation and can be modified using molecular grafting and/or evolution techniques to target extracellular and intracellular protein-protein interactions [[28], [29], [30], [31]], making them ideal molecular platforms for the design of novel peptide-based therapeutic leads [32,33].

Despite their significant resistance to biological degradation, cyclotides, can still be efficiently eliminated through renal clearance due to their relatively small size [21,34]. For example, the cyclotide-based CXCR4 antagonist MCo-CVX-5c presents high ex-vivo serum stability to proteolytic degradation (τ1/2 ≈ 60 h) [18] but is rapidly eliminated in mice in <90 min by renal clearance as determined by micro-PET bioimaging [21].

The principle of fatty acid derivatization has been widely used to increase the half-life of biomolecules by facilitating binding to serum albumin [35]. More recently, the use of several fatty acids in combination with a pegylated γ-Glu spacer has been reported to successfully extend the half-life of GLP-1-like peptides such as liraglutide and semaglutide. This groundbreaking and seminal work lead by the researchers at Novo Nordisk, where the effect of peptide lipidation was systematically studied to extend the half-life of GLP-1-based peptides allowed the development of the FDA-approved drugs, liragluide and semaglutide [36].

Here we report the synthesis, lipid conjugation, biological activity, and pharmacokinetic profile in rats, of several lipidated versions of cyclotide MCo-CVX-5c using a pegylated ε-Lys spacer. We have shown that lipidation of this cyclotide exhibited a significant increase in the half-life when compared to the unlipidated form. The half-life extension of the corresponding cyclotide-derivative, however, was strongly dependent on the nature of the lipid, with di-carboxylic fatty acids providing the longer half-life extension when compared to mono-carboxylic fatty acids. The biological activity of the lipidated versions of cyclotide MCo-CVX-5c was also affected by the nature of the lipid used for extending half-life. In this case, derivatization with dicarboxylic fatty acids significantly decreased the biological activity of the modified cyclotide, while the use of monocarboxylic fatty acids had little effect on the activity. The longer pharmacokinetic profiles, together with maintaining biological activity of some of the lipidated cyclotides support the potential of cyclotides as valuable tools for drug development.

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