Improving CTPs

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The affinity and/or avidity of CTPs for their receptor can be improved in different ways. In general, cyclization and multimerization of the peptide have been used to boost the interaction with the target receptor. These aspects are developed in the following sections through one of the best studied examples, the RGD tri-peptide that presents a specific interaction with the integrin receptors. We think that the wealth of research on this peptide is likely to be transferable to any other short peptide showing specific affinity for a given cell receptor.

The RGD peptide: a “scholar study”

The avb3 integrin receptor plays a role in angiogenesis of solid tumors, cell migration, invasion and also metastatic activity (for a review [135]) and it expression depends on the type and stage of the tumor. Therefore, it became rapidly a target of choice for cancer treatment. In the early 80’s, the Arg-Gly-Asp (RGD) sequence, which is present in various circulating proteins, was shown to be responsible for the binding to the avb3 receptor either on its own or included in a penta-peptide. Then, much work was focused on the definition of the requirements needed to produce a ligand with very strong affinity/selectivity towards avb3. This led to the development of a molecule, Cilengitide, which is now in phase II study. Researches are still trying to find more specific ligands to reduce the currently used pharmacologic doses. As explained in a very recent and exhaustive review about the design and chemical synthesis of integrin ligands [118], small changes within the natural sequence of these molecules could deeply improve their activity profiles. For instance, in the c-RGDfV peptide, where f stands for a D-amino acid, each amino acid was replaced by its a-N-methylated form [136]. Depending on the structure-activity refinement of the N-methylation position for instance, the specific binding (IC50) varied from 10^-5M for the c(RGD(NMe)fV) to 10^-8M for the c(RGDf(NMe)V), which represents a gain of more than 1000 folds [118]. This is also illustrated by the replacement of the aspartate (D) residue by glutamate (E) within the RGD peptide. The resulting RGE sequence has an additional methylene group and is used as a negative control peptide for monitoring integrin binding. Another peptide used as a negative control is RAD in which the alanine (A) residue replaces the glycine (G) of RGD.

Cyclization

It is commonly admitted that the interaction between a peptide ligand and its receptor shows higher affinity when the peptide is under a constrained conformation rather than simply linear [137]. Moreover, resistance against proteases is also largely increased upon cyclization [138]. One of the nicest examples to illustrate these aspects is probably again the Arg-Gly-Asp (RGD) tri-peptide. The linear RGD peptide recognizes its target receptor, and has been initially used as a targeting peptide [139]. However, this peptide is inserted in the turn localized at the extremity of a b-sheet within two of the natural ligands of the integrin receptor, fibronectin and vitronectin. The bend of such a turn involves 3 to 4 residues. Therefore, the RGD peptide was rapidly synthesized in a cyclized form in order to mimic its natural environment within the protein. One of the first “cyclization” was performed by bridging two cysteine side chains placed at the two extremities of the peptide [140, 141]. Around the same time, cyclization using chemical closure, mainly through the formation of an amide bond within the N-ter a-amine and the C-ter a-carboxyl functions, was also introduced [142]. Cyclo-peptides with five to six amino acid residues represent the best compromise between flexibility and reactivity to obtain cyclization with good yields. Other types of ring closure have been also performed through various chemical strategies, and peptido-mimetics have been inserted at the place of different native residues. This sometimes leads to molecules apparently far away from the native tri-peptide, in which the charge locations of the ionic side chains (Arg (R) and Asp (D)) have to be placed and oriented accordingly (for recent reviews, see [118, 143].).

Multimerization

The affinity of a dimeric ligand for its receptor is usually higher than that of the corresponding monomeric molecule [144]. In most examples, the increased avidity is the mechanical result of a higher local concentration of ligands because of the direct linkage between two moieties. Since the binding of a ligand to its receptor is a dynamic event, if one molecule becomes detached from the receptor, it is statistically more likely to observe the instantaneous re-attachment of the second half of the dimer to the same receptor molecule. On the other hand, a dimeric ligand could also bind to two adjacent receptors, thus increasing the overall strength of the apparent affinity of the two receptors. It has to be pointed out that initial multimerization of targeting sequences have been made by repeating two or more adjacent recognition sequences [145] and this feature strengthens the hypothesis of a higher local concentration, since it appears unlikely that two adjacent receptors could bind to two sequences so closely linked along the same primary sequence. One might definitely solve this issue by modifying the length of the linker joining the two monomers. The crystal structure of the avb3 receptor in its activated state allowed the observation that the a and b sub-units assemble into an ovoid volume of 60Å to 90Å [146]. Since the avb3 binding site is located in a central cleft between the two subunits, the minimum length of a linker between two ligands should be at least of 60Å to allow binding to two adjacent receptors. In most studies with cyclic divalent RGD peptides, the spacer length between the two ligands was much shorter than the length required for binding two contiguous receptors [147-149]. Moreover, in a recent study, a dimeric RGD with a spacer compatible in length with a putative attachment of the dimer to two adjacent receptors did not have a stronger affinity than a molecule with a shorter spacer length, or the monomer [150]. This study revealed also that dimers with intermediate length spacers (from 29A to 43A) provided the best cell uptake, thus indirectly confirming that binding to two neighbor receptors could not explain the better affinity. In some cases, homo-bivalent ligands can cross-link two adjacent receptors when bound through an optimal linker length which is correlated to the inter-receptor distance [151]. Moreover, the flexibility of the linker can also influence the kinetics of the binding as the presence of flexible PEG moieties within the linker significantly modifies the level of recognition [151]. This data stresses again the plethora of possible modifications within a single molecule.

With the aim to investigate the limit of multimerization, several study further evaluated the affinity of tetra- and octa-meric RGD structures [147, 152-154]. The monomeric c(RGDfV) peptide was grafted on a multi-branched core made of different functional groups. In most of the cases, the ligand exposing scaffold also carried a second type of functional group, able to link another molecular entity ranging from a radionuclide for tumor localization during diagnostic phase, to a drug to promote the specific killing of the targeted cells. For instance, Dumy et al. synthesized a planar cyclodecapeptide scaffold with a face harboring four RGD cyclopeptides, and the other one a fluorescein [154]. However, the ultimate goal of such a strategy is to deliver a cytotoxic molecule into cells which harbor avb3 receptors and preliminary encouraging in vivo experiments have been very recently published [155, 156]. Kessler’s group evaluated the requirement of multimerization to improve the binding affinity of the monomeric RGD moiety [153]. To this aim they used a scaffold made of lysine moieties, that is known under the acronym of MAP (i.e., multiple antigen peptide) and was conceived in the 90’s to expose several antigenic peptides in order to avoid their coupling to a carrier protein [157]. RGD peptides were attached to the MAP structure via PEG chains.

In conclusion, the use of multimeric RGD structures improves the apparent affinity, or more precisely the avidity of the ligand for the integrin receptor compared to the monomeric ligand. The majority of the reported studies limited the investigation to 8 RGD moieties at most, and in general tetra- di- and monomers were used. The efficacy was 4 > 2 > 1 RGD peptide [147], but it has been also described a 2 > 4 > 1 RGD peptide efficiency [158]. These discrepancies suggest that the results depend also on other structural and compositional parameters within the macromolecular complex. Four targeting moieties seem sufficient to increase the binding forces over the monomer. At the same time, this also limits the complexity of the molecule and the potential solubility problems which could be encountered when injecting them in vivo.

In some particular cases the number of targeting peptides could be higher. For instance, liposomes or nanoparticles aimed at delivering drugs into targeted tissues can be coated with up to several hundred of targeting units. Indeed, in this case, the overall surface of the drug-transporter is important to define its degree of functionalization.