Chapter 1

The Basics of Peptidomimetics

1.1 Introduction

During the last three decades an important number of biologically active peptides has been discovered and characterized, including hormones, vasoactive peptides and neuropeptides. As a consequence of interaction with their membrane-bound receptors, these bioactive peptides influence cell–cell communication and control a series of vital functions. Thus, they are of great interest in the biomedical field, and the number of native and modified peptides used as therapeutics is ever increasing. Many bioactive peptides have been prepared on a large scale and tested both in pharmacology and the clinic, thus allowing for the development of new therapies for various pathologies.

However, the use of peptides as therapeutics is limited due to several factors, including low metabolic stability towards proteolysis in the gastrointestinal tract, poor absorption after oral ingestion, low diffusion in particular tissue organs (i.e. the central nervous system, CNS), rapid excretion through liver and kidneys and undesired effects due to interaction of flexible peptides with several receptors (Figure 1.1) [1]. In particular, the flexibility of medium-sized polypeptides (<30 amino acids) is due to the multiple conformations that are energetically accessible for each residue constituting the peptide. The flexibility of each residue constituting a peptide is due to two degrees of conformational freedom addressed by N-Cα and Cα-CO rotational bonds and described by ϕ and ψ dihedral angles, respectively, which result in a population of local conformations, all contributing to the overall flexibility of the peptide in dynamically interconverting equilibria in aqueous solution.

Figure 1.1 Conformational flexibility of peptides and their affinity with proteases cause off-target interactions and degradation, respectively, resulting in undesired biological effects, and inactive fragments from proteolytic events. (Reproduced with permission from Reference [1]. Copyright 1993 Wiley-VCH Verlag GmbH & Co. KGaA.)

Besides all these drawbacks, biomedical research is constantly oriented towards the development of new therapeutics based on peptides and proteins, by introducing both structural and functional specific modifications and maintaining the features responsible for biological activity.

These requirements are all matched in the development of peptidomimetics [2, 3]. In this approach, peptides and proteins are considered as tools for the discovery of other classes of compounds.

1.2 Definition and Classification

A peptidomimetic compound may be defined as a substance having a secondary structure, besides other structural features, similar to native peptide, such that it binds to enzymes or receptors with higher affinity than the starting peptide. As an overall result, the native peptide effects are inhibited (antagonist or inhibitor) or increased (agonist). Since their introduction as a new concept for developing drug candidates, peptidomimetics have shown great promise both in organic and medicinal chemistry. Apart from being much more selective and efficient than native peptides, thus resulting in fewer side effects, peptidomimetics show greater oral bioavailability and the biological activity is prolonged due to lowered enzymatic degradation [4, 5]. The generation of peptidomimetics is basically focussed on knowledge of the electronic and conformational features of the native peptide and its receptor or active site of an enzyme. Thus, the development of peptidomimetics as compounds with potential biological activity must take account of some basic principles [6], including:

• Replacement of peptide backbone with a non-peptide framework: if an amide bond substitution does not change the biological activity or amide bonds are not exposed to the active site, then the template may be designed to eliminate peptide bonds.
• Preservation of side-chains involved in biological activity, as they constitute the pharmacophore. In the development of second-generation mimetics, several modifications may be introduced to improve biological activity, including chain length modification, introduction of constraints, cyclopeptide bond replacement with a covalent one and introduction of isosteric replacements [7].
• Maintenance of flexibility in first-generation peptidomimetics: if biological activity is observed for a flexible mimetic, then the introduction of elements of rigidity to side-chains is a rational approach to improve the preliminary activity observed.
• Selection of proper targets based on a pharmacophore hypothesis. In other words, knowledge of the structure–activity relationship or the three-dimensional structure of bioactive conformation is a promising route to rapidly achieve the best compound, without generating a huge number of compounds with poor biological activity.

Peptidomimetics may be divided into three classes depending on their structural and functional characteristics [8]:

• Type I mimetics, or structural mimetics, show an analogy of a local topography with the native substrate, and they carry all the functionalities responsible for the interaction with an enzyme or a receptor in a well-defined spatial orientation.
• Type II mimetics, or functional mimetics: here the analogy with the native compound is based on the interaction with the target receptor or enzyme, without apparent structural analogies.
• Type III mimetics, or functional-structural mimetics, are generally conceived as possessing a scaffold with a structure different from that of the substrate, in which all the functional groups needed for biological interactions are mounted in a well-defined spatial orientation. Many examples have been reported in the literature in which an unnatural framework substitutes the peptide backbone and carries the required functional groups for biological activity.

An elegant example of a peptidomimetic scaffold is given by a thyrotropin-releasing hormone (TRH) mimetic based on a cyclohexane scaffold (1, Figure 1.2), which replaces the peptide backbone, and the three functional groups that constitute the pharmacophore are placed on the scaffold with the same spatial orientation of amino acid side-chains found in TRH hormone [5]. Other examples include replacement of peptidic fragment in somatostatin receptor binding cyclopeptide with a d-glucose scaffold (2) [9], and steroidal scaffold 3 to mimic the type II′ β-turn structure of RGDfV cyclopeptide (Figure 1.2) [10].

Figure 1.2 Peptidomimetic compounds consisting of cyclohexane (1), glucose (2) or steroid (3) scaffolds. (Reproduced with permission from Reference [2]. Copyright 1994 Wiley-VCH Verlag GmbH & Co. KGaA.)

The workflow towards the development of peptidomimetics has been proposed within the drug discovery process in the case of peptide molecules as hit compounds towards an identified target [11].

Accordingly, the first step in a drug discovery process is hit identification; this is generally carried out by scanning peptide libraries for binding affinity (i.e. by phage display or combinatorial chemistry of synthetic peptide libraries). Molecular biology techniques, such as sequencing, cloning and site-directed mutagenesis experiments, are essential to achieve structural information regarding receptor residues responsible for peptide recognition in combination with molecular modelling calculations. Such information is very important in selecting a bioactive peptide to be successively processed in a hierarchical way, also taking advantage of structural data. The hierarchical approach takes advantage of several steps, which are important in giving insight into structure–activity relationships with respect to the starting bioactive peptide hit compound to be converted into a peptidomimetic lead:

• alanine scanning
• size reduction
• d-amino acid scanning
• introduction of local and global constraints to define the bioactive conformation.

The so-obtained first-generation peptidomimetics are then subjected to further conformational studies aimed at defining the rationale for ligand–receptor (or enzyme–inhibitor) key interactions. The results are then applied for the optimization of hit peptidomimetics towards improved compounds possessing a non-peptide framework.

Chapter 2 presents a detailed overview of the design principles and applications to peptidomimetics.

1.3 Strategic Approaches to Peptidomimetic Design

A major effort in peptidomimetic chemistry is connected to the development of compounds capable of replacing one or more amino acids in a peptide sequence without altering the biological activity of the native peptide. The overall result of this structural intervention is to stabilize the molecule with respect to metabolic processes that occur in vivo, thus giving access to orally available drugs and compounds with improved pharmacokinetics/pharmacodynamics (PK/PD) properties.

The development of peptidomimetics has generally been approached by synthesizing novel amino acids possessing several features, including synthetic accessibility from commercially available enantiopure reagents, such as amino acid and sugar derivatives, or straightforward synthetic methods for asymmetric synthesis, to access a wide array of novel compounds. Moreover, the need to achieve partially rigid compounds has been pursued to probe a limited number of conformations with the aim of understanding the...