Conformation of the Pharmacophore

Most peptides are highly flexible in a conformational manner in aqueous solution, but upon interacting with another biologically relevant molecule they adapt a preferred conformation. Thus, the reduction of conformational freedom may eventually lead to insights regarding the receptor/ acceptor-bound conformation, and can also result in selective interaction of a ligand with a receptor. Conformationally constrained peptides can provide crucial information about biologically active conformations. A major goal of using conformational constraints is to determine which pep-tide conformation is required for binding to the receptor. Conformational constraint of flexible bioactive peptides can significantly improve potency, selectivity, stability, and bioavailability compared with endogenous peptides. The determination of a biologically active conformation of peptide is a tedious process. However, general strategies have been developed and tested in many laboratories. In many cases, the relevant conformation would be one from the major low energy secondary structures such as a-helix, P-sheet, a reverse (P-turns or y-turns) or extended structures.

Turns are important conformational motifs of peptides and proteins, besides a-helix and P-sheets. Reverse turns contain a diverse group of structures with well-defined three-dimensional (3D) orientation of amino acid side chains. Turns represent the most important subgroup. A P-turn is formed from four amino acids and is stabilized by a hydrogen bond between the carbonyl group of the first amino acid residue and the amino group of the fourth amino acid residue (Figure 8.3).

The correlation of biological activity with peptide conformation provides useful information about the best fit to the corresponding finding region of the receptor. Once the primary structure of

nh o

FIGURE 8.3 General structure of a ß-turn.

a biologically active peptide has been identified, the next step is to determine the key amino acid residues that are involved in the interaction with a receptor/acceptor via alanine scan, d-amino acid scan, etc., and evaluation of the truncated sequences of the biologically active peptide as described. Once the minimal peptide sequence and key amino acid residues have been determined, the next step involves further modification of peptide conformation. There are two general strategies to constrain the peptide conformations. The flexibility of a peptide chain can be restricted either by global or local constraints.

(1) Local conformational constraints. Local conformational constraints can often provide important insights into the structural basis of agonist, antagonist, and inverse-agonist biological activity. The most informative local conformational constraints are those that constrain the backbone y, and œ torsional angles. Local constraints can be achieved by introduction of unnatural a- or P-substituted amino acids, or modification of amino acid side chains, and/or modification of the peptide backbone.

(2) The modification of amino acid side chains. If the conformational flexibility of the side chain groups of key pharmacophore are restricted to varying degrees in a bioactive peptide, important insights into their biologically active 3D topography can be obtained. Usually, the side chain conformation can be controlled in several ways. One general approach is to introduce an alkyl group at the P-position or on the 3' and/or 5' position of the aromatic ring of an aromatic amino acid residue. These kinds of modifications can constrain x1 and X2 angles; on the other hand, they generally do not perturb the backbone conformation drastically, and still allow the peptides to have some degree of flexibility. In a similar manner, substitution on the aromatic ring of an aromatic amino acid in the 3' and/or 5' positions will limit the conformational flexibility of a peptide to varying degrees depending on the nature of substitution. Furthermore, the introduction of alkyl groups, halogens, or other functional groups can enhance the lipophilicity or other chemical properties and thus help the peptide bind to receptors and/or cross membrane barriers. Incorporation of these highly constrained amino acids into peptides and studies of such peptidomimetics have provided a valuable approach to probe the stereochemical requirements of binding pharmacophore for recognition of receptors, and sometimes such changes alone can lead to completely different biological activities. Some examples from the Hruby research group are given in Figure 8.4 as illustration of the many possibilities. Among natural amino acids, proline is unique with a constrained cyclic system and substituted versions of this amino acid can be used as a semirigid template in design of conformationally constrained peptidomimetics.

(3) The modification of the peptide backbone. Another strategy in the design of peptide drugs is the peptide backbone modifications, which generally refer to the isosteric or isoelec-tronic exchange of NHCO units in the peptide chain or introduction of additional groups. Some of the most frequent modifications to the peptide backbone are listed in Figure 8.5.

1co2h nh2

h3c x1co2h nh2

h3c x1co2h h n

nh nh

2 1 CO2H xch3X

2 1 CO2H xch3X

r nh2


FIGURE 8.4 %-Constrained amino acids synthesized in the Hruby group. (From Kazmierski, W.M. et al., J. Org. Chem. 22, 231, 1994; Boteju, L. et al., Tetrahedron 50, 2391, 1994; Xiang, L. et al., Tetrahedron, 6, 83, 1995; Qian, X. et al., Tetrahedron, 51, 1033, 1995; Liao, S. and Hruby, V.J., Tetrahedron Lett, 37, 1563, 1993; Han, Y. et al., Tetrahedron Lett., 38, 5135, 1997; Wang, S. et al., Tetrahedron Lett., 41, 1307, 2000; Qiu, W. et al., Tetrahedron, 56, 2577, 2000.)

The modification to the peptide backbone can also serve to introduce local backbone constraints. For example, N-alkylation restricts the ^ torsional angle but eliminates the hydrogen bonding capability of the amide bond. N-Methyl amino acids have been incorporated into bioactive mimetics of opioid peptides, bradykinin, thyrotropin releasing hormone (THR), angiotensin II, and cholecystokinin (CCK), and many others. a-Methyl (and a-alkyl or a-anyl) substituted amino acids often can induce or stabilize particular turn and helical structures.

(4) Globally constrained peptide conformation. Cyclization of a peptide is another general approach to constrain the conformation by limiting the flexibility of the peptide. In this approach, the amino acid side chain groups and backbone moieties that are unimportant in biological activity are chosen as the sites to construct a cyclic structure. The cyclization can be formed between side chains through different types of bonds, such as disulfides, lactams, and thioethers (Figure 8.6). Other kinds of cyclic constraint are also possible between side chains and C- or N-termini or between side chain and backbone nitrogens (Figure. 8.7). By making an appropriate covalent bond between two groups one can stabilize a particular conformer and get a relatively rigid structure that adopts a particular secondary structure.

Side chain modifications at His, Phe, and Trp

Side chain modifications at His, Phe, and Trp

FIGURE 8.5 The most frequent modifications employed to the peptide backbone.

rSr hn'





Enkephalin mimetic

FIGURE 8.6 Examples of converting biologically active linear peptides to potent cyclic peptides.

hn oh o

hn o ho

Broken bonds indicate an alternate site for cyclization







FIGURE 8.7 Other cyclic constrains.

By restricting the flexibility the number of conformations of linear peptides can be reduced. To reduce these dynamic degrees of freedom, cyclization is an excellent protocol that has revolutionized the discipline of peptide chemistry. Such global modifications within a linear biologically active peptide offer essential advantages such as: (a) increasing agonist and antagonist potency; (b) reducing proteolytic degradation; (c) increasing receptor selectivity; (d) enhancing bioavailability; and (e) providing conformational insight for receptor/acceptor binding and drug design.

Global constraints can be established by introducing a covalent bond between any two given positions along the peptide chain. The classic examples include, lactam bridges, disulfide bonds, or by introduction of spacers like a succinyl [ CO (CH2)2-CO ] moiety. These linkages or bridges can be broadly categorized into four distinct ways: (1) side chain to side chain; (2) side chain to N- or C-terminus; (3) N- to C-terminal; and (4) backbone residue to backbone residue (for instance, N or Ca of the backbone).

A classic and natural example is oxytocin, which has a disulfide bridge that is necessary for its full biological activity. Among synthetic constrained peptides, DPDPE (H-Tyr-c[D-Pen-Gly-Phe-D-Pen]-OH) and MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2) are well-known examples of side chain to side chain cyclized bioactive superagonists toward the 5-opioid and melanocortin receptors-respectively (Figure 8.6). DPDPE, which is derived from the linear enkephlin pharma-cophore, bears the disulfide linkage from the side chains of D-penicillamine. Ligands like DPDPE are of high interest for development of potent and selective ligands that can exhibit high efficacy and facilitate development of drugs toward neuropathic pain. Another example of side chain cyclization is MT-II and SHU9119 (Figure 8.6), which exhibit totally opposite biological profiles toward certain melanocortin receptors. These ligands were the result of a careful structure-activity relationship

(SAR) studies of the linear ligands, starting from the natural a-melanocyte stimulating hormone (a-MSH), which led first to a potent and stable linear peptide NDP-a-MSH and then to the cyclized ligands (Figure 8.6, MT-II and SHU9119). Melanocortin receptors are involved in many critical physiological actions such as pigmentation, feeding behavior, and sexual behavior. As shown in Figure 8.6, MT-II and SHU9119, bear the lactam ring using the side chain groups of lysine and aspartic acid. SHU9119 was derived from superagonist MT-II, by the replacement of the amino acid D-phenylalanine to D-2'-naphthylalanine. This introduction of four carbons (a local modification) on the side chain of the phenyl ring led to SHU9119, a superpotent antagonist at the melanocortin 3 and 4 receptors, which demonstrates the drastic change in biological action that a small change in structure can induce. Cyclic RGD analogues are a class of avP3 antagonists and are of particular interest in human tumor metastasis and in angiogenesis. Kessler's group has demonstrated an excellent example of N- to C-terminal cyclized bioactive peptides. Incorporation of D-amino acids to induce a P-turn structure within the cyclized moiety has resulted in very potent avP3 antagonists.

Examples of side chain to the C-termini are well demonstrated by Schiller's group in exploring opiate receptor selectivity by altering the conformational restriction on the modified enkephalin sequences. The series of cyclic enkephalins that were synthesized exhibited the subtle variation in conformational restriction thus disclosing the conformational space exhibited by the opiate receptors. Many other constraining moieties exist including alkylation, trans-guanidation, acylations, thioether bridges, and metal complexed peptides, are not discussed here (Figure 8.7).

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