Figure 8.6. Molecular building blocks of the /3-lactam antibiotic cephalexin. The three amino acids phenylalanine, valine, and cysteine are shown to merge through (pseudo)peptide bonds, to form a peptidomimetic compound with the molecular features of a tripeptide.

ers (212), is well conserved between mammalian and C. elegans peptide transporters CPTA and CPTB, but is not specific to or well conserved in other members of the POT super-family. Structure-Transport Relationship of PepT1. Structural information on PepTl has been limited to its primary sequence and predicted structural membrane topology. Hydropathy analysis of the human, rabbit, and rat PepTl isoforms have predicted the presence of 12 transmembrane domains (TMD) in each isoform (212). This model has been partially proved by other investigators (144,213).

The human PepTl sequence has been predicted to contain a 2127 base-pair (bp) open reading frame that encodes a 708 amino acid, 79-kDa protein, with an estimated pI of 8.58 (212). Hydropathic analysis also predicts that the N- and C-termini are located on the cyto-plasmic side of the membrane. Site-directed mutagenesis, using EYMPME (EE)-epitope tag insertion at different locations of the human PepTl transmembrane regions have identified the COOH-terminal to be intracellular, whereas epitope tags at positions 106 and 412 showed that the predicted loops between 3TM and 4TM. and 9TM and 10TM, respectively, were extracellularly localized

(144).These results support the predicted loop and TMD numbers and orientations from TMD4 through the C-terminus (144). The results using EE epitope insertions in the amino terminal region were inconclusive, possibly because of EE effects on function, leaving ambiguity to the predicted structure from the N-terminus to TMD3.

Because no three-dimensional structure of the transporter is available, design of new substrates for PepTl has relied on indirect structure-affinity relationships. Early studies were directed to define a pharmacophore pattern for this transporter (214-216). A pharma-cophore or "recognition site" is defined based on a common arrangement of essential atoms or groups of atoms appearing in each active molecule. In many published studies the active analog approach (AAA), originally developed by Marshall and coworkers (217), has proved to be useful in rationalizing and predicting pharmacological data of active and inactive substrates. In the AAA, the structural requirements common to a set of compounds showing affinity for the same transporter may be used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space, which are necessary for binding to this transporter. Two factors have to be considered for the successful application of this technique: the included compounds should be structurally homogeneous and the individual compounds should have a low level of conformational flexibility. Because the natural substrates, di- and tripeptides, contain many freely rotatable bonds, most studies have concentrated on substrates with more rigid backbones, such as a j3-lactam nucleus (214, 215, 218, 219). Therefore, the AAA is limited, in that it requires structurally similar rigid molecules. One study used 0-lactams and showed that a carboxylic carbon (likely to position in a positively charged pocket), two carbonyl oxygen atoms (hydrogen bond acceptors), a hy-drophobic site, and finally an amine nitrogen atom (hydrogen bonding region) were important features of substrates (218).

Another study examined the three-dimensional structural features of three structurally closely related PepTl substrates: enalapril, enalaprilat, and lisinopril (Fig. 8.7) (220). Enalapril is an ester prodrug of the pharmacologically active enalaprilat. After oral administration of enalapril, the active compound (enalaprilat) is formed by bioconversion of enalapril. Enalaprilat, a diacid, binds slowly and tightly to ACE, producing well-defined clinical effects, but is poorly absorbed from the gastrointestinal tract (3-12% bioavailability) (221). The prodrug approach of esterifying enalaprilat to enalapril is required to enhance the oral bioavailability to 60-70%. By use of a combined in vitro and molecular modeling approach, we showed that intramolecular hydrogen bond formation between the lysyl side chain in enalaprilat and its carboxylic acid groups may explain decreased affinity for PepTl (220).

Structural studies of PepTl substrates were extended using comparative molecular field analysis (CoMFA) of 10 known substrates for the peptide transporter with data derived from an in situ rat model (222). The CoMFA approach required manual alignment of relatively rigid molecules; however, it allowed explanation of the variation in the permeability with respect to steric and electrostatic interaction energies of the molecules (222).This 3D-QSAR produced by our laboratory provided a valuable starting point for future prediction of




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