Grafting Bioactive Functionalities onto Scaffolds Protein-protein Interaction-inhibiting b-Peptides

Protein p53 is a transcriptional activator critical for stress-induced cell cycle arrest and apoptosis [222]. The overexpression of hDM2 in cancer cells results in unchecked cell growth [223], and is therefore an important target for anticancer therapy. The protein hDM2 inhibits p53 transcriptional activity by binding the p53 transactivation domain [224], exporting p53 from the nucleus [225], resulting in ubiquitination of p53 and subsequent degradation [226, 227]. The binding interface between p53 and hDM2 is well characterized. Three hydrophobic residues of the a-helical activation domain of p53 (F19, W23, L26) are key for hDM2 binding [228, 229]. a-Peptide inhibitors of hDM2 based on the a-helical activation domain of p53 have been reported to induce apoptosis in tumor cells overexpressing hDM2 in vivo [230].

The protein-protein interaction hDM2-p53 has been targeted by b-peptides [40-44]. Recapitulation of the three key hydrophobic side chains one turn apart

on a b-peptide 14-helix scaffold as shown to have good overlap with the p53 activation domain bioactive side chains as determined by computational methods (Fig. 8.17) [40]. The designed b-peptide, b53-1, adopted a 14-helix structure as determined by CD and NMR [40]. Fluorescence polarization studies showed that b53-1 bound directly to hDM2 with submicromolar affinity (KD = 368-583 nM), only 1.6-2.5-fold lower in affinity than an a-peptide analog of the p53 activation domain. b53-1 also competed with a p53-derived peptide for the hDM2 binding site, displacing the peptide with an IC50 of 94.5 G 4.4 mM [40]. b53-1 variants which displayed altered orientations ofb3-hPhe, b3-hTrp, and b3-hLeu side chains demonstrated the importance of these residues for binding affinity and specificity [40].

Combinatorial libraries of b-peptides have also been created to screen antagonists of the p53-hDM2 interaction [43, 44]. An efficient synthesis of a 14-helical b-peptide library on polystyrene macrobeads has been achieved using microwave irradiation, yielding an inhibitor containing cyclically-constrained residues with an IC50 of 250 mM [44]. Another library that was created using modified peptide synthesis solid phase protocols resulted in a b-peptide with an inhibitory potency nearly 8-fold greater than b53-1 [43].

14-Helical inhibitors have also been designed to target the N-terminal region of GP-41, a protein implicit to HIV viral fusion to host cell membranes [45]. Three residues comprising the WWI domain of the GP41 C terminus (Trp628, Trp631, and Ile635) were presented on short 14-helical scaffolds consisting of b3-amino acids (Fig. 8.18). Fluorescence polarization studies demonstrated four inhibitor designs that bound to a GP-41 based model peptide system with micromolar affinity (0.75-2.4 mM), which is comparable to the highest affinity a-peptide of similar size (1.2 mM). The inhibition was also specific for targeting GP41 over carbonic anhydrase II and calmodulin, two proteins that interact with hydrophobic helices. Inhibitor designs were shown to inhibit cell fusion in vivo, with EC50 values in the micromolar region (5.3-27 mM).

Human cytomegalvirus (HCMV) viral fusion and cell entry is another therapeutic target [231-233] that has been inhibited by b-peptides [49]. HCMV requires interaction between the helical components of two proteoglycans, gB and gH, which presumably bind one another through a coiled-coil [234]. 12-Helical b-peptides were designed to display a series of nonpolar side chains with a spacing similar to coiled coils to mimic the gB heptad and bind gH. Cell-based assays

showed that the b-peptide induced inhibition of HCMV with greater potency than the a-peptide model (IC50 = 30 mM) [49]. Protein-protein Interaction-inhibiting Peptoids

Using structure-based design, several peptoids were designed to target hDM2 [235]. Three peptoids designed to form a right-handed type-I polyproline helical conformation were found to weakly bind the N-terminal domain fragment of hDM2 (Fig. 8.19) [235]. Synthetic modifications to the side chains to enhance water solubility and improve hydrophobic and nonhydrophobic contacts resulted in a peptoid decamer with improved binding affinity that competed against a peptide analog of the N-terminal region of p53. The IC50 for the peptoid was only approximately 2-fold higher than the p53 peptide (6.6 mM versus 3 mM). However, hydrophobic peptoid side chains that mimicked the Phe, Trp, and Leu of p53 may not be limited to binding in the nonpolar cleft of hDM2 based on computer generated models [235]. Interestingly, achiral peptoid designs were found to bind hDM2 with higher affinity than helical peptoid designs, indicating that the helical sense may not be important for binding hDM2.

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