Limitations of Peptide Retro-inverso Isomerization in Molecular Mimicry*

A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an α-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288–16289) made a surprising finding that the retro-inverso isomer of p53(15–29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the d-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15–29) binding to MDM2 or MDMX by 3.2–3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that d-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level.

Protein-protein interactions govern a great variety of biological processes and present important targets for therapeutic intervention (1,2). Small peptides emulating the activity of one binding partner to antagonize the other play instrumental roles in drug screening and design. Despite their ability to bind proteins with high affinity and unsurpassed specificity, peptides themselves are rarely used as therapeutic agents due primarily to their poor in vivo stability. Even for in vitro applications, efficacy often necessitates peptide resistance to proteolytic degradation. To tackle peptide susceptibility to proteolysis, various peptidomimetic chemistries have been developed, involving the use of D-amino acids, unnatural amino acids, peptide backbone modifications, cyclizations, and secondary structure-inducing templates, among others (3). Peptide retro-inverso isomerization, pioneered by Chorev and Goodman (4), represents an elegant solution to functional peptides stable under physiological conditions.
It is postulated that a retro-inverso peptide (a peptide of the reversed sequence made up of D-amino acids, also known as a retro-all-D-or retro-enantio-peptide) assumes a side chain topology, in its extended conformation, similar to that of its native L-sequence, thus emulating biological activities of the parent molecule while fully resistant to proteolytic degradation (4). Some success has been achieved immunologically in using retro-inverso peptides toward antigenic mimicry of their parent L-peptides (5). Failures, however, have also been noted for retro-inverso isomers to elicit antibodies that cross-react with native immune epitopes (6). In fact, retro-inverso peptides are not isofunctional to their parent L-peptide molecules with respect to binding energetics even in some successful immunological applications of antigenic mimicry (7). This mixed outcome comes as no surprise, because antibody-antigen recognition is notoriously lenient at the structural level to tolerate conformational plasticity (8).
These conceptually conflicting reports motivated us to carry out a comprehensive study of functional effects of peptide retro-inverso isomerization on the p53-MDM2 interaction, using a combination of biochemical, biophysical, and structural tools. The interactions of p53 (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) with MDMX (a MDM2 homolog) and of PMI (a high affinity, dual specific peptide ligand of MDM2 and MDMX) with MDM2 were also subjected to investigation. To determine whether or not conclusions from the p53/PMI-MDM2/MDMX interactions are applicable to others, we expanded our study to include two additional, well characterized peptide-protein interacting systems: the C-terminal domain of HIV-1 2 capsid protein ( C CA) and the Src homology 3 (SH3) domain of Abl tyrosine kinase with their respective peptide ligands.

MATERIALS AND METHODS
Chemical Synthesis of Peptides and Proteins-Total chemical synthesis of highly pure and correctly folded p53-binding domains of MDM2 (residues 25-109, referred to hereafter as syn MDM2) and MDMX (residues 24 -108 or syn MDMX) has been described elsewhere (15). Abl SH3 domain was prepared as described (16). Folding of Abl SH3 domain was initiated by dissolving the polypeptide at 3 mg/ml in 0.2 M phosphate buffer containing 6 M guanidine HCl and 1 mM dithiothreitol, pH 7.5, followed by extensive dialysis (molecular weight cut-off 3000) in water at 4°C. HIV C CA (S 146 PTSILDIRQG 156 PKEPFRDYV-D 166 RFYKTLRAEQ 176 ASQEVKNWMT 186 ETLLVQNANP-196 DCKTILKALG 206 PGATLEEMMT 216 ACQGVGGPGH 226 -KARVL) was made using native chemical ligation (17,18). The two peptide fragments of C CA, H 2 N-CA(146 -197)␣COSR (where R represents CH 2 CO-Leu-OH) and H 2 N-CA(198 -231)COOH, were individually synthesized on t-butoxycarbonyl-Leu-OCH 2 -PAM resin using an optimized HBTU activation/DIEA in situ neutralization protocol developed by Kent and co-workers (19). Crude peptides, after hydrogen fluoride cleavage and deprotection in the presence of 5% p-cresol at 0°C, were precipitated with cold ether and purified by preparative C18 reversed-phase HPLC, and their molecular masses were ascertained by electrospray ionization mass spectrometry. The determined molecular mass of 9518.7 Da of the full-length ligation product agreed with the expected value of 9519.0 Da calculated on the basis of the average isotopic compositions of C CA. Spontaneous folding of C CA and formation of the disulfide bond between Cys 198 and Cys 218 was achieved through air oxidation in aqueous buffer. The determined molecular mass of 9517.1 Ϯ 0.6 Da of oxidized C CA, differing by 2 mass units from the calculated value of 9519.0 Da of reduced C CA, confirmed the disulfide formation.
The binding affinity of Y4W-P40 and RI-Y4W-P40 for Abl SH3 domain was determined essentially as described (16). Briefly, different concentrations of Y4W-P40 (0 -20 M) or RI-Y4W-P40 (0 -200 M) were incubated with SH3 (5 M for Y4W-P40 and 15 M for RI-Y4W-P40) for 15 min in 5 mM phosphate buffer, pH 7.0, and ligand-induced changes of Trp fluorescence of SH3 were measured at 350 nm in a cuvette. The K d values were obtained using a four-parameter non-linear regression analysis as previously described (16).
Inhibition ELISA-GST-MDM2(1-150) and His 6 -p53 were expressed in Escherichia coli and purified by binding to glutathione-agarose and Ni 2ϩ -nitrilotriacetic acid beads under nondenaturing conditions. ELISA plates were incubated with 2.5 g/ml His 6 -p53 in PBS for 16 h. After washing with PBS plus 0.1% Tween 20 (PBST), the plates were blocked with PBS plus 5% nonfat dry milk plus 0.1% Tween 20 (PBSMT) for 30 min. GST-MDM2(1-150) (5 g/ml) was mixed with compounds in PBSMT plus 10% glycerol plus 10 mM dithiothreitol and added to the wells. The plates were washed with PBST after incubation for 1 h at room temperature and incubation with MDM2 antibody 5B10 in PBSMT for 1 h, followed by washing and incubation with horseradish peroxidase-rabbit anti-mouse Ig antibody for 1 h. The plates were developed by incubation with TMB peroxidase substrate (KPL) and measured by absorbance at 450 nm.
Crystal Structure Determination of Oxidized C CA-Crystals were grown in 24-well plates at room temperature using the hanging drop, vapor diffusion method. 1 l of 10 mg/ml C CA in water was mixed with an equal volume of crystallization solution and equilibrated against 800 l of mother liquor consisting of 0.1 M HEPES, pH 7.5, 0.8 M sodium phosphate monobasic monohydrate, and 0.8 M potassium phosphate monobasic. Crystals were soaked briefly in reservoir solution plus 30% (v/v) glycerol as cryoprotectant and subsequently flash-frozen in liquid nitrogen.
X-ray diffraction data were collected using a rotating anode x-ray generator Rigaku-MSC Micromax 7 and a Raxis-4ϩϩ image plate detector (at the X-ray Crystallography Core Facility, University of Maryland, Baltimore, MD) and were integrated and scaled with the HKL2000 package (27). The structure was solved by the molecular replacement method with the program Phaser from the CCP4 suite (28), based on the Protein Data Bank entry 1A43 model (29), and refined with Refmac and coupled with manual refitting and rebuilding with COOT (30,31). Data collection and refinement statistics are summarized in supplemental Table S1. Molecular graphics were generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).

Retro-inverso Isomerization Diminishes CAI Binding to the C-terminal Domain of HIV-1 Capsid Protein by 3.0 kcal/mol-
HIV capsid protein (CA) assembles into a cone-shaped core structure encasing the viral RNA (42). The C-terminal onethird of CA ( C CA) adopts a four-helix bundle conformation and dimerizes in solution through hydrophobic packing of its second ␣-helix (29,43). C CA mediates viral assembly and maturation (44) and is a significant yet largely unexploited antiviral target. Sticht et al. (45) identified via phage display a duodecimal peptide inhibitor, termed CAI (ITFEDLLDYYGP), that inhibited assembly of immature-and mature-like particles in vitro. CAI bound at micromolar affinity as an amphipathic ␣-helix to a conserved hydrophobic groove of C CA, forming a compact five-helix bundle with altered dimeric interactions (46).
We chemically synthesized C CA via native chemical ligation and determined its crystal structure at 2.05 Å resolution. As expected, dimerization of synthetic C CA is mediated primarily by hydrophobic interactions between the second ␣-helices (Fig. 4A). Comparative structural analysis indicates that the synthetic C CA dimer is nearly identical to the recombinant CA(146 -231) dimer (root mean square deviation(C␣) ϭ 0.47 Å) (29); the latter fits well as a dimeric unit into the cryoelectron microscopy map of the full-length HIV CA capsid lattice (47). To quantify interactions of CAI and RI-CAI with C CA, we immobilized the synthetic protein on a CM5 biosensor chip and obtained steady-state binding kinetics of the two peptides at different concentrations. A non-linear regression analysis yielded K d values of 13.6 M and 2.2 mM for CAI and RI-CAI, respectively (Fig. 4B). Thus, retro-inverso isomerization of CAI decreased its binding affinity for C CA by 162fold or 3.0 kcal/mol.
Retro-inverso Isomerization Diminishes Y4W-P40 Binding to Abl SH3 Domain by 3.4 kcal/mol-SH3 domains are small eukaryotic protein modules of ϳ60 amino acid residues that intramolecularly regulate the activity of Src family tyrosine kinases and, more generally, target their parent protein molecules to cellular sites of their recognition partners (48,49). Selectively interfering with SH3-dependent signaling events with peptide ligands is of great interest in biology (50,51). Most SH3 domains recognize proline-rich peptides that adopt an extended, left-handed polyproline II helical conformation in the complexes (52,53). The Serrano laboratory previously reported a micromolar affinity decapeptide ligand, termed P40 (APTYSPPPPP), of the SH3 domain of Abl tyrosine kinase (54). We subsequently improved the binding affinity of P40 for Abl SH3 domain through a Y4W mutation, which was identified by affinity panning and mass spectrometric decoding of targeted synthetic peptide libraries (16).
For this study, we measured the binding affinity of Y4W-P40 and its retro-inverso isomer RI-Y4W-P40 for Abl SH3 domain using a previously detailed Trp fluorescence titration method (16). As shown in Fig. 4C, Y4W-P40 bound to Abl SH3 domain at an affinity of 0.79 M, contrasting with a K d value of 262 M of RI-Y4W-P40 for the same protein under identical conditions. Therefore, retro-inverso isomerization weakened Y4W-P40 binding to Abl SH3 domain by 332-fold or 3.4 kcal/mol.  (59). We used a reverse retro-inverso strategy, converting D PMI-␤ to its retro-all-L-isomer (RLLKEFNAYWAT-NH 2 ). Quantification of the binding of RI-D PMI-␤ to syn MDM2 by competition SPR resulted in a K d value of greater than 1 mM, at least a 25000-fold decrease in binding affinity (supplemental Fig. S2).

DISCUSSION
The tumor suppressor protein p53 transcriptionally regulates growth inhibitory and apoptotic responses to prevent stressed cells from proliferating and passing mutations on to the next generation (41,60,61). Dubbed the "guardian of the genome," p53 is critical for maintaining genetic stability and preventing tumor development (62). Not surprisingly, in 50% of human cancers, p53 is either deleted or carries missense mutations primarily in its DNA-binding domain. In many other tumors harboring wild type p53, the oncoprotein MDM2 and its homolog MDMX negatively regulate the activity and stability of the tumor suppressor protein (41,60), resulting directly in p53 inactivation and malignant progression (41,60). It has been validated both in vitro and in vivo that MDM2/MDMX antagonists disrupt the p53-MDM2/MDMX interactions and selectively kill tumor cells by reactivating the p53 pathway (63,64). Due to their high potency and specificity (thus low toxicity), peptides and/or peptidomimetics capable of antagonizing MDM2 to activate p53 are of potential therapeutic value (65). D-Peptide antagonists are particularly attractive because they are fully resistant to proteolytic degradation in vivo, thereby ensuing maximal bioavailability and optimal therapeutic efficacy.
Retro-inverso peptides as antigenic mimicry of their parent L-peptides succeed in some cases yet fail in others (5,6). By contrast, the retro-inverso strategy has garnered a much less impressive track record in mimicking small, biologically active peptides that become helical upon target binding (9,10,66). For these reasons, the report by Sakurai et al. (11) that retro-inverso p53 (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) retains the same biological activity as its parent L-form is highly unusual and somewhat provocative. Obviously, if the retro-inverso strategy works for the p53-MDM2 interacting system, protease-resistant D-peptide activators of p53 can be readily designed through enantiomeric conversion of high affinity L-peptide ligands of MDM2 selected from phage-displayed peptide libraries (15,38). More importantly, this simple but powerful approach, if borne out, will have a far reaching impact on the design of potent and stable D-peptide inhibitors for a great variety of therapeutic applications.
Unfortunately, we completely failed on multiple accounts to replicate the finding by Sakurai et al. (11) after subjecting four peptide-protein interacting systems to painstaking scrutiny  (29). Disulfide bonds are displayed as ball-and-stick representations. The overall structures of dimers are very similar, with the root mean square deviation between equivalent C␣ atoms of 0.47 Å. Dimerization is primarily mediated by hydrophobic interactions between the ␣2 helices in an anti-parallel fashion, which buries 1,500 -1,800 Å 2 of the surface area. B, SPR quantification of direct binding of CAI and RI-CAI to immobilized synthetic HIV C CA (500 RUs) based on steady-state kinetic assays. C, fluorescence titration of the synthetic Abl SH3 domain by Y4W-P40 and RI-Y4W-P40 in 5 mM phosphate buffer, pH 7.0. Progressive subtractions of the background signal contributed by the Trp-containing peptides were carried out as described (16).
using a battery of biochemical, biophysical, and structural tools. Compelling evidence from the multifaceted experiments allows us to conclude that retro-inverso isomers are not isofunctional to their parent L-peptides with respect to target protein binding. As has been shown repeatedly in this work, the energetic penalty of peptide retro-inverso isomerization amounts to 3.0 -4.9 kcal/mol in binding free energy. The lack of general success with the retro-inverso strategy in molecular mimicry is attributed to the fact that a retro-inverso peptide, with inverted amide peptide bonds, cannot possibly be equivalent to its parent sequence at the secondary and tertiary structure levels, despite similar side chain topologies at the primary structure level (66).