Metabolic Stability of Peptidomimetics: N-Methyl and Aza Heptapeptide Analogs of a PKB ⁄ Akt Inhibitor Yftah Tal-Gan 1,  , Noam S. Freeman 1,  , Shoshana Klein 2 , Alexander Levitzki 2 and Chaim Gilon 1, * 1 Institute of Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel 2 Unit of Cellular Signaling, Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel *Corresponding author: Chaim Gilon, gilon@vms.huji.ac.il   Both authors contributed equally to this work. à The abbreviations for amino acids are according to the IUPAC-IUB Commission of Biochemical Nomenclature, http:// www.chem.qmul.ac.uk/iupac/AminoAcid/. Linear peptides suffer from poor pharmacokinetic and pharmacodynamic properties. Peptidomimet- ics are designed to overcome these pharmacologi- cal drawbacks while maintaining the biological effects of the parent peptides. Aza-peptides, in which an alpha carbon is replaced with nitrogen, are promising peptidomimetic analogs; however, little is known about the stability of these analogs toward enzymatic degradation. We performed sys- tematic aza and N-methyl scans of a PKB ⁄ Akt inhibitor, PTR6154. We evaluated the stability of the aza-scan and N-methyl scan libraries toward enzymatic degradation by trypsin ⁄ chymotrypsin. Our results indicate that the modification site is important for metabolic stability and that aza-pep- tides have a more global effect than N-methyla- tion, affecting cleavage sites distant from the modification site. Key words: aza-peptide, chymotrypsin, N-methylation, peptidomi- metics, PKB ⁄ Akt, trypsin Received 6 February 2011, revised 14 July 2011 and accepted for publi- cation 2 August 2011 Abbreviations à : ACN, acetonitrile; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; PKB ⁄ Akt, Protein kinase B; RP-HPLC, reverse-phase high-pressure liquid chromatography; TDW, triple-distilled water; TFA, trifluoroacetic acid; TOF, time of flight. Linear peptides have critical drawbacks as drug candidates; typi- cally, rapid metabolism by proteolysis, nonselective receptor binding and poor bioavailability. Peptidomimetics are designed to retain or enhance the biological effects of natural peptides while, at the same time, overcoming these undesirable properties. Many types of local and global modifications have been developed in attempts to obtain peptidomimetics with improved pharmacological properties (1–4). The local modifications include N a -methylated derivatives and aza-peptides. N a -methylation (Figure 1) is a powerful and common tool for struc- ture–activity relationship studies. It is often used to examine the effects of local backbone modifications and hydrogen bonding on the potency of a known peptide sequence. N-methylation has been shown to improve important pharmacological parameters such as lipophilicity (5), bioavailability (5–10), proteolytic stability (4,6– 9,11,12), conformational rigidity (7,8,13), and duration of action (6,7). N-methylation may also result in enhanced potency (7–9,14– 16), new receptor subtype selectivity (9,12,17–21), and conversion of an agonist into an antagonist (6). N-methylation induces local backbone constraints caused by steric hindrance (6–8,22). However, the reduced number of potential hydrogen bonds formed by the backbone and the lower tendency to exhibit trans conformations may result in subsequent secondary structural changes (23). Hence, a single N-methylation may influence the ability of the peptide to recognize its binding site, as well as its selectivity and its pharma- cological properties (7–9,11,12,16,18–21,24). The synthesis of N-methyl peptides is well established (25–31), and these peptidomimetics often display favorable pharmacokinetic properties. N-methylation is usually one of the first modifications applied when attempting to enhance the proteolytic resistance of a bioactive peptide (4,32–34). There are several examples of system- atic N-methylation scans of active peptides to obtain superior ana- logs that exhibit longer duration of action (5,11,12,35,36). Aza-peptides are peptides in which one (or more) of the a-carbons bearing the side chain residues has been replaced by a nitrogen atom (Figure 1). Aza-amino acid residues impart special conforma- tional properties to the peptide structure, owing to the loss of stere- ogenicity and the reduction in flexibility of the parent linear peptide (37). Insertion of an aza-amino acid residue imparts both local and global effects. Locally, the substitution of a Ca by N results in the potential for additional hydrogen bonding and affects the acidity of the neighboring amide N-H bonds (38). A global reduction in the flex- ibility of aza-peptides results from the generation of hydrazide and urea structural elements and from lone pair repulsion of the adjacent amines. Notably, this reduced flexibility has been shown to induce 887 Chem Biol Drug Des 2011; 78: 887–892 Research Letter ª 2011 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2011.01207.x