Peptide stability assay

In Vivo Peptide Stability Assay/Pharmacokinetics in Mice... 

Synthetic peptides often lack the conformational stability required for a successful drug therefore determination of peptide stability in serum constitutes a powerful and important screening assay for the elimination of unstable peptides in the pipeline of drug development (see Note 1). Peptide stability in serum can rather easily be determined by reverse phase-high-performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS) from both in vitro and in vivo studies. 

In a general serum stability assay, the peptide is subjected to human serum (see Note 3) at realistic temperature conditions and incubated for various time intervals, comparable to traditional protease assays. The reactions are stopped by TCA or ethanol, precipitating larger serum proteins while leaving peptides... 

Early SAR investigations revealed that the amide of Met-enkephalin was several times as active as the parent with a longer duration of action and that replacement of Gly2 by D-Ala had a similar influence on peptide stability/62,63 It was later shown that a variety of D-amino acids in place of Gly2 caused a marked increase in potency both in GPI and MVD assays, results presumed... 

Table 11.2. Ranking of amino acids according to results from stabilization assays of 0/X7 libraries on MHC H-2K1 molecules (31). Amino acids O are classified for Iheir influence on stabilization (destabilizing upper part (a) stabilizing lower part (b)). Motif amino acids identified by sequence analysis of natural peptide libraries [33] are underlined.

Strickland E, Hakala K, Thomas PJ, DeMartino GN (2000) Recognition of misfolding proteins by PA700, the regulatory subcomplex of the 26S proteasome. J Biol Chem 275 5565-5572 Stuber G, Dillner J, Modrow S, Wolf H, Szekely L, Klein G, Klein E (1995) HLA-A0201 and HLA-B7 binding peptides in the EBV-encoded EBNA-1, EBNA-2 and BZLF-1 proteins detected in the MHC class I stabilization assay. Low proportion of binding motifs for several HLA class I alleles in EBNA-1. Int Immunol 7 653-663... 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

The hydrolysis of peptide bonds catalyzed by the serine proteases has been the reaction most extensively studied by low-temperature trapping experiments. The reasons for this preference are the ease of availability of substrates and purified enzymes, the stability of the proteins to extremes of pH, temperature, and organic solvent, and the existence of a well-characterized covalent acyl-enzyme intermediate. Both amides and esters are substrates for the serine proteases, and a number of chromo-phoric substrates have been synthesized to simplify assay by spectrophotometric techniques. 

Today peptide synthesis is a mainstream operation in many big laboratories. Several companies have in addition specialized in providing custom-ordered peptides, e.g., large-scale peptides (made in solution), peptide libraries (made on cellulose membranes), or most commonly small peptide quantities (made on resin, solid phase synthesis). Although peptides are considered rather stabile in many test assays, concerns have been raised regarding their stability in presence of human serum. 

While this approach has limitations related to the peptidic nature of the linker such as stability to harsh reaction conditions and requirement of specific functional groups to be coupled with the linker arms, its application to particular libraries and chemistries may be useful. Its biological utility was assessed in a bead-based antimicrobial assay on bacterial cells [54], which produced good correlations between the MIC (minimal inhibitory concentration) values for the compounds released in situ in the culture medium from the beads or tested as standard solutions. 

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