Controlled Peptide Solvation in Portion-Mixing Libraries of FRET Peptides:  Improved Specificity Determination for Dengue 2 Virus NS2B-NS3 Protease and Human Cathepsin S

• Published in: Journal of combinatorial chemistry Vol. 9; no. 4; pp. 627 - 634
• Main Authors: Alves, Fabiana M, Hirata, Izaura Y, Gouvea, Iuri E, Alves, Marcio F. M, Meldal, Morten, Brömme, Dieter, Juliano, Luiz, Juliano, Maria A
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 01.07.2007; Amer Chemical Soc
• Subjects: Amino Acid Sequence; Cathepsins - metabolism; Chemistry; Chemistry, Applied; Chemistry, Medicinal; Chemistry, Multidisciplinary; Cross-Linking Reagents - chemistry; Dengue Virus - enzymology; Dengue virus type 2; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Life Sciences & Biomedicine; Peptide Library; Peptides - chemistry; Peptides - metabolism; Pharmacology & Pharmacy; Physical Sciences; Polyethylene Glycols - chemistry; Science & Technology; Serine Endopeptidases - metabolism; Solutions - chemistry; Substrate Specificity; Viral Nonstructural Proteins - metabolism; SCANNING COMBINATORIAL LIBRARIES; SUBSTRATE-SPECIFICITY; CYSTEINE PROTEASE; VIRUS; ENZYME; COLLAGENASE; PHAGE DISPLAY; OPTIMIZATION; SUBSITE; ENERGY-TRANSFER PEPTIDES
• ISSN: 1520-4766; 1520-4774
• DOI: 10.1021/cc070042k

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the d-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K−R, R−R, or Q−R/K at P2−P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.