Identification of efficient pentapeptide substrates for the tyrosine kinase pp60c-src

Abstract

The development of inhibitors of protein tyrosine kinases (PTKs) is a promising approach to obtaining new therapeutic agents for a variety of diseases, particularly cancer. However, the discovery of peptide-based inhibitors has been hindered by the lack of small peptide substrate sequences (i.e. five residues or less) with which a variety of inhibitor designs could be readily evaluated by replacing the Tyr with natural and unnatural amino acids. These prototypical small peptide inhibitors could then form the basis for designing analogous conformationally constrained, peptide-mimetic or non-peptide inhibitors with improved therapeutic potential. In this study we have identified the best known small peptide substrate for the PTK pp60c-src, which is the parent of the src family of nonreceptor PTKs. This pentapeptide substrate, Ac-Ile-Tyr-Gly-Glu-Phe-NH2, has a Km of 368 microM and Vmax of 1.02 mumol/min/mg when tested utilizing the assay methodology of Budde et al. (Anal. Biochem. 1992, 200, 347-351) after a series of modifications were made to more closely simulate the conditions inside a typical mammalian cell. This substrate was designed from information obtained by Songyang et al. (Nature 1995, 373, 536-539) with a 2.5 billion member combinatorial library of peptide substrates for pp60c-src. A second pentapeptide substrate, Ac-Glu-Asp-Ala-Ile-Tyr-NH2, with a weaker binding affinity (Km = 880 microM) but improved Vmax (1.86 mumol/min/mg), was also identified. This peptide was designed from the pp60c-src autophosphorylation sequence and information obtained by Songyang et al. (Ibid.) and Till et al. (J. Biol. Chem. 1994, 269, 7423-7428) with combinatorial libraries of peptide substrates. These new substrates provide sufficient binding affinities and rates of phosphorylation to be utilized for evaluating the relative effectiveness of various reversible and mechanism-based irreversible inhibitor designs for pp60c-src while appended to easily prepared small peptides.

Catalytic Specificity of Phosphotyrosine Kinases Blk, Lyn, c-Src and Syk as Assessed by Phage Display

“…The related PTK c-Src strongly required glutamic acid in position −3, isoleucine, leucine or valine in position −1, tryptophan or glycine in position +1, and one of the aromatic residues phenylalanine, tryptophan or tyrosine in position +3, The resulting consensus substrate E-X-L/I-Y-W/G-X-F/W-X resembles the findings from the chemical library approach, where the best substrates for both v-Src and c-Src appeared to be -E/D-E-E-I-Y-G-E-F . The K m of the corresponding peptide A-E-E-E-I-Y-G-E-F-E-A-K-K-K was reported to be between 33 and 100 mM Nair et al, 1995) which corresponds well to a K m of 67 mM reported here for the c-Src peptide E-E-P-L-Y-W-S-F-P-A-K-K-K. In agreement with the chemical library approach we observed a strong preference for glutamic acid in position −3, but we rarely observed negatively charged residues in position −2.…”

“…Previous studies on peptidic substrates and homology-based molecular models suggested that about 9 to 13 residues of a peptidic substrate could presumably contact the active site cleft of the kinase domain (Knighton et al, 1991;Bossemeyer et al, 1993), but this range of amino acid may not be fully needed, as pentapeptides have been reported to be modest substrates of PTK c-Src (Nair et al, 1995). Former investigations on substrate specificity of the catalytic kinase domains utilized a limited number of peptidic and non-peptidic substrates mostly derived empirically (Pearson & Kemp, 1991;Cheng et al, 1992;Brunati et al, 1995).…”

Cross‐linked coatings for electrophoretic separations in poly(dimethylsiloxane) microchannels

“…Peptides were synthesized by the Beckman Peptide and Nucleic Acid Facility at Stanford University (Stanford, CA, USA) and labeled with fluorescein as described previously [20]. The peptide sequences were F-src (fluorescein-AEEEIYGEFEAKKKK) [21], F-PKB (fluorescein-GRPRAATFAEG) [22], PF-PKB (fluorescein-GRPRAA(T-PO 3 )FAEG), F-calc (fluorescein-DLDVPIPGRFDRRVSVAAE) [23,24], PF-calc (Fluorescein-DLDVPIPGRFDRRV(S-PO 3 ) VAAE). All peptides were amidated on the carboxy terminus.…”

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

“…The agents that inhibit the enzymatic process of a PTK have been conceptually classified by Burke: (i) inhibitors preventing the binding of substrate; (ii) inhibitors preventing the binding of ATP; and (iii) inhibitors that decrease the catalytic efficiency of the enzyme by some other mechanisms [10]. Based on the motif (EEIYGEFF) discovered by Songyang et al [36], Nair et al [37] have developed a pentapeptide substrate (Ac-IYGEF) for pp60 ...... . Conceptually, pseudosubstrate-based inhibitors that compete at the substrate binding site may have a high degree of selectivity towards PTKs.…”