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  • Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing alpha-methylphenylalanyl based phosphotyrosyl mimetics.

Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing alpha-methylphenylalanyl based phosphotyrosyl mimetics.

Journal of medicinal chemistry (2005-02-04)
Shinya Oishi, Rajeshri G Karki, Sang-Uk Kang, Xiangzhu Wang, Karen M Worthy, Lakshman K Bindu, Marc C Nicklaus, Robert J Fisher, Terrence R Burke
ABSTRACT

Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c) or alpha-methyl-p-phosphonophenylalanine ((alpha-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximal position (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. The tetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneously presents structural features of both (alpha-Me)Ppp and Ac6c residues. The current study compares the affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (alpha-Me)Ppp residues when incorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptide platform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (alpha-Me)Ppp-containing parent, with the corresponding Ac6c-containing peptide being nearly 2-fold less potent (KD = 23.8 nM). The lower KD value was attributable primarily to a 50% increase in off-rate. Replacement of the Ac6c residue with the tetralin-based hybrid resulted in a further 4-fold decrease in binding affinity (KD = 97.8 nM), which was the result of a further 6-fold increase in off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation of the key structural components found in (alpha-Me)Ppp into the Ac6c residue, the tetralin hybrid does enhance binding on-rate. However, net binding affinity is decreased due to an associated increase in binding off-rate. Alternatively, global conformational constraint of an (alpha-Me)Ppp-containing peptide by beta-macrocyclization did result in pronounced elevation of binding affinity, which was achieved primarily through a decrease in the binding off-rate. Mathematical fitting using a simple model that assumed a single binding site yielded an effective KD of 2.28 nM. However this did not closely approximate the data obtained. Rather, use of a complex model that assumed two binding sites resulted in a very close fit of data and provided KD values of 97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformational constraint in the pY + 1 residue proved to be deleterious, global conformational constraint through beta-macrocyclization achieved higher affinity. Similar beta-macrocyclization may potentially be extended to SH2 domain systems other than Grb2, where bend geometries are required.

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Sigma-Aldrich
α-Methyl-DL-phenylalanine, 98%
Sigma-Aldrich
α-Methyl-D-phenylalanine, ≥98.0% (HPLC)