| Abstract
| - Previous work has shown that incorporation of either 1-aminocyclohexanecarboxylic acid (Ac6c)or α-methyl-p-phosphonophenylalanine ((α-Me)Ppp) in the phosphotyrosyl (pTyr) C-proximalposition (pY + 1 residue) of Grb2 SH2 domain binding peptides confers high affinity. Thetetralin-based (S)-2-amino-6-phosphonotetralin-2-carboxylic acid (Atc(6-PO3H2)) simultaneouslypresents structural features of both (α-Me)Ppp and Ac6c residues. The current study comparesthe affinity of this tetralin hybrid Atc(6-PO3H2) versus Ac6c and (α-Me)Ppp residues whenincorporated into the pY + 1 position of a high-affinity Grb2 SH2 domain binding tripeptideplatform. The highest binding affinity (KD = 14.8 nM) was exhibited by the (α-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-folddecrease in binding affnity (KD = 97.8 nM), which was the result of a further 6-fold increasein off-rate, offset by an approximate 45% increase in on-rate. Therefore, by incorporation ofthe key structural components found in (α-Me)Ppp into the Ac6c residue, the tetralin hybriddoes enhance binding on-rate. However, net binding affinity is decreased due to an associatedincrease in binding off-rate. Alternatively, global conformational constraint of an (α-Me)Ppp-containing peptide by β-macrocyclization did result in pronounced elevation of binding affinity,which was achieved primarily through a decrease in the binding off-rate. Mathematical fittingusing 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 modelthat assumed two binding sites resulted in a very close fit of data and provided KD values of97 pM and 72 nM for the separate sites, respectively. Therefore, although local conformationalconstraint in the pY + 1 residue proved to be deleterious, global conformational constraintthrough β-macrocyclization achieved higher affinity. Similar β-macrocyclization may potentiallybe extended to SH2 domain systems other than Grb2, where bend geometries are required.
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