title: Design and synthesis of conformationally constrained Grb2 SH2 domain binding peptides employing alpha-methylphenylalanyl based phosphotyrosyl mimetics.
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.
authors.affiliation: Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.
authors.forename: Shinya
authors.initials: S
authors.lastname: Oishi
authors.forename: Rajeshri G
authors.initials: RG
authors.lastname: Karki
authors.forename: Sang-Uk
authors.initials: SU
authors.lastname: Kang
authors.forename: Xiangzhu
authors.initials: X
authors.lastname: Wang
authors.forename: Karen M
authors.initials: KM
authors.lastname: Worthy
authors.forename: Lakshman K
authors.initials: LK
authors.lastname: Bindu
authors.forename: Marc C
authors.initials: MC
authors.lastname: Nicklaus
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D001665:Binding Sites; D003500:Cyclization; D051380:GRB2 Adaptor Protein; D008958:Models, Molecular; D008968:Molecular Conformation; D018716:Molecular Mimicry; D063065:Organophosphonates / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D010748:Phosphopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry*; D011485:Protein Binding; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013764:Tetrahydronaphthalenes / Q000737:chemistry; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D063065:Organophosphonates; D010748:Phosphopeptides; D013764:Tetrahydronaphthalenes; D019000:Phosphotyrosine; C012436:alpha-methylphenylalanine; D010649:Phenylalanine