TI - High-affinity Src-SH2 ligands which do not activate Tyr(527) -phosphorylated Src in an experimental in vivo system . AB - The Src-SH2 domain has been determined to play a key role in many signaling pathways , especially in osteoclast-mediated bone resorption . Therefore , non-peptidic small molecules , mimicking the natural pYEEI peptide ligand , have been designed , to inhibit SH2 -mediated protein - protein interactions and provide therapeutic treatment of certain diseases such as osteoporosis . However it has been shown in vitro that phosphopeptidic ligands of the SH2 domain are able to increase Src kinase activity by disrupting the intramolecular interactions between the Tyr(521) -phosphorylated C-terminal tail and the SH2 domain , thereby inducing a change from a "closed" inactive to an "open" active conformation of Src . Thus it was not clear whether non-peptidic ligands would limit their action to the inhibition of the signaling cascade by interfering with the intermolecular SH2 binding , or would activate the enzyme as do phosphopeptides . To address this question we have investigated the effects of a series of both peptidic and non-peptidic ligands of the SH2 domain on Src kinase activation , both in vitro in an ELISA based assay and in vivo using csk and src double transformed Schizosaccharomyces pombe . We found that , in the peptide series , the extent of c-Src activation is directly correlated to the respective binding affinity for Src-SH2 . By contrast such correlation is not valid for non-peptidic ligands , some high-affinity SH2 binders showing no detectable Src activation in vivo . These results have significant implications for the design of SH2 binders , as they allow a way to inhibit Src-SH2 -mediated signal transduction in target cells , without activating Src in non-target cells , thereby reducing the possibility of side effects .