Probing SH2-domains using Inhibitor Affinity Purification (IAP)

Abstract

BackgroundMany human diseases are correlated with the dysregulation of signal transduction processes. One of the most important protein interaction domains in the context of signal transduction is the Src homology 2 (SH2) domain that binds phosphotyrosine residues. Hence, appropriate methods for the investigation of SH2 proteins are indispensable in diagnostics and medicinal chemistry. Therefore, an affinity resin for the enrichment of all SH2 proteins in one experiment would be desirable. However, current methods are unable to address all SH2 proteins simultaneously with a single compound or a small array of compounds.ResultsIn order to overcome these limitations for the investigation of this particular protein family in future experiments, a dipeptide-derived probe has been designed, synthesized and evaluated. This probe successfully enriched 22 SH2 proteins from mixed cell lysates which contained 50 SH2 proteins. Further characterization of the SH2 binding properties of the probe using depletion and competition experiments indicated its ability to enrich complexes consisting of SH2 domain bearing regulatory PI3K subunits and catalytic phosphoinositide 3-kinase (PI3K) subunits that have no SH2 domain.ConclusionThe results make this probe a promising starting point for the development of a mixed affinity resin with complete SH2 protein coverage. Moreover, the additional findings render it a valuable tool for the evaluation of PI3K complex interrupting inhibitors.

Affinity Capture Enrichment versus Affinity Depletion: A Comparison of Strategies for Increasing Coverage of Low-Abundant Human Plasma Proteins

“…These proteins include cAMP-dependent protein kinase A proteins [21,22], cyclic nucleotide gated ion channels [23], and guanine nucleotide exchange factors [24]. By contrast, the pTYR based affinity probe is supposed to enrich for phosphor-amino acid binding proteins such as phosphatases and SH-domain-containing proteins [25] The ABA-based probe enriches for trypsin-like proteases including thrombin, kallikrein, the former being a key enzyme in the blood coagulation cascade [4].…”

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

“…Designing novel probes or tags for affinity purification is critical for efficient identification of the interactomes of target proteins. For example, a recent study has synthesized a probe to specifically enrich several Src homology 2 (SH2) domain containing proteins by inhibitor affinity purification approach [ 22 ]. In another example, affinity probes have been designed and validated to isolate the Hsp70 complexes [ 23 ].…”

Recent advances in synthetic and medicinal chemistry of phosphotyrosine and phosphonate-based phosphotyrosine analogues

“…A similar copper-mediated cross coupling of phosphonodifluoromethyl-zinc bromide was employed to make F 2 Pmp-containing building block 8, which was used for the production of an affinity resin to enrich SH2 proteins from cell lysates. 33 To perform the inhibitor affinity 79 This methodology was successfully applied to a synthesis of AcNH-F 2 Pmp-OMe (Fig. 8B).…”

“…Recently, the F 2 pmp scaffold was successfully employed in the discovery of cell-permeable PTP inhibitors [30][31][32] and for affinity purification of SH2 proteins from cell lysates. 33 Targeting of phosphotyrosine-binding domains by small molecules has attracted particular attention over the last decades, and is now seeing a renaissance as a promising approach in drug discovery. 6,9,34 The syntheses of pY containing molecules and pY mimetics are well established and have been previously reviewed.…”