Potent and selective inhibition of SH3 domains with dirhodium metalloinhibitors

Abstract

Src-family kinases (SFKs) play important roles in human biology and are key drug targets as well. However, achieving selective inhibition of individual Src-family kinases is challenging due to the high similarity within the protein family. We describe rhodium(ii) conjugates that deliver both potent and selective inhibition of Src-family SH3 domains. Rhodium(ii) conjugates offer dramatic affinity enhancements due to interactions with specific and unique Lewis-basic histidine residues near the SH3 binding interface, allowing predictable, structure-guided inhibition of SH3 targets that are recalcitrant to traditional inhibitors. In one example, a simple metallopeptide binds the Lyn SH3 domain with 6 nM affinity and exhibits functional activation of Lyn kinase under biologically relevant concentrations (EC₅₀ ∼ 200 nM).

Fig. 1. (a and b) Structures of the SH3 domains of representative Src-family kinases, Lyn (PDB ID: 1W1F) and Lck (PDB ID: ; 2IIM) with a peptide ligand (PDB ID: ; 4EIK). Histidine residues in the SH3 domain are shown explicitly. (c) Alignment of core SH3 residues for a variety of human Src-family (1–7) and other SH3 domains, highlighting histidine residues. (d) Affinity of designed rhodium(ii) metallopeptides for three Src-family SH3 domains.

Fig. 2. (a) Structure of the optimal Lyn-binding peptide, S2E^Rh. (b and c) ITC analysis for affinity determination of S2E^Rh (b) and a negative control (c).

Fig. 3. Computational models of S2E^Rh bound to Lyn SH3. (a) QM/MM-optimized structure of Nγ–Nγ isomer of Lyn–S2E^Rh with overlayed native SH3-binding peptide ligand (magenta). (b) Depiction of the histidine-flanked cleft of Lyn where dirhodium binding occurs. (c) Top slice view of an overlay of native Lyn SH3 (yellow) and the Nγ–Nγ isomer of Lyn–S2E^Rh (green). (d) Overview of the QM/MM optimization. High layer (DFT): tube. Low layer (MM): stick. See text and ESI for details.

Fig. 4. ITC analysis of Abl SH3 binding to designed metallopeptides.

Fig. 5. Potent sequestration of a metallopeptide catalyst by Lyn SH3. (left box) In the absence of Lyn, a metallopeptide (R5E^Rh) catalyzes covalent attachment of an alkyne-containing small molecule to the Yes SH3 domain (expressed as a fusion with MBP) in cell lysate, visualized after reaction with a fluorogenic azide. The activity of the rhodium metallopeptide catalyst is inhibited by added Lyn SH3, indicating selective binding in lysate. (right box) Total protein (Ponceau) stain of the lysate reactions. Conditions: MBP–Yes fusion (2 μM), metallopeptide (10 μM), and MBP–Yes fusion (2 μM) in E. coli lysate, diluted 2× with tert-butylhydroxylamine buffer at pH 6.2 at 4 °C.

Fig. 6. Activation of Lyn kinase activity by a metallopeptide, S2E^Rh, and the parent peptide, S2E. The negative control, Rh₂(OAc)₄, had no effect on kinase activity. Full-length Lyn kinase (74 nM) was treated with substrate peptide and ATP. Kinase activity was measured after 5 min in an adaptation of reported methods. See ESI for details.