Reply to Alarcon and Borroto: Small molecule AX-024 reduces T cell proliferation independently of CD3ε-Nck1 interaction at SH3.1

We thank Alarcon and Borroto for commenting on our work (1, 2). Modulation of TCR signaling by AX-024 was shown previously by ZAP70 phosphorylation data (1, 3, 4). We deliberately used low concentrations of anti-CD3 to weakly stimulate primary human T cells but could not reproduce the published effect of AX-024 on ZAP70 phosphorylation, possibly due to technical differences. In line with this finding, we observed that AX-024 does not impede the CD3ε/Nck1-SH3.1 interaction as measured by SPR and NMR (2). In Ref. 3, the response unit scale of the SPR data suggests very low protein-binding activity. In our opinion, the observed lag phase during association and the incomplete signal return to baseline upon dissociation preclude extraction of kinetic and thermodynamic parameters of CD3ε peptide binding to Nck1-SH3.1. Our SPR and ligand-observed NMR studies did not reveal interaction of AX-024 with Nck1-SH3.1 (based on resonances, line widths, and chemical shifts). We noted that the average changes in ¹H and ¹⁵N NMR chemical shifts at millimolar AX-024 concentrations are markedly smaller compared with the chemical shift perturbations observed with CD3ε in Ref. 3. In addition, we could not recapitulate the binding mode of AX-024 to Nck1-SH3.1 proposed in Ref. 3. Together with the tendency for AX-024 to bind to other proteins (2), the mode of action of AX-024 remains to be established. Thus, publication of the announced structure-activity relationship (SAR)-enabling co-crystal structure of AX-024 bound to Nck will generate valuable insights.