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SPSB2 mediates the proteasomal degradation of iNOS. Inhibitors of SPSB2-iNOS interaction are expected to prolong iNOS lifetime and thereby enhance killing of persistent pathogens. Here, we describe the synthesis and characterization of two redox-stable cyclized peptides containing the DINNN motif required for SPSB2 binding. Both analogues bind with low nanomolar affinity to the iNOS binding site on SPSB, as determined by SPR and 19F NMR, and efficiently displace full-length iNOS from binding to SPSB2 in macrophage cell lysates. These peptides provide a foundation for future development of redox-stable, potent ligands for SPSB proteins as a potential novel class of anti-infectives. [ABSTRACT FROM AUTHOR]

SPRY domain-containing SOCS box protein 2 (SPSB2) regulates inducible nitric oxide synthase ( iNOS) by targeting it for proteasomal degradation. Inhibiting this interaction prolongs the intracellular lifetime of iNOS, leading in turn to enhanced killing of infectious pathogens such as bacteria and parasites. SPSB2 recognizes a linear motif (DINNN) in the disordered N-terminus of iNOS, and ligands that target the DINNN binding site on SPSB2 are potentially novel anti-infective agents. We have explored 19F NMR as a means of probing ligand binding to SPSB2. All six Trp residues in SPSB2 were replaced with 5-fluorotryptophan (5-F-Trp) by utilizing a Trp auxotroph strain of Escherichia coli. The labeled protein was well folded and bound a DINNN-containing peptide with similar affinity to native SPSB2. Six well-resolved 5-F-Trp resonances were observed in the 19F NMR spectrum and were assigned using site-directed mutagenesis. The 19F resonance of W207 was significantly perturbed upon binding to DINNN-containing peptides. Other resonances were perturbed to a lesser extent although in a way that was sensitive to the composition of the peptide. Analogues of compounds identified in a fragment screen also perturbed the W207 resonance, confirming their binding to the iNOS peptide-binding site on SPSB2. 19F NMR promises to be a valuable approach in developing inhibitors that bind to the DINNN binding site. [ABSTRACT FROM AUTHOR]