The SPRY domain–containing SOCS box (SPSB) proteins (SPSB1, 2 & 4) are negative regulators of inducible nitric oxide synthase (iNOS) in macrophages. Deletion of the Spsb2 gene causes prolonged expression of iNOS, which leads to increased production of nitric oxide (NO) and the enhanced killing of persistent pathogens such as Mycobacterium tuberculosis and Leishmania major.1 This implies that inhibitors of the SPSB2-iNOS interaction have potential as a novel class of anti-infective agents.
Previous studies have demonstrated that the SPSB2-iNOS interaction is mediated by a conserved DINNN motif present in the disordered N-terminal region of iNOS and the SPSB2-SPRY domain.1,2 . Our group has further demonstrated that the linear DINNN peptide binds SPSB2-SPRY domain with 13 nM affinity as part of a longer peptide.3 Two redox stable cystathionine and lactam-bridged analogues inhibitors of SPSB2-iNOS interaction were developed through the macrocyclisation of linear DINNN peptide precursors into cyclic peptides.4 Both analogues bind to human SPSB2 with KD values of 36 and 27 nM, respectively. We have determined the crystal structures of the lactam-bridged analogue, c[WDINNNβA] in complex with the human SPSB2-SPRY and the human SPSB4-SPRY domain as a basis for structure-based design of additional peptide analogues.
The delivery of the c[WDINNNβA] analogues to macrophages was assessed using peptide conjugates. The accumulation of fluorescently-tagged peptide inside the endosomes/lysosomes suggests that intracellular delivery requires further optimisation. Accordingly, we are currently reengineering our cyclic peptide to improve endosomal escape and cytosolic delivery through reduction of the size and polarity of these peptides. Novel DINNN analogues should be potentially valuable leads in the treatment of persistent microbial infections.