Nitric oxide, produced by inducible nitric oxide synthase (iNOS) is utilized by macrophages to kill phagocytosed microbes. Deletion of iNOS renders mice vulnerable to microbial infections such as Mycobacterium tuberculosis, Salmonella enterica and Listeria monocytogenes.1 We have shown that endogenous degradation of iNOS occurs through a ubiquitination pathway initiated by SPRY containing SOCS box (SPSB) proteins. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in enhanced nitric oxide levels following challenge by pathogens and enhanced killing of these pathogens.2 Thus, inhibitors of the SPSB-iNOS interaction in macrophages are potential anti-infective agents for bacterial and parasitic diseases.
We have shown that SPSB proteins bind to a highly conserved DINNN sequence in iNOS and that peptides corresponding to these residues bind SPSB2 with high affinity (KD 13 nM).2 Cyclization of this peptide sequence generated protease-resistant peptides whilst maintaining low nM affinity.3 Unfortunately, these inhibitors showed no active uptake or passive penetration into the macrophage. This lack of penetration represents a major hurdle as SPSB2 and iNOS are known to be cytosolic.
Accordingly, we have commenced a program focused on enhancing passive membrane penetration. This program seeks to reengineer our lead inhibitors to allow passive diffusion through the cell membrane. Key findings from this program show that penetration of phospholipid membranes can be achieved through strategies including N-methylation of the amide backbone, reduction in molecular weight and replacement of polar side chains with lipophilic bioisosteres.