Inducible nitric oxide synthase (iNOS) is a key component of the innate immune system and produces nitric oxide (NO), which is responsible for killing of pathogens in macrophages. iNOS is down-regulated by the protein SPSB2, which promotes its proteasomal degradation by forming an E3 ligase complex1. Inhibition of SPSB2 prolongs the lifetime of iNOS and thereby enhances the macrophage killing of pathogens in prolonged infections. Current inhibitors of the SPSB have a strong affinity for this target but are highly polar, and unable to access the cytoplasmic SPSB22,3. Cell-penetrating peptides (CPPs) are a class of peptides that are able to improve permeability of their cargo by uptake through the endocytic pathway.
In this study, several SPSB inhibitor-CPP conjugates were designed and synthesised to enable cellular uptake. Affinity measurements by SPR showed that addition of CPP moieties did not compromise peptide binding to SPSB2. Cellular uptake studies using sulfo-Cyaninine 5-labelled peptides showed that the CPP analogues were taken up efficiently, although localised mainly in endosomes. Using a Griess assay, the ability of these conjugates to enhance NO production in macrophages was explored, with cyclic CPP conjugates showing the greatest ability to enhance prolonged NO production. These data suggest that these cell-permeable peptide conjugates represent a major step forward for this new class of antibiotic agents.