Inducible nitric oxide synthase (iNOS) is a key factor of the innate immune system and produces nitric oxide (NO), which is critical for the killing bacteria of pathogens in macrophages. iNOS is down-regulated by the adapter protein SPSB2, which promotes its proteasomal degradation by forming an E3-ubiquitin 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.
We designed and synthesised several SPSB inhibitor-CPP conjugates to promote cellular uptake. CPP’s with a variety of physicochemical properties were selected to probe the structure activity relationship, including polycationic and amphiphilic sequences. Cyclic CPP’s were also employed, and the synthesis of these peptides optimised for increased yield, and the inclusion of a sulfo-Cyanine 5 fluorophore.
Affinity measurements by SPR showed that addition of CPP moieties did not reduce peptide binding to SPSB2. Cellular uptake studies using fluorophore-labelled peptides showed that the CPP analogues were taken up efficiently, although localised mainly in endosomes. The ability of these conjugates to enhance NO production in macrophages was explored using a Griess assay, 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.