Nitric oxide (NO) is a key effector molecule of the innate immune response, and plays a crucial role in macrophage killing of infective agents.1 NO is produced by inducible NO synthase (iNOS) in macrophages in response to foreign stimuli. The lifespan of iNOS is regulated by proteasomal degradation, which is mediated by binding to SPRY domain containing SOCS box proteins (SPSB).2 Our earlier studies revealed that the DINNN sequence at the N-terminus of iNOS is the key motif for SPSB binding.2 Disruption of the iNOS-SPSB interaction resulted in enhanced lifespan of iNOS, increased NO production and pathogen killing.2 A series of linear (7-mer, 13-mer) and cyclic (CP0-3) peptide inhibitors of the iNOS-SPSB interaction have been designed based on the DINNN motif and refined for increased binding affinity, stability and drug likeness.3-5
In this study we have conjugated the inhibitors with cell-penetrating peptides (CPPs) and demonstrated that binding to SPSB is not compromised by this conjugation. We have assessed the successful uptake of fluorophore tagged inhibitor-CPP conjugates by RAW 264.7 and immortalised bone marrow derived macrophage (iBMDM) cell lines. We have designed and optimised an assay to evaluate the potential of CPP-cargo for enhance NO production and found that these inhibitors elevated NO level. We have also shown that these inhibitors are not toxic to macrophages. The findings of this study will be useful in further optimising the design of SPSB inhibitor-CPP conjugates.
1 Bogdan, C. et al. Immunol. Rev. 173, 17-26 (2000)
2 Kuang, Z. et al. J. Cell Biol. 190, 129-141 (2010)
3 Yap, B. K. et al. FEBS Lett. 590, 696-704 (2016)
4 Yap, B. K. et al. J. Med. Chem. 57, 7006-7015 (2014)
5 Sadek, M. M. et al. ACS Chem. Biol. 13, 2930-2938 (2018)