We previously reported dipeptidomimetic compounds as inhibitors of neuronal and/or inducible NO synthases (n/iNOS) with significant selectivity against endothelial NOS (eNOS). They were composed of an S-ethylisothiocitrullin-like moiety linked to an extension through a peptide bond or a 1,2,4-oxadiazole link. Here, we developed two further series where the extension size was increased to establish more favorable interactions in the NOS substrate access channel. The extension was introduced on the solid phase by the reductive alkylation of an amino-piperidine moiety or an aminoethyl segment in the case of dipeptide-like and 1,2,4-oxadiazole compounds, respectively, with various benzaldehydes. Compared to the previous series, more potent inhibitors were identified with IC₅₀ in the micromolar to the submicromolar range, with significant selectivity toward nNOS. As expected, most compounds did not inhibit eNOS, and molecular modeling was carried out to characterize the reasons for the selectivity toward nNOS over eNOS. Spectral studies showed that compounds were interacting at the heme active site. Finally, selected inhibitors were found to inhibit intra-cellular iNOS and nNOS expressed in RAW264.7 and INS-1 cells, respectively.

Protein inhibitor of neuronal NO synthase (PIN) or dynein light chain 8 (LC8) is a highly conserved protein interacting with multiple partners, like neuronal NO synthase (nNOS) or myosin Va to modulate a variety of cellular functions. As PIN is expressed in skeletal muscle, our aim was to investigate a possible role of PIN in glucose uptake in L6 and primary muscle cells. PIN overexpression resulted into a decrease in glucose uptake with reduced GLUT4 expression and translocation at the plasma membrane, similarly to the pharmacological blockade of nNOS with L-NAME. PIN effect is mediated by a reduction in nNOS protein level and a direct interaction with nNOS leading to a reduced NO production in L6 myocytes. Surprisingly, a siRNA targeting PIN decreased glucose uptake and GLUT4 translocation, suggesting the involvement of nNOS-independent effects. We therefore focused on myosin Va which interacts with PIN in L6 myocytes. Myosin Va silencing provoked a decrease in glucose uptake. As PIN siRNA also reduced myosin Va expression, this confirms the essential role of myosin Va in the observed effects of PIN silencing on glucose uptake. We conclude that PIN modulates glucose uptake and GLUT4 translocation in rat muscle cells, through NO-dependent and -independent mechanisms.