NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms

(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)-labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear-cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.