VIP/PACAP-Based Drug Development: The ADNP/NAP-Derived Mirror Peptides SKIP and D-SKIP Exhibit Distinctive in vivo and in silico Effects.

Shlomo Sragovich,Illana Gozes,Adva Yeheskel,Noy Amram

doi:10.3389/fncel.2019.00589

Abstract

Activity-dependent neuroprotective protein (ADNP) was discovered and first characterized in the laboratory of Prof. Illana Gozes to be regulated by vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) toward neuroprotection. Importantly, ADNP is a master regulator of >400 genes, essential for brain formation, while its haploinsufficiency causes cognitive impairments. Recently, de novo mutations in ADNP were identified as leading to the autism-like ADNP syndrome, mimicked by the Adnp-deficient mouse model. Furthermore, novel peptide derivatives of the neuroprotective ADNP-snippet NAP (NAPVSIPQ), developed in our laboratory, include SKIP and the mirroring all D-amino acid SKIP (D-SKIP). We now extended previous evidence suggesting potential antagonistic features for D-SKIP, compared with the neuroprotective peptide SKIP, as was observed by NMR analysis and social/olfactory functional testing. Here, an impact of the Adnp genotype was observed in the Morris Water Maze (MWM) test measuring cognition, coupled with improvement by SKIP, opposing the inert/exacerbating effect of D-SKIP. In the elevated plus-maze and open field tests measuring anxiety-related behaviors, contrasting effects of SKIP and D-SKIP were found, with SKIP improving/preserving the normal phenotype of the mouse, and D-SKIP causing alterations. Lastly, an in silico analysis suggested that SKIP and D-SKIP bind the microtubule end binding (EB) proteins EB1 and EB3 in different conformations, thereby indicating distinctive natures for the two peptides, potentially mediating differential in vivo effects. Altogether, our findings corroborate the notion of D-SKIP acting as an antagonist, thus distinguishing it from the neuroprotective SKIP.

Highlights

Activity-dependent neuroprotective protein (ADNP) and activity-dependent neuroprotective factor (ADNF) were originally discovered and characterized in our laboratory (IG) in collaboration with the laboratory of Dr Douglas E
Latency measured in seconds to reach the hidden platform in its new daily location is depicted. (A) For Trial 1 [a non-significant value was observed for main interaction effect (F(20,208) = 1.0301, p = 0.427)], Tukey post hoc tests showed that Adnp+/+ male mice learned the task on day 5 (∗p = 0.034), compared with the impaired Adnp+/− mice
SKIP-treated Adnp+/− mice learned the task on days 2, 4–5 (∗∗p = 0.003, ∗∗p = 0.009, ∗p = 0.034, respectively). (B) For Trial 2 [a non-significant value was observed for main interaction effect (F(20,208) = 1.119, p = 0.332)], Tukey post hoc effects showed that vehicle-treated Adnp+/+ male mice learned the task on days 4–5 (∗∗p = 0.003, ∗∗∗p < 0.001, respectively), compared with the impaired Adnp+/− mice

Summary

Introduction

Activity-dependent neuroprotective protein (ADNP) and activity-dependent neuroprotective factor (ADNF) were originally discovered and characterized in our laboratory (IG) in collaboration with the laboratory of Dr Douglas E. NAP was shown to enhance cognitive function in Adnp+/− mice (Vulih-Shultzman et al, 2007; Hacohen-Kleiman et al, 2018; Sragovich et al, 2019a), by interacting with tubulin/microtubule end binding (EB) proteins through its SIP motif (NAPVSIPQ; Oz et al, 2014), eventually leading to microtubule stabilization and fortification (Gozes et al, 2005). Additional peptides developed in our lab were derived from ADNF, namely ADNF-14 (VLGGGSALLRSIPA) and ADNF-9 (SALLRSIPA = SAL), both sharing the SIP motif with NAP (Gozes et al, 2000b), and exhibiting neuroprotective activity (Brenneman and Gozes, 1996; Brenneman et al, 1998). Both D- and L-amino acid forms of ADNF-9 (SAL) exhibited similar neuroprotective potency, with the D-amino acid form being protease stable and suitable for oral administration (Brenneman et al, 2004; Wilkemeyer et al, 2004; Parnell et al, 2007), further found to be neuroprotective in vitro and in vivo (Gozes et al, 2008; Shiryaev et al, 2011)

Methods

Results

Conclusion