Influence of the N-terminus acetylation of Semax, a synthetic analog of ACTH(4-10), on copper(II) and zinc(II) coordination and biological properties (2023)

Adrenocorticotrophic hormone (ACTH), melanocyte-stimulating hormone (MSH) fragments and synthetic analogues thereof, belong to a class of endogenous regulatory peptides [1], [2] with a marked activity on the functions of the central nervous system (CNS). These compounds showed neurotrophic, nootropic and neuroprotective effects [3], [4], [5] and can affect human and animal behaviour [6], [7], [8], [9]. This action is not associated with the ACTH hormonal activity and likely is the result of a direct action on the CNS [10]. Several studies showed that both behavioural and neurotrophic activity of ACTH are ascribable to the N-terminal part of the ACTH molecule [4], with the fragment ACTH(4-10), which mimic some features of the full-length ACTH molecule, showing the most strong effects on behaviour [11], [12], [13], [14]. Several analogues of the ACTH natural peptides, showing more effectiveness than the natural N-terminal ACTH fragments, have been synthesized [15], [16], [17].

Semax is an ACTH(4-10) analog peptide that lacks hormonal effects but has marked neurotrophic activity and protease stability [18]. In vitro and in vivo studies demonstrated a high efficacy of Semax in the treatment of cognitive/memory disorders [19] and ACTH-like and anti-inflammatory effects [20], [21], [22]. These properties can be explained by taking into account that Semax: i) modulates the vascular endothelial growth factor (VEGF) family gene expression in focal ischemia of rat brain [23]; ii) influences the morphology and proliferative activity of rat brain cells during experimental ischemia [24]; iii) regulates the expression of brain derived neurotrophic factor (BDNF) and of the tyrosine receptor kinase (TrkB) in the rat hippocampus [25].

Transition metal ions are important players involved in neuromodulation/neurotransmission [26], [27], neurodegenerative diseases, memory and cognitive processes [28], [29], [30]. In particular, it is well known the key role of essential transition metals such as copper(II) and zinc(II) in the neuropathology of Alzheimer disease (AD) [31], [32], [33] and their effects on neurotoxicity [34], [35], [36] and rupture of the redox-active metal ions homeostasis in the brain and in senile plaques [37], [38]. More recently, metal ions have been suggested to play an essential role in the mechanisms of memory formation [28].

A large body of evidence suggests a cross-talk between copper and zinc dyshomeostasis and dysregulation of expression of proteins related to learning and memory, including nerve growth factor (NGF) and BDNF [39], [40], [41], [42], [43]. The mechanisms of interaction of these metal ions with various neurotrophins and their peptide fragments have gained increasing interest [44], [45], [46], [47].

N-terminal peptide acetylation is commonly used to make the peptide similar to the protein backbone, especially to study the chelating properties of a specific protein region. For example, proteins like α-synuclein are naturally N-terminal acetylated and this acetylation reduces the Cu²⁺ affinity for the N-terminal binding site and modulates the copper mediated aggregation kinetics of the protein [48].

Recently, we demonstrated that Semax possesses a high affinity for Cu(II) ions and a protective ability against metal-induced cell toxicity, suggesting that this peptide might have a role in copper homeostasis [49]. Ac-Semax can be a promising drug to obtain the prolongation of the Semax nootropic effect on the CNS, owing to the higher stability with respect to Semax in biological fluids [50].

In the present work, the copper(II) complexes formed by Ac-Semax are described. The results obtained are compared with those previously reported for Semax [49] in order to discern the effects of acetylation of the N-terminal amino group on copper(II) chelation properties. Moreover, since there is a cross-talk between BDNF and zinc [51], and Semax has a regulatory action on BDNF, the chelation ability towards Zn²⁺ was also characterized for both acetylated and non-acetylated derivatives.

Moreover, the effect of Semax acetylation on the copper-induced cytotoxicity on SH-SY5Y neuroblastoma cell line was also studied in comparison with the previously reported Semax protective activity. Finally, the functional interaction of Zn(II) with both peptides were tested by evaluating the effects on the viability of the SH-SY5Y neuroblastoma cells.