Neuroprotective Peptides and New Strategies for Ischemic Stroke Drug Discoveries

Ischemic stroke (IS) continues to be one of the leading causes of death and disability in the adult population worldwide. In the early stages after the onset of an ischemic attack, effective treatment for stroke still includes the use of thrombolytic agents or mechanical thrombectomy [ 1 , 2 , 3 , 4 ]. Timely successful reperfusion is the most effective treatment for patients with acute IS, but vascular recanalization can lead to ischemia-reperfusion (IR) injury, and thrombolytic drugs have no effect on protecting or reversing neuronal damage [ 2 , 5 , 6 ]. The treatment of IS is not limited to reperfusion therapy. Drug therapy includes antiplatelet agents, anticoagulants, antioxidants, antihypertensives, anti-excitotoxic calcium-stabilizing drugs, and other drugs [ 7 , 8 , 9 , 10 ]. Despite the versatility and diversity of the drugs used, the currently existing pharmacological methods for the treatment of IS are not effective enough and require further study of the molecular basis of ischemic damage, the development of new therapeutic agents, and approaches to identify potential neuroprotectors. The use of neuroprotectors to protect nerve cells from damage and death and to improve the activity of the nervous system is one of the main directions in the pathogenetic treatment of many neuropathologies. Among them are encephalopathies of various origins, neurodegenerative diseases, the consequences of traumatic brain injuries, chronic cerebrovascular accidents in the elderly, and acute cerebral ischemia. Neuroprotectors have heterogeneous chemical structures and different mechanisms of action. Herbal drugs, antioxidants and vitamins, calcium channel blockers, and agents that improve cerebral metabolism and affect neurodegeneration have a neuroprotective effect [ 11 , 12 , 13 , 14 , 15 ]. Promising candidates for neuroprotector roles are agents with pleiotropic activity, which have antioxidant, anti-inflammatory, regenerative, antiplatelet, and antiapoptotic properties. Peptide drugs are used as neuroprotectors to treat many diseases [ 16 , 17 , 18 ]. Peptides are a unique class of pharmaceutical compounds with high potency, clear specificity, low immunogenicity, biocompatibility, mild action, and no side effects. Efforts and achievements in the discovery, production, and modification of peptide drugs in recent years, as well as their current application in various pathologies, have been summarized in several reviews [ 19 , 20 , 21 , 22 , 23 ]. In the search for new therapeutic agents for stroke prevention, peptides are of great interest. Today, in the development of neuroprotective drugs for the treatment of stroke, special attention is paid to peptides that act in several key areas: (1) in blocking the cascade of pathological processes caused by the restriction of blood flow to the brain tissues, (2) in preventing repeated violations of the blood supply to the brain and complications that aggravate the course of the disease, and (3) in restoring nerve tissue and brain function after ischemia [ 24 , 25 , 26 ]. A significant contribution to the development of new neuroprotective drugs is made by transcriptome studies based on genome-wide sequencing of cellular RNAs. This approach makes it possible to detail the changes in gene expression during the development of IS in order to identify the signaling pathways involved in the molecular mechanisms of damage. As a result, genome-wide analysis makes it possible to detect potential therapeutic targets, the impact of which can serve as a new approach for the treatment of the pathological process. The use of the transcriptomic approach has made it possible to study the mechanisms of action of many natural medicinal compounds and peptide drugs successfully used for the treatment of IS [ 27 , 28 , 29 ]. Current problems in the development of neuroprotective strategies, shortcomings in preclinical modeling, lessons from earlier approaches in clinical trials, and ways to overcome these problems have been discussed in previous reviews [ 8 , 26 , 30 , 31 ]. Previously, we have described, in detail, medical technologies for the treatment of IS using drugs based on natural regulatory peptides [ 27 , 28 , 32 ]. Additionally, the effects of several peptides on brain impairment conditions, including NPY, substance P, galanin, nocistatin, and sectroneurin, have also been described in reviews [ 33 , 34 , 35 ]. In this review, we consider the latest achievements and trends in the development of new biologically active peptides, as well as the role of genome-wide transcriptome studies in identifying the molecular mechanisms of action of potential drugs for the development of methods for the treatment of IS. The properties of some potential neuroprotective regulatory peptides are listed in Table 1 .

3.1. Peptides Are New Potential Neuroprotective Agents for the Treatment of Acute IS 3.1.1. Interfering Peptides It is well known that protein–protein interactions (PPI) are involved in normal physiological processes. It is estimated that, in the human interactome, PPI networks include about 650,000 contacts [ 38 ]. To date, more than half a million forms of PPI dysregulation have been associated with pathological events, and addressing such dysregulation is considered a new therapeutic approach for the treatment of many diseases [ 17 ]. It has been found that short peptides that interfere with PPIs may have therapeutic effects. These peptides are called interfering peptides (IP). They are able to bind to the surfaces of proteins, thus blocking their interaction. Among the pathologies associated with PPI is IS. As is known, excessive release of glutamate from synaptic endings and associated excitotoxicity is one of the main mechanisms causing neuronal death in stroke. A large number of studies have significantly expanded the understanding of the mechanisms underlying excitotoxicity in cerebral ischemia and identified many molecular targets for action, and thus, opened up new possibilities for neuroprotective strategies for ischemia [ 39 , 40 , 41 , 42 ]. The development of IPs that prevent excitotoxic stress during cerebral ischemia has become one of the strategies aimed at preventing neuronal death in the penumbra region surrounding the damage zone [ 43 , 44 , 45 ]. IPs include the recently developed synthetic R1-Pep (SETQDTMKTGSSTNNNEEEKSR) and PP2A-Pep (FQFTQNQKKEDSKTSTSV) peptides ( Table 1 ), which inhibit the interaction of γ-aminobutyric acid type B (GABA B ) receptors with enzymes involved in their phosphorylation [ 46 , 47 ]. Under physiological conditions, GABA B receptors control the excitability of neurons in the brain through prolonged inhibition, and thereby counteract neuronal overexcitation and death. However, during cerebral ischemia, excitotoxic states rapidly downregulate GABA B receptors through phosphorylation/dephosphorylation processes mediated by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 2A (PP2A) [ 46 , 48 , 49 ]. After phosphorylation, GABA B receptors undergo lysosomal degradation rather than being recycled back to the plasma membrane [ 48 , 50 ]. The R1-Pep peptide penetrating into cells inhibits the interaction of GABA B receptors with CaMKII, preventing its phosphorylation. Administration of this peptide to cultured cortical neurons exposed to excitotoxic conditions, as well as its addition to mouse brain slices after middle cerebral artery occlusion (MCAO), restores the function of GABA B receptors [ 46 ]. Another small interfering PP2A-Pep peptide, according to in vitro and ex vivo studies, inhibits the interaction of GABA B receptors with PP2A and restores the expression of GABA B receptors on the cell surface of neurons under normal physiological conditions and after excitotoxic stress [ 47 ]. The model of regulation for GABA B receptor activity with the participation of R1-Pep and PP2A-Pep peptides is shown in Figure 1 . A current drug candidate for disrupting PPI in IS is the cell-penetrating peptide nerinetide (NA-1, YGRKKRRQRRRKLSSIESDV) ( Table 1 ). The peptide consists of nine C-terminal residues of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor, which selectively binds glutamate and aspartate. It prevents the post-synaptic density protein 95 (PSD-95) from binding to the NR2B subunit of the NMDA receptor and the PDZ domain of neuronal nitric oxide synthase (nNOS) [ 8 , 51 ]. Disruption of this PPI by NA-1 attenuates neurotoxic signaling cascades that lead to excessive calcium ion entry into neurons. NA-1 also prevents excitotoxic cell death and subsequent brain damage. Long-term evaluation of stroke outcomes in rodent and primate models of focal ischemia has also shown that NA-1 can reduce infarct size, along with an improvement in the neurological deficit. However, clinical studies have shown that treatment with a single dose of NA-1 along with endovascular thrombectomy did not improve long-term stroke outcome compared to patients treated with placebo [ 52 ]. At the same time, the cohort of patients treated with NA-1 without any thrombolytics had a lower risk of mortality, a significant reduction in infarct volume, and an improvement in functional outcome. This finding will require confirmation but suggests that neuroprotection in human stroke might be possible [ 52 ]. Thus, according to the data of in vitro and ex vivo studies, the developed interfering peptides exerted neuroprotective activity and inhibited excitotoxic neuronal death. IPs are believed to have great therapeutic potential for inhibiting progressive neuronal death in patients with acute stroke.

Another new therapeutic strategy for acute IS is the use of peptides as carriers (shuttles) that ensure the permeability of drugs through the blood–brain barrier (BBB) [ 60 , 61 ]. It is well known that the BBB is a structural barrier that ensures the supply of nutrients to the brain, protects it from harmful substances, and, at the same time, effectively blocks the entry of most neuroprotective agents. A useful property of peptides is their ability to overcome the BBB, which allows them to be used as a carrier for drug delivery. Brain-permeable peptide–drug conjugates, consisting of BBB shuttle peptides, linkers, and drug molecules, directly cross the BBB via an adsorption-mediated transcytosis pathway or through interaction with receptors or other proteins on the surfaces of BBB cells to initiate endogenous transcytosis or other means of transport [ 62 , 63 , 64 ]. It is well known that glycine has a neuroprotective effect in cerebral IR but has a low permeability through the BBB. A tripeptide, H-Gly-Cys-Phe-OH (GCF) ( Table 1 ), which can permeate through the BBB, acts as a BBB shuttle and prodrug, delivering the amino acid glycine to the brain to provide neuroprotection [ 65 ]. A well-known cellular antioxidant enzyme is superoxide dismutase (SOD). However, exogenous SOD cannot be used to protect tissues from oxidative damage due to the low permeability of the cell membrane. The recombinant CPP-SOD fusion protein, which combines the SOD protein with short cell-penetrating peptides (CPPs) ( Table 1 ), can cross the BBB and alleviate severe oxidative damage in various brain tissues by scavenging reactive oxygen species, reducing the expression of inflammatory factors, and inhibiting NF-κB/MAPK signaling pathways [ 61 ]. Thus, the clinical application of CPP-SOD impacts the damage associated with oxidative stress and new therapeutic strategies. Based on their physical and chemical properties, CPPs are classified as cationic, amphipathic, and hydrophobic peptides. CPPs typically contain more than five positively charged amino acids. Most cationic CPPs are derived from natural TAT (YGRKKRRQRRR) and penetratin (RQIKIWFQNRRMKWKK) peptides [ 53 , 66 ].

At present, transcriptomics has become an effective approach for studying the mechanisms of pathological processes in various diseases and searching for molecular targets for their drug treatment. High-throughput mRNA sequencing (RNA-Seq) reveals information about the expression of individual genes and makes it possible to identify signaling pathways involved in the development of many diseases. Transcriptomic analysis has made a significant contribution to the study of the molecular mechanisms of brain damage as a result of IR [ 99 , 100 , 101 ]. This approach has also made it possible to reveal the mechanisms of therapeutic effects of many potential drugs at the genetic level, creating a theoretical basis for the treatment of IS [ 102 , 103 , 104 , 105 , 106 ]. RNA-Seq analysis revealed the molecular mechanisms by which regulatory peptides and peptide-related drugs perform a neuroprotective role in cerebral ischemia. There are many examples of the use of transcriptome analysis to study the mechanisms of action of a number of peptides, including Orexin-A [ 107 ], Semax [ 27 ], VR-10 [ 29 ], and semaglutide [ 101 ]. Recently, using RNA-Seq, we studied the protective properties of the Semax peptide at the transcriptome level under transient MCAO conditions. Previously, we studied the gene expression in rat brain under conditions of incomplete global ischemia of the rat brain and permanent MCAO using real-time reverse transcription polymerase chain reaction (RT-PCR). Thereby, we showed that the peptide affects the expression of a limited number of genes encoding neurotrophic factors and their receptors [ 108 , 109 ]. Using RatRef-12 BeadChips, it was shown that Semax affects the expression of genes associated with the immune and vascular systems in the brain of rats after permanent MCAO [ 92 , 97 ]. Using RNA-Seq analysis, we identified several hundred differentially expressed genes (DEGs) (>1.5-fold change) in the brains of rats 24 h after transient MCAO treated with Semax compared with control animals treated with saline [ 27 , 110 ]. We found that Semax suppressed the expression of genes associated with inflammatory processes (e.g., Hspb1, Fos, iL1b, iL6, Ccl3, Socs3 ) and activated the expression of genes associated with neurotransmission (e.g., Cplx2, Chrm1, Gabra5, Gria3, Neurod6, Ptk2b ), while IR, on the contrary, activated the expression of genes involved in inflammation, immune response, apoptosis, and stress response and suppressed the expression of genes associated with neurotransmission. Analysis of signaling pathways associated with Semax-induced DEGs in the transient MCAO rat model using a web server for functional enrichment analysis (g:ProfileR) and Gene Set Enrichment Analysis (GSEA) data showed that DEG activation in Semax-treated rats 24 h after MCAO was associated with calcium signaling, dopaminergic, cholinergic, the glutamatergic synapse, and G-protein coupled receptor (GPCR) signaling through chemical synapses, whereas DEG suppression was associated with the phagosome, interleukin 17 (IL-17), tumor necrosis factor (TNF), p53 signaling pathways, innate immune system, neutrophil degranulation, and cytokine signaling in the immune system [ 27 ]. Thus, according to RNA-Seq data, 24 h after transient MCAO, Semax initiated a neurotransmitter and anti-inflammatory response that compensated for mRNA expression patterns that were disturbed under IR conditions. Perhaps, Semax can mediate its influence on gene expression indirectly through interaction with receptors on the cell membrane. Moreover, the action of Semax, which is able to reduce the disturbances caused by ischemia, can be explained by joint action of peptide on receptors in an allosteric manner, together with hormones and mediators of the ischemic response. The model of the influence of Semax on the transcriptome of brain cells and the spectrum of Semax effects identified at the gene expression level at 24 h after transient MCAO in rats is shown in Figure 3 . Semax, as noted above, contains an ACTH(4–7) fragment flanked at the C-terminus by the PGP tripeptide. There is evidence that PGP not only ensures the resistance of peptides against biodegradation, but also exhibits a wide range of biological activity. Namely, PGP is involved in the formation of the immune response, and the PGP peptide has antiplatelet, anticoagulant, fibrinolytic, anxiolytic, antiapoptotic, and antistress activity [ 108 , 111 , 112 , 113 , 114 , 115 , 116 ]. Using real-time RT-PCR, we studied the effects of PGP and another PGP-containing the Pro-Gly-Pro-Leu (PGPL) peptide on the expression of a number of inflammatory cluster (IC) and neurotransmitter cluster (NC) genes in rat brain under transient MCAO. Then, we compared the studied peptide effects with the action of Semax under IR conditions [ 96 , 117 ]. Both PGP and PGPL peptides showed an effect dissimilar to the effects of Semax at 24 h after transient MCAO. The administration of peptides did not have a

It can be assumed that the latest developments in the creation of neuroprotective agents, combined with new possibilities for their delivery to the brain, an understanding of the pathogenesis of stroke in general, and consideration of the previous shortcomings in preclinical and clinical studies, will provide a breakthrough in the treatment of IS. Here, we reviewed the most recent strategies to search for neuroprotectors for the treatment of IS, advances in the development of biologically active neuroprotective peptides, and the role of genome-wide transcriptome studies in identifying the molecular mechanisms of action of potential drugs.