论文总字数：36756字

2020 届毕业设计（论文）

题 目： Study of alloferon, a novel

immunomodulatory antimicrobial

peptide (AMP), and its potential

effects on Covid-19

专 业： Pharmacy

班 级： L003160102

姓 名： Vladimir Retyunskiy

指导老师： Ye Zhao

起讫日期： 2020.03-2020.06

2020 年 6 月

Study of alloferon, a novel immunomodulatory antimicrobial peptides (AMPs), and its potential effects on Covid-19 (SARS-Cov-2)

Abstract

Alloferons are a group of naturally occurring antimicrobial peptides (AMPs) primarily isolated from insects. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cells cytotoxicity and interferon (IFN) synthesis in mouse and human models, as well as to enhance antiviral and antitumor activities in mice. Also, the anti-viral effects of alloferon and its synthesized analogues undergone examinations regarding enhancement of apoptotic activity of cells, cytotoxicity parameters and viral cells using human larynx carcinoma cells (HEp-2), African green monkey kidney cells (Vero) and Rhesus monkey kidney cells (LLC-MK2) in-vitro tested on various types of viruses. COVID-19, as a novel virus, requires versatile approach in search of efficient drugs. Currently, as the efficient drug against COVID-19 is not found yet, it is necessary to test as many potentially effective biologically active agents as possible. In this article, the biological effects of alloferon and its analogues are reviewed and several research achievements are shown, which might broaden the understanding on the novel AMPs. The potential application of the peptide as antiviral agent in treatment of SARS-CoV-2 is considered and analysed as well.

Key words: Alloferon, antimicrobial peptides, NK cell, IFN, COVID-19

Anti-microbial peptides (AMPs) are bioactive-peptides with a low molecular mass of less than 10kDa, which have aroused attention for its safety, efficiency, specificity, relatively easy synthesis and modification^1,2. Some of AMPs has been found to inhibit the tumor growth, which is also known as anti-cancer peptides (ACPs) ^3,4. Both ACPs and AMPs are cationic and amphipathic, with high net positive charge⁵. For host defense against exogenous pathogen, AMPs are secreted from various organisms, including animals (Rock bream), plants (fermented Suan cai) and microbes (Bacillus) ⁵. It is generally accepted that AMPs impact bacterial function via changes in cell wall synthesis and composition, the inhibition of proteases and/or enzyme activity and nucleic acid and/or protein synthesis, promoting DNA binding as well as transmembrane pore formation⁶. Additionally, AMPs have a suppressive effect on cancer cells through cell apoptosis and necrosis pathways by interacting with cell membranes ^1,3. Currently, more than 2500 APMs has been collected in the Antimicrobial Peptide Database (AMPD, http://aps.unmc.edu/AP/main.php). Antimicrobial peptides divide into subgroups, each isolated from one of six kingdoms including mammals, bacteria, plants, fungi, protists and archaea. One of the bactenecin, antibacterial peptide, is a cyclic dodecapeptide isolated from Bos Taurus having anti-Gram and anti-Gram- influence.⁴⁶ Another AMP belonging to anti-viral class is Cecropin A, isolated from Giant silk moth, Hyalophora cecropia, has HIV inhibiting effect through suppressing of gene expression.⁴⁷ Among the AMPs, alloferons attract growing attention because of the wide range of positive effects: immunomodulatory activity against tumor, inhibition of viruses multiplication, interferon synthesis stimulation.

Structural character of alloferon with a potential regular arrangement

Alloferon is originally isolated from a hemolymph of bacteria challenged maggots of the blow fly Calliphora vicina (Diptera)⁷. Sergei Chernish purified the Calliphora acid extract of Vickers hemolymph and two peptides of 13 and 12 amino acids were isolated and their amino acid sequences were identified as HIS-GLY-VAL-SER-GLY-HIS-GLY-GLN-HIS-GLY-VAL-HIS-GLY (Alloferon-1) and GLY-VAL-SER-GLY-HIS-GLY-GLN-HIS-GLY-VAL-HIS-GLY (Alloferon-2) which stimulate in-vitro natural cytotoxicity of mouse spleen lymphocytes and human blood mononuclear cells⁷. Fig.1 shows the basic structure of alloferon. Alloferon is a linear peptide with a unique amino acid sequence represented by the following general formula: x1 hisgly-x2-his-gly-val-x3, where x 1 does not exist or represents at least one amino acid residue, x 2 is a peptide bond or represents at least one amino acid residue, and x 3 does not exist or represents at least one amino acid residue⁸. The primary structure of alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His- Gly-Val-His-Gly-OH) is similar to some functionally relevant proteins such as precursors of influenza virus B haemagglutinin, bovine prion protein I and II and Sarcophaga peregrina antifungal protein⁹. (Table 1, Table 2)

Table 1. Primary structure of alloferon 1 and 2 and sequence alignment with influenza virus B hemagglutinin precursor (fragment 382–392)⁷

Table.2 The primary structures of alloferon and its synthesized analogues⁹

The antiviral identification of alloferon analogues showed that the first aromatic ring of peptide chain could play a role in the expression of antiviral properties in vitro and the truncated analogue of alloferon without the N-terminal dipeptide His-Gly exhibited the strongest antiviral activity⁹.

Recent works on the biological activity of alloferon, including its analogues with point mutations containing 3 histidine residues in peptide molecule and their Cu (II) complexes, showed the importance of the histidine residue position in the peptide chain of alloferon on its cytotoxic activity¹⁰. The existence of hydrophilic residues at position 1 of alloferon is proved to be not important for inhibition of the virus replication¹¹. In addition, cellular and humoral defense of the mealworm against Staphylococcus aureus infection was significantly weakened by the [Phe(p-NH2)1]- and [Phe(p-OMe)1] sectors of alloferon.¹⁰ By analyzing the structure of alloferon, it has been found that there might be a potential regular arrangement for the amino acid sequence, which is the repeated fragments of “HGVSG” or “VHG”.

Alloferon potentiates immune cells by activating the NF-KB signaling pathway

Some reports have demonstrated that its mechanism for cancer treatment is not the direct killing of tumor cells but rather activating natural killer (NK) cells in the tumor microenvironment. NK cells release interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) to reverse tumor cell-mediated immune system inhibition⁷. IFN-γ and TNF-α are biological factors that can kill a large amount of tumor cells and activate other lymphocytes (such as T and B lymphocytes). When NK cells are activated, the amount of IFN-γ and TNF-α released from the cells is significantly increased ¹². NKG2D is an activating receptor that is highly expressed when NK cells are activated, and CD94 is an inhibitory receptor that is highly expressed when NK cells are inhibited¹². Therefore, Yi Huang et al. used ELISA kit to study the levels of IFN - γ and TNF - α in the peripheral blood of mice. The results showed that the concentration of IFN - γ and TNF - α in blood increased in the group containing alloferon-1.¹³

NK cell-based immunotherapy has been extensively studied. The most common NK cell applied to immunotherapy of cancer is the lymphokine-activated killer (LAK) cell ¹⁴. It is known that LAK cell is induced by the treatment of several kinds of cytokines and shows an increased cellular cytotoxicity against cancer^14,15. IL-2, -12, -15, -18 and -21 are commonly used cytokines to increase cellular cytotoxicity of NK cells^{16 17}; Parrish-Novak et a some natural substances, such as vitamin C and resveratrol, show the potent effect on the induction of the anticancer effect of NK cells ^18,19 It has been recently reported that alloferon has significant anti-inflammatory effect not only on UVB-induced inflammation in the normal human keratinocyte cell line, HaCaT, but also on mouse skin. Therefore, the anti-tumor activity of alloferon is mediated by the up-regulation of 2B4 expression followed by the increase of IFN-γ and TNF-α production and granule exocytosis.

Alloferon’s anti-tumoral activity is based on the enhancement of the cytotoxicity of NK cells by the increase of perforin/granzyme B secretion. NK cells are the main effectors in areas affected by inflammation processes as of being the components of innate immune system, thus becoming potential sites for the targeted drug treatment. NK cells participate in the formation of strong adaptive immune response through their cytokine production capacities. Moreover, eliminating autologous immune cells, NK cells are capable of limiting and controlling autoimmunity.⁵⁰ It may be speculated that alloferon-1 activated NK cells causing release of a considerable amount of IFN-γ and TNF-α in vivo, which is consistent with the aforementioned results.

Enhanced antiviral and antitumor activities by stimulation of NK cytotoxic activity and interferon synthesis

As an immunomodulatory peptide, alloferon has antiviral capability in mice ^7,23,24. It has been showed that allereron had antitumor effect by inducing cytotoxic activity of NK cells and stimulating interferon synthesis by activating nuclear factor kappa B in mice and human. In addition, alloferon has antiviral activity against DNA and RNA viruses (including herpesvirus and several Coxsackie viruses) ¹⁰. Alloferon can treat herpes virus infection by increasing the immune effect of natural killer (NK) cells and stimulating the synthesis of interferon (IFN). The experimental data showed that alloferon had a strong stimulating effect on the cytotoxic activity of human peripheral blood lymphocytes. Importantly, preclinical studies have shown that alloferon is not toxic to normal cells and does not affect their growth. Alloferon was identified to have the similar pharmacological effects with IFNα, which can stimulate the synthesis of interferon and the immune response of NK to kill harmful defective cells. Alloferon is not cytotoxic, immunogenic, mutagenic or carcinogenic, embryotoxic or reproductive, which means it is indeed stronger than other chemicals ¹⁰. Furthermore, alloferon rapidly enters the bloodstream and interacts with immune cells. It has showed that the level of interferon increased significantly within 2 hours, while the cytotoxicity of NK cells increased within 7 days. Report has also showed that alloferon enhance immune cells by down regulating antioxidant protein and IKB α to activate NF KB signaling pathway. The increase of NK cell killing activity was followed by the up regulation of NK activated receptor 2B4. At the same time, alloferon increased the IFN - γ, TNF - APLHA and granular exocytosis produced by NK cells, thus antagonizing cancer cells²⁰. However, it is not clear how alloferon regulates NF KB signaling pathway and how alloferon precisely regulates cell secretion.

Alloferon can be used to treat or prevent various infectious or tumor diseases, in which the improvement of innate immunity (including interferon system and natural cell-mediated cytotoxicity) can have therapeutic significance ²¹. Alloferons is used as an antiviral agent in the treatment of infections caused by influenza, herpes, papillomas, viral hepatitis, AIDS and AIDS related secondary infections. Based on the result of proteomic analysis, the activativation of the NF-kB signaling pathway including increased IKK, higher phosphorylation of IkBa, and a lower level of total IkBa has been demonstrated upon alloferon treatment. Rearch has also demonstrated that NF-kB activation is involved in IFN synthesis and provides a clue to the basis of alloferon’s capacity to stimulate IFN synthesis²².

Suppression of tumor immunity by Type II NKT Cells

Alloferons are useful in the treatment or prophylaxis of various infectious or oncological diseases where improvement of innate immunity, including interferon system and natural cell mediated cytotoxicity can have therapeutic significance ²¹. They are known to have anti-tumor property, used in the treatment of oncological conditions like acute and chronic leukemia. It was showed that synthetic alloferon in picomolar concentrations exerts an anti-tumor effect via the induction of cytotoxic activity of NK cells and the stimulation of interferon synthesis through activation of nuclear factor κB in mice and humans.²³ Alloferons mediate signalling by NF-kB pathway to boost recognition of viral and tumor antigens²². Endogenic interferons responsible for CD25 receptor expression upregulation and activation of defense responses are also stimulated by alloferons. Alloferons are capable to stimulate lymphocytes with cytotoxic characteristics after the detection of nonself or aberrant cells causing a further lysis of these cells.⁵¹ Based on the results, alloferon-1 has shown no observable toxic effects on nervous system of rats. Considering the aforementioned processes, alloferon may be potentially used as an antitumoral drug in addition to its antinociceptive activity.⁷

Alloferon potentiates immune cells by activating the NF-KB signaling pathway

Some reports have demonstrated that its mechanism for cancer treatment is not the direct killing of tumor cells but rather activating natural killer (NK) cells in the tumor microenvironment. NK cells release interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) to reverse tumor cell-mediated immune system inhibition. As known, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are biological factors that can kill a large amount of tumor cells and activate other lymphocytes (such as T and B lymphocytes). Natural killer (NK) cells release IFN-γ and TNF-α to reverse tumor cell-mediated immune system inhibition. NK cells were identified as the peptide pharmacological target responding to alloferon-1 with immediate growth of cytotoxic activity ^9,11,24-26. When NK cells are activated, the amount of IFN-γ and TNF-α released from the cells is significantly increased. NKG2D is an activating receptor that is highly expressed when NK cells are activated, and CD94 is an inhibitory receptor that is highly expressed when NK cells are inhibited.¹⁶ Yi Huang has used ELISA kit to study the levels of IFN - γ and TNF - α in the peripheral blood of mice. The results showed that the concentration of IFN - γ and TNF - α in blood increased in the group containing alloferon-1. It may be speculated that alloferon-1 activated NK cells causing release of a considerable amount of IFN-γ and TNF-α in vivo, which is consistent with the aforementioned results.

Potential effects of alloferon on COVID-19 induced immune reactions

COVID-19 is explored to be biding to ACE-2 membrane protein as a binding cite, which is expressed in most of the tissues in human body, including human brain cells, arterial and venous endothelial cells, type II pneumocites, erythrocytes of small intestine and smooth muscle tissue of most of the organs. However, considering the spread of the virus occurring by airborne droplets, lungs become the first and the most vulnerable target for the novel coronavirus. After the viral particles enter the lungs, the disease subsequently proceeds in 2 phases: the incubation period and gradual multiplication of virus, the immune resistance of an organism takes place, and the leading excessive propagation of virus in case of insufficient endogenous immune system response.⁴⁸ Alloferon’s stimulation of NK cells to produce IFN- α and IFN – γ may be particularly useful for the enhancement of the general state of organism’s immunity. Moreover, the minimum effective concentration of alloferon is 0.0005 ng/ml with the optimal concentration was of 0.05-0.5 nanogram/ml for immunomodulatory activity, which is 40 times less than the effective dose of rimantadine, a strong antiviral drug that is especially effective against Influenza virus A.⁹ Alloferon However, the most efficient ways of alloferon introduction as well as the fitting sites in treatment of COVID-19 have yet to be tested.

Figure 1. Representation of SARS-CoV-2 infection progress⁴⁸

Enhanced signaling of ROS dependent IFN induction

The conclusion of alloferon having both antitumoral and antiviral effects came from the in-vivo experiments in mice. The clinical trials on patients with human papilloma virus (HPV) and herpes simplex virus (HSV) demonstrated the therapeutic efficacy of alloferon-1.⁵¹ The upregulated IFN synthesis in patients with viral infection were displayed in Namalya cells treated with alloferon. Alloferon treatment induced downregulation of several antioxidant proteins such as PDIA3, TR1, IDPc, PRX4, and GSTP1. These proteins play critical roles in maintaining a reducing environment within the cell to protect against ROS. PRX4 has been shown to regulate NF-KB activity via a modulation of IkBa phosphorylation. Various reports have indicated that perturbation of redox balance toward the oxidized state, as well as ROS production, activates NF-kB signaling. It has been demonstrated the increased IKK activity, higher phosphorylation of IkBa, and a lower level of total IkBa upon alloferon treatment.

Preclinical Studies of alloferon

Allokine-alpha is the Russian peptide drug, the main component of which is alloferon-1, was designed to treat herpes-viral infection by increasing the immune-effect of natural killer(NK) cells and stimulating the synthesis of interferon(IFN).³⁴ However, how the alloferon exactly works is still unknown. According to the experimental results conducted in-vivo, the considerable stimulating effect of alloferon on peripheral blood lymphocytes’ cytotoxicity was detected in the lymphocytes set towards the lysis of tumor cells. Importantly, preclinical studies have shown that alloferon has no toxicity to normal cells and does not affect the growth of normal cells.³⁵ Anna Majewska team used the mice lethal pulmonary infection with human influenza viruses A and B as model to study the anti-microbial function of alloferon in vivo³⁶ . It can be seen that alloferon could prevented mortality of tested mice challenged by influenza viruses A and B. Also, Anna Majewska team used the Human lung fibroblasts and Human lung carcinoma infected with HHV-1MC ,found that alloferon successfully decrease the level of TCID50/ml¹². For another thing, cell lines including African green monkey kidney cells/vero, Rhesus monkey kidney cells/LLC-MK2, human larynx carcinoma cells/HEp-2, and virus including the standard strains of HHV-1MC and 971 PT Coxsackie type B2, the clinical strain of HHV-1/Coxsackie type B2 were applied for anti-viral function study in Vitro²⁶. Alloferon has more potency to inhibit the replication of HHV-1MC and HHV-1(IC50=305.50 and 479.00 ug/ml, respectively) in Vero cells, compared to HHV-1（IC50=252.20ug/ml）in HEp cell. But this peptide failed to inhibit the replication of virus HHV-1MC in HEp-cells in this model which the anti-viral activity was assessed using Vero, LLC-MK2, or HEp-2 cells lines infected with 0.01TCID50%/cell of respective virus. Together, it has been found that alloferon inhibited the herpes virus multiplication and failed to affect the coxsackievirus replication, whereas [Lys1]-alloferon exhibited a high inhibitory action towards both viruses³⁷. The research shown, however, that alloferon was successful in suppression of the herpes virus replication, but did not have an influence coxsackievirus replication, while [Lys1]- modification of alloferon affected both types of viruses.

Clinical Trials of alloferon and analogues

Although differing from known immunotropic biologicals like cytokines, chemokines, interferons and therapeutic antibodies by molecular structure, mechanism of action and origin, alloferon-1 can be classified as an immunomodulatory peptide with experimental and clinical studies taken as a basis.

Currently alloferon-1 is used as a drug against viral infections, however, the full anti-tumoral spectrum requires additional preclinical assessments. Alloferon-1 and especially allostatine are worthy of further consideration as potential anticancer drugs. Moreover, Allostatine, based on the obtained data, has its potential as an agent in adjuvant cancer immunotherapy. Human and mouse immunoglobulins were found to contain allostatine-like pattern formed in the process of evolution in their CDR3 region based on sequence similarity search. By analogy with allostatine, the pattern may execute unknown so far functions in anti-tumor immune response regulation.³⁸ Alloferon-1 was applied alone or in combination with conventional cytotoxic chemotherapy (a mixture of cyclophosphamide, doxorubicin and vincristine). The comparable with a low dose chemotherapy tumoristatic and tumoricidal activities were observed during alloferon-1 monotherapy. The combinated antitumoral activity of alloferon and cytotoxic drugs used as a pulse immunochemotherapy had a visible enhancement of the effect compared to the separate treatment with corresponding agents. Alloferon-1 antiviral efficacy was clinically proven in the treatment of genital herpes and oncogenic human papilloma virus infections³⁹.

Alloferons are used in medicine for treatment of herpes-viral infections and viral hepatitis B.³³ Paulson have demonstrated that modified sialic acid, a kind of carbohydrate derivative, can be presented on the cell surface and combine with specific antigen CD22, which highly express in the B Cell lymphoma, to regulate the immune activity of B cell^{40, 41}. CD22 belongs to the Siglect family, besides Siglect-4、Siglect-15、CD33⁴². Lieping Chen have suggested that Siglect-15 was an immune suppressor and potential target for cancer immunotherapy⁴³. The therapeutic antibody NC318 has been implemented for I/II phase clinical trials. Moreover, recent research suggested that CD24 signaling through macrophage Siglect-10 is a target for immunotherapy⁴⁴. Sialic acid has its potential in the future study of alloferon. Briefly, sialic acid may be combined with Alloferon and its analogues (Table 3) to obtain new analogues for database. Also, immune cells, NK cell and PBMC may be used as a model to express the complex in cell surface and test the anticancer activity. It was showed that synthetic alloferon in 45 picomolar concentrations exerts an antitumour effect via the induction of cytotoxic activity of NK 46 cells (Chernysh et al., 2002).

Improvement of drug design based on analogues of alloferon

Recently, a new biological activity of alloferon and its structural analogues in insects were discovered (Kuczer et al., 2013a). These peptides, similar to a Neb-colloostatin and its analogues exert a pro-apoptotic action on haemocytes of Tenebrio molitor (mealworm beetles), thus it may be suggested that apoptosis induced by these peptides may have important implications for the insect’s immune defense, resulting in weakening of the immune system due to loss of the haemocyte activity (Czarniewska et al., 2012; Kuczer et al., 2013a). Recent works on the biological activity of alloferon, including its analogues with point mutations containing 3 histidine residues in peptide molecule and their Cu(II) complexes, showed the importance of the histidine residue position in the peptide chain of alloferon on its cytotoxic activity (Kuczer et al., 2013b; Matusiak et al., 2014). Moreover, it was demonstrated that, generally, Cu(II) complexes of analogues tested so far significantly increased biological activity of these peptides (Kuczer et al., 2013b; Matusiak et al., 62 2014). Based on the previous reports in respect to alloferon and its analogues, the further studies were performed to investigate immunological activities of alloferon and its 3 structural analogues, which were characterized by the highest pro-apoptotic activities in T. molitor (Kuczer et al., 2013). In these analogues of alloferon, the His residue at position 1 of the alloferon molecule was replaced by para-substituted phenylalanine derivatives: Phe(p-NH2), Phe(p-OMe), and Phe(p-Cl). Haemocytes The novelty of this study lies in showing the diverse long-term immunological effects of alloferon and its structural analogues in haemolymph of T. molitor. Recent studies also shown the effect of pro-apoptotic peptides on insects’ immune system. The previous structure-activity studies showed that alloferon and its analogues modified at His residue at position one by para-substituted phenylalanine derivatives, Phe(p-NH2) or Phe(p-OMe) and Phe(p-Cl), may imply their possible immunoinhibitory effects in insects because they strongly induced T. molitor haemocytes to undergo apoptosis just one hour after peptide injection (Kuczer et al., 2013a; Matusiak et al., 2014; Matusiak et al., 2015). It was shown that the N-terminal His1 and His6 residues are more important for alloferon pro-apoptotic properties in insects than those at positions 9 and 12 in the peptide chain (Matusiak et al.,2015).

Table 3. Summary of alloferon and analogues

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