Peptides: Prospects for Use in the Treatment of COVID-19

There is a vast practice of using antimalarial drugs, RAS inhibitors, serine protease inhibitors, inhibitors of the RNA-dependent RNA polymerase of the virus and immunosuppressants for the treatment of the severe form of COVID-19, which often occurs in patients with chronic diseases and older persons. Currently, the clinical efficacy of these drugs for COVID-19 has not been proven yet. Side effects of antimalarial drugs can worsen the condition of patients and increase the likelihood of death. Peptides, given their physiological mechanism of action, have virtually no side effects. Many of them are geroprotectors and can be used in patients with chronic diseases. Peptides may be able to prevent the development of the pathological process during COVID-19 by inhibiting SARS-CoV-2 virus proteins, thereby having immuno- and bronchoprotective effects on lung cells, and normalizing the state of the hemostasis system. Immunomodulators (RKDVY, EW, KE, AEDG), possessing a physiological mechanism of action at low concentrations, appear to be the most promising group among the peptides. They normalize the cytokines’ synthesis and have an anti-inflammatory effect, thereby preventing the development of disseminated intravascular coagulation, acute respiratory distress syndrome and multiple organ failure.

2.1. Prospective Molecular Mechanisms for the Pathogenesis of Coronavirus Infection Caused by SARS-CoV-2 The structure, life cycle and molecular targets of SARS-CoV-2 in host cells are shown in the diagram ( Figure 1 ). Each stage of the SARS-CoV-2 coronavirus life cycle represents a potential target for drug therapy. The penetration of the virus into the host cell membrane occurs in two stages: the first stage involves contacting the receptor on the surface of the target cell; the second stage is the fusion of the virus with the cell membrane, either on the cell surface or inside of it. In case of SARS-CoV-2, the first step requires that the spike proteins undergo biochemical modification, the so-called priming of protein S [ 11 , 12 , 13 ]. The viral spike SARS-CoV utilizes angiotensin-converting enzyme 2 (ACE2) as an input receptor [ 14 ] and uses cell serine proteases TMPRSS2 and cathepsin L for priming [ 15 , 16 , 17 ]. The functions of the TMPRSS2 protein remain insufficiently studied. Overexpression of the TMPRSS2 gene under the action of androgens in prostate cancer cells and reduced expression of the TMPRSS2 gene in androgen-independent prostate cancer were detected [ 18 ]. In the case of SARS-CoV-2, TMPRSS2 is responsible for the priming of the viral protein spike, which entails cleavage of the spike at two sites: Arg685/Ser686 and Arg815/Ser816. The TMPRSS2 and virus spike binding model was created. It was found that TMPRSS2 interacts with two spike loops. The key functional residues of TMPRSS2 (His296, Ser441 and Ser460) interact with the flanking residues of the spike cleavage sites. It is assumed that the TMPRSS2 region, which interacts with the C-terminal cleavage site (Arg815/Ser816) of the spike, is relatively more suitable for creating targeted drugs compared to the TMPRSS2 region, which interacts with the N -terminal cleavage site (Arg685/Ser686) [ 19 ]. The role of cathepsin L in the SARS-CoV-2 spike priming is suggested based on the data on its participation in the SARS-CoV priming [ 20 , 21 , 22 ]. At high (alkaline) pH, the fusion of the viral membrane and the target cell does not occur. Thus, protonation can indirectly facilitate the penetration of SARS-CoV into the cell. Inhibitors of cathepsin L activity block the introduction of SARS-CoV into target cells [ 22 ]. Probably, protonation is necessary for the activation of cathepsin L, rather than a spike protein. These results showed that cathepsin L is a SARS-CoV-activating protease. The efficiency of ACE2 receptor recruitment is a key factor in determining the ability of SARS-CoV to penetrate the cell [ 23 ]. Viral spikes SARS-CoV and SARS-CoV-2 have an amino acid identity of 72%; therefore, it is assumed that the mechanisms of penetration of SARS-CoV and SARS-CoV-2 are similar. However, there is a characteristic loop with flexible glycine residues in the SARS-CoV-2 structure, as opposed to hard proline residues in the same SARS-CoV region. Molecular modeling revealed a stronger interaction of RBD SARS-CoV-2 with the ACE2 receptor. Phenylalanine F486, located in a flexible loop, probably plays a major role in this interaction, since it penetrates the hydrophobic “pocket” of ACE2 [ 24 ]. The SARS-CoV-2 viral spike has higher affinity for the human ACE2 receptor compared to the Bat-CoV viral spike [ 25 , 26 ]. ACE2′s active extracellular domain is exposed on the surfaces of the alveolar epithelial cells of the lungs, and on the endothelial cells of arteries and veins, smooth muscle arterial cells, renal tubule epithelium and small intestine epithelium [ 27 , 28 ]. In addition, ACE2 gene expression was found in the epithelium of the upper respiratory tract, central nervous system (CNS) organs, pigmented epithelial cells of the eyes and liver hepatocytes [ 17 ]. In patients with COVID-19, an increase in the level of angiotensin II (Ang II) in the blood was detected, which indicates a suppression of the ACE2 expression in the tissues. This leads to dysfunction of the renin-angiotensin system (RAS). It should be noted that RAS is a hormonal system in humans and mammals that regulates blood pressure and blood volume in the body through a peptide cascade. Several key molecules are distinguished in RAS: renin, angiotensin I and its fragment Ang I (1–10), angiotensin-converting enzyme (ACE), angiotensin II. Renin is released from the secretory granules of juxtaglomerular cells in the event of a decrease in perfusion pressure or NaCl concentration. ACE hydrolyses Ang I (1–10) to angiotensin II, a polypeptide with potent vasoconstricting properties. It should be noted that the activation of RAS raises blood pressure. ACE2 is an ACE homolog and a negative regulator of RAS. The function of ACE2 is the degradation of angiotensin II, which is a vasoconstrictor and has pro-fibrous and pro-inflammatory properties, to Ang (1–7), a vasodilator with antiproliferative and apoptotic properties. In addition to the systemic function in maintaining blood pr

Patients with COVID-19 and concurrent diseases (cardiovascular disease, diabetes mellitus) are offered treatment with RAS blockers: ACE inhibitors (ACEIs) or angiotensin receptor blockers (ARBs). Patients treated with ACEIs or ARBs had increased ACE2 expression [ 56 ]. It is believed that the binding of SARS-CoV-2 to ACE2 can reduce the residual activity of ACE2, disrupting the balance of ACE/ACE2 towards increased ACE activity. This leads to pulmonary vasoconstriction and inflammatory and oxidative organ damage, and this, in turn, increases the risk of acute lung damage and systemic inflammation [ 36 ]. In this case, ACEIs and ARBs will help restore ACE/ACE2 balance. However, the use of ACEIs or ARBs can increase the binding of SARS-CoV-2 virus to ACE2 and stimulate disease progression [ 57 ]. It has been suggested that increasing the level of the soluble form of ACE2 in the blood can act as a competitive SARS-CoV-2 interceptor and inhibit the penetration of the virus into alveolar epithelial cells, protecting them from damage [ 58 ]. A clinical trial of Phase 2 administration of losartan (specific angiotensin II receptor antagonist of type AT1) is currently underway in patients with COVID-19 in an outpatient setting (ClinicalTrials.gov ID: NCT04311177 ) and an inpatient settings (ClinicalTrials.gov ID: NCT04312009 ) [ 59 ]. However, additional clinical and prospective studies are needed to find out if using ACEIs and ARBs can reduce the incidence and mortality rate of COVID-19.

Ribavirin is an inhibitor of viral RNA-dependent RNA polymerase, which explains the interest towards this inhibitor as a potential pharmacological agent during epidemics caused by SARS and MERS viruses. However, when studying the effectiveness of ribavirin against SARS-CoV, high concentrations of the drug were required to inhibit viral replication. Moreover, ribavirin had dose-dependent hematological toxicity, which makes it impossible to use it in high doses with a high frequency [ 68 ].

Currently, there are data available on peptides which are capable of inhibiting various phases of the SARS-CoV-2 life cycle, increasing the immune cells’ activity and normalizing the bronchopulmonary system functions in COVID-19 ( Table 1 ). Non-structural proteins SARS-CoV nsp10 and nsp16 were discovered to form a complex, in which nsp10 induces the methyltransferase activity of nsp16 by stabilizing the SAM-binding pocket and expanding the RNA-binding groove. In the nsp10 protein, the aa65–107 domain is necessary for binding to nsp16, and the aa42–120 domain is associated with nsp16 stimulation. The peptides GGASCCLYCRCH and FGGASCCLYCRCHIDHPNPKGFCDLKGKY obtained from the aa65-107 nsp10 domain can inhibit the 2-O-methyltransferase activity of the SARS-CoV complex nsp16/10. These data prove the relevance of the development of peptide drugs against SARS-CoV [ 74 ]. Heptad repeats (HR1 and HR2) are highly conserved sequences, located in the glycoproteins of the viral envelopes [ 75 ]. Peptides obtained from the HR regions of some viruses have been shown to inhibit the penetration of these viruses into the cell [ 76 , 77 ]. Based on this model, cell penetration inhibitors were identified for some viruses [ 78 , 79 , 80 , 81 ]. Molecular modeling showed that the S2 subunit in the spike protein SARS-CoV contains the HR1 and HR2 regions [ 82 ]. Based on these data, 25 peptides potentially capable of inhibiting the SARS-CoV penetration into the cell were constructed; their effects on the penetration of SARS-CoV viruses into 293T cells were investigated. The addition of peptides HR1-1 (NGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTA) and HR2-18 (IQKEIDRLNEVAKNLNESLIDLQELGK) at concentrations of 0.14 and 1.19 μM led to 75% inhibition of SARS-CoV penetration into cells. It is possible that HR1-1 and HR2-18 can serve as functional probes for disrupting the mechanism of SARS-CoV-2 fusion with the membrane of the target cell [ 83 ]. During the COVID-19 pandemic in China, an immunomodulatory pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY, TP5), which is the active center of the thymus hormone, thymopoietin, was used as one of the therapeutic agents [ 84 , 85 ]. The main function of thymopentin is to normalize immunological parameters in cases of tumor, immunodeficiency and autoimmune diseases [ 86 , 87 ]. In vitro experiments showed that TP5 affects the functions of T cells and monocytes, increasing the level of cGMP secondary mediator and activating intracellular signaling cascades [ 88 ]. The ability of TP5 to normalize the functions of the immune system in viral diseases has been established [ 89 ]. In the context of SARS-CoV-2, thymopentin is considered a 3CLpro inhibitor. This peptide has a high affinity for the active site of this protease according to molecular modelling [ 90 ]. In 2003, thymopentin was used to treat patients with chronic bronchitis and SARS-CoV [ 90 ]. In Russia medicinal immunomodulatory drugs thymalin (registration number LS-000267 dated 26 February 2010, Ministry of Health of the Russian Federation) and thymogen (EW dipeptide, registration number LS-002304 dated 13 September 2011, Ministry of Health of the Russian Federation) have been used for a long time in cases of various diseases associated with impaired immune system functions [ 91 ]. Indications for thymalin application in children and adults: acute and chronic viral and bacterial infections; infectious purulent and septic processes; impaired thymus function, impaired regenerative processes; immune system and blood formation suppression after chemotherapy or radiation therapy in cancer patients. Indications for the administration of Thymogen are as follows: prophylaxis and complex therapy of acute and chronic viral and bacterial diseases of the upper respiratory tract; prevention of immune function and clot formation, and regeneration process suppression in the post-traumatic and postoperative periods; complex therapy of acute and chronic infectious and inflammatory diseases accompanied by a decrease in immunity. The use of thymalin reduces the incidence of acute respiratory infections in senior patients by 2.0–2.4 times [ 92 ]. Studies on the effect of thymalin on the human respiratory system during early embryogenesis revealed its ability to accumulate in the epithelial cells of the respiratory tract [ 93 ]. Later studies showed that the use of thymalin and thymogen for the treatment of patients with acute lung abscess led to a decrease in the systemic inflammatory response syndrome, a decrease in plasma fibrinolytic activity and discontinuation of hypercoagulability [ 94 ]. In accordance with these results, thymalin and thymogen appear to be promising agents for COVID-19 therapy. A double-blind, randomized, placebo-controlled study on the efficacy of thymogen in elderly patients after solid tumor removal in the abdominal cavity and retroperitoneal space was performed. The preoperative use of thymogen restored the structural and function

Short peptides can prevent the development of the pathological process with COVID-19 in three ways: virus protein inhibitor peptides (HR1-1, HR2-18, peptides based on aa65-107 domain structure analysis of nsp10 protein) are believed to inhibit virus replication; immunomodulating peptides (thymalin, RKDVY, EW, KE, AEDG) contribute to the normalization of innate and adaptive immunity, the hemostatic system and the synthesis of cytokines, and have an anti-inflammatory effect, thereby preventing the development of distress syndrome and multiple organ failure; additionally, peptides-bronchoprotectors (EDG, AEDL) can be used to reduce apoptosis, and activate proliferation and differentiation of bronchial epithelial cells. Thus, one of the safest and most effective classes of substances suitable for further research and application in the treatment of coronavirus infection consists of three groups of peptides: SARS-CoV-2 protein inhibitors, immunomodulators and bronchoprotectors with a physiological mechanism of action.