key: cord-0681904-gq8mqfrv authors: Zhirnov, O. P. title: Molecular Targets in the Chemotherapy of Coronavirus Infection date: 2020-05-18 journal: Biochemistry (Mosc) DOI: 10.1134/s0006297920050016 sha: d01ccc2773eb85cf5173842a708e6a374b4ef027 doc_id: 681904 cord_uid: gq8mqfrv In the pathogenesis of the infectious process in the respiratory tract by SARS, MERS, and COVID-19 coronaviruses, two stages can be distinguished: early (etiotropic) and late (pathogenetic) ones. In the first stage, when the virus multiplication and accumulation are prevalent under insufficient host immune response, the use of chemotherapeutic agents blocking the reproduction of the virus is reasonable to suppress the development of the disease. This article considers six major chemotherapeutic classes aimed at certain viral targets: inhibitors of viral RNA polymerase, inhibitors of viral protease Mpro, inhibitors of proteolytic activation of viral protein S allowing virus entry into the target cell, inhibitors of virus uncoating in cellular endosomes, compounds of exogenous interferons, and compounds of natural and recombinant virus-neutralizing antibodies. In the second stage, when the multiplication of the virus decreases and threatening pathological processes of excessive inflammation, acute respiratory distress syndrome, pulmonary edema, hypoxia, and secondary bacterial pneumonia and sepsis events develop, a pathogenetic therapeutic approach including extracorporeal blood oxygenation, detoxification, and anti-inflammatory and anti-bacterial therapy seems to be the most effective way for the patient’s recovery. The family Coronaviridae is comprised of numerous viruses infecting human and diverse animals including farm livestock and wild animals (cats, dogs, bats, cows, camels, pigs, birds, etc.). It consists of two virus subfami lies (Letovirinae and Orthocoronavirinae) including five genera and around 40 virus species [1] . The subfamily Orthocoronavirinae that contains human coronaviruses consists of four genera: Alphacoronavirus, Betacorona virus, Gammacoronavirus, and Deltacoronavirus. Corona viruses (CoV) affect various organs and tissues and act as pathogens causing a broad range of diseases including severe human respiratory infection called atypical pneu monia. Usually, viruses of this family induce acute infec tion manifested by signs of inflammation featured with properties of cytokine storm syndrome [2, 3] . Coronaviruses are enclosed by a lipid envelope (enveloped viruses) and carry genomic positive sense RNA, which is translated by host ribosomes and guides synthesis of viral proteins as well as sub genomic RNAs and subsequent replication of the viral genome and assembly of viral particles [1, 4] . Depending on species, coronavirus genomic RNA consists of 25 30 · 10 3 nucleo tides and bears 22 29 viral genes encoding relevant pro teins, four of which (N, S, M, E) play the major structur al role in viral particles (Table) . Moreover, several acces sory viral proteins functioning as ion channels (viro porins) may also be found in virions [5] . Great interest in Coronaviridae has now been raised due to emergence of the dangerous type of human pneu monia caused by the novel Betacoronavirus strain SARS CoV 2 [4] . This strain turned out to be close to bat SARS like coronavirus as well as those inducing SARS (Severe Acute Respiratory Syndrome) and MERS (Middle East Respiratory Syndrome), which caused in 2003 and 2012, severe pneumonia outbreaks in humans, referred as atypical pneumonia. Such infections did not induce a wide pandemic spread but showed a threatening pattern due to high mortality rate reaching up to 9.6 35.5% [2, 36] . Hence, the threat of the emerging coronavirus pandemic corroborates the need to develop high efficacy pharmaceuticals against coronaviruses, refining principles for using available antivirals and devel opment of pathogenetic approaches to the treatment of disease. Currently, there may be highlighted six essential chemical classes of drugs acting on diverse viral targets able to block coronavirus replication and suppress the development of disease. Such drug classes were designed based upon current knowledge about coronavirus replica tion and the pathogenetic mechanisms underlying corona virus infection, and include: (1) viral polymerase inhibitors; (2) inhibitors of the viral protease Мpro, which participates in generation of active viral poly merase; (3) inhibitors of cell proteases involved in activa tion of CoV S protein that drives virus entry into target cells; (4) endosomal inhibitors of virus deproteinization; (5) preparations containing recombinant interferons α2 and β1; (6) preparations containing antiviral antibodies. Viral polymerase is a standard therapeutic target, and its blockade inhibits replication of the viral genome and thus suppression of replication of the virus. By now, there are diverse multi specific RNA polymerase inhibitors acting on various viruses due to marked struc tural and functional similarities of this enzyme existing among different viruses [37] . Ribavirin (furanosyl car boxamide) is among inhibitors of this type because it exhibits high activity against diverse viruses [10] (includ ing coronaviruses) at concentration of 10 25 nM (IC 50 ), with selectivity index of more than 100 [ 10 12] . Because SARS, MERS, and COVID 19 (coronavirus disease 2019) mainly develop in the respiratory tract cells, riba virin aerosol inhalations that could create effective antiviral activity at non toxic concentrations in the air way epithelial layer and thus might serve as the most appropriate drug formulation. This is based on low pul monary bioavailability shown for oral vs. aerosol riba virin (1% and more than 70%, respectively) and its sub sequent activation via a phosphorylation reaction occur ring in the respiratory epithelium [12] . Of note, direct aerosol delivered action on respiratory epithelium might be most active and efficient at an early stage after the onset of infection, which is accompanied by virus repli cation at eclipse phase when pathological events of inflammation and edema would have not reached a dan gerous level. Favipiravir and its ribosylated derivatives might be other candidate drugs fighting against coronaviruses [13] ; they exhibit high antiviral potential and selectivity index with regard to diverse RNA bearing viruses [13] . On the other hand, remdisivir derived from phosphorylated 1′ cyano substituted adenosine is a broad spectrum drug displaying high antiviral potential at IC 50 ranging within 50 70 nМ against diverse viruses including coronaviruses [14, 15] . This drug is undergoing the final phase of clini cal trials [6] . Comments. 1) Genes and relevant protein names (or domains) in virus SARS CoV 2 listed in order starting from the 5′ end in genomic RNA [5] . GenBank data were used to determine the size of the protein (the number of amino acid residues) (ac.n. YP 009725301.1). 2) Classes of inhibitor agents with identified mode of action are shown. 3) Functions for proteins nsp1 nsp16 (proteolytic products derived from polyprotein 1ab) are considered elsewhere [5, 33, 34] . 4) SARS CoV 2 lacks in protein nsp3 one of two papain like protease domains but preserves ubiquitin like domains [35] . 5) A question mark (?) denotes gene products with unidentified function (no data). 6) Protease inhibitors (camostat, aprotinin, lutevirin, etc.) indirectly suppress S protein driven entry by inhibiting its proteolytic cleavage into active subunits S→S1/S2. 7) Fusion inhibiting oligopeptides targeting S protein upon entry into host cells [23] . Translation of viral RNA generating a lab polypep tide (MM ~750 kDa) that undergoes autoproteolytic cleavage into 14 16 fragments (nsp1 nsp16), depending on viral type, may function as an active viral polymerase and regulate replication of the viral genome and subse quent synthesis of viral proteins in the first stage of coro navirus replication after entering target cells [1] . It turned out that cleavage of the polyprotein 1ab (pp1ab) is medi ated by its own domain 5 (nsp 5) (called protease domain Mpro) exerting specificity of the target proteolytic sites close to picornavirus and HIV proteases [38] and being sensitive to the binary protease inhibitor lopinavir/ritona vir ("Kaletra") [6 9 ]. In some viruses, two initial breaks within the polyprotein 1ab are performed by protein nsp3 (PLpro), also bearing cysteine protease papain like domain [35] . Lopinavir/ritonavir simulating proteolytic target sites in viral proteins exhibited pronounced thera peutic effect during SARS and MERS, displaying IC 50 at the level of 5 20 nM [6, 9] . Structural similarity in pro tease domain nsp5 from CoVs causing COVID 19, SARS, and MERS allows it to be unequivocally recommend it for treatment of atypical pneumonia during COVID 19 [7, 9] . Use of this drug might be specifically efficient at the initial phase of CoV replication during an early stage of infection. Viral S glycoprotein (MM ~150 kDa) drives entry of coronavirus into target cells [1] . For this, CoV S protein undergoes targeted cleavage into two distinct subunits, S1 and S2, within the proteolytic cleavage site (amino acid positions 641 687) [38, 39] . Cell transmembrane bound protease TMPRSS2 is involved in such activation of S protein [15, 38] , whereas inhibitors targeting this pro tease can suppress cell infection and virus spread at the site of infection [16] . This therapeutic approach might be applied in clinical practice by recommending approved protease inhibitors camostat [16] and aprotinin [17 19 ]. Moreover, these antiprotease agents exert marked anti inflammatory activity via inhibiting some pro inflammatory cytokines and related signaling pathways [19, 40] that may ensure pathogenetic effects to reduce lung inflammation and edema, which might act benefi cially during the second phase of the infectious process, wherein pathogenetic mechanisms related to excessive protease activation and subsequent rise in active pro inflammatory cytokines could exert dominant effects on disease severity [41, 42] . Recent reports from China suggest that along with protease TMPRSS2 [16, 38] , cellular proteases furin and endosomal cathepsin L may also be involved in proteo lytic activation of S protein resulting in COVID 19 [16, 39, 42] . If a role for furin assumingly activating SARS CoV 2 would be confirmed, it might then be recom mended to use natural plant derived inhibitors [20] as well as synthetic oligopeptide inhibitors [21, 22] as antivi rals. Chloroquine has been widely used in medical prac tice for treatment of malaria. This drug exhibits pro nounced activity against coronaviruses in cell culture and animal models [24] . The mechanism of its action against multiple viruses including Betacoronaviruses is mediated by elevated acidic pH value inside cell endosomes that interferes with pH dependent conformational transition of viral fusion proteins (coronavirus S protein) into their active state thus resulting in retarded virus deproteiniza tion (viral uncoating) inside cell endosomes and prevents further infection of target cells; additionally, this drug may alter glycosylation of cell receptors including ACE2 used by SARS CoV and SARS CoV 2 for entry [25] . Based on this platform, chloroquine was recommended for treatment of COVID 19, and it demonstrated positive effects in some patients in China [26] . This approach to therapy of coronavirus infection implies administration of antibodies able to neutralize infectious properties of this virus. In addition, inoculation of antibodies might also be used for early disease preven tion called passive immunization. Two essential opportu nities are available for using antiviral antibodies [28 30 ]: (i) design and generation of tailored virus neutralizing antibodies (or their active antiviral domains) by using gene engineering and biotechnology. Such preparations specif ic to coronaviruses including SARS CoV 2 have not yet been created [27] ; (ii) a specific antiviral immunoglobulin preparation obtained via a more traditional and simpler technique from convalescent subjects who recovered after coronavirus infection including COVID 19 or from ani mals vaccinated with SARS CoV 2 or its components [31, 32] . The first observations have been reported of success fully administered antiviral immunoglobulins purified from convalescent subjects with MERS and COVID 19, which were used for treatment of atypical pneumonia in China during 2020 SARS CoV 2 outbreak [43] . Preparations containing antiviral antibodies should be used with some caution, because coronavirus infectiveness to immune cells was noted to be augmented by some types of artificial antibodies targeting CoV S protein [44] . Fortunately, such antibodies have not been identified yet in sera from convalescent subjects [31] . Moreover, successful administration of such preparations should require that convalescent serum immunoglobulins would contain high titer (1/80) of anti CoV antibodies assessed by HI test [32] . Preparations containing human recombinant inter feron α2 and β1 classes were used in therapy of closely related infections caused by SARS CoV, MERS CoV, and SARS CoV 2 [45, 46] . It was found that interferon β1 exerted slight curative effects, whereas interferon α2 revealed no activity, but the most prominent activity was observed after using interferon β1 in combination with ribavirin [6, 11, 46] . While choosing a treatment strategy, it should be taken into consideration that interferon preparations may exhibit peak efficacy solely at early dis ease stages, when host reaction has not yet been aug mented too much or culminated [2, 47, 48] . Moreover, use of exogenous interferon based preparations would hardly be rational at later disease stages due to the high quantity of endogenous interferons produced in response to acute coronavirus infection. Finally, inoculation of exogenous interferon based preparations at late stages of infection by further elevating pre set high level of endoge nous interferons could promote cytokine storm syndrome and inflammation at the site of infection, thereby deteri orating the disease course [3, 48] . The combined use of several drugs (pharmaceuticals) acting on various phases of the virus reproductive cycle or disease pathogenesis called a combination therapy was also administered to treat coronavirus infection. A posi tive curative effect was observed while administering riba virin, interferon β1, and lopinavir/ritonavir [6, 11, 46] . This approach allows: (i) improving therapeutic efficacy, (ii) reducing drug dosing, (iii) preventing emergence of dangerous viral mutants with augmented virulence. It is known that a risk of developing viral mutants in an infect ed host is minimized when applying combination therapy [49] . Two major stages can be distinguished within the timeframe of developing acute viral disease. The early (etiotropic) phase dominated by virus replication is called the exponential phase of virus propagation and accumu lation (day 7 10 from the onset of viral infection). It is characterized by emergence of developing virus specific defense reaction to infected host cells (production of immunoglobulins, antigen specific T and B cell clones, and interferons) and general inflammatory response due to cytokines and chemokines synthesized at the site of infection. Later, growth of the virus declines and this results in developing a pathogenetic phase associated with formation of pathologic mechanisms critically affecting disease outcome as well as posing a threat of potential complications. Among the latter are exuberant inflamma tion, developing acute respiratory distress syndrome, lung edema and hypoxia, as well as emergence of infections caused by pathogenic microbes and sepsis [2, 3, 50] . Owing to a two phase pathogenesis in viral diseases, it is reasonable to build a proper therapeutic strategy. In particular, specific antivirals should be available in the therapeutic arsenal and dominate in treatment during the etiotropic phase. In the case of coronavirus it can be referred to as the phase for use of lopinavir/ritonavir, aerosol ribavirin inhalation, injection of virus specific antibodies and inhibitors of host proteases, and interfer on preparations (particularly interferon β1, see above the corresponding sections). On the contrary, the patho genetic phase should rely on a therapeutic strategy aimed at restriction or relief of pathological life threating mech anisms by taking into consideration patient condition and severity of pathological events, mainly to eliminate intox ication, reduce lung edema, and improving blood oxy genation to compensate for lung failure, mostly by extra corporeal membrane oxygenation of blood (ECMO) for avoiding rupture of swollen lungs in case of involuntary inhaled oxygen therapy. In addition, anti inflammatory drugs should be also used during this phase to recover res piratory function by paying special attention to antibac terial therapy to prevent emerging secondary bacterial pneumonia and concomitant sepsis [51, 52] . On the other hand, it might not seem very reason able to use antivirals during a pathogenetic phase of infection for two main reasons: (i) specific antiviral anti bodies as well as B and T cell clones inhibiting virus growth and removing host infected cells have been already formed by the onset of this phase [3, 32, 48] , and (ii) avoiding use of such drugs would contribute to lower ing their toxic side effects on developing mature immune response that includes specific antibodies, T and B cell clones, as well as interferon response. However, it should be noted that coronavirus disease might potentially be exacerbated due to virus evolution and continuous replacement of the initial parental virus in a single host. Such a phenomenon was described for Betacoronavirus, so that a more virulent within patient virus strain may emerge during disease progression that could markedly aggravate it and pose a threat to the patient's (or ani mal's) life [53 55] . Hence, in the case of developing impaired immune response and signs of residual viral infection, administration of antivirals should be contin ued to lower a risk of developing higher virulence viral ZHIRNOV BIOCHEMISTRY (Moscow) Vol. 85 No. 5 2020 mutants. It seems important that a combination therapy affecting various targets in viral growth should be appro priate to use even at early the phase of infection in order to efficiently prevent emergence of highly virulent viral mutants. Betacoronavirus, including SARS CoV 2, elicits infection of the respiratory tract often ending with the development of lung edema, severe hypoxia, and sepsis. Two phases referred as etiotropic and pathogenetic can be highlighted in disease pathogenesis. During the first stage, virus growth and accumulation dominate, which is accompanied by appearance of initial pathological distur bances in the respiratory tract. However, during the sec ond stage virus propagation declines, but pathological events mainly manifested as excessive inflammation and lung edema develop as a secondary consequence of virus induced cytopathic effects. Whereas in the first stage it is justified to use pharmaceuticals and their combinations (aerosol ribavirin inhalation, lopinavir/ritonavir, protease inhibitors, interferon compounds, antiviral antibodies) aimed at suppressing diverse targets during virus propaga tion, during the second disease stage it might be impor tant and reasonable to rely on administration of patho genetic drugs to restrict life threatening events resulting in marked inflammation, intoxication, hypoxia, second ary pneumonia, and sepsis. 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I am sincerely grateful to Dr. V. O.