2009 Drug Targets in Severe Acute Respiratory Syndrome (SARS) Virus and other Coronavirus Infections

Infectious Disorders - Drug Targets 2009, 9, 223-245 223

1871-5265/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd.

Drug Targets in Severe Acute Respiratory Syndrome (SARS) Virus and other Coronavirus Infections

Tommy R. Tong*

Department of Pathology, Montefiore Medical Center, Bronx, NY

Abstract: Coronaviruses are important human and animal pathogens of the order Nidovirales. Several new members were discovered following the emergence of SARS-CoV in human populations, including two human coronaviruses and several animal coronaviruses. They cause respiratory and gastrointestinal illnesses and have been found in the brains of patients with multiple sclerosis. The high mortality of SARS, the identification of a natural reservoir, and the well-founded fear of provoking antibody-enhanced disease as a result of vaccination fueled the ongoing efforts in anti-coronavirus drug discovery. This review presents the results of current research.

Keywords: Coronavirus, SARS-CoV, Severe acute respiratory syndrome, SARS, main protease, 3CLpro, polymerase, helicase, interferon, interferon-inducer, antibody

INTRODUCTION

The coronaviruses, together with the arteriviruses, belong to the order Nidovirales, by virtue of sharing the trans-criptome of a nested set of subgenomic mRNAs [1]. They are enveloped positive-sense single-stranded RNA viruses (Baltimore group IV). The coronaviruses have the largest genome (around 30K) among the RNA viruses. They infect humans, mammals and birds. Phylogenetically, the coro-naviruses can be divided into three subgroups, two of which are mammalian (groups 1 and 2) and the remaining avian (group 3). This corresponds to the serotype classification. The previously well-studied human coronaviruses (HCoV), -OC43 and -229E, and the novel HCoV-NL63 and -HKU1, and SARS-CoV, are found in groups 1 and 2, along with various other mammalian coronaviruses. The recently discovered SARS-CoV belongs to subgroup 2b, along with bat-CoV, the putative ancestor [2-5], There is evidence that H-CoV-OC43 is also a zoonosis, probably arising from bovine CoV [6].

Diseases caused by the generally epitheliotropic viruses include respiratory and gastrointestinal illnesses. However, neurotropism is demonstrated in brains of patients suffering from multiple sclerosis [7, 8]. Other tissues may also be susceptible, as seen in SARS [9, 10].

Severe acute respiratory syndrome (SARS), caused by SARS-CoV [11-16], emerged in late 2002 in southern China, spreading to 29 countries within a few weeks, and infected ~8,000 people resulting in close to 800 deaths. It is a pneumonia with 10% fatality rate [11, 12, 17-19] and pathogenesis encompassing viral infection of pneumocytes in the lower respiratory tract [20-23], disturbance of the rennin-angiotensin system [24-29] and alterations in the innate [30] and adaptive immune systems [31]. After the epidemic, the natural reservoir was identified, the viral life

*Address correspondence to this author at the Department of Pathology, Jack D. Weiler Hospital, 1825 Eastchester Road, Bronx, NY 10461; Tel: 661-889-8218; Fax: 661-885-5297; E-mail: [email protected]

cycle studied for therapeutic intervention, and many drug leads are identified.

THE LIFE CYCLE OF CORONAVIRUSES

As with all viruses, coronavirus infection begins with viral attachment to specific cellular receptors. The receptor for SARS-CoV [32] and HCoV-NL63 [33] is angiotensin I converting enzyme 2 (ACE2), to which the viral spike protein attaches. The HCoV-229E receptor is CD13 or human aminopeptidase N [34], homologous versions of which are employed by transmissible gastroenteritis virus (TGV) and feline infectious peritonitis virus (FIP) [35], whereas HCoV-OC43, bovine coronavirus (BCoV) and the porcine hemagglutinating encephalomyelitis virus (HEV) employ N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) as receptor [36]. HCoV-OC43 also binds to MHC class I molecule [37], whereas SARS-CoV also binds the HIV-attachment factor dendritic cell-specific intercellular adhe-sion molecule-grabbing non-integrin (DC-SIGN or CD209), a C-type lectin expressed on dendritic cells, and CD209L (L-SIGN or DC-SIGNR) [38, 39]. Another coronavirus, mouse hepatitis virus (MHV) utilizes a member of the immuno-globulin superfamily, carcinoma embryonic protein (CEA) homologue, mCEACAM or CE66a, as receptor [40, 41]. ACE2, a topologically type I transmembrane protein, is the principal receptor for SARS-CoV, and is expressed in the respiratory and gastrointestinal tracts, liver, and kidneys, as reflect in tissue tropism. Following viral attachment to the receptor, conformational change of the S2 region of the spike protein takes place, a process involving heptad repeats (HR1 and HR2). Thus coronavirus S protein belongs to the class I fusion protein [42]. In many coronaviruses, such as murine hepatitis virus (MHV), the S proteins are cleaved by cellular furin-like proteases ~200 residues upstream of the fusion peptide, giving rise to a receptor binding S1 unit and a membrane-anchored fusion unit (S2). In other coronaviruses, this furin cleavage does not occur. However, SARS-CoV entry involves an endosomal protease, cathepsin L (CTSL), in both clathrin-dependent [43] and clathrin- and caveolae-

224 Infectious Disorders - Drug Targets 2009, Vol. 9, No. 2 Tommy R. Tong

independent manners [44]. Recently, there is experimental evidence of cleavage of SARS-CoV S at position T678 between the S1 and S2 domains by CTSL. The cleavage site maps to the same region of furin-cleavage sites in MHV [45]. The conformational change of S2 is instrumental to viral entry by approximating the viral envelop and the cell membrane that leads to fusion of these lipid layers and the access to the cytosol by the viral nucleocapsid. Following entry of the positive-sense, single-stranded RNA genome, translation of polyprotein 1a and polyprotein 1ab begins without delay. The latter is achieved by programmed -1 ribosomal frameshifting just 5’ of the termination codon of ORF1a, first published as a novel mechanism of gene expression in Rous sarcoma virus [46]. This process requires a hepta-nucleotide slippery site sequence of X XXY YYZ (X - any nucleotide, Y - A or U, Z - A, U or C) and a 6 - 7 nucleotide downstream “kissing loop”, or in the case of SARS-CoV, a three-stemmed pseudoknot [47, 48]. More recently, fine-tuning of frame shift efficiency in SARS-CoV through an in cis upstream attenuation sequence is identified [49]. The polyproteins thus formed are co- and post-translationally processed by two proteases into sixteen nonstructural proteins (nsp). These proteases are encoded in ORF1a - a papain-like (accessory) cysteine proteinase (PLpro, nsp3) and a 3C-like (main) proteinase (3CLpro or Mpro, nsp5) [50-53]. Pp1a is processed into nsp1 to nsp11, and pp1ab into nsp1 to nsp10 and nsp12 to nsp16 [54]. These nsp assemble into the multiple subunit viral replicase and include such enzymes as RNA-dependent RNA polymerase (RdRp, nsp12), helicase (nsp13), exonuclease (nsp14), endoribonuclease (nsp15), and methyltransferase (nsp16), and two putative membrane proteins (nsp4 and nsp6). The next phase of the life cycle involves transcription of a nested set of functionally monocistronic subgenomic mRNA (sgRNA) from a negative-strand intermediate, an attribute common to the order Nidovirales. These are translated into viral structural proteins for the reproduction of virions. Also translated are possibly pathogenic accessory proteins that differ among the Coronaviridae and which are not essential to viral viability. Transcription of the sgRNAs are achieved through a unique discontinuous mechanism resulting in the acquisition of a common 5’-terminal leader sequence from the 5’ end of the genome [52]. RNA synthesis is protected and sequestered within “parasitic vacuoles” - endoplasmic reticulum/Golgi or autophagosome-derived [55, 56] mem-brane-associated replication/transcription complexes [57]. The assembly of virions is triggered by genome-associated Nucleocapsid (N) and lipid membrane-associated Membrane (M) protein in membrane-bound endoplasmic reticulum/ Golgi intermediate compartment. Post-translational modifi-cations (glycosylation, acylation, and cleavage) of structural proteins take place within Golgi apparatus/ endoplasmic reticulum, and mature virions are released after trafficking to the cell surface.

The SARS-CoV genome is replicated by a complex series of time-consuming steps (generation time of 17 – 19 hours in FRhK cells [58]) and is reflected in the complexity of its proteome, which contains RNA processing activities not found in other RNA viruses and yet to be identified in the less well studied members of the Coronaviridae. This includes 3’-to-5’ exonuclease activity (ExoN, nsp14) that is

possibly involved in RNA proofreading and repair [59]. Also identified are poly(U)-specific endoribonuclease (XendoU, nsp15) involved in small nucleolar RNA processing and utilization, and 2’-O-methyltransferase (2’-O-MT, nsp16) involved in the capping of viral RNA [59].

The host cell is not defenseless. Indeed, the host range of coronaviruses is dictated by the affinity of the viral spike protein for the cellular receptor, without which the virus cannot gain entry into the cytoplasm. Moreover, RNAi [60-64], the interferon system [65], the major histocompatibility (MHC) system, and various cellular proteins and enzymes (e.g. MxA, 2’,5’ oligoadenylate synthetase, RNA-specific adenosine deaminase (ADAR1 [66]), PKR (p68 kinase), RNase L, and APOBEC [67]) serve to retard the march of the virus and “buy time” for the development of specific adaptive immunity against the virus. These together with viral proteins and other cellular processes utilized by the virus represent legitimate drug targets.

In addition, the host organism employs various molecules of the innate immune system (e.g. mannose-binding lectin [68, 69]), and the adaptive immune system to prevent viral dissemination.

TARGETS FOR INTERRUPTION OF CORO-NAVIRUS LIFECYCLE

Interferon and Innate Antiviral Defense

The interferons (IFN) are cytokines that work both locally and systemically to prevent viral dissemination [65, 70]. The interferon system is coupled with the sensory arm of pathogen pattern recognition receptors (PRR), including toll-like receptors (TLR), RNA-dependent protein kinase (PKR), RNA helicases such as retinoic acid inducible gene-I (RIG-I) and the homologous melanoma differentiation-associated gene 5 (mda-5) [71] and the effector arm of antiviral molecules including, PKR, 2’,5’-oligoadenylate synthetase (OAS), RNase L, RNA-specific adenosine deaminase (ADAR1), protein Mx GTPase, inducible nitric oxide synthase, among others. PKR is constitutively present and is activated by dsRNA and ssRNA. It inhibits mRNA translation by phosphorylation of eIF-2 . OAS works with RNase L to degrade RNA. OAS is activated by dsRNA. It has several isoforms encoded by three genes on chromosome 12. The product, several different oligoadenylates of the general structure ppp(A2’p)nA, causes dimerization and activation of the endonuclease RNase L. In-vivo mouse studies have demonstrated the importance of the OAS/RNase L antiviral system by showing a more rapidly fatal course in RNase-/- mice as compared with RNase+/+ mice infected with encephalomyocarditis virus. Many in-vitro studies have demonstrated that upregulation of OAS correlates with an antiviral state (reviewed in [65]). ADAR1 edits dsRNA by deamination of adenosine to yield inosine, which indis-criminately base-pairs with adenine, thymine or cytosine.

SARS-CoV employs various strategies to intercept the information in the interferon system. Thus, induction of INF-

by IRF-3 in response to infection is abrogated by re-localization of IRF-3 from the nucleus back to the cytoplasm [72]. SARS-CoV ORF6 antagonizes STAT1, and hence interferon function, by sequestering nuclear import factors

Drug Targets in Severe Acute Respiratory Syndrome (SARS) Infectious Disorders - Drug Targets 2009, Vol. 9, No. 2 225

on the rough endoplasmic reticulum/Golgi membrane [73]. Nsp1 promotes host mRNA degradation [74] and reduces STAT1 phosphorylation [75]. In addition, the nucleocapsid (N) protein inhibits synthesis of interferon [76], and OF3b inhibits the synthesis of interferon and signaling [76].

Despite all this, several uncontrolled clinical studies during the SARS epidemic have suggested activity of interferons against SARS-CoV [77-82]. Alfacon-1, an FDA-approved anti-HCV inhibitor tested in clinical trials, is a consensus molecule synthesized and assembled artificially from sequences of several natural IFN- by assigning the most frequently observed residues in each corresponding position. In an uncontrolled, open-label clinical trial involving 22 SARS-infected patients during the epidemic in Canada, its use together with corticosteroids resulted in significant reduction of morbidity and no mortality [79]. This is corroborated by in-vitro studies showing significant antiviral activity in neutral red uptake and virus yield reduction assays with pre-incubation of the cell cultures with Alfacon-1. Importantly, a human bronchial epithelial cell line (Calu-3) [83] showed better IC90 and SI compared with African Green Monkey cell lines (Vero 76, Vero E6, and MA104) [84], at 0.046 ± 0.011 ng/ml and 2173, respectively [84].

Additional in-vitro studies showed that IFN- and IFN- selectively inhibited SARS-CoV replication in cell culture [77, 84-90], a finding corroborated by the identification of IFN- in a drug screen employing a novel non-cytopathic, selectable, HCoV-229E-based replicon RNA [91]. It was also found that natural IFN- and IFN- have more potent in-vitro activity than recombinant IFN- [88]. Alferon N injection, a mixture of several species of IFN- ( 2, 4, 7,

8, 10, 16, 17), is the only approved natural alpha-interferon available in the US. Animal studies with macaque monkeys (Macaca fascicularis) demonstrated protection from SARS-CoV by prophylactic use of pegylated IFN-α [92]. Post-exposure treatment with IFN yielded intermediate results [92].

VIRAL ENTRY INHIBITORS

Coronavirus Spike protein (S) is a type I fusion protein, binding to the receptor as well as enabling fusion of the viral envelope with the host cell membrane. Thus both the receptor binding S1 and the fusion S2 domains are targets for antiviral therapy. Here we discuss monoclonal antibodies targeting the RBD, peptide drugs targeting the S2 domain, CTSL inhibitors, and several small molecule inhibitors of viral entry.

Engineered Monoclonal Antibodies (MAb)

Medicinal therapy was much enriched since the use of antibodies over a century ago. Whereas applications for bacterial infections are mostly probable or unproven, the list of viral diseases that are amenable to antibody therapy is increasing, mostly in the area of prophylaxis [93]. Anti-biotics have mostly displaced antibodies in bacterial infections and small molecule drugs are competing with antibodies for viral infections. Antibody as medicine has recently been reviewed [94, 95]. As in the case of vaccines, antibodies need to be refrigerator (2 – 8 C) during

distribution and storage. However, because of its long in-vivo half-life of 20 days and a prolonged shelf-life (Pali-vizumab lyophilized form has shelf-life of 3 years), MAb definitely has a place in the pharmacy. Fully humanized antibodies, with elimination of unwanted immunological side effects, and other recent advances are promising to expand our repertoire of drugs as diverse as that of our adaptive immune system [95, 96]. Signs of changing times are found in the FDA-approval of passive immunopro-phylaxis of neonatal respiratory syncytial virus (RSV) infections, with the new addition of a humanized monoclonal antibody (MAb) Palivizumab (MEDI-493, Synagis ) to the existing polyclonal hyperimmune globulin (RespiGam) [97, 98]. Even more potent and longer-lasting second and third generation MAbs against RSV are currently undergoing phase III clinical trials (motavizumab) and development (Numax-YTE) [98].

Although antibody-dependent enhancement (ADE) of infection occurs in coronavirus infection, as will be addressed later, it is reassuring that neutralizing convalescent or engineered antibodies have shown therapeutic potential [99]. Convalescent serum has been used in SARS patients during the epidemic, and in mice without ill effect [100-102]. Experimental studies employing HIV(S), a novel SARS-CoV S protein-pseudotyped retrovirus, demonstrated virus neutralization with convalescent sera, even before the cellular receptor for SARS-CoV is identified [103].

Antibodies from SARS-Immune Patients

An efficient approach to developing MAb against SARS-CoV is from immune patients. Traggiai et al. interrogated the B-cell memory repertoire of a convalescent SARS patient and identified thirty five neutralizing MAbs. IgG+ memory B-cells elaborating SARS-CoV-specific antibodies were isolated by a combination of magnetic and fluorescence-activated cell sorting (FACS). The B-cells were directly immortalized by EBV, in the presence of a polyclonal memory B-cell activator, CpG 2006 (a CpG oligonucleotide that activates toll-like receptor 4) and irradiated allogeneic mononuclear cells. One of the antibodies, S3.1, an IgG1-kappa neutralizing antibody, prevented SARS-CoV repli-cation in mice lungs [104]. This MAb, along with two others (S111, S127), were subsequently found to exhibit ADE [105].

Also starting from SARS convalescent patients, Duan et al. constructed an immune antibody phage-display library with diversity of 1.85 x 106 from four SARS convalescent patients [106]. B1, a human scFv recognizing an epitope on S2, was characterized. It has high binding affinity for SARS-CoV virions (K(d) = 105 nM) and potent neutralizing activities against infection by pseudovirus expressing SARS-CoV S protein.

The technique of generating MAbs from a patient’s immune repertoire has several advantages, including the delivery of large numbers of potential antibodies for selection according to criteria such as affinity, epitope specificity, and propensity to generate escape mutants [104]. However, the repertoire is not unlimited. For example, a patient who recovered from humanized SARS-CoV does not produce antibodies specific for civet-SARS-CoV.


Antibodies from Non-Immune Libraries

Without using immune B-cells, Sui et al. employed two highly diverse (2.7 x 1010) non-immune human single-chain variable region fragment (scFv) libraries constructed from 57 human donors for the identification of MAb against recombinant SARS-CoV S1 domain (residues 12-672, 12-327, or 261-672) [107]. One of the eight scFvs identified neutralized SARS-CoV and competed with soluble ACE2 for binding to S1 with high affinity (equilibrium dissociation constant, Kd = 32.3 nM). 80R IgG1, a monoclonal antibody engineered from the fragment possessed 20 times the neutralizing activity of the scFv fragment with a Kd of 1.70 nM with S1. It was further demonstrated to confer resistance to infection in a mouse model when given prophylactically [108]. Since not all strains of SARS-CoV (e.g. GD03) tested are sensitive to the 80R MAb, it is strategically important to monitor the viral genotype for prediction of efficacy [107].

In a similar fashion, a human IgG1 antibody, CR3014, was generated with antibody phage display technology from a large naïve semisynthetic [109] antibody library [110, 111]. CR3014 exhibited potent in-vitro viral neutralization and in-vivo protection of ferrets from macroscopic lung pathology [110]. It apparently recognizes a non-linear conformational epitope within the S1 domain which cannot be resolved by PEPSCAN analysis and which is probably different from that recognized by 80R [111]. Another neutralizing MAb, CR3006, is susceptible to viral escape. It loses its binding affinity with S with the introduction of naturally occurring amino acid substitutions of residues Y442 or F360, L472, D480, and T487, as are present in two different SARS-CoV isolates [111]. Because of ADE and viral escape, this group employed a combination of mono-clonal antibodies. They showed that the subsequently identified neutralizing MAb, CR3022, is active against CR3014 escape mutants and is not prone to new escape variants. It is also synergistic to CR3014, permitting a lower dose of either antibody for passive immunoprophylaxis of SARS-CoV infection [112].

Antibodies from Vaccinated Animals

Vaccinated transgenic mice (Medarex, Inc.) developed a humoral immune response to SARS-CoV [113]. Using these animals as a starting point, Greenough et al. identified two neutralizing antibodies, MAb 201 (IgG1) and MAb 68 (human heavy chain and murine light chain). Immuno-precipitation analysis defined the neutralizing epitopes as within (aa 490-510) and outside (aa 130-150) the RBD, respectively. The fully humanized MAb 201 and the chimeric MAb 68 were both effective in protection of the mouse model from SARS-CoV. In the Golden Syrian hamster model of SARS-associated pulmonary pathology, MAb 201 given postexposure afforded alleviation of pulmonary pathology as well as reduction in viral titer [114]. The epitope recognized by MAb 201 is apparently invariant [115-117]. It is planned for clinical trial in the event that SARS should return [113].

Recently, IgG2 human MAbs have been generated from transgenic mice (XenoMouse®), with neutralizing activity against SARS-CoV S protein [118]. These IgG2 antibodies are less susceptible to ADE because of the lack of Fc

receptors on macrophages and phagocytes. They are also more readily available in extracellular fluids and do not activate the classical complement pathway. Although more laborious because transgenic mice are immunologically less robust and require more immunizations and antibody screen-ings, a successful immunization can yield a multiplicity of MAbs that could be combined as a cocktail to more effec-tively combat the pathogen. No further antibody engineering is required with this approach.

Antibody-Dependent Enhancement, Escape Variants and other Considerations

Although antibodies have therapeutic potential, antibody-dependent enhancement (ADE) of coronavirus pathogenicity is a concern, as researchers of feline infectious peritonitis virus have shown [119]. An experimental animal model for SARS, the ferrets, exhibited a more severe hepatitis when first given a vaccinia-based recombinant SARS vaccine [120, 121], although a mild response elicited by a formalin-inactivated SARS-CoV vaccine given to ferrets showed no evidence of enhanced liver or lung disease [122]. There is also evidence that antibodies raised against the human strain of SARS-CoV mediated enhanced entry of a lentiviral vector expressing civet S [123].

Yang et al. demonstrated that immunoglobulins (e.g. S111, S127 and S3.1) isolated from patients and directed against a human strain could potentiate infection by civet-SARS-CoV [105].

Antibody-Enhancement of Infection and Viral Escape can

be Overcome

Additional steps during screening may produce antibodies that do not enhance infection, as shown by the identification of neutralizing MAb S110, which did not enhance entry of civet-SARS-CoV [105]. Other antibodies, such as m396 and S230.15, neutralized all known epidemic and zoonotic strains except bat-SARS-CoV [124]. The MAb m396 had IC50 of 0.1 and 0.01 μg/ml, respectively, against GD03 and Tor2 S-glycoprotein pseudotyped-HIV-1 viruses. In laboratory mice, the serum neutralizing antibody titers correlated with in-vitro neutralizing activity and conferred protection against replication-competent recombinant SARS-CoV from 2002/2003 (icUrbani) and 2003/2004 (icGD03), as well as civet-SARS-CoV (icSZ16). Two highly conserved residues, Ile-489 and Tyr-491, likely accounted for the broad-spectrum neutralizing activity, as determined by alanine-scanning site directed mutagenesis of S protein in combination with analysis of the crystal structure of the RBD.m396 complex. The relative potency (IC50) of m396 and S230.15 compared favorably with other published MAbs, as tabulated by Zhu et al. [124].

Summary and Perspective

Could MAb therapy be harmful? The case of TGN1412 illustrates the potential for disaster [125], demanding careful pre-clinical studies before the clinical trial. Even well tested MAb may have unexpected side effects, as recognized after widespread application of the highly successful therapeutic breast cancer MAb against HER2 [126, 127].

Acknowledging the potential for unexpected side-effects, antibody-enhanced viral infection [105] and anti-antibody


response [96], passive immunoprophylaxis with carefully selected fully human MAbs or combination holds great promise against SARS-CoV and other viral infections, such as rabies [109]. The specificity however, precludes its appli-cation to other coronaviruses, such as bat-SARS-CoV. Indeed, genotype monitoring is imperative as part of the overall strategy.

The rise of the research community with medicinal MAb to the challenge of SARS has proved not only that MAbs effective against an emerging pathogen can be precisely engineered but also that the drug can potentially be brought to the shelf in a timely fashion to make an impact. The technology is not without challenge, however, as previously discussed and reviewed [96], including the ability of the virus to quickly evolve and mutate into a drug resistant variety. Thus, a comprehensive strategy against an emerging virus would include not only production of effective antiviral drugs, but also the continued surveillance for drug resistant mutants. The challenges posed to advocates of MAbs hold true also for other specific antivirals drugs.

In the decades to come, we could expect a proliferation of know-how, a race to commercialize, and a dizzying increase in the virtually limitless number of medicinal MAb. The creative combinations of MAb cocktails [112, 118], MAb with other anti-infectives or vaccine, and fine-tuning of the Fc-effector and other molecular attributes, such as glycosylation, permit even broader applications.

Membrane Fusion Inhibitors

Peptides - Heptad Repeat Inhibitors

The enveloped coronaviruses utilize a similar mechanism as HIV-1 in achieving membrane fusion with the host cell to effect entry of the nucleocapsid. Following binding of the RBD of S1 with ACE2, the S2 domain undergoes a confor-mational change, with the N-terminal HR1 and C-terminal HR2 heptad repeats (HR) interacting with each other in an oblique anti-parallel manner. The resulting hairpin configuration and oligomerization of S proteins into a six-helix bundle fusion core approximates the viral envelope and the lipid cell membrane, resulting in fusion [128-130]. Inhibitors that block the hairpin formation between HR1 and HR2 can prevent the formation of the fusogenic complex and viral entry [131]. In addition, the conservation of the HR regions suggests that it is a good drug candidate. As predic-ted, spike protein HR-derived peptides have been shown to inhibit SARS-CoV infection of Vero cells [132]. These peptides have -helical structures and show coiled-coil interaction with the S2 heptad region. Bosch et al. evaluated peptides derived from both heptad repeat regions. HR1-derived peptides are generally ineffective as inhibitors of SARS-CoV (strain 5688, fourth passage) entry into Vero 118 cells, with EC50 exceeding 50 μM. This is also noted by other investigators [133, 134]. Four HR2-derived peptides (sHR2-1, sHR2-2, sHR2-8 and sHR2-9) had EC50 ranging from 17 – 43 μM. The inhibitory activity of these peptides is inferior when compared with the peptide inhibitor for MHV [135]. CP-1, also derived from HR2, had similar inhibitory activity of 19 μM on entry of Vero E6 cells by SARS-CoV (WHU strain) [133]. Another report evaluating 25 HR-derived peptides for entry inhibition of HIV-luc/SARS

pseudotyped virus identified HR1-1 and HR2-18 as being active, with EC50 of 0.14 and 1.19 μM, respectively [136]. Against wild-type SARS-CoV, the corresponding EC50 were 3.68 and 5.2 μM, respectively,

In an effort to reduce the cost of production and improve the stability, stable recombinant proteins were engineered. HR121 and HR212, comprising HR1 and HR2 linked in HR1-HR2-HR1 and HR2-HR1-HR2 configurations, respec-tively, demonstrated IC50 of 4.13 and 0.95 μM, respectively, on entry of the HIV/SARS pseudoviruses [137].

Peptides that Bind S and Block Interaction with ACE2

Ho et al. identified a putative RBD of SARS-CoV S protein (residues 660-683) using peptide scanning [138]. A small synthetic peptide, SP10 (STSQKSIVAYTM, derived from residues 668-679 of SARS-CoV S) was shown to inhibit binding of the 138-kDa recombinant S to ACE2 in ELISA study. It also inhibited the entry of S protein-pseudotyped retrovirus into Vero E6 cells. Inexplicably, two other peptides binding outside of the putative novel RBD also exhibited significant inhibitory activity (SP-4 [residues 192–203], SP-8 [residues 483–494]).

Peptides Analogous to Loop Region Prevent Fusion of

Viral Envelope with Cell Membrane

Sainz et al. posited that peptides analogous to the hydrophobic membrane fusion region of S2 might be inhibitory of viral entry. They used a computational appr-oach to identify five regions of high interfacial hydro-phobicity in S2 that might interact with lipid membranes. Novel, randomly coiled peptides based on these regions are synthesized and tested in plaque-reduction assay using SARS-CoV (Urbani strain) and murine hepatitis virus (MHV). Those analogous to the N-terminus or pre-trans-membrane domain of the S2 subunit showed modest inhibitory activity (40-70% inhibition by SARS-CoV plaque reduction assay at 15-30 μM) [139]. However, peptides analogous to the loop region (WW-III and WW-IV) are more potent (>80% plaque reduction at 30 μM; IC50=2-4 μM), establishing a new class of peptide inhibitors directed to regions outside the HR regions. These peptides did not affect the viability of Vero E6 cells treated for 2 hours with 30 μM of SARS-CoV peptides.

Peptides that Prevent Interaction of S Monomers and

Peplomer Formation

Another group investigating SARS-CoV S sequence differences between animal and human strains noted inhibitory activity of 20-mer synthetic peptides designed against these areas. Three of these peptides mapped to the interface between the three monomers of the trimeric peplomers [140]. These regions might be involved in virion-cell membrane binding sites, as co-receptor binding sites, or as proteinase cleaving sites. P8, corresponding to residues 737-756 of S protein (S2 domain), showed the strongest inhibitory activity on SARS-CoV (strain GZ50) cytopathic effect in FRhK-4 cells. No cytotoxic effect was observed at the concentration used (up to 100 μg/ml). The IC50 was 2.9 μg/ml. Proof of inhibition of viral entry as the antiviral mechanism was established by showing higher viral titers without than within the cells when peptides were given prior


to viral inoculation of the cell culture, and by showing lack of inhibitory activity when applied 1 hour after viral inoculation. The peptides showed dose-dependent inhibitory activity and synergism, with three-peptide combinations more efficacious than two. However, the peptides are specific to the animal species rather than showing broad-spectrum inhibitory activity.

Peptide Against other Regions of SARS-CoV S

Yet another group linked two peptides derived from two regions of the SARS-CoV receptor, ACE2, and obtained potent inhibitory activity against entry of a pseudovirus [141], with IC50 of ~ 0.1 μM [142]. The peptides were designed to bind SARS-CoV S. One segment was based on a region spanning residues 22 - 57, where charged residues, in particular K26 and D30, critical for infection, are located, as determined by alanine scanning mutagensis. Modest antiviral activity, with IC50 of 50 μM and 6 μM were obtained with single peptides derived from residues 22 – 44 and 22 – 57, respectively. The activity was specific (viruses pseudotyped with MuLV envelope protein and VSV-G-pseudotyped MuLV were not inhibited, even at 100 μM), and no cytotoxicity was observed with 200 μM.


Peptide drugs have come of age, with over 40 already available for clinical use. These include the blockbuster Humalog (insulin lispro) from Eli Lilly. Enfurvitide (Fuzeon ), approved by the FDA in March 2003, is another example. It is a 38-residue peptide based on HR2 of the HIV-1 glycoprotein (gp41).

Apart from the disadvantage of parenteral administration, peptides and proteins compete with small molecule drugs, which have better stability, improved delivery across hydrophobic cell membranes, longer half-lives, and low risk of immunogenic effects. However, peptides and proteins are more active and specific and therefore have less nonspecific side effects, have minimal drug interactions, do not accumulate in the body, and have fewer toxicology issues from xenobiotic metabolism.

Like other antiviral drugs, peptides are not exempt from viral escape, as reported recently [132]. Fortunately, it appears that the virus has limited options for escape from heptad inhibitors because the same A1006V substitutions that determined the resistance phenotype appeared first in four independently passaged viruses.

Enfurvitide for HIV is expensive because of long-term prescription. Anti-coronaviral peptide drugs, however, will mainly be employed for prophylaxis or short-term therapy, as there is no persistent infection. As such, peptides are useful drugs that should meet the requirements as a drug for rapid deployment and stockpiling in the event of a new outbreak.

Other Viral Entry Inhibitors

Cathepsin L Inhibitors

The details of coronavirus entry have not been completely elucidated, but it is now known that cathepsin L (CTSL) is required for SARS-CoV entry into the cytosolic

compartment via the endosomal pathway [143]. In this model, SARS-CoV S1 engages the receptor ACE2. S2 then mediates conformational change. It is followed by the third step of proteolysis by an endosomal enzyme, CTSL. The last step, however, is not required by the subsequently discovered HCo-V NL63, which also utilizes ACE2 as the cellular receptor [144].

Cathepsins are endosomal and lysosomal proteases with endo- and exopeptidase activities and diverse functions. These enzymes have been known to play a role in cellular entry of reovirus and recently, Ebola virus [145, 146]. They can be divided according to their catalytic activities as aspartyl, serine, or cysteine proteases. Cathepsin S is inducible by interferon (IFN)- in MHC-class II expressing cells and plays a pivotal role in the maturation and peptide-binding competency of class II molecules [147]. CTSL is one of the major proteases in mammalian cells with broad activity against intracellular proteins, as well as a variety of extracellular matrix proteins. It is implicated in the malignant transformation because of its co-regulation with cell growth [148]. Recently, CTSL was found to be required by endothelial progenitor cells for neovascularization after ischemia [149]. Because malignant tumors are dependent on neovascularization, CTSL might be a novel oncologic drug target.

Because of its role in SARS-CoV entry, CTSL was investigated as a novel target. A protease inhibitor, MDL28170 (also known as calpain inhibitor III or Z-Val-Phe-CHO), was identified in a chemical library of 1,000 pharmacologically active compounds. MDL28170 has an IC95 of 2.0 mM against HIV-luc(SARS-S) entry of ACE2-transfected 293T cells, as well as SARS-CoV replication (data not provided) [143]. Several other commercially (Calbiochem, San Diego) available CTSL inhibitors, Z-FF-fluoromethylketone, Z-FY-CHO, Z-FY(tBu)-dimethyl-ketone, and 1-Naphthalenesulfonyl-IW-CHO are also active, with IC95 of 0.8 – 1.8 mM. Cytotoxicity data is not available.

The fact that HCoV-NL63 does not require CTSL for entry suggests that this class of medicine does not have broad spectrum activity against the coronaviruses. It is also possible that SARS-CoV may mutate under selection pres-sure into escape mutants independent on CTSL for cellular entry.

Moreover, CTSL enhances HIV-1 infection by disrupting lysosomal interference with productive HIV-1 infection [144], raising the concern of similar effect with some other viruses, and mandating virological diagnosis before insti-tution of CTSL inhibitor therapy. Furthermore, the endo/ lysosomal system is vital in antigenic processing and the adaptive immune system, as highlighted by recent findings that administration of a CTSL inhibitor, CLIK148 [150], shifted a protective Th1 anti-parasitic (to Leishmania) response to a devastating Th2 response in laboratory mice, pointing to potential dangers with targeting host proteins [151].

VE607

This molecule blocks SARS-CoV S protein-ACE2-mediated viral entry [152]. It is identified with a “chemical genetics” approach to the discovery of protein/chemical


interactions among a library of diverse small molecules. VE607 (Fig. 1) blocked SARS-CoV S protein pseudotyped HIV-1 virus from infection of 293T cells expressing ACE2, with an EC50 of 3 μM, and SARS-CoV plaque formation with an EC50 of 1.6 μM. It is specific and did not inhibit poliovirus plaque formation. The cytotoxicity on Vero cells (TC50) was low (>50 μM).

Fig. (1). VE607. Identified by a “chemical genetics” approach, this compound blocked SARS-CoV S protein pseudotyped HIV-1 virus from entry of 293T cells expressing ACE2, with an EC50 of 3 μM.

Tetra-O-Galloyl- -D-Glucose (TGG) and Luteolin

These are drug-like small molecules that bind to the S2 domain of SARS-CoV S protein identified in a library containing extracts of 121 Chinese herbs [153]. They are identified through a two-step screening consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on HIV-luc/SARS pseudotyped virus. The two molecules (Figs. 2 and 3) have EC50 of 2.86 and 9.02 μM, and identified by structural analysis as TGG and luteolin, respectively. These molecules are ineffective in preventing another pseudotyped virus (HIV-luc/VSV) from entry, and are therefore specific. With wild-type SARS-CoV, the EC50 values are 4.5 and 10.6 μM, respectively. This contrasts with an EC50 of >607.6 μM for glycyrrhizin under the same conditions. The compounds appeared safe on cells and mice.

Fig. (2). Tetra-O-galloyl- -D-glucose (TGG). It has an EC50 of 4.5 μM against entry of wild-type SARS-CoV. Emodin

Emodin, an anthraquinone derived from Chinese medi-cinal herbs from genus Rheum and Polygonum, was found to significantly block S from binding ACE2 in a dose-depen-

Fig. (3). Luteolin. It has an EC50 of 10.6 μM against entry of wild-type SARS-CoV.

dent manner. Furthermore, it inhibits S protein-pseudotyped retrovirus from infecting Vero E6 cells, thus providing another potential lead therapeutic agent that blocks viral entry [154].

Chloroquine and Derivatives and Inhibitors of p38 MAPK

Also acting on viral entry is chloroquine, apparently at the level of endosomal stage by increasing the pH. The inhibitory activity is thus similar to ammonium chloride, a lysosomotropic agent that is also inhibitory against SARS-CoV. Chloroquine (Fig. 4) is a 9-aminoquinoline, used for malaria, amebiasis, and autoimmune diseases, such as rheumatoid arthritis. It is being tested for its anti-HIV effect in clinical trials [155]. When chloroquine phosphate was tested in-vitro for inhibitory effect against SARS-CoV-induced cytopathic effect in Vero E6 cells with IFN- 1a as positive control, an IC50 of 8.8 +/- 1.2 μM was recorded, with CC50 of 261.3 +/- 14.5 μM (SI = 30). The IC50 was achieved at concentrations well below the plasma levels attained during treatment of acute malaria [156]. There was no significant loss of antiviral effect even with delayed application of chloroquine to infected cultures for up to 5 hours post-infection.

Employing an immunofluorescence assay for SARS-CoV antigen expression in infected cells, Vincent et al. demons-trated an antiviral activity of chloroquine in Vero E6 cells beginning at 0.1 μM (28% reduction). At 10 μM concen-tration, achievable in-vivo at dosages used for malaria prophylaxis and treatment, viral inhibition was complete [157]. The antiviral mechanism of chloroquine may be more complicated, as it is also known to modulate immune response by reducing secretion of tumor necrosis factor and interleukin 6 in some viral infections [155]. Recently, organometallic compounds closely mimicking hydroxych-loroquine, with less cytotoxic effect compared with the parent ferroquine compound (Fig. 5), were found to have antiviral effect on SARS-CoV replication in Vero cells, with EC50 of 1.9 and 3.6 μM for compounds 3 and 4, respectively [158]. Nevertheless, chloroquine did not reduce lung infection by SARS-CoV in the BALB/c mouse model, as determined by CPE titration assay [159].

Recently, Kono et al. studied the effect of chloroquine on HCoV-229E infection in human lung epithelial cells (L132). Chloroquine inhibited viral replication but had no effect on viral entry. Further investigations showed that both chloroquine (previously found to selectively abolish CpG DNA-mediated MAPK activation [160]) and SB203580 (inhibitor of p38 MAPK) prevented phosphorylation of p38 mitogen-activated protein kinase (MAPK) and reduced viral titers in a dose-dependent manner. The findings suggest that

O

NHO

O

HO

N

O

GOOG

OG

OG

OH

G: galloyl C

OOH

OH

OH

OHO

OH O

OH

OH


HCoV-229E replication in L132 cells is dependent on activation of host p38, a process inhibited by chloroquine. It would be interesting to see if this could be extrapolated to SARS-CoV and perhaps other viruses. If confirmed, it would add p38 to the class of host molecules, including inosine 5’-monophosphate (IMP) dehydrogenase, S-adenosylhomo-cysteine (SAH) hydrolase, orotidine 5’-monophosphate decarboxylase, CTP synthetase, and others, as possible targets for antiviral therapy [161, 162].

Fig. (4). Chloroquine (7-Chloro-4-(4-Diethylamino-1-Methyl-butylamino) Quinoline) and Hydroxychloroquine.

Fig. (5). Ferroquine.

PROTEASES AS DRUG TARGET

Coronavirus proteases, of which there are two in SARS-CoV and three in several other coronaviruses cleave the ORF-1 polypeptide as it is translated, enabling the formation of the viral replication complex (Fig. 6). It is therefore crucial for the early stage of the viral life cycle and a rational drug target [50, 163-168]. The clinical experience with protease inhibitors was reviewed earlier [169].

Fig. (6). Polyprotein 1a and polyprotein 1ab cleavage sites.

The Main Coronavirus Protease - 3CLpro

3CLpro, the main SARS-CoV protease responsible for catalytic cleavage of 11 sites in polyprotein 1a (pp1a) and 1ab (pp1ab), also releases RNA-dependent RNA polymerase (RdRp) and helicase [59]. The 11 sites are conserved, and contain a large hydrophobic residue (preferably Leu) in the P2 position, a Gln in the P1 position, and a small aliphatic amino acid residue (Ser, Gly, Ala) in the P1′ position [50]. Because of conservation across the coronaviridae, 3CLpro is the focus of much effort for finding a broad-spectrum inhi-bitor, relevant to the recent discovery of novel coronaviruses in animal reservoirs [2, 3]. Critical to rational drug design is the availability of the crystal structures of several coronaviral 3CLpro [50, 170-172], including the newly discovered group 2A coronavirus, HCoV-HKU1 [173], and the availability of expression systems and functional assays. The availability of different 3CLpro expression constructs and kinetic assays lead to the need for standardization, in order that inhibitors being developed be compared [174].

SARS-CoV 3CLpro is a 33 kDa cysteine protease (Fig. 7). Its enzymatic activity apparently resides in the catalytic dyad of Cys145 and His41 within a chymotrypsin-like fold formed from the double -barrel domains I and II. Asp187 may also be involved, with enhanced catalytic efficiency, after a molecule of water trapped by Asp187 and His41 is displaced by substrate binding [175].

3CLpro functions as a dimer and associates via the third unique -helical domain, with contribution by the amino terminus of the other monomer (“N-finger”), which maintains the shape of the oxyanion loop and of the S1 pocket through hydrogen bonds [170, 172, 176]. Domain III of 3CLpro is recently proposed as a new target for specific inhibitors because of its critical importance in the function of the viral enzyme [51, 177].

Fig. (7). X-ray crystal structure of the dimeric SARS-CoV

3CLpro (Bonanno JB et al. PDB: 1q2w).

Conservation of 3CLpro Substrate-Recognition Pocket

The catalytic site is the main area of research. Compari-son of the previously known coronavirus cleavage sites with those identified in the SARS-CoV-3CLpro suggests conservation of P4 (Ser, Thr, Val, Pro, Ala [STVPA]), P2 (Leu, Ile, Val, Phe, Met[LIVFM]), P1 (Gln[Q]) and P1' (Ser,

NCl

HN

CH3

NX

CH3

Chloroquine - X=HHydroxychloroquine - X=OH

N

HN

Fe

N CH3

CH3

Cl


Ala, Gly, Asn, Cys[SAGNC]). Trans-cleavage assays with expressed SARS-CoV 3CLpro and in-vitro translated substrates or synthetic 15-mer peptides derived from the N-terminal TGEV (group 1) and MHV (group 2) 3CLpro autoprocessing sites have confirmed these findings. Expec-ted cleavage products were found and cleavage was prevented by targeted mutation of the Cys-to-Ala at the active site [52]. Because of substrate conservation, a drug with Gln (Ser,Ala,Gly) specificity ( denotes cleavage site) against SARS 3CLpro may also have activity against the other coronaviruses [178].

Recently, it was discovered that SARS-CoV uniquely showed equal preference for P1-His in addition to the expec-ted P1-Gln, with implications for inhibitor design [179].

Structure-Based Identification and Derivation of 3CLpro Inhibitors

A 3-D model of the enzyme was created computationally before the crystal structure of SARS-CoV was made available, by homology modeling technique, based on the close sequence similarity between the 3CLpro of SARS-CoV and porcine transmissible gastroenteritis virus (TGEV) and the crystal structure of TGEV [180].

Another 3-D model of SARS-CoV 3CLpro bound to a substrate, with a catalytic triad in the active site, was deposited in the Protein Data Bank (PDB) before the release of the first X-ray structure, in this instance with multiple molecular dynamics simulations [175].

With the publication of the crystal structure of SARS-CoV 3CLpro, it was suggested that Ruprintrivir (AG7088), a rhinovirus medication in clinical trials, be used as starting point for a specific inhibitor [50]. However, ruprintrivir did not inhibit SARS-CoV in in-vitro studies [181, 182]. A derivative, KZ7088, was found by in-silico studies to interact with the active site through six hydrogen bonds [183-185], and led to three other drug candidates [185]. Similarly, a screen of 29 approved and experimental drugs against the SARS-CoV 3CLpro as well as the structure of TGEV identified the existing HIV-1 protease inhibitor, L-700,417, as a starting point for specific 3CLpro inhibitors [186]. Another potent inhibitor against rhinovirus, an isatin compound, was derivatized to yield several compounds with low micromolar range IC50 values [187].

The crystal structure of 3CLpro also resulted in the identification of a herbal product, sabadinine, as another drug lead, in a search of 140,000 chemicals in the National Cancer Institute diversity set, using an automated docking computer software [188]. However, sabadinine is a complex molecule and difficult to modify, and did not exhibit antiviral effect on murine hepatitis virus (MHV), as evaluated for syncytium formation and cytopathic effects.

The crystal structures of HCoV-HKU1 and infectious bronchitis virus (IBV) 3CLpro, in complex with a Michael acceptor inhibitor, N3, were recently published [171, 173]. N3 was recently discovered by Yang et al. (see below).

“Distorted Key” and Potential Peptide Inhibitors

Because proteases cut peptides, modified peptides that cannot be cleaved but which fit in the active site represents a

class of inhibitors, as elaborated by the “distorted key” approach [189]. An octapeptide that hydrogen bonds with six residues in the SARS-CoV 3CLpro active pocket, AVLQSGFR, was reported by Chou et al. [183, 190]. Chemical synthesis of the peptide and in-vitro testing in Vero cells infected with SARS-CoV (BJ-01 strain), provided evidence of antiviral activity (EC50 = 2.7 x 10-2 mg/L; CC50 = >100 mg/L, SI = >3704) [191]. Two other octapeptides (ATLQAIAS and ATLQAENV), identified through statis-tical analysis of 11 candidates extracted from 396 cleavage sites of 36 SARS-CoV examined bioinformatically, may also be potential inhibitors [192].

Protein Flexibility and Substrate-Active Site Interaction

Proteins fold and associate with other molecules into tertiary and quaternary configurations, as well as undergo molecular motions. The flexibility in the case of 3CLpro is shown to be pH-dependent and affects the substrate-binding site, as shown by multiple X-ray structures and MD simulations [176].

To delineate the properties of the active site of 3CLpro in complex with the substrate and to address the flexibility of protein molecules, Pang employed terascale computing and 420 different molecular dynamics simulations at 2.0 ns for each simulation with a 1.0-fs time step [175]. Surprisingly, the predicted model of the active site comprises a catalytic triad of Cys145 and His41 and the newly predicted Asp187, which was revealed after a water molecule was displaced by the substrate. The addition of the aspartate residue could solve the enigma posted by Savarino [193], of aspartate protease inhibitors of HIV-1, such as lopinavir and ritonavir, exhibiting anti-SARS-CoV activity [194, 195]. This finding, however, apparently was not confirmed by others [176]. Nevertheless, the expanded active-site pocket has impli-cations in anti-SARS drug design. Indeed, a small molecule inhibitor, CS11, was identified through virtual screening of 36,143 mall molecules. CS11 (Fig. 8) inhibited the Toronto-2 strain SARS-CoV 3CLpro with EC50 of 23 μM and was nontoxic to cells at that concentration [196]. Because CS11 is easy to synthesize and derivatize, it is considered an excellent inhibitor lead of 3CLpro. The drug discovery process can be shortened to 1 month by the available 3.4 teraflops computing resource and further shortened by petascale computing in the near future.

Fig. (8). CS11. This molecule docks well into an expanded 3CLpro active site predicted computationally. It inhibits SARS-CoV (Toronto-2 strain) with an EC50 of 23 μM.

Subsites Within 3CLpro Active Site

Further studies revealed adjacent subsites such as a cluster of serine residues (Ser139, Ser144 and Ser147), espe-

-O2C N

HO

O


cially Ser147, which when replaced with alanine, inhibited dimerization and resulted in loss of enzymatic activity [197]. These subsites are also conserved in coronaviruses and are susceptible to inhibition by compounds containing boronic acid, in particular bifunctional aryl boronic acid compounds (Fig. 9), with inhibition constants as low as 40 nM. The strong binding with the protease by these inhibitors was reversible in enthalpically favorable fashion, as determined through isothermal titration microcalorimetry, arguably more readily optimized, as opposed to entropically driven ones. FL-166 has a Ki (inhibitor concentration that supports half the maximal rate of inactivation) of 40 nM. This novel molecular scaffold could be further optimized as a drug candidate [163].

Fig. (9). Bifunctional aryl boronic acid compound FL-166. This compound inhibits 3CLpro by reversible binding to a subsite having a cluster of serine residues (Ser139, Ser144 and Ser147).

Competitive Inhibitors Against SARS Main Protease (3CLpro)

MP576

Two SARS-CoV 3CLpro inhibitors are among several other drug leads identified by a shot gun-like “chemical genetics” approach that Kao et al. employed [152]. A structurally diverse library of 50,240 small molecules was screened. One of the 3CLpro inhibitors, MP576 (Fig. 10), was reported to have potent effects on the enzyme (IC50=2.5 μM, EC50=7 μM by plaque-reduction assay), and not showing cytotoxicity at 50 μM. The molecule docks into the active site and did not nonspecifically inhibit human neutrophil elastase at 100 μM [152]. The authors discussed the advantages of their approach to the problem.

Fig. (10). MP576. Also identified by “chemical genetics” approach. This reversible SARS-CoV 3CLpro inhibitor has an IC50 of 2.5 μM.

MAC-8120 and MAC-13985

Hundreds of compounds with 3CLpro inhibitory activity were found in a high-throughput screen of 50,000 drug-like small molecules employing a quenched fluorescence reso-nance energy transfer assay. After a series of filtering, two promising leads with incompletely characterized mechanism of action were found. MAC-8120 (Fig. 11) and MAC-13985 (Fig. 12) have good activity against 3CLpro (IC50 4.3 and 7 μM, respectively) and show no nonspecific inhibition of chymotrypsin and papain [168]. Blanchard et al. have made the data set available to the public.

Fig. (11). MAC-8120. This SARS-CoV 3CLpro competitive inhibitor has an IC50 of 4.3 μM.

Fig. (12). MAC-13985. This SARS-CoV 3CLpro competitive inhibitor has an IC50 of 7 μM.

Keto-Glutamine Analogues

The substrate specificity of coronavirus 3CLpros is mainly determined by the P2, P1 and P1’ positions [198]. Since the substrate P1 is a conserved glutamine [52], keto-glutamine analogues with a phthalhydrazido group at the -position were synthesized and tested as reversible (com-petitive) inhibitors. A continuous quenched fluorescence resonance energy transfer assay [168] identified compound 8c (Fig. 13) as having IC50 of 0.6 μM [166]. The Ki value is not yet published. Of note is that the parent compound is a reversible inhibitor of hepatitis A virus (HAV) 3C enzyme, with a Ki of 9 μM.

Fig. (13). Keto-glutamine analogue (8c) with IC50 of 0.6 μM.

Zinc-Containing Compound

Among 960 compounds screened in a high-throughput fluorogenic substrate assay, two mercury-containing com-

HN

NH

O

NO2

BHO OH

O

O2N

BHO OH

N+

OO-

O

NH

O

O

S

N

NH2

SO

O

OH

O

O ON

OH

F

F

F

AcHNNH

O

HN

O

NH

O

N

HNO

HN

O

O NO2

H

H

H

OBnO


pounds (phenylmercuric acetate and thimerosal) exhibited potent anti-SARS-CoV 3CLpro activity (Ki = 0.7 and 2.4 μM, respectively) [199]. To avoid using mercury, zinc-conjugated compounds were investigated. One such com-pound, 1-hydroxypyridine-2-thione zinc (fig. 14) was found to be a competitive inhibitor, having Ki of 0.17 μM. Zn2+ ions alone is less potent (Ki = 1.1 μM) and acted non-competitively. The in-vitro antiviral activity was confirmed by Western-blot analysis of SARS-CoV spike protein expression in cell lysate. Although cytotoxicity data for 1-hydroxypyridine-2-thione zinc was not reported, it was noted that oral zinc supplement was used without ill effects in patients with Wilson’s disease, and that zinc is used as a remedy for common cold.

Fig. (14) 1-hydroxypyridine-2-thione zinc. A competitive SARS-CoV 3CLpro inhibitor with Ki of 0.17 μM.

Cinanserin

An old drug, cinanserin [2-(3-dimethylaminopropylthio) cinnamanilide] (Fig. 15), was identified as an inhibitor of 3CLpro by a genome-to-drug-lead approach [200]. Cinanserin had undergone preliminary clinical testing as a serotonin antagonist. It inhibits the catalytic activity of both SARS-CoV and HCoV-229E 3CLpro with an IC50 of 5 μM on a fluorogenic substrate. In tissue culture assays with a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E, cinan-serin demonstrated an antiviral activity with IC50 of 19 - 34 μM and reduction of viral yield of up to 4 logs. Because cinanserin caused hepatoma in rats, and has anti-serotonin activity, it will need to be modified before being tested in clinical studies.

Interestingly, cinanserin and the expanded active site of 3CLpro were discovered before the availability of the crystal structure of SARS-CoV 3CLpro [175, 196], illustrating the utility of genome-to-drug approach in not only fast-tracking drug development but also in addressing the flexibility of protein molecules.

Fig. (15). Cinanserin [2-(3-dimethylaminopropylthio) cinnama-

nilide]. This serotonin antagonist inhibits SAS-CoV and HCoV-229E 3CLpro with IC50 of 19-34 μM.

Hexachlorophene

Liu et al. developed a FRET technique for high-through-put screening for anti-SARS CoV 3CLpro drugs having an excellent Z0-factor value for the assay of 0.81. Screening of

1,000 compounds lead to the discovery of a competitive inhibitor, hexachlorophene (Fig. 16), with Ki of 4 μM and IC50 of 5 μM against recombinant SARS-CoV 3CLpro [201]. Several analogs of hexachlorophene have IC50 ranging from 7.6 to 84.5 μM. No cellular toxicity was found in Vero E6 cells at 10 μM (CC50 = 100 μM) [199]. Computer docking of hexachlorophene with SARS-CoV 3CLpro (PDB: 1UK4) revealed hydrogen bonding with a catalytic residue Cys145, further suggesting its applicability for treating human SARS disease. Hexachlorophene is experimentally used as a cholinesterase inhibitor.

Fig. (16). Hexachlorophene. This cholinesterase inhibitor has an IC50 of 5 μM against recombinant SARS-CoV 3CLpro.

Anilide Inhibitor of 3CLpro

Peptide nitroanilides were explored as potential inhibitors because of possible activity of niclosamide and because p-nitroaniline-containing chromogenic peptide substrates not only failed to be cleaved by, but also mildly inhibited 3CLpro, an example of “distorted key”. Anilide 2a (Fig. 17), derived from 2-chloro-4-nitroaniline, L-phenylalanine, and 4-(dimethylamino)benzoic acid proved to be a potent competitive inhibitor (Ki = 0.03 μM, IC50 = 0.06 μM). It also has the lowest binding free energy in docking experiments compared with other anilides [167].

Fig. (17). Anilide 2a, derivative of 2-chloro-4-nitroaniline, L-

phenylalanine and 4-(dimethylamino)benzoic acid. This potent competitive inhibitor of SARS-CoV 3CLpro has an IC50 = 0.06 μM.

Non-Covalent HIV Protease Inhibitor Derivative

As reported by Wu et al, a 3CLpro inhibitor was found through a cell-based screen of >10,000 agents. This non-covalent HIV protease inhibitor, TL-3 (Fig. 18), previously developed by the same group, has Ki of 0.6 μM against SARS-CoV 3CLpro [181]. Following the report, TL-3 underwent a series of optimizations. Two compounds, 4 and 9 (Fig. 19), showed strong competitive inhibitory activities, with Ki values of 0.34 and 0.073 μM, respectively [202]. Of note is the switch of selectivity of compound 9 from HIV-1 protease inhibition to SARS-CoV 3CLpro inhibition. The exercise increased the inhibition constant of the agent by one order of magnitude.

N+

SZn

S

O-

N+

O-

N

O

S N

Cl

OH

Cl

Cl

Cl

Cl

OH

Cl

Me2N

NH

HN Cl

NO2

O

O


Fig. (18). TLC-3. This compound is developed as a non-covalent HIV-1 protease inhibitor. The Ki of this compound against 3CLpro is 0.6 μM.

Fig. (19). Compound 9. Modified from TLC-3, the Ki of this compound is 0.073 μM.

Non-Competitive 3CLpro Inhibitors

Noncompetitive (covalent) inhibitors loaded with reactive warheads such as diazo compounds or haloketones suffer from inherent reactivity towards non-target molecules, making them unsuitable for in vivo studies. The discovery of Michael acceptors such as Ruprintrivir and naturally occurring epoxysuccinyl cysteine protease inhibitor such as E64, (containing thiol-reactive (2S,3S)-oxirane-2,3-dicar-boxylic acid group) with lesser reactivity, and the entry of Ruprintrivir into clinical trial, has revived interest in developing irreversible noncompetitive inhibitors for cysteine proteases [203].

Cysteine Protease Inhibitor E64-d

(2S,3S)-transepoxysuccinyl-l-leucylamido-3-methylbu-tane ethyl ester (E64-d) is a derivative of E64, a cysteine protease inhibitor. It inhibits MHV and foot and mouth disease virus replication and is not cytotoxic. Yount et al. tested it as a one-time dose of 0.5 mg/ml against an infectious recombinant SARS-CoV (icSARS-CoV) as well as wild-type SARS-CoV (Tor2 and Tor7) in Vero E6 cells 1 hour after infecting the cultures with a multiplicity of infection of 0.1. The viral titer as determined by plaque assay showed a 3-4 log reduction, with accompanying reduction of cytopathic effect and healthy intact monolayers [204].

HIV Protease Inhibitors

Rajnarayanan et al. also identified several covalent (non-competitive) and non-covalent inhibitors of 3CLpro and derivatized covalent irreversible inhibitors with enhanced affinity in-silico [203]. Based on an initial virtual screen of old drugs, 9 of 33 HIV protease inhibitors were observed to share similar pharmacophoric features. These fit well into the 3CLpro active site and were subjected to secondary rule-based screening to select the small molecules that lie within 2 – 3 Å away from the S of Cys145. Following in silico

derivatization by addition of thiol-reactive warheads and analysis with Occluded Surface Program (for tightness of fit), differences between covalent and non-covalent analogs were found. The covalent inhibitors are being synthesized ex silico and tested with the non-covalent ones in in-vitro studies.

Recently, Ge et al. produced a non-infectious SARS-CoV replicon cell line and applied it to screen for viral inhibitors. Incubation of SCR-1 cells with E64-d at 0.4 mg/ml resulted in observable inhibition of reporter gene expression, replicon RNA copy number, and the overall percentage of green fluorescent cells. No cytotoxicity was observed. Two other agents tested, ribavirin and glycyrrhizin, showed no to low inhibitory effect, respectively [205].

Michael Acceptor Inhibitor N3 and Derivatives

Several compounds with fast in vitro inactivation of multiple CoV 3CLpros were developed and shown to have extremely low cellular toxicity [178]. These are irreversible inhibitors with Michael acceptors, designed based on the structure of the conserved region of 3CLpro, with the mechanism of inhibition elucidated from the structures of the inhibitors complexed with SARS-CoV and porcine TGV 3CLpro.

Preliminary studies showed that N3 (Fig. 20) exhibited in vitro inhibition of all CoV 3CLpros tested, including TGEV, HCoV-229E, FIPV, HCoV-NL63 (group I), MHV, HCoV-HKU1 (group IIa), SARS-CoV (group 2b), and IBV (group III). The IC50 values were 4.0, 8.8, and 2.7 μM against HCoV-229E, FIPV and MHV-A59, respectively. This is in spite of high multiplicity and single-cycle infection condi-tions. In-vitro plaque-reduction assay showed that N3 had EC50 of 3.4 μM. Cellular cytotoxicity was extremely low, with 28.3% inhibition of cell growth at 500 μM.

Further development of broad-spectrum coronaviral 3CLpro inhibitors based on structural insights gained from the enzyme-inhibitor complex of IBV and SARS-CoV 3Clpro mutant (H41A), resulted in new generations of inhibitors with longer side chains at P3, such as N27 (Fig. 21) and H16 (Fig. 22). Both of these inhibitors have impro-ved inhibitor potency against SARS-CoV 3CLpro [171].

Fig. (20). N3. This non-competitive inhibitor is active against all CoV 3Clpro tested.

Benzotriazole Esters

Recently, Wu et al. reported several stable benzotriazole esters as being noncompetitive inhibitors of SARS-CoV 3CLpro. Benzotriazole esters are intermediates in the synthesis of lopinavir and cause acylation of Cys145 at the

Cbz-Ala-Val-HN NH-Val-Ala-Cbz

HO OH

TL-3

Cbz-Ala-Val-HN NH-Val-Ala-Cbz

HO OH

Compound 9

HN

HN

ON

HN

O

O

NH

HN

O

NH

O

NH

O

OBn

O


Fig. (21). N27. A new inhibitor derived from N3, with longer side chain at P3. It has improved inhibitor potency against SARS-CoV 3CLpro.

Fig. (22). H16. Another new derivative of N3 with longer side chain at P3, and having more potent inhibition of SARS-CoV 3CLpro.

catalytic site of 3CLpro, as revealed by mass spectrometry, an activity abolished in C145A mutants. The most potent of these, compound 8, has reported kinact (maximal rate of enzyme inactivation) of 0.0011 sec-1 and Ki (inhibitor con-centration that supports half the maximal rate of inactivation) of 7.5 nM (kinact/Ki = 146.7) [206]. Significantly, these irreversible inhibitors are nontoxic to Vero cells (CC50 >100 μM). Even so, they are likely to cross inactivate cellular cysteine proteases and will require derivatization of the sites occupying the S1 and S2 pockets of the enzyme [207]. Replacement of the bridged ester-oxygen group with carbon results in several competitive, albeit relatively weak, inhibitors compared with compound 8, with <1% of its potency (Ki of compound 14 = 1.0 μM).

The ester bound to 3CLpro is visualized in a new crystal structure, which suggests that inhibitor potency and specificity might be improved by modifying substituents that occupy the S1 and S2 specificity pockets of 3CLpro [208]. These studies show that benzotriazole esters are important milestones towards a 3CLpro medicine.

WRR 183 ( , -Epoxyketone)

Recently, Goetz et al. identified a novel preference of SARS-CoV 3CLpro for P1-His containing substrates equal to the expected preference for P1-Gln [179]. The full knowledge of the tetrapeptide substrate specificity of 3Clpro was achieved with the use of a fully degenerate peptide library consisting of all 160,000 possible naturally occurring tetrapeptides. This information was utilized to develop optimal substrates for a high-throughput screen of a 20,000 compound library of small molecules. Following two rounds of screening and based on the reproducible time-dependent inhibition of proteolytic activity, the most promising scaffold identified was the dipeptidyl epoxyketone. Among the

synthesized dipeptidyl derivatives of the parent compound, WRR 183 (an , -epoxyketone, Fig. 23) was further characterized. The 1.8-Å X-ray crystal structure of 3Clpro bound to the irreversible inhibitor of 3Clpro was reported. It was shown that a favorable binding configuration resulted, with Cys145 opening the epoxide ring by nucleophilic attack, forming an alcohol group at P1, which directly and indirectly hydrogen-bonded with residues at the bottom of the S1 pocket. This promising scaffold provides improve-ments for a rational small-molecule inhibitor design based upon the pharmacophore identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.

WRR 183 has kinact of 0.004 ± 0.0003 s-1, Ki of 2.2 ± 0.2 μM and rate of inhibition/inhibition constant (k3/Ki) of 0.002 μM-1 s-1 (for irreversible inhibitors). In in-vitro studies, WRR 183 was found to inhibit SARS-CoV (Tor2) in Vero E6 cell culture with an EC50 of 12 μM, while exhibiting 50% toxicity at ~60 μM.

Fig. (23). WRR 183. An , -epoxyketone, with non-competitive inhibition of SARS-CoV 3CLpro (EC50 = 12 μM, CC50 = 60 μM, SI = 5).

Halomethyl Ketone Inhibitors

Although as noted earlier, inhibitors with haloketones warheads are inherently reactive, they are amenable to careful modification. Bacha et al. [209] reported the identification of a pharmacophore based on structure activity relationship studies of a new class of irreversible 3CLpro inhibitors containing a halomethyl ketone warhead. The compounds are highly potent and have Ki as low as 300 nM. Compound 4, with Ki of 400 nM and novel Pn-Sn interactions, inhibits 3CLpro by first forming a reversible complex followed by a much slower irreversible reaction between Cys145 and the adjacent bromomethyl resulting in a thioether linkage. The covalent linkage takes up to 12 hours or more and at high concentration of the inhibitor. When examined for specificity against similar serine and cysteine proteases, the compounds showed some affinity for calpain (Ki of 8 – 20 μM; 15 μM for compound 4). No reactivity with trypsin or thrombin was found. Further optimization to improve their potency and specificity towards 3CLpro are warranted. No in vivo testing is reported.

Fig. (24). Compound 4. This halomethyl ketone non-competitive SARS-CoV 3CLpro inhibitor has Ki of 15 μM.

ON

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NH

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O

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The Auxiliary Coronavirus Protease - Papain-Like Protease (PLpro)

The auxiliary proteases of coronaviruses are monomeric, zinc-ribbon-containing, papain-like, transmembrane pro-teases, of which there are two in most members but only one in SARS-CoV and the group 3 infectious bronchitis virus (IBV) [59]. From the genomic location, SARS-CoV PLpro is considered to be paralogous with PL2pro. The absence of a paralogous PL1pro is thought to confer increased conser-vation of cleavage sites and narrower substrate specificities of the remaining PLpro, attributes that could be exploited for the development of selective inhibitors. On the basis of sequence comparison, all coronavirus PLpros are considered papain-like cysteine proteases and have catalytic dyads of Cys and His residues, but show low sequence homology within the genus (25%) and even lower when compared with cellular papains. In SARS-CoV, the protease function resides in the middle portion of nsp3, spanning the residues 1541 to 1855. The predicated location of the catalytic dyad is Cys1651 and His1812. An additional Aspartic acid might be involved in a functional triad [210]. The enzyme shows conservation of both position and sequences of polyprotein cleavage sites, differentially cleaving between the amino acids Gly180 and Ala181, Gly818 and Ala819, and Gly2740 and Lys2741 of the viral polypeptide pp1a, as determined by reversed-phase high-performance liquid chromatography analysis coupled with mass spectrometry [211]. It is demonstrated that an LXGG motif at the P4 -P1 positions of the substrate is essential for recognition and cleavage. There appears to be no preference for the P’ position or for residues N-terminal of P4.

Zinc Inhibits Papain-Like Cysteine Protease (PLpro)

A fluorogenic assay with the substrate Abz-FRLKGGAPIKGV-EDDNP was developed to enable high-throughput screening for specific inhibitors. Only Zinc and zinc conjugates, including N-ethyl-N-phenyldithiocarbamic acid Zn (Fig. 25) and hydroxypyridine-2-thione Zn were active, with IC50 of 1.3, 3.3 and 3.7 μM, respectively. Inhibition cannot be obtained with other divalent ions. Other compounds tested that are without inhibitory activity include the known cysteine protease inhibitors, such as E64 at 0.1 mM, N-ethylmaleimide at 1mM, cystatin at 10 μg/mL, leupeptin at 0.1 mM, antipain at 0.1 mM, and chymostatin at 1 mM.

Fig. (25). N-ethyl-N-phenyldithiocarbamic acid Zn. This zinc conjugate inhibits the SARS-CoV accessory protease PLpro with IC50 of 3.3 μM.

Deubiquitining and de-ISGylating Activities of SARS-CoV

PLpro

Ubiquitination is the cellular process of adding small proteins such as ubiquitin and ubiquitin-like proteins (such as ISG15) in linear or branched chains to label other protein

molecules for proteasomal degradation. The same process in different contexts are involved in membrane protein trafficking, in activation of the transcription factor NF B, in DNA repair, and in autophagy, a starvation-induced process where double membrane vesicles are assembled and which are susceptible to being usurped by the virus for its own replication [215]. The product of the cellular interferon-stimulated gene 15 (ISG15) is interesting in that this 15kD ubiquitin-like protein conjugates with other cellular protein as part of an innate anti-viral response.

In addition to cleavage of pp1a, SARS-CoV PLpro was found to have pathogenic properties, such as deubiquitining (DUB), and de-ISGylating activities and interference with the cellular interferon signaling pathway. Thus SARS-CoV PLpro is similar to adenoviral protease and influenza NS1, which inhibits protein ISGylation [210, 212-214]. Because PLpro recognizes the motif LXRR, it is not surprising that it cleaves ubiquitin after the four C-terminal residues, LRGG [210]. Protein de-ISGylation is a property of PLpro recog-nizing and cleaving the C-terminal sequence -LRLRGG of ISG15 [213].

PLpro Interferes with the Host Interferon System

SARS-CoV PLpro also binds IRF-3, a latent type I IFN transcription factor essential in innate anti-viral immunity. It does so by preventing phosphorylation and nuclear trans-location of IRF-3, thereby interception the signal for activation of type I IFN responses through either Toll-like receptor 3 or retinoic acid-inducible gene I (RIG-I)/mela-noma differentiation-associated gene 5 (MDA5) pathways [72, 216].


The auxiliary protease is another essential machinery in the viral life cycle. Its pathogenic properties and its narrow substrate specificities are enticing researchers to refocus on it as a target for antiviral drug development. Zinc conjugates have shown some promise and should be further investigated.

SARS-COV POLYMERASE AS DRUG TARGET

RNA viruses except retroviruses encode RNA-dependent RNA polymerase (RdRp) for genomic replication. The enzyme is neither encoded nor has homolog in mammalian cells, although plants and lower animal species do encode RdRp, putatively involved in gene silencing [217, 218]. Sequence comparisons and mutagenesis studies of a wide range of RdRps have revealed conserved motifs with few exceptions. The crystal structures of several of these enzymes also reveal a common architecture and catalytic function. RdRp is therefore an attractive antiviral drug target because of its absence in humans.

Widespread interest in coronaviruses is relatively recent. As a result, there is little experimental data on the charac-teristics of coronaviral RdRp other than that of murine hepatitis virus (MHV) and a consequent lack of inhibitors for this enzyme. This contrasts with infections by hepatitis B and C, HIV-1 and herpes viruses, where polymerase inhi-bitors are very successful.

N+

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The first coronaviral RdRp to be structurally and func-tionally elucidated is that of SARS-CoV [219]. Following localization of the SARS-CoV RdRp motif conserved by all RdRps, a 3-D model of the catalytic domain is constructed, with the prediction of structural attributes of potential anti-SARS-CoV RdRp nucleotide analog inhibitors. These include hydrogen-bonding capability with Asp623 and Asn691 of SARS-CoV RdRp by the 2’ and 3’ groups of the sugar ring and C3’ endo sugar puckering conformation to maintain its ability for making a hydrogen bond at the 3’ position and to avoid steric conflicts at the 2’position. Thus, nucleoside analogs 2’-C-methyladenosine and 2’-O-methylcytidine may be potential inhibitors of SARS-CoV RdRp. This was somewhat borne out by experiments showing that -D-N4-hydroxycytidine (Fig. 26) has modest selective activity, with EC50 of 4 μM by virus yield reduction assay and CC50 (neutral red assay) of 50 μM (SI = 10) [220]. Also predicted is the absence of a hydrophobic binding pocket near the catalytic site of SARS-CoV RdRp for non-nucleoside analog inhibitors, suggesting that non-competitive binding of such inhibitors that retard HIV-1 and HCV polymerases will not work for SARS-CoV RdRp [219]. Non-nucleoside HIV-1 RT inhibitors were indeed found to be inactive against SARS-CoV RdRp [221].

Fig. (26). -D-N4-hydroxycytidine. This nucleoside analog has

selective activity against SARS-CoV, with EC50 of 4 μM by virus yield reduction assay and CC50 (neutral red assay) of 50 μM (SI = 10).

Ribavirin

Ribavirin (1-( -D-Ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide, Fig. 27) is a long half-life purine nucleoside analog prodrug derived from D-ribose, with broad-spectrum anti-viral activities against influenza virus, flaviviruses, and many agents of viral hemorrhagic fevers. It is activated by cellular kinases into the pharmacologically active 5’-triphosphate nucleotide. It is incorporated by RdRp into viral RNA as base analogs of either adenine or guanine, resulting in lethal “error catastrophe”. It also inhibits certain viral RdRps, as well as having other activities, such as depletion of intracellular GTP by inhibition of cellular inosine monophosphate (IMP) dehydrogenase [222] and enhancing anti-viral Th1 function in the treatment of Hepatitis C virus infection [223].

However, in SARS-CoV infection, ribavirin has a low selectivity index for SARS-CoV of <1 [80]. It also lacks demonstrable clinical benefit in uncontrolled series [224, 225]. In the SARS-CoV proteome, a conserved domain with

presumed 3'-to-5' exonuclease (ExoN) function is mapped to the N-terminal domain of nsp14. Drawing parallel with cellular DNA-processing homologs such as exonuclease I and the exonuclease domain of DNA polymerases, SARS-CoV nsp14 ExoN activity is speculated to perform viral RNA proofreading, repair and/or recombination [59]. If confirmed, this unusual capability among RNA viruses may account for the failure of ribavirin in SARS-CoV therapy. Recently, ribavirin and other IMP dehydrogenase inhibitors were found to prolong and enhance SARS-CoV replication in the lungs of BALB/c mice [226], suggesting that ribavirin and IMP dehydrogenase inhibitors be avoided in SARS-CoV infection in humans [225, 227].

Fig. (27). Ribavirin. Ribavirin has low selectivity index (<1) against SARS-CoV.

Aurintricarboxylic Acid

Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases with 10 and 100 times more potency against SARS-CoV than IFN- and IFN- , respectively [228]. ATA binds with HIV integrase as well as cellular Ca2+-activated neutral protease (m-calpain) and protein tyrosine phos-phatase, among other viral and cellular proteins including vWF/platelet GP1b [229-233]. Molecular docking study suggests that it inhibits SARS-CoV RdRp by binding to a region in the palm sub-domain (residues 754-766), where two of the three catalytic residues (Asp 760, Asp 761) are located [231]. Because ATA interferes with the interferon pathway of antiviral response [234], as well as being nonspecific and protean in activity, there is doubt about its role as an antiviral agent. Thus, although ATA has a selec-tivity index (SI) of 187 [228], more investigations will need to be carried out before pre-clinical studies.

A Second RdRp - Nsp7-nsp8 Supercomplex

Eight nsp7 and eight nsp8 form a hollow cylinder-like structure of 30 Å internal diameter and positive electrostatic properties. This nsp7-nsp8 supercomplex confers pro-cessivity on RdRp [235]. In addition, nsp8 (see below) is shown to be a second RdRp with proposed primer generation function for the primer-dependent nsp12 RdRp [236]. The high degree of conservation of nsp7 and nsp8 among coronaviruses suggests that this viral machinery is common to all coronaviruses. Disrupting the interaction between nsp7 and nsp8 by peptides or nonpeptidyl compounds is one strategy for broad-spectrum anti-coronaviral inhibitors.


It can be seen that despite the attractiveness of this group of drugs, little data is available to support their applicability in coronavirus infection. One possible exception is -D-N4-

O

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hydroxycytidine (Fig. 26), which is the only nucleoside analog among 26 tested that has an ED90 of 6 μM by virus yield-reduction assay [220]. Unfortunately, the drug did not reduce lung virus titer when tested in BALB/c mice [159].

SARS-COV HELICASE (NSP13) AS DRUG TARGET

SARS-CoV helicase (nsp13) is a superfamily 1 helicase with an N-terminal zinc-binding domain. The quaternary structure is recently predicted [237]. In common with the previously characterized HCoV-229E helicase, it unwinds DNA as well as RNA in a 5’ to 3’ direction [52, 58]. It has remarkable processivity that is probably conferred by the nsp7-nsp8 supercomplex [235]. This property facilitates the search for inhibitors by way of high-throughput DNA-based helicase assays [52]. Optimism for a coronavirus helicase inhibitor stems from the success of helicase inhibitors in animal models of herpes simplex virus infection [238, 239] and the multiple mechanisms of interfering with HCV helicase [240]. In addition to RNA and DNA helicase activities, nsp13 has other enzymatic functions, including deoxynucleoside triphosphatase (dNTPase), NTPase and RNA 5’-triphosphatase activity that may be involved in the formation of the 5’ cap of viral genomic RNA [54, 241]. It is speculated that the helicase might release the 3’ end of the nascent negative strand from its template during negative-strand RNA synthesis, thereby permitting the addition of the antileader sequence at the 3’ end and the synthesis of 5’ leader-containing mRNAs, which are the hallmark of coronaviruses [54]. The multiple crucial roles of helicase make it an attractive drug target.

Adamantane Derivatives as Helicase Inhibitor

Recently, the adamantane-dervied anti-viral bananins (pyridoxal-conjugated trioxa-adamantanes), were shown to be potent noncompetitive inhibitors of the ATPase activity of SARS-CoV helicase. Iodobananin and vanillinbananin (Fig. 28) have IC50 values of 0.54 and 0.68 μM, respectively, whereas the parent compound, bananin (Fig. 29), has IC50 of 2.3 μM [242]. Bulky side groups of some bananins (ansa-bananin [Fig. 30] and adeninobananin) appear to curtail the anti-ATPase activity. Similarly, a FRET-based DNA helicase assay showed IC50 of 2.7 to 7.0 μM for bananin and its derivatives without bulky side groups. When inhibition of viral replication was measured in cell culture, bananin has an EC50 of <10 μM (drug added postinfection at 48 hr). Interestingly, prophylactic administration of bananin appears to affect other cellular pathways that in the absence of viral infection may negate the antiviral property of the drug. The cytotoxicity of bananins to FRhK cells as measured by MTT assay yielded a CC50 of 390 μM (SI of >39). Since the adamantane derivatives have been in use for treatment of viral infections and as muscle relaxants, the bananins are proposed as having significant therapeutic potential against SARS-CoV.

Helicase Inhibitor HE602

The same “chemical genetic” approach that identified two 3CLpro inhibitors and a number of viral entry inhibitors yielded HE602, a helicase inhibitor with substantial inhibitory activity at 2 μg/ml [152]. It inhibits the poly-

Fig. (28). Vanillinbananin.

Fig. (29). Bananin.

Fig. (30). Ansabananin.

nucleotide-stimulated ATPase activity and not the non-stimulated activity (IC50=6.9 μM). In helicase assay, it is active at 20 μM but not at lower concentration. These pharmacological profiles of HE602 are similar to those of herpes simplex virus helicase inhibitors. HE602 also inhibited SARS-CoV plaque formation in Vero cells (EC50=6 μM). The cytotoxicity on Vero cells (TC50) is >50 μM.

NTPase inhibitors

This enzymatic activity makes available the energy from ATP hydrolysis for unwinding of nucleic acid. It is the target of Hoffmann et al., who employed the 3-D jury method of model selection as an aid in the design of small molecule inhibitors. They found a unique amino acid environment in the ATP-binding pocket of SARS-CoV helicase. Compounds with two phosphonic acid moieties or phosphates located at the distal end of the molecule appear to be promising candidates. No in vitro data is reported [243].

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Fig. (31). HE602. Identified by a “chemical genetics” approach, this compound inhibited SARS-CoV plaque formation in Vero cells (EC50=6 μM). The cytotoxicity on Vero cells (TC50) is >50 μM.

RNA Aptamers Targeting SARS-CoV Helicase

RNA aptamers, a new research tool with newly disco-vered biological counterpart as riboswitches [244-247], has found medicinal application in an FDA-approved locally-delivered treatment (Pegaptanib specifically binds to VEGF165) to inhibit angiogenesis in wet (neovascular) age-related macular degeneration.

An RNA aptamer targeting SARS-CoV helicase was recently reported, which inhibits DNA helicase activity by up to 85% with an IC50 of 1.2 nM. The stimulated ATPase activity was also slightly inhibited [248]. Unmodified RNA aptamers have short half-lives because of RNases in body fluids and the environment. However, PEGylation (addition of polyethylene glycol) of the molecules, as in Pegaptanib, results in much increased stability.

OTHER VIRAL TARGETS

RdRp, helicase and proteases are found in all RNA viruses. Unique among the coronavirus, and to some extent among its relatives in the Nidovirales, the viral replicase has enzymatic activities involved in RNA processing. These catalytic activities include ADP-ribose 1”-phosphatase (nsp3), 3’-to-5’ exoribonuclease (nsp14), endoribonuclease (nsp15), and 2’-O-ribose methyltransferase (nsp16) [59, 249].

Nsp8-Primase

Coronavirus RdRps are phylogenetically clustered with those of (+)RNA viruses whose 5’-end of the genome are covalently linked with the viral protein genome-linked (VPg). In poliovirus, this serves as the primer for the syn-thesis of RNA by RdRp [250]. Coronavirus Vpg is replaced by a cap and was shown instead to be dependent on nsp8 for synthesis of a primer for the purpose of RNA synthesis by nsp12 RdRp [236]. Nsp8 is further shown to be sequence specific (5’-(G/U)CCNN-3’), although not very stringent, and synthesizes short primers (<6 nucleotides) at multiple internal initiation points within the genome, with a relatively low fidelity similar to that of known DNA-dependent RNA primases. In light of the fact that SARS-CoV nsp12 RdRp is

found to be primer-dependent [221], and thus requiring the primer-synthesizing capacity of nsp8 RdRp, a new drug target may be reasonable. Thus nsp8 RdRp was tested against several nucleotide analogs (3’-dGTP, ddGTP and 2’-O-methyl-GTP). While most are inactive as inhibitors, 3-dGTP, which also inhibits eukaryotic DNA primase, showed efficient inhibition of RNA synthesis at concentrations ranging from 100 nM to 10 μM [236].

Nsp14-Exoribonuclease (ExoN)

One coronavirus enzyme involved in RNA processing is a 3’ to 5’ exonuclease (ExoN) belonging to the DEDD super-family, predicted in SARS-CoV ppa1b [59]. The enzyma-tically active residues are conserved among the Nidovirales and reside in the N-terminal domain of nsp14. The enzymatic activity of SARS-CoV ExoN has been charac-terized biochemically [251]. The enzyme’s putative role in RNA proofreading and repair [59] is recently demonstrated in HCoV-229E, mutants of which revealed severe defects in RNA synthesis and replication failure [252]. Another coronavirus, murine hepatitis virus (MHV), exhibited 15-fold more mutations than wild-type virus when active-site residues are replaced with alanine, illustrating the impor-tance of the enzyme in coronavirus replication fidelity [253]. Further studies should enable exploitation of inhibitors of this enzyme for therapy of coronaviral infections.

Nsp15-Endoribonuclease (NendoU)

NendoU is one of the two unique genetic markers common to the nidoviruses, thought to mediate a nidovirus-specific step in RNA synthesis [59]. A distant cellular homolog, XendoU, a poly(U)-specific endoribonuclease is involved in small nucleolar RNA processing and utilization [254]. Both XendoU and NendoU depend on Mn2+ and share similar chemistry of endonucleolytic cleavage and specificity to uridylates in both ssRNAs and dsRNAs and generate products with 2'-3' cyclic phosphate ends. NendoU preferen-tially cleaves GU(U) sequences in dsRNA [254]. This short sequence is complementary to the strictly conserved core element, ACC, of nidovirus transcription regulatory sequences, which play a critical role in synthesis of subgenomic mRNA at the stage of discontinuous synthesis of nascent negative strand template whereupon body and leader TRS undergo fusion. The critical role of NendoU in coronaviral RNA synthesis is demonstrated in HCoV-229E by ablation of the activity with the substitution of alanine for Asp-6408 in pp1ab [254]. These findings of conservation of NendoU among nidoviruses, its essential role in coronavirus RNA synthesis, and lack of close cellular homolog, make NendoU an attractive drug target [254, 255]. Renzi et al. have determined the crystallographic structure of XendoU, demonstrated a unique fold in refined searches and identified the active site. Conservation of structural determinants of the active site provides a framework for the design of antiviral drugs for nidoviruses and coronaviruses [256].

2’-O-Ribose Methyltransferase (Nsp16)

This functional domain belongs to an ancient family of AdoMet-dependent ribose 2’-O-methyltransferases that is appropriated by many viruses before the three domains of life evolved from the last universal common ancestor (LUCA) [257]. Capping of the mRNA increases the stability

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and is performed co-transcriptionally in the nucleus. RNA viruses complete their life cycles in the cytoplasm and encode their own methyltransferases. Although the end product is the same, the viral and eukaryotic methyl-transferases differ in structure and catalytic mechanism. The vital role in the viral life cycle makes the methyltransferases attractive antiviral targets.

The predicted S-adenosylmethionine-dependent ribose 2’-O-methyltransferase (2’-O-MT) of SARS-CoV has 298 residues and is located on nsp16 at the 3’ end of ORF1b [59]. It is conserved among the Nidovirales. Recently, Decroly et al. demonstrated for the first time that feline coronavirus (FCoV) nsp16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity [249]. The crystal structures of several viral 2’-O-MT, including the double-stranded DNA vaccinia virus, the double-stranded RNA reovirus, and recently, the positive-sense, single-stranded RNA Dengue virus (a Flavivirus), are available [258]. A 3-D model of SARS-CoV nsp16 was constructed by von Grotthuss, illustrating the side chains of the conserved tetrad of residues (K-D-K-E) essential for cap-1 methylation and the docked AdoMet cofactor [241]. As they noted, the preceding cap-0 (mGpppN) formation is more critical than cap-1 formation, suggesting utility of inhibiting both steps by carbocyclic analogs of adenosine, such as Neplanocin A or 3-deazaneplanocin.

Luzhkov et al. found a new scaffold with modest inhi-bitory activity against Dengue virus 2′-O-methyltransferase. This was achieved by high-throughput structure-based vir-tual screening followed by bioassay tests of selected com-pounds. Potential inhibitors for the S-adenosylmethionine binding site were further explored in silico. The inhibitory activities of 15 top-ranking compounds were tested on a recombinant methyltransferase with the RNA substrate 7MeGpppAC5. This novel scaffold has an IC50 value of 60 μM [259].

Coronavirus Envelope (E) Protein

The envelope (E) protein of coronaviruses mediates viral assembly and morphogenesis [260]. SARS-CoV E protein, as well as E proteins from coronaviruses of all three taxonomic groups (HCoV-229E, mouse hepatitis virus, infectious bronchitis virus), were recently shown to form cation-selective ion channels in planar lipid bilayers [261]. It appears that coronavirus E protein belongs to the virus ion channels family, similar to influenza virus M2, HIV-1 Vpu protein, Hepatitis C virus p7, and Dengue virus M protein, among others. Virus ion channels are the latest antiviral targets known as “viroporins”. Pharmacological blockage inhibits viral budding and replication. Thus, hexamethylene amiloride (HMA) blocks Vpu ion channel activity in planar lipid bilayers and inhibits replication of HIV-1 in cultured human macrophages [262, 263]. It was recently shown to inhibit HCoV-229E and MHV E protein ion channel conductance in bilayers and viral replication in cell cultures [264]. The antiviral effect of HMA acts through the E protein because it has no antiviral effect on a recombinant MHV with the E gene deleted (MHV E) [264]. HMA is well tolerated at 10 μM by cultured HeLa cells, CEMT4 cells, primary T lymphocytes, and human blood monocyte-derived macrophages [263]. The EC50 of HMA as measured

by plaque reduction assay was 3.91 μM for MHV in L929 cells and 1.34 μM for HCoV-229E in human MRC5 cells [264].

Fig. (32). 5-(N,N-hexamethylene)amiloride. This “viroporin” has EC50 as measured by plaque reduction assay of 3.91 μM for MHV in L929 cells and 1.34 μM for HCoV-229E in human MRC5 cells.

CONCLUSION

In the 5 years since the conclusion of the SARS epidemic, much has been learned about the life cycle of SARS-CoV and new coronaviruses identified. The know-ledge is being actively translated into novel antiviral drug targets. The most promising of these are existing drugs that show anti-coronaviral activity, such as chloroquine and interferon, and engineered monoclonal antibody combi-nations. The search for a broad-spectrum 3CLpro inhibitor has made considerable progress, and promises to be fruitful in the near future. Although a renewed threat from SARS cannot be predicted, the identification of a natural reservoir of the predecessor of SARS-CoV in bats makes it imperative that we not only research, but also stockpile a number of different anti-coronaviral medications. This will enable us to meet the demands, in the event of a new outbreak, of the needs of the public, health care personnel, contacts, and others groups, who demand different levels of prophylaxis, with the attendant risks stemming from these medications.

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Received September 8, 2008 Accepted September 25, 2008

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