Abstract:
The invention relates to a method of developing a main proteinase SARS inhibitor. Human coronaviruses are major causes of upper respiratory tract illnesses in humans, in particular, the common cold. Recent investigations have shown that a novel coronavirus causes the Severe Acute Respiratory Syndrome (SARS), a disease that is characterized by high fever, malaise, rigor, headache, non-productive cough or dyspnea, which is rapidly spreading. Within the scope of the invention, based on the structural analysis of the binding mode of the SARS M pro  enzyme, a group of prototype inhibitors is provided that acts as suitable drugs targeting a majority of viral infections of the respiratory tract, including SARS.

Description:
PRIOR APPLICATIONS 
       [0001]    This application is a continuation-in-part of International Patent Application Ser. No. PCT/EP2004/005109, filed May 13, 2004, which in turn bases priority on U.S. Provisional Application Ser. No. 60/469,818, filed May 13, 2003. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The invention relates to human coronavirus 229E main proteinase (M pro ) and uses in the development of inhibitors for Severe Acute Respiratory Syndrome (SARS). 
         [0004]    2. Description of the Prior Art 
         [0005]    Human coronaviruses (HCoV) are major causes of upper respiratory tract illness in humans, in particular, the common cold. To date, only the 229E strain of HCoV has been characterized in detail because it used to be the only isolate that grows efficiently in cell culture. It has recently been shown that a novel coronavirus causes the Severe Acute Respiratory Syndrome (SARS), a disease that is rapidly spreading from its likely origin in Southern China to several countries in other parts of the world. SARS is characterized by high fever, malaise, rigor, headache, non-productive cough or dyspnea and may progress to generalized, interstitial infiltrates in the lung, requiring incubation and mechanical ventilation. The fatality rate among persons with illness meeting the current definition of SARS is around 15% (calculated on outcome, i.e. deaths/deaths+recovered patients). Epidemiological evidence suggests that the transmission of this newly emerging pathogen occurs mainly by face-to-face contact, although airborne transmission cannot be fully excluded. By May 5, 2003, more than 6400 cases of SARS had been diagnosed world-wide, with the numbers still rapidly increasing. At present, no efficacious therapy is available. 
         [0006]    Coronaviruses are positive-stranded RNA viruses featuring the largest viral RNA genomes known to date (27-31 kb). The human coronavirus 229E replicase gene, encompassing more than 20,000 nucleotides, encodes two overlapping polyproteins, pp1a (≈450 kD) and pp1ab (≈750 kD) that mediate all the functions required for viral replication and transcription Expression of the COOH-proximal portion of pp1ab requires (−1) ribosomal frame shifting. The functional polypeptides are released from the polyproteins by extensive proteolytic processing. This is primarily achieved by the 33.1-kDa HCoV main proteinase (M pro ), also called 3C-like proteinase or 3CL pro , which cleaves the polyprotein at 11 conserved sites involving mostly Leu-Gln↓(Ser,Ala,Gly) sequences, a process initiated by the enzyme&#39;s own autolytic cleavage from pp1a and pp1ab. The functional importance of M pro  in the viral life cycle makes this proteinase an attractive target for the development of drugs directed against SARS and other coronavirus infections. 
         [0007]    The design of anticoronaviral drugs directed against the viral main proteinases requires the availability of data on the three-dimensional structures of the target enzymes. In 2002, we determined the crystal structure of the M pro  of transmissible gastroenteritis virus (TGEV), a coronavirus infecting pigs. The structure revealed that coronavirus M pro  consists of three domains, the first two of which together distantly resemble chymotrypsin. However, the catalytic site comprises a Cys-His dyad rather than the Ser-His-Asp triad found in typical chymotrypsin-like serine proteinases. 
       SUMMARY OF THE INVENTION 
       [0008]    We determined the crystal structure, at 2.6 Å resolution, of the free enzyme of human coronavirus (strain 229E) M pro . Further, we constructed a three-dimensional model for the M pro  of SARS coronavirus (SARS-CoV), based on our crystal structures for HCoV and TGEV M pro s, and on the genomic sequence of SARS-CoV. SARS-CoV M pro  shares 40 and 44% amino-acid sequence identity with its TGEV and HCoV counterparts, respectively. We also analyzed the putative cleavage sites of M pro  in the viral polyprotein as derived from the genomic sequence, and found them to be highly similar to those of M pro s of HCoV, TGEV and other coronaviruses. Further, we developed a method to produce recombinant SARS-CoV M pro  and modifications (mutants), thereof. We show that the recombinant wild-type enzyme exhibits proteolytic activity while an active-site mutant (C145A) does not. We demonstrate that recombinant SARS-CoV M pro  cleaves a pentadecapeptide representing the NH 2 -terminal autocleavage site of TGEV main proteinase. Comparison of the crystal structures for HCoV and TGEV M pro  and the model for SARS-CoV M pro  shows that the substrate-binding sites are well conserved among coronavirus main proteinases. 
         [0009]    In order to determine the exact binding mode of the substrate and to enable the structure-based design of drugs directed at coronavirus M pro , we have synthesized the substrate-analog chloromethyl ketone inhibitor Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK, the sequence of which was derived from the P4-P1 residues of the NH 2 -terminal autoprocessing site of HCoV M pro . We have determined the 2.37 Å crystal structure of a complex between this inhibitor and porcine transmissible gastroenteritis (corona) virus (TGEV) main proteinase. Analysis of the binding mode of this inhibitor shows that it is similar to that seen for an inhibitor of the distantly related human rhinovirus 3C proteinase. On the basis of the combined structural information, a group of prototype inhibitors 1, is proposed that should block all these enzymes and thus be suitable drugs targeting a majority of viral infections of the respiratory tract, including SARS. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0010]    The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
           [0011]    Further advantages and features of the invention can be gathered from the following description of a preferred embodiment of the invention with reference to the attached drawings, wherein: 
           [0012]      FIG. 1A  shows a three-dimensional structure of coronavirus M pro  (Monomer of HCoV M pro ) of the present invention, wherein domains I (top), II, and III (bottom) are indicated (helices are red and strands are green); 
           [0013]      FIG. 1B  shows the structure-based sequence alignment of the main proteinases of coronaviruses from all three groups including HCoV, human coronavirus 229E (group I); TGEV, porcine transmissible gastroenteritis virus (group I); MHV, mouse hepatitis virus (group II); BCoV, bovine coronavirus (group II); SCoV, SARS coronavirus (between groups II and III); and IBV, avian infectious bronchitis virus (group III); 
           [0014]      FIG. 1C  shows a Cα plot of a monomer of M pro  as a model-built on the basis of the crystal structures of HCoV 229E M pro  and TGEV M pro , wherein residues identical in HCoV M pro  and M pro  are indicated in red; 
           [0015]      FIG. 2  shows a dimer of HCoV M pro  , wherein the NH 2 -terminal residues of each chain squeeze between domains II and III of the parent monomer and domain II of the other monomer. NH 2 - and COOH-termini are labeled by cyan and magenta spheres, and letters N and C, respectively; 
           [0016]      FIG. 3A  shows a refined model of the TGEV M pro -bound hexapeptidyl chloromethyl ketone inhibitor built into electron density (2∥Fo|−|Fc∥, contoured at 1σ above the mean). There was no density for the Cbz group and for the Cβ atom of the P1 Gln. The inhibitor is shown in red, protein is shown in gray, and Cys 144  is shown in yellow (Inhibitors will bind to different coronavirus M pro s in an identical manner); 
           [0017]      FIG. 3B  shows a superimposition (stereo image) of the substrate-binding regions of the free enzymes of HCoV 229E M pro  (shown in blue), SARS-CoV M pro  (shown in magenta), and TGEV M pro  (shown in green), in complex with the hexapeptidyl chloromethyl ketone inhibitor (shown in red), with the covalent bond between the inhibitor and Cys 144  of TGEV M pro  shown in orange; 
           [0018]      FIG. 4  shows a TGEV M pro  cleavage site which is recognized and cleaved by recombinant SARS-CoV M pro . The peptide H 2 N-VSVNSTLQ↓SGLRKMA-COOH (vertical arrow indicates the cleavage site), representing the NH 2 -terminal autoprocessing site of TGEV M pro , was efficiently cleaved by M pro , but not by an inactive catalytic-site mutant of this enzyme. HPLC elution profiles of uncleaved peptide (incubated with buffer) in the absence of proteinase are represented in graph A, peptide incubated with M pro  are represented in graph B, and peptide incubated with M pro -C145A are represented in graph C; 
           [0019]      FIG. 5  shows derivatives of the antirhinoviral drug AG7088 that should inhibit coronavirus M pro s. Superimposition (stereo image) of the substrate-binding regions of TGEV M pro  (shown in green) are in complex with the hexapeptidyl chloromethyl ketone inhibitor (shown in red), and HRV2 3C pro  (shown in marine) are in complex with the inhibitor AG7088 (shown in yellow); 
           [0020]      FIG. 6  shows derivatives of AG7088, compounds 1, proposed for inhibition of coronavirus main proteinases, including SARS coronavirus (SARS-CoV) M pro  (P2=p-fluoro-benzyl, AG7088). 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0021]    As shown in  FIG. 1A , α-helices are labeled A to F according to occurrence along the primary structure, with the additional one-turn A′ α-helix in the N-terminal segment (residues 11-14). β-strands are labeled a to f, followed by an indication of the domain to which they belong (I or II). NH 2 -and COOH-terminus are labeled N and C, respectively. Residues of the catalytic dyad, Cys 144  and His 41 , are indicated. 
         [0022]    As shown in  FIG. 1B , the autocleavage sites of the proteinases are marked by vertical arrows above the sequences. In addition to the sequences of the mature enzymes, four residues each of the viral polyprotein NH 2 -terminal to the first and COOH-terminal to the second autocleavage site are shown. Note the conservation of the cleavage pattern, (small)-Xaa-Leu-Gln↓(Ala,Ser,Gly). Thick bars above the sequences indicate α-helices (numbered A′, A to F), and horizontal arrows indicate β-strands (numbered a-f, followed by the domain to which they belong). Residue numbers for HCoV M pro  are given below the sequence (3-digit numbers are centered about the residue). Symbols in the second row below the alignment mark residues involved in dimerization of HCoV and TGEV M pro  including open circle “ 602  ” (only main chain involved), asterisk “*” (only side chain involved), and plus “+” (both main chain and side chain involved). From the almost absolute conservation of side chains involved in dimerization, it can be concluded that SARS-CoV M pro  also has the capacity to form dimers. In addition, side chains involved in inhibitor binding in the TGEV M pro  complex are indicated by triangles (Δ), and catalytic-site residues Cys 144  and His 41 , as well the conserved “Y 160 MH 162 ” motif, are shaded. 
         [0023]    Referring to  FIG. 1A , the crystal structure of HCoV M pro  shows that the molecule comprises three domains. Domains I and II (residues 8-99 and 100-183, respectively) are six-stranded antiparallel β-barrels, and together resemble the architecture of chymotrypsin and of picornavirus 3C proteinases. The substrate-binding site is located in a cleft between these two domains. A long loop (residues 184 to 199) connects domain II to the COOH-terminal domain (domain III, residues 200-300). This latter domain, a globular cluster of five helices, has been implicated in the proteolytic activity of M pro . The HCoV M pro  structure is very similar to that of TGEV M pro . The r.m.s. deviation between the two structures is ˜1.5 Å for all 300 Cα positions of the molecule, but the isolated domains exhibit r.m.s. deviations of only ˜0.8 Å. The construction of HCoV M pro  used in this work lacks two amino acid residues from the COOH-terminus. HCoV M pro  Δ(301-302) has the same enzymatic properties as full-length HCoV M pro  but yields much superior crystals. In the structure of full-length M pro , residues 301 and 302 are disordered and not seen in the electron density. With HCoV 229E and TGEV both being group I coronaviruses, their main proteinases share 61% sequence identity. 
         [0024]    For comparison of its enzymatic properties with those of the HCoV and TGEV M pro s, we have expressed SARS-CoV (strain TOR2) M pro  in  E. coli  (SARS-CoV M pro  from strain TOR2, and acc. AY274119, SARS-CoV pp1a/pp1ab residues 3241 to 3544), and preliminarily characterized the proteinase. The amino-acid sequence of SARS-CoV M pro  displays 40% and 44% sequence identity to HCoV 229E M pro  and TGEV M pro , respectively (see  FIG. 1B  for a structure-based alignment). Identity levels are 50% and 49%, respectively, between SARS-CoV M pro  and the corresponding proteinases from the group II coronaviruses, mouse hepatitis virus (MHV), and bovine coronavirus (BCoV). Finally, M pro  shares 39% sequence identity with avian infectious bronchitis virus (IBV) M pro , the only group III coronavirus for which a main proteinase sequence is available. These data are in agreement with the conclusion deducible from the sequence of the whole genome that the new virus is most similar to group II coronaviruses, although some common features with IBV (group III) can also be detected. 
         [0025]    The level of similarity between SARS-CoV M pro  and HCoV, as well as TGEV M pro s, allowed us to construct a reliable three-dimensional model for SARS-CoV M pro  (See  FIG. 1C ). There are three 1- or 2-residue insertions in M pro , relative to the structural templates, and as to be expected, these are all located in loops and do not present a problem in model building. Interestingly, domains I and II show a higher degree of sequence conservation (42%-48% identity) than domain III (36%-40%), between SARS-CoV M pro  and the coronavirus group I enzymes. 
         [0026]    HCoV 229E M pro  forms a tight dimer (contact interface, predominantly between domain II of molecule A and the NH 2 -terminal residues of molecule B, ˜1300 Å 2 ) in the crystal, with the two molecules oriented perpendicular to one another, as indicated in  FIG. 2 . Our previous crystal structure of the TGEV M pro  revealed the same type of dimer. We could show by dynamic light scattering that both HCoV and TGEV M pro  exist as a mixture of monomers (˜65%) and dimers (˜35%) in diluted solutions (1-2 mg proteinase/ml). However, since the architecture of the dimers, including most details of intermolecular interaction, are the same in both TGEV M pro  (three independent dimers per asymmetric unit) and HCoV M pro  (one dimer per asymmetric unit), i.e., in completely different crystalline environments, we believe that dimer formation is of biological relevance in these enzymes. In the M pro  dimer, the NH 2 -terminal amino-acid residues are squeezed in between domains II and III of the parent monomer, and domain II of the other monomer, where they make a number of very specific interactions that appear tailor-made to bind this segment with high affinity after autocleavage. This mechanism would immediately enable the catalytic site to act on other cleavage sites in the polyprotein. However, the exact placement of the amino terminus also seems to have a structural role for the mature M pro , since deletion of residues 1 to 5 lead to a decrease in activity to 0.3% in the standard peptide-substrate assay. Nearly all side chains of TGEV M pro  and HCoV M pro  involved in formation of this dimer (marked in  FIG. 1B ) are conserved in the SARS-CoV enzyme so that it is safe to assume a dimerization capacity for the latter, as well. 
         [0027]    In the active site of HCoV M pro , Cys 144  and His 41  form a catalytic dyad. In contrast to serine proteinases and other cysteine proteinases, which have a catalytic triad, there is no third catalytic residue present. HCoV M pro  has Val 84  in the corresponding position (Cys in SARS-CoV M pro ) with its side chain pointing away from the active site. A buried water molecule is found in the place that would normally be occupied by the third member of the triad. This water is hydrogen-bonded to His 41  Nδ1, Gln 163  Nε2, and Asp 186  Oδ1 (His, His, and Asp in both SARS-CoV and TGEV M pro ). 
         [0028]    To allow structure-based design of drugs directed at coronavirus M pro s, we sought to determine the exact binding mode of M pro  substrates. To this end, we synthesized the substrate-analog chloromethyl ketone inhibitor Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK (hereinafter ‘CMK’), and soaked it into crystals of TGEV M pro  because these were of better quality and diffracted to higher resolution than those of HCoV M pro . The sequence of the inhibitor was derived from the P6-P1 residues of the NH 2 -terminal autoprocessing site of TGEV M pro  (SARS-CoV M pro  and HCoV M pro  have Thr-Ser-Ala-Val-Leu-Gln and Tyr-Gly-Ser-Thr-Leu-Gln, respectively, at the corresponding positions (see  FIG. 1B )). X-ray crystallographic analysis at 2.37 Å resolution revealed difference densities for all residues of the inhibitor, except the benzyloxycarbonyl (Cbz) protective group, in two (B and F) out of the six TGEV M pro  monomers in the asymmetric unit (See  FIG. 3A ). In these monomers, there is a covalent bond between the Sγ atom of Cys 144  and the methylene group of the chloromethyl ketone. 
         [0029]    Referring still to  FIG. 3A , there are no significant differences between the structures of the enzyme in the free and in the complex state. The substrate-analog inhibitor binds in the shallow substrate-binding site at the surface of the proteinase, between domains I and II. The residues Val-Asn-Ser-Thr-Leu-Gln occupy, and thereby define, the subsites S6 to S1 of the proteinase. Residues P5 to P3 form an antiparallel β-sheet with segment 164-167 of the long strand eII on one side, and they also interact with segment 189-191 of the loop linking domains II and III on the other. The functional significance of this latter interaction is supported by the complete loss of proteolytic activity upon deletion of the loop region in TGEV M pro . 
         [0030]    In coronavirus M pro  polyprotein cleavage sites, the P1 position is invariably occupied by Gln. At the very bottom of the M pro  S1 subsite, the imidazole of His 162  is suitably positioned to interact with the P1 glutamine side chain (See  FIGS. 3A and 3B ). The required neutral state of His 162  over a broad pH range appears to be maintained by two important interactions, 1) stacking onto the phenyl ring of Phe 139 , and 2) accepting a hydrogen bond from the hydroxyl group of the buried Tyr 160 . In agreement with this structural interpretation, any replacement of His 162  completely abolishes the proteolytic activity of HCoV and feline coronavirus (FIPV) M pro . Furthermore, FIPV M pro  Tyr 160  mutants have their proteolytic activity reduced by a factor of &gt;30. All of these residues are conserved in M pro  and, in fact, in all coronavirus main proteinases. Other elements involved in the S1 pocket of the M pro  are the main-chain atoms of Ile 51 , Leu 164 , Glu 165 , and His 171 . In M pro , Ile 51  becomes Pro and Leu 164  is Met, although this is less relevant since these residues contribute to the subsite with their main-chain atoms only, as shown in  FIG. 3B  (side chains involved in specificity pockets are marked by “Δ” in  FIG. 1B ). 
         [0031]    Apart from a few exceptions, coronavirus M pro  cleavage sites have a Leu residue in the P2 position. The hydrophobic S2 subsite of the proteinase is formed by the side chains of Leu 164 , Ile 51 , Thr 47 , His 41  and Tyr 53 . The corresponding residues in SARS-CoV M pro  are Met, Pro, Asp, His and Tyr. In addition, residues 186-188 line the S2 subsite with some of their main-chain atoms. The Leu side chain of the inhibitor is well accommodated in this pocket. As shown in  FIG. 3B , it is noteworthy that M pro  has an alanine residue (Ala 46 ) inserted in the loop between His 41  and Ile 51 , but this is easily accommodated in the structural model and does not change the size or chemical properties of the S2 specificity site. 
         [0032]    There is no specificity for any particular side chain at the P3 position of coronavirus M pro  cleavage sites. This agrees with the P3 side chain of our substrate analog being oriented towards bulk solvent. At the P4 position, there has to be a small amino-acid residue, such as Ser, Thr, Val, or Pro, because of the congested cavity formed by the side chains of Leu 164 , Leu 166 , and Gln 191 , as well as the main-chain atoms of Ser 189 . These are conserved or conservatively substituted (L164M, S189T) in SARS-CoV M pro . The P5 Asn side chain interacts with the main chain at Gly 167 , Ser 189 , and Gln 191  (Pro, Thr, Gln in the enzyme), thus involving the loop linking domains II and III, whereas the P6 Val residue is not in contact with the protein. Although the inhibitor used in the present study does not include a P1′ residue, it is easily seen that the common small P1′ residues, Ser, Ala, or Gly, can be easily accommodated in the S1′ subsite of TGEV M pro  formed by Leu 27 , His 41 , and Thr 47 , with the latter two residues also being involved in the S2 subsite, Leu, His, and Asp in M pro . Superimposition of the structures of the TGEV M pro -CMK complex and the free enzyme of HCoV M pro  shows that the two substrate-binding sites are basically the same (See  FIG. 3B ). All residues along the P site of the cleft are identical, with the exception of the conservative M190L replacement, Ala in SARS-CoV M pro . In other coronavirus species including the SARS pathogen, M pro  residues 167 and 187-189 show some substitutions, but since these residues contribute to substrate binding with their main-chain atoms only, the identity of the side chains is less important. Indeed, the substrate-binding site of the SARS-CoV M pro  model matches those of its TGEV and HCoV counterparts perfectly. Thus, there is no doubt that the CMK inhibitor will bind to the HCoV M pro  and SARS-CoV M pro , as well as all other coronavirus homologs with similar affinity and in the same way as it does to TGEV M pro . 
         [0033]    We developed a method to express SARS-CoV M pro  in  E. coli , as a fusion protein with maltose-binding protein (MBP). The free SARS-CoV M pro  was released from this fusion protein by cleavage with factor Xa. We demonstrated that the purified, recombinant SARS-CoV M pro  processes the peptide H 2 N-VSVNSTLQ↓SGLRKMA-COOH. Referring now to  FIG. 1B , this peptide, which represents the NH 2 -terminal autoprocessing site of TGEV M pro  (cleavage site indicated by ↓), and contains the sequence of our CMK inhibitor, is efficiently cleaved by SARS-CoV M pro  but not by its inactive catalytic-site mutant C145A, as shown in  FIG. 4 . 
         [0034]    While peptidyl chloromethyl ketone inhibitors themselves are not useful as drugs because of their high reactivity and their sensitivity to cleavage by gastric and enteric proteinases, they are excellent substrate mimetics. With the CMK template structure at hand, we compared the binding mechanism to that seen in the distantly related picornavirus 3C proteinases (3C pro ) . The latter enzymes have a chymotrypsin-related structure, similar to domains I and II of HCoV M pro , although some of the secondary-structure elements are arranged differently, making structural alignment difficult (sequence identity &lt;10%). Also, they completely lack a counterpart to domain III of coronavirus M pro s. Nevertheless, the substrate specificity of picornavirus 3C pro s for the P1′, P1 and P4 sites is very similar to that of the coronavirus M pro s. 
         [0035]    Referring still to  FIG. 4 , we found similar interactions between inhibitor and enzyme in case of the human rhinovirus (HRV) serotype 2 3C pro  in complex with AG7088, an inhibitor carrying a vinylogous ethyl ester instead of a CMK group. Only parts of the two structures can be spatially superimposed (r.m.s. deviation of 2.10 Å for 134 pairs of Cα positions out of the ˜180 residues in domains I and II). Both inhibitors, the hexapeptidyl chloromethyl ketone and AG7088, bind to their respective target proteinases through formation of an antiparallel β-sheet with strand eII. However, completely different segments of the polypeptide chain interact with the substrate analogs on the opposite site, with residues 188-191 of the loop connecting domains II and III in M pro , as opposed to the short β-strand 126-128 in HRV 3C pro . As a result, the architectures of the S2 subsites are entirely different between the two enzymes, hence, the different specificities for the P2 residues of the substrates (Leu vs. Phe). The inhibitor AG7088 has a p-fluorophenylalanine side chain (p-fluorobenzyl) in this position. Based on molecular modeling, we believe that this side chain might be too long to fit into the S2 pocket of coronavirus M pro , but an unmodified benzyl group would probably fit, as evidenced by Phe occurring in the P2 position of the COOH-terminal autocleavage site of the SARS coronavirus enzyme (deduced from the genomic sequence)(See  FIG. 6 ). Apart from this difference, the superimposition of the two complexes suggests that the side chains of AG7088 binding to subsites S1 (lactone derivative of glutamine) and S4 (5-methyl-isoxazole-3-carbonyl), can be easily accommodated by the coronavirus M pro . Thus, AG7088 could well serve as a starting point for modifications which should quickly lead to an efficient and bioavailable inhibitor for coronavirus main proteinases. Such modifications could incorporate all derivatives of the AG7088 compound with any P2 group, and more distantly related compounds such as AA1-AA2-AA3-AA4-P2-Gln-vinylogous ester (also the methyl and isopropylester, and other alkyl), with AA1, AA2, AA4 (any amino acid or absent), AA3 (small, such as Thr, Val, Ser, Ala), P2 (Leu, Phe, Met), and derivatives thereof. 
         [0036]    Since AG7088 is already clinically tested for treatment of the “common cold” (targeted at rhinovirus 3C pro ) and since there are no cellular proteinases with which the inhibitors could interfere, prospects for developing broad-spectrum antiviral drugs on the basis of the structures presented here are good. Such drugs can be expected to be active against several viral proteinases exhibiting Gln↓(Ser,Ala,Gly) specificity, including the SARS coronavirus enzyme. 
         [0037]    The structural information provided herein can be utilized to design or identify novel peptide drugs using, for example, a rational drug design (RDD) approach. Software applications typically utilized for such purposes include RIBBONS, Carson, M., 1997,  Methods in Enzymology,  277, P. 25; O, Jones, T A., et al., 1991,  Acta Crystallogr , A47, P. 110; DINO,  DINO: Visualizing Structural Biology,  2001, http://www.dino3d.org; and QUANTA, CHARMM, INSIGHT, SYBYL, MACROMODE, ICM, MOLMOL, RASMOL and GRASP, reviewed in Kraulis, J., 1991,  Appl. Crystallogr.,  24, P. 946. Additional information regarding RDD can be found in  Rational Drug Design  by Truhlar, et al, 1999, Springer-Verlag, Incorporated, New York. 
         [0038]    The term “peptide” as used herein encompasses native peptides, either degradation products, synthetically synthesized peptides or recombinant peptides, and peptidomimetics, typically, synthetically synthesized peptides, as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to, N terminus modification, C terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in  Quantitative Drug Design , C. A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press, 1992. Further details in this respect are provided hereinbelow. 
         [0039]    Peptide bonds (—CO—NH—) within the peptide may be substituted, for example, by N-methylated bonds (—N(CH3)—CO—), ester bonds (—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), and α-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds (—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds (—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—), or peptide derivatives (—N(R)—CH2-CO—), with R being the “normal” side chain naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time. Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted for synthetic non-natural acid such as TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. 
         [0040]    In addition to the above, the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers, e.g., fatty acids, complex carbohydrates, etc. The term “amino acid” or “amino acids” is understood to include the 20 naturally occurring amino acids, those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine, and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, the term “amino acid” includes both D- and L-amino acids. 
         [0041]    The peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized. The peptides may be synthesized by any techniques that are known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young,  Solid Phase Peptide Synthesis , W. H. Freeman Co., San Francisco, 1963, and J. Meienhofer,  Hormonal Proteins and Peptides , vol. 2, P. 46, Academic Press, New York, 1973. For classical solution synthesis see G. Schroder and K. Lupke,  The Peptides , vol. 1, Academic Press, New York, 1965. 
         [0042]    In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently to afford the final peptide compound. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, i.e., by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide, to form, after deprotection, a pentapeptide, and so forth. A further description of peptide synthesis is disclosed in U.S. Pat. No. 6,472,505. 
         [0043]    A preferred method of preparing the peptide compounds of the present invention involves solid phase peptide synthesis. Large scale peptide synthesis is described by  Andersson Biopolymers,  55, P. 227-250, 2000. The peptides can be provided to the subject per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. 
         [0044]    As used herein, a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism. Herein the term “active ingredient” refers to the preparation accountable for the biological effect. Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be interchangeably used, refer to a carrier or a diluents that does not cause significant irritation to an organism, and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases. 
         [0045]    Since activity of peptides is directly correlated with a molecular weight thereof, and measures are taken to conjugate the peptides of the present invention to high molecular weight carriers. Such high molecular weight carriers include, but are not limited to, polyalkylene glycol and polyethylene glycol (PEG), which are biocompatible polymers with a wide range of solubility in both organic and aqueous media, (Mutter et al, 1979). 
         [0046]    Alternatively, microparticles, such as microcapsules or cationic lipids can serve as the pharmaceutically acceptable carriers of this aspect of the present invention. As used herein, microparticles include liposomes, virosomes, microspheres and microcapsules formed of synthetic and/or natural polymers. Methods for making microcapsules and microspheres are known to the skilled in the art and include solvent evaporation, solvent casting, spray drying and solvent extension. Examples of useful polymers which can be incorporated into various microparticles include polysaccharides, polyanhydrides, polyorthoesters, polyhydroxides and proteins and peptides. Liposomes can be generated by methods well known in the art such as those reported by Kim, et al.,  Biochim. Biophys. Acta.,  728, P.339-348, 1983; Liu et al.,  Biochim. Biophys. Acta.,  1104, P. 95-101, 1992; Lee et al.,  Biochim. Biophys. Acta,  1103, P. 185-197, 1992; and Wang et al.,  Biochem.,  28, P. 9508-9514, 1989. Alternatively, the peptide molecules of this aspect of the present invention can be incorporated within microparticles, or bound to the outside of the microparticles, either ionically or covalently. 
         [0047]    As mentioned hereinabove the pharmaceutical compositions of this aspect of the present invention may further include excipients. The term “excipient”, refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in  Remington&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., latest edition. 
         [0048]    Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Alternately, one may administer a preparation in a local rather than systemic manner, for example, via injection of the preparation directly into a specific region of a patient&#39;s body. 
         [0049]    Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making,  levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The peptide or peptides can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. 
         [0050]    Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. 
         [0051]    For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank&#39;s solution, Ringer&#39;s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. 
         [0052]    For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose, and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt such as sodium alginate. 
         [0053]    Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. 
         [0054]    Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin, as well as soft-sealed capsules made of gelatin, and a plasticizer such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration. 
         [0055]    For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. 
         [0056]    For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. 
         [0057]    Capsules and cartridges, e.g., gelatin for use in a dispenser, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. 
         [0058]    The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. 
         [0059]    Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. 
         [0060]    Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethyl cellulose, sorbitol or dextrin. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. 
         [0061]    The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas using conventional suppository bases such as cocoa butter or other glycerides. 
         [0062]    Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art. 
         [0063]    For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient&#39;s condition. (See Fingl, et al., 1975,  The Pharmacological Basis of Therapeutics , Ch. 1, P. 1). 
         [0064]    Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations with course of treatment lasting from several days to several weeks, or until a cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent upon the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. 
         [0065]    Compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. 
         [0066]    Pharmaceutical compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. 
       EXAMPLES 
     Materials and Methods 
     Protein Expression and Purification: 
       [0067]    Recombinant HCoV 229E M pro Δ(301-302) (residues 1 to 300; COOH-terminal residues 301 and 302 deleted) was expressed and purified essentially as described previously for the FIPV and full-length HCoV main proteinases. Briefly, fusion proteins in which the HCoV pp1a/pp1ab amino acids 2966 to 3265 had been fused to the  E. coli  maltose-binding protein (MBP), were expressed in  E. coli  TB1 cells (New England Biolabs). The fusion protein MBP-HCoV-M pro Δ(301-302) was purified by amylose-affinity chromatography and cleaved with factor Xa to release HCoV M pro Δ(301-302). Subsequently, the recombinant proteinase was purified to homogeneity using phenyl Sepharose HP (Amersham Biosciences), Uno-Q (Bio-Rad Laboratories), and Superdex 75 (Amersham Biosciences) columns and concentrated to ≧15 mg/ml (Centricon-YM3, Millipore). SARS-CoV M pro Δ(305-306), which also had its two COOH-terminal residues deleted, was produced in an analogous way. As a control, a SARS-CoV M pro  mutant (SARS-CoV M pro Δ(305-306)-C145A) was expressed and purified in an identical manner. In the latter, the active-site nucleophile, Cys 145  (corresponding to Cys 3385  of the pp1a/pp1ab polyprotein), was replaced by Ala. TGEV M pro  was expressed and purified as described. 
       Preparation of Selenomethionine-Derivatized HCoV M pro : 
       [0068]    To produce selenomethionine (SeMet)-substituted protein, the coding sequence of the MBP-HCoV-M pro Δ(301-302) fusion protein was amplified by PCR and inserted into the unique NcoI site of pET-11d plasmid DNA (Novagen). The resulting plasmid, pET-HCoV-M pro Δ(301-302), was used to transform the methionine auxotrophic 834(DE3)  E. coli  strain (Novagen), which was propagated in minimal medium containing 40 μg/ml seleno-L-methionine. The SeMet-substituted HCoV M pro ΔA(301-302) was purified as described above and concentrated to ≧7.1 mg/ml (Centricon-YM3, Millipore). 
       Dynamic Light Scattering: 
       [0069]    DLS experiments were performed using a DynaPro 801 device (Protein Solutions) with sample volumes of 15 μl. 
       Cleavage of a TGEV M pro  Cleavage Site by Recombinant SARS-CoV M pro : 
       [0070]    The peptide used in this assay was H 2 N-VSVNSTLQSGLRKMA-COOH which represents the NH 2 -terminal autocleavage site of TGEV M pro , and corresponds to TGEV pp1a/pp1ab residues 2871 -2885. The SARS-CoV M pro Δ(305-306) and M pro (305-306)-C145A proteins (each at 0.5 μM) were incubated with 0.25 mM of the peptide for 45 min at 25° C. in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol. HPLC analysis of the cleavage reactions was done on a Delta Pak C 18  column as described previously. 
       Synthesis and Purification of the Hexapeptidyl Chloromethyl Ketone (Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK): 
       [0071]    Peptide synthesis was performed on an Applied Biosystems 433A peptide synthesizer using standard Fmoc-solid phase peptide synthesis protocols. The reverse-phase HPLC chromatogram showed well-resolved peaks corresponding to the free NH 2 -terminal peptide, and the desired peptide carrying the Cbz group at the NH 2 -terminal valine. The identity of the product was confirmed by mass spectrometry. Conversion of the free COOH-terminal of the purified, NH 2 -protected peptide to the chloromethyl ketone functionality was performed as previously reported. The product was then again purified by RP-HPLC, and its identity confirmed by mass spectrometry. 
       Crystallization: 
       [0072]    Selenomethionine-HCoV M pro Δ(301-302) crystals were grown at 10° C. in hanging drops by equilibration of 7.1 mg/ml protein in 11 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 1% 1,6-hexanediol, and 10% polyethylene glycol 10,000 against 20% polyethylene glycol 10,000, 2% 1,6-hexanediol, 5 mM DTT, 12% dioxane and 100 mM HEPES, pH 8.5. Within about a week, fragile, plate-like crystals (˜0.2×0.2×0.05 mm 3 ) were obtained. Crystals displayed space group P2 1  with unit cell dimensions a=53.3 Å, b=76.1 Å, c=73.4 Å, β=103.7° , and two proteinase monomers per asymmetric unit. TGEV M pro  crystals were grown as described previously, and soaked for 16 h in a fivefold molar access of Cbz-Val-Asn-Ser-Thr-Leu-Gln-CMK, dissolved in a 1:1 mixture of dimethyl sulfoxide and acetonitrile. These crystals displayed space group P2 1  with unit cell dimensions a=72.4 Å, b=158.5 Å, c=88.2 Å, β=94.4°, and six proteinase molecules per asymmetric unit. 
       Collection of Diffraction Data: 
       [0073]    Using a Mar345 detector (X-ray Research), diffraction data from crystals of SeMet-HCoV M pro Δ(301-302) were collected at 100 K using synchrotron radiation at the XRD beamline of ELETTRA (Sincrotrone Trieste, Italy) at four different wavelengths around the selenium absorption edge (see Table 1). Due to the high concentration of polyethylene glycol in the mother liquor, these crystals did not require any cryoprotectant. Crystals of TGEV M pro  that had been soaked with hexapeptidyl chloromethyl ketone inhibitor were rinsed with mustard oil before cryo-cooling in liquid nitrogen. A full diffraction data set was collected at 100 K, using the Joint IMB Jena/University of Hamburg/EMBL synchrotron beamline X13 at DESY (Hamburg, Germany) at a wavelength of 0.802 Å and equipped with a MarCCD detector (X-ray Research). For both proteins, diffraction data were processed using the DENZO and SCALEPACK programs. Diffraction data statistics are given in TABLE 1. 
       Structure Solution: 
       [0074]    The anomalous signal from selenium in crystals of HCoV M pro Δ(301-302) was weak and did not provide sufficient phase information for solving the structure. Therefore, data collected at all four wavelengths were merged and used for structure elucidation by molecular replacement using AMoRe, with a monomer of TGEV M pro  as the search model (See TABLE 2). The structure of TGEV M pro  in complex with the hexapeptidyl chloromethyl ketone inhibitor was determined by different Fourier methods. The maps showed density for all residues (except the benyzyloxycarbonyl (Cbz) protective group) of the inhibitor in the substrate-binding sites of monomers B and F. Density was weak at the Cβ atom of the P1 Gln residue, but the orientation of this side chain was still well defined due to the strong density for the carboxamide group. Density was also relatively weak for the side chains of the P5 and P6 residues of the inhibitor, indicating high mobility (particularly in the complex with monomer F). There was only little difference density near the S2 subsite in the substrate-binding clefts of the remaining four monomers, A, C, D, and E, indicating that these sites were occupied by 2-methyl-2,4-pentanediol (MPD) molecules from the crystallization medium, as in the free TGEV M pro . 
       Model Building and Refinement: 
       [0075]    Both the HCoV M pro  and TGEV M pro  CMK complex models were refined using CNS. A random set of reflections containing 4% of the total data was excluded from the refinement for calculation of R free . Model building was carried out using the program ‘O’. σ A -weighted maps were used to avoid model bias. All residues of the HCoV M pro Δ(301-302) dimer were in unambiguous electron density. The final model comprises 600 amino-acid residues, 2 dioxane molecules and 221 water molecules. For the TGEV M pro  complex structure, all amino-acid residues in all six copies of the protein had well-defined electron density, with the exception of residues 301 and 302. The final model comprises 1799 amino-acid residues, 2 hexapeptidyl chloromethyl ketones, 4 MPD molecules, 27 sulfate ions, and 925 water molecules. Refinement statistics are summarized in TABLE 3. 
       Homology Model Building: 
       [0076]    Insight II (Molecular Simulations) was used to construct the three-dimensional model for SARS-CoV M pro  on the basis of the sequence alignment with HCoV M pro  and TGEV M pro , and the crystal structures of these two enzymes. The model was energy-minimized in Insight II and inspected for steric consistency. 
       Analysis of the Structural Models: 
       [0077]    Overall geometric quality of the models was assessed using PROCHECK. For HCoV M pro  and TGEV M pro , respectively, 85.1% and 89.0% of the amino-acid residues were found in the most favored regions of the Ramachandran plot, and 15.5% and 10.5% were in additionally allowed regions. The corresponding numbers for the homology model of SARS-CoV M pro  were 87.1% and 11.3%. The agreement between structure-factor data and the atomic model was analyzed using SFCHECK. Solvent accessibilities were calculated using the algorithm of Lee and Richards as implemented in the program NACCESS (probe radius 1.4 Å). Molecular diagrams were drawn using the programs MOLSCRIPT), PyMol, and RASTER 3D. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Crystal Parameters and Statistics of Diffraction Data 
               
             
          
           
               
                 Diffraction data 
                   
                 TGEV M pro -CMK 
               
               
                 statistics 
                 HCoV M pro   
                 complex 
               
               
                   
               
               
                 Crystal Information 
                   
                   
               
             
          
           
               
                 Space group 
                 P2 1   
                 P2 1   
               
             
          
           
               
                 Unit cell parameters 
                 a = 53.3, b = 76.1, 
                 a = 72.4, b = 158.5, 
               
               
                 (Å, °) 
                 c = 73.4, β = 103.7 
                 c = 88.2, β = 94.4 
               
               
                 Estimated solvent 
                 44   
                 51   
               
               
                 content a  (%) 
               
               
                 Diffraction data 
                   
                   
               
               
                 statistics 
               
               
                 X-ray source 
                 Synchrotron 
                 Synchrotron 
               
               
                   
                 radiation b   
                 radiation c   
               
               
                 Detector 
                 Mar 345 
                 MarCCD detector 
               
               
                 No. of frames 
                 600   
                 720    
               
               
                 Crystal oscillation (°) 
                  1.0 
                 0.5 
               
             
          
           
               
                 Wavelength (Å) 
                 0.980 
                 (average) 
                  0.802 
               
             
          
           
               
                 Temperature (K) 
                 100   
                 100    
               
             
          
           
               
                 Resolution (Å) d   
                 25-2.60 
                 (2.69-2.60) 
                 50-2.37 
                 Å (2.41-2.37) 
               
             
          
           
               
                 Completeness (%) 
                 98.9 
                 99.8  
               
             
          
           
               
                 R merge  (%) d,e   
                 14.2 
                 (41.2) d   
                 8.0 
                 (28.0) d   
               
               
                 R rim  (%) d,f   
                 14.2 
                 (43.9) d   
                 2.2 
                 (8.5) d   
               
               
                 R pim  (%) d,g   
                 3.0 
                 (13.0) d   
                 0.058 
                 (22.0) d   
               
             
          
           
               
                 Redundancy 
                 12.3 
                 7.1 
               
               
                 I/σ(I) 
                  9.1 
                 9.9 
               
               
                 Mosaicity (°) 
                  1.80 
                  0.49 
               
               
                 No. of reflections 
                 216,984    
                 569,126     
               
               
                 measured 
               
               
                 Unique reflections 
                 17,533    
                 79,667    
               
               
                   
               
               
                   a Solvent content estimated according to B. W. Matthews, J. Mol. Biol., 33, P. 491, 1968. 
               
               
                   b X-ray diffraction beamline at ELETTRA, Trieste, equipped with a Mar345 detector. 
               
               
                   c Joint IMB Jena/University of Hamburg/EMBL synchrotron beamline X13 at Deutsches Elektronen-Synchrotron (DESY), Hamburg, equipped with a MarCCD detector. 
               
               
                   d Highest resolution bin shown in parentheses. 
               
               
                   e R merge  = 100 × Σ i Σ hkl |I i  − &lt;I&gt;|/Σ i Σ hkl I i , where I i  is the observed intensity and &lt;I&gt; is the average intensity from multiple measurements. 
               
               
                   f R rim  = 100 × Σ i  (N/N − 1) 1/2 Σ hkl |I i  − &lt;I&gt;|/Σ i Σ hkl I i  where N is the number of times a given reflection has been measured. This quality indicator corresponds to an R sym  that is independent of the redundancy of the measurements, M. S. Weiss, R. Hilgenfeld, J. Appl. Crystallogr., 30, P. 203, 1997. 
               
               
                   9 R pim  = 100 × Σ i  (1/N − 1) 1/2 Σ hkl |I i  − &lt;I&gt;|/Σ i Σ hkl I i . This factor provides information about the average precision of the data, M. S. Weiss, R. Hilgenfeld, J. Appl. Crystallogr., 30, P. 203, 1997. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Structure solution by molecular replacement: HCoV M pro   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Resolution range 
                 10.0-4.0 Å 
               
             
          
           
               
                 Rotation and translation function (1 st  monomer) 
               
             
          
           
               
                   
                 Best solution 
                 □ = 21.64°, β = 59.58°, γ = 256.95° 
               
               
                   
                   
                 tx = 0.483, ty = 0.000, tz = 0.250 Å 
               
               
                   
                 Correlation coefficient 
                 0.217 
               
               
                   
                 R-factor 
                 51.9% 
               
             
          
           
               
                 Rotation and translation function (2 nd  monomer) 
               
             
          
           
               
                   
                 Best solution 
                 □ = 319.92°, β = 79.38°, γ = 5.39° 
               
               
                   
                   
                 tx = 0.054, ty = 0.481, tz = 0.785 Å 
               
               
                   
                 Correlation coefficient 
                 0.213 
               
               
                   
                 R-factor 
                 52.1% 
               
             
          
           
               
                 Refinement of combined solution 
               
             
          
           
               
                   
                 Monomer 1 
                 □ = 21.80°, β = 60.40°, γ = 257.02° 
               
               
                   
                   
                 tx = 0.478, ty = −0.002, tz = 0.250 Å 
               
               
                   
                 Monomer 2 
                 □ = 320.45°, β = 79.89°, γ = 5.89° 
               
               
                   
                   
                 tx = 0.057, ty = 0.482, tz = 0.784 Å 
               
               
                   
                 Correlation coefficient 
                 0.30 
               
               
                   
                 R-factor 
                 48.8% 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Phasing and refinement statistics, and model quality 
               
             
          
           
               
                   
                   
                   
                 TGEV M pro - 
               
               
                 Phasing 
                   
                 HCoV M pro   
                 CMk complex 
               
               
                   
               
             
          
           
               
                 Refinement 
                   
                   
               
               
                 Resolution range (Å) 
                 25-2.6 
                 50-2.37 
               
               
                 R factor a   
                 0.219 
                 19.1 
               
               
                 R free   
                 0.283 
                 23.5 
               
               
                 No. of non-hydrogen atoms 
               
               
                 (average B value (Å 2 )) 
               
             
          
           
               
                 Protein 
                 4594 
                 (28.12) 
                 13,819 
                 (43.0) 
               
               
                 Water 
                 221 
                 (24.9) 
                 925 
                 (51.3) 
               
             
          
           
               
                 MPD 
                 — 
                 32 
                 (78.6) 
               
               
                 Sulfate 
                 — 
                 135 
                 (59.8) 
               
             
          
           
               
                 Dioxane 
                 12 
                 (58.39) 
                 — 
               
             
          
           
               
                 Substrate-analog inhibitor 
                 — 
                 92 
                 (71.0) 
               
             
          
           
               
                 Bonds (Å) 
                 0.012 
                   0.006 
               
               
                 Angles (°) 
                 1.5  
                  1.3 
               
               
                   
               
               
                   a R-factor = Σ (|F o | − k|F c |)/Σ |F o |