SARS-CoV (severe acute respiratory syndrome-associated coronavirus) caused infection of ~8000 people and death of ~800 patients around the world during the 2003 outbreak. In addition, picornaviruses such as enterovirus, coxsackievirus and rhinovirus also can cause life-threatening diseases. Replication of picornaviruses and coronaviruses requires 3Cpro (3C protease) and 3CLpro (3C-like protease) respectively, which are structurally analogous with chymotrypsin-fold, but the former is a monomer and the latter is dimeric due to an extra third domain for dimerization. Subtle structural differences in the S2 and S3 pockets of these proteases make inhibitors selective, but some dual inhibitors have been discovered. Our findings as summarized in the present review provide new potential anti-coronavirus and anti-picornavirus therapeutic agents and a clue to convert 3CLpro inhibitors into 3Cpro inhibitors and vice versa.
Viral infections are renowned as a major leading cause of acute morbidity in individuals of all ages worldwide . Respiratory viral infective disease is the major threat to all ages, particularly children under 5 years of age . The primary viral pathogens associated with acute respiratory infections include picornaviruses, CoVs (coronaviruses), adenoviruses, parainfluenza viruses, influenza viruses and respiratory syncytial viruses [3,4]. Recently, a human CoV emerged as life-threatening and highly transmissible disease causing SARS (severe acute respiratory syndrome) and thus the virus was named SARS-CoV. SARS-CoV is a positive-stranded RNA virus with a relatively large genome of ~30 kb. The genome constitutes five major open reading frames, namely replicase polyproteins, nucleocapsid proteins, spike, envelope and membrane glycoproteins. SARS-CoV typically causes respiratory and enteric diseases, pneumonia, exacerbation of asthma, neurological symptoms and myocarditis in humans and domestic animals [5–7]. The initial outbreak of SARS was first identified in Guangdong Province, China, in November 2002. This outbreak spread to many countries and had a significant impact on health and economies. The mortality rate is nearly 10% . The resulting structural and functional studies of the coronaviral life cycle has provided a number of significant targets for stopping viral replication. During SARS-CoV replication, the replicase polyproteins undergo extensive processing by two viral proteases, namely a chymotrypsin-like protease called 3CLpro (3C-like protease) since it is analogous to 3Cpro (3C protease) in picornavirus and PLpro (papain-like protease), which reside within the polyprotein. They catalyse their own release from the polyprotein and the maturation of other NSPs (non-structural proteins) to initiate virus-mediated RNA replication . These proteases, especially 3CLpro, are attractive targets for developing anti-SARS agents .
Analogous to CoV, Picornaviridae family members such as CV (coxsackievirus), EV (enterovirus) and RV (rhinovirus) also contain positive-sense single-stranded RNA, but with smaller genomes (~7.5 kb) . These picornaviral infections are responsible for causing fever, headache, sore throat and gastrointestinal distress, as well as chest and muscle pain, known as pleurodynia or Bornholm disease in many areas. In some cases, the symptoms progress to myocarditis or pericarditis, which can result in permanent heart damage or death. Similarly to SARS-CoV, CV contains a virally encoded chymotrypsin-like protease named 3Cpro responsible for viral replication .
To date, no effective antiviral drugs for CV and SARS-CoV infections are available except for over-the-counter drugs for symptomatic relief. Also the existing drugs approved for other viral infections have failed against CV and SARS-CoV. Hence it is necessary to find new leads or drugs to meet medical needs. From our efforts, several classes of inhibitors of 3CLpro have been identified (see below), among which one class of compounds also inhibit 3Cpro. Crystal structures of both 3Cpro and 3CLpro with the same inhibitors have been solved and compared to reveal the subtle differences in the active sites. This provides an opportunity to convert 3CLpro inhibitors into 3Cpro inhibitors and vice versa.
SARS-CoV 3CLpro inhibitors
Using a fluorigenic peptide as a substrate for assay , we have reported various SARS-CoV inhibitors obtained from screening of compound libraries and rational design based on the crystal structures of the product-bound form of 3CLpro . These inhibitors, as shown in Figure 1, include zinc-conjugates (1) [15,16], C2-symmetric diols (2a and 2b) [17,18], peptidomimetic α,β-unsaturated esters (3) , anilides (4) , benzotriazole (5) , N-phenyl-2-(2-pyrimidinylthio)acetamide (6) , biphenyl sulfone (7) , natural products including tea polyphenols (8)  and plant terpenoids and lignins (9 and 10) , as well as glutamic acid and glutamine peptides possessing a trifluoromethylketone group (11) , pyrimidinone (12) , four hits (13–16) from a chemical library , and pyrazole analogues (17–19) that also inhibit 3Cpro (dual inhibitors), as described in more detail below [28,29]. Their IC50 or Ki values against either only 3CLpro or both 3Cpro and 3CLpro are shown below their structures in Figure 1.
Inhibitors of SARS-CoV 3CLpro and dual inhibitors for 3CLpro/3Cpro derived from our studies
Dual 3CLpro and 3Cpro inhibitors
Among the 3CLpro inhibitors identified so far, we identified pyrazoles 17 and 18 (Figure 1) as dual inhibitors for 3Cpro of CV-B3, EV-71 and RV-14 as well as 3CLpro of SARS-CoV and CoV-229E . Compound 18 showed better inhibition against 3CLpro (IC50 2.5 μM) and 3Cpro (IC50 1.7 μM) than 17 (IC50 8.0 μM and 7.7 μM for inhibiting 3CLpro and 3Cpro respectively), indicating that the lengthy butyl-benzimidazole group in compound 17 did not provide any other additional interaction with the proteases and that the additional interaction is provided by the imidazole ring of 18. The diphenyl 4,5-dihydro-1H-pyrazole moiety of 17 fits well at the S1′ and S2 sites in the SARS-CoV 3CLpro, with the rest of the molecule at the S3 site and beyond. With this binding mode, the compound was predicted to also bind well in the 3Cpro, consistent with the inhibition data. In fact, RV 3Cpro prefers a phenyl group at the S2 site as is evident from its strong inhibition by AG7088 which has a P2-fluorophenylalanine (see Figure 2A for its structure).
AG7088 and its analogue as a potent 3C and 3CL protease inhibitor respectively
With the help of further modelling studies, we designed and synthesized some pyrazolones as dual inhibitors of 3CLpro and 3Cpro . Compound 19 showed inhibitory activity against both SARS-3CoV 3CLpro (IC50 8.4 μM) and CV-B3 3Cpro (IC50 9.6 μM) with no cytotoxicity at 200 μM . The SARS-CoV 3CLpro-inhibiting activity of the above analogues is dependent on the carboxy group bound at the S3 site and a nitro group at S1 site to form hydrogen bonds with Gln192 and Gly143 respectively. The C3 phenyl ring forms a hydrophobic interaction at the S2 site. The same compound was docked with CVB3 3Cpro and shows that the C3 phenyl ring of 19 is pointed to the S1 site and the carboxyl benzylidene group is relocated to S2 in order to form the hydrogen bond in 3Cpro due to the subtle differences between the structures of 3CLpro and 3Cpro .
Structural differences between 3Cpro and 3CLpro
Peptidomimetic inhibitors  and zinc-conjugated inhibitors  have been co-crystallized with both 3Cpro and 3CLpro to reveal the structural differences of the two proteases, as a basis for rationalizing inhibitor specificities . As mentioned above, unlike 3CLpro, which is dimeric containing three domains in each subunit, 3Cpro is a monomer with only two domains forming catalytic site. However, domains I and II from both proteases superimpose well, despite subtle differences. On the basis of structure-based sequence alignment, 3CLpro has a large loop between β-strands C1 and D1, whereas 3Cpro has smaller loops inserted between E1 and F1 and between B2 and C2 . The CVB3 3Cpro E1–F1 loop makes the S2 site more shallow and open. Therefore AG7088 with planar fluorophenylalanine at P2 and the smaller valine at P3 designed for inhibiting 3Cpro failed to inhibit SARS-CoV 3CLpro  and TG0204998 with the non-planar leucine at P2 and larger t-butylcarboxamide at P3 shows remarkably improved inhibitory activity against 3CLpro (Figure 2A). As shown in the left-hand panel of Figure 2(B), AG7088 binds well to CV-B3 3Cpro as expected, since it was originally developed as potent inhibitor against 3Cpro of human RV, but TG0204998 binds poorly to CV-B3 3Cpro as compared with its binding with SARS-CoV 3CLpro, as shown in the right-hand panel of Figure 2(B). Thus the bulky P3 residue of TG0204998 is actually relocated to the hydrophobic environment in the S4 site formed by the CVB3 3Cpro B2–C2 loop, leaving the unbound P4-benzoxy group facing the bulk solvent (see left-hand panel of Figure 2B). AG7088 fails to bind SARS-CoV 3CLpro due to the steric hindrance exerted by the fluorophenylalanine group against Arg188 in the S2 pocket and by the isoxazoyl moiety against the hydrophobic residues if overlaying AG7088 with TG0204998 in the right-hand panel of Figure 2B. These two Figures rationalize the different inhibitor specificities of 3Cpro and 3CLpro.
No effective therapy has been developed so far, and the recent isolation of strains of SARS-CoV emphasizes the possibility of a re-emergence. Therefore it is still a great challenge to explore new chemical classes of SARS-CoV 3CLpro inhibitors that can be used in anti-SARS therapy in case the disease re-emerges. There is also no anti-picornaviral drug targeting 3Cpro in the market yet, although AG7088 (now named rupintrivir), which contains a lactam ring to mimic glutamine at the P1 position and an α,β-unsaturated ester at P1′ as a Michael acceptor to form a covalent bond with the active-site cysteine residue, shows promising results against a broad spectrum of picornaviruses . We have identified several classes of 3CLpro inhibitors and some can also inhibit 3Cpro. Structural studies have revealed that 3Cpro is a monomer, whereas 3CLpro is a dimeric protein which has an extra third domain for dimer formation, although their active sites are superimposed very well . Moreover, 3Cpro has a more open but shallow S2 site, as well as a smaller S3 site, which account for different inhibitory specificity. The information discussed in the present review helps to ultimately develop drugs against infectious CoVs and picornaviruses, which should be continuously pursued.
Joint Sino–U.K. Protein Symposium: a Meeting to Celebrate the Centenary of the Biochemical Society: A Biochemical Society Focused Meeting held at Shanghai University, Shanghai, China, 5–7 May 2011. Organized by Tom Blundell (Cambridge, U.K.), Zengyi Chang (Peking University, China), Ian Dransfield (Edinburgh, U.K.), Neil Isaacs (Glasgow, U.K.), Glenn King (University of Queensland, Australia), Sheena Radford (Leeds, U.K.), Zihe Rao (Nankai University, China), Yi-Gong Shi (Tsinghua University, China), Chihchen (Zhizhen) Wang (Institute of Biophysics, Chinese Academy of Sciences, China), Jiarui Wu (Shanghai Institute of Biological Sciences, China) and Xian-En Zhang (Ministry of Science and Technology, China). Edited by Zengyi Chang and Neil Isaacs.
This research was supported by National Science Council and Academia Sinica of Taiwan.