key: cord-0809139-bkkkltdy
authors: Shih, Shin‐Ru; Chen, Shu‐Jen; Hakimelahi, Gholam Hossein; Liu, Hsing‐Jang; Tseng, Chen‐Tso; Shia, Kak‐Shan
title: Selective human enterovirus and rhinovirus inhibitors: An overview of capsid‐binding and protease‐inhibiting molecules
date: 2004-04-07
journal: Med Res Rev
DOI: 10.1002/med.10067
sha: 11650b1552206bab37e5a751670a94aa3c38137c
doc_id: 809139
cord_uid: bkkkltdy

The absence of effective vaccines for most viral infections highlights an urgent necessity for the design and development of effective antiviral drugs. Due to the advancement in virology since the late 1980s, several key events in the viral life cycle have been well delineated and a number of molecular targets have been validated, culminating in the emergence of many new antiviral drugs in recent years. Inhibitors against enteroviruses and rhinoviruses, responsible for about half of the human common colds, are currently under active investigation. Agents targeted at either viral protein 1 (VP1), a relatively conserved capsid structure mediating viral adsorption/uncoating process, or 3C protease, which is highly conserved among different serotypes and essential for viral replication, are of great potential to become antipicornavirus drugs. © 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 4, 449–474, 2004

syndromes such as hand, foot, and mouth disease, and herpangina. However, the same viruses also cause potentially severe and life-threatening infections such as meningitis, encephalitis, myocarditis, polio-like syndrome, and neonatal sepsis. Human rhinoviruses infections cause nasopharyngeal syndrome (the ''common cold'') in general population of all ages. Although rhinovirus infection is self-limited, complications could still occur in patients with asthma, congestive heart failure, bronchiectasis, and cystic fibrosis.

To date, no antiviral agent has been approved by FDA for the treatment of either enterovirus or rhinovirus infection. Clinical treatments are directed toward symptomatic relief of the most prominent symptoms of each clinical syndrome. Steps such as viral attachment, uncoating, viral RNA replication, and protein synthesis in the replication cycle of enteroviruses/rhinoviruses can serve as potential targets for antiviral agents. The following sections briefly review the virology and clinical diseases of enteroviruses and rhinoviruses, and the capsid-binding/protease-inhibiting molecules that are potential agents for drug development.

Enterovirus and rhinovirus are two important pathogens within the family of picornaviridae. The human enteroviruses, so-called because most inhabit the enteric tract, include the polioviruses (types 1, 2, and 3), coxsackieviruses A (23 serotypes), coxsackieviruses B (6 serotypes), the echoviruses (32 serotypes), and the numbered enteroviruses 68-73 (Table I) . [1] [2] [3] [4] Enterovirus 72 has been reclassified as the hepatitis A virus. 5, 6 The rhinoviruses, so-called because of their special adaptation to the nasopharyngeal region, are the most important etiologic agents of the common cold in adults and children. There are more than 100 serotypes of rhinoviruses in existence.

The picornaviral genome consists of a single-stranded, positive sense (messenger-active) RNA. This viral RNA has a small protein called VPg covalently attached to its 5 0 -end and is polyadenylated at its 3 0 -terminus. 7, 8 The genomic RNAs vary in length from 7,200 to 7,500 bases. 7, [9] [10] [11] [12] [13] The 5 0 -non-coding region (5 0 -NCR) is long and highly structured, containing a cloverleaf-like structure that is important for negative strand viral RNA synthesis 14, 15 and an internal ribosome entry site (IRES) that is essential for directing translation of mRNA. [16] [17] [18] The 3 0 -NCR is short, ranging in length from 47 to 125 bases. The 3 0 -NCR also contains a secondary structure, notably a pseudoknot that plays a role in controlling viral synthesis. 19 Replication of picornaviruses takes place entirely in cytoplasm. After attachment to the host cell, the viral genomic RNA is uncoated from the viral capsid. The positive stranded viral RNA is translated to viral proteins that are essential for viral gene replication and production of new viral particles. Genome replication and mRNA synthesis occur in small membranous vesicles that are induced by several viral proteins. A single replication cycle ranges from 5 to 10 hr. The speed of viral replication depends on many factors, such as virus strain, environmental temperature, pH, host cell type, and multiplicity of infection.

Picornavirus virions are spherical in shape with a diameter of about 40 nm. The viral particle has no lipid envelope. Enteroviruses are acid stable and retain infectivity at pH lower than 3.0. Rhinoviruses, in contrast, are labile at pH less than 6.0. The capsids of picornaviruses are composed of four structural viral proteins, namely, VP1, VP2, VP3, and VP4. The capsid contains 60 structural proteins arranged into an icosahedral lattice. 20, 21 The basic building block of the picornaviral capsid is the protomer, which contains one copy of each VP1, VP2, VP3, and VP4. The shell is formed by VP1, VP2, and VP3, VP4 lies on its inner surface. VP1, VP2, and VP3, though with no homology in sequence, form a common structure: the b-barrel jelly roll. The main structural difference between VP1, VP2, and VP3 is the loop that connects the b-strands and the N-and C-terminal sequences that extend from the bbarrel domain. 22 The amino acid sequences give each picornavirus its distinct antigenicity. The surface of the virion has a prominent star-shaped plateau at the fivefold axis of symmetry, surrounded by a deep depression (''canyon''). It has been proven, in poliovirus and rhinovirus, that the canyon serves as a receptor-binding site. [23] [24] [25] [26] C

The viral RNA is translated into a long polyprotein. This single polyprotein then undergoes proteolysis by virus-encoded protease 2A and 3C (Fig. 1) . Cleavage of the Tyr-Gly pairs which connect coat precursors P1 to P2-P3 and 3C 0 -3D 0 in enterovirus is accomplished by viral proteinase 2A, 27 but the cleavage of 3C 0 -3D 0 by protease 2A is not essential for viability of the virus. 28 The remaining cleavage in P2-P3 at Gln-Gly pair is executed by viral protease 3C, which is essential for enterovirus replication. 29, 30 Sequence alignment for enterovirus 3C protease reveals no homology with mammalian protease. Therefore, 3C protease is a potential target for drug discovery. In addition to the cleavage of viral polyprotein, it has been shown that 2A pro cleaves the host cell protein eIF4G. [31] [32] [33] [34] [35] Cleavage of eIF4G prevents eIF4F from recruiting 40S ribosomal subunits to capped mRNAs, because the cleavage releases the N-terminal domain of eIF4G which binds to eIF4F which in turn binds to the 5 0 cap of cellular mRNA. This event shuts off the translation of host cellular mRNA. The viral proteases 2A and 3C also contribute to poliovirus-induced apoptosis. [36] [37] [38] Poliovirus 2A induces apoptosis through the cleavage of translation initiation factor eIF4G, 36 whereas poliovirus 3C kills cells by apoptosis through the activation of caspase. 38, 39 Similar apoptotic pathways have also been demonstrated in enterovirus 71 (EV71). Transient expression of EV71 2A protease results in triggering apoptosis. 40 EV71 3C protease induces apoptosis in the human neural cells via the activation of caspase. 41 

Rhinoviruses usually cause ''common cold,'' while enteroviruses may cause very different clinical manifestations as listed in Table II .

The common cold (summer cold), pharyngitis, tonsillitis, and croup have been frequently reported. 42 Most of the respiratory illnesses caused by human rhinovirus and enterovirus are benign, but symptoms may persist for several days, and the resultant interruption in school and work days may be substantial. In an etiology study of viral respiratory illness, rhinoviruses infection has been found to be the top cause (35.8%). 43 The enteroviruses are responsible for approximately 15% of upper respiratory infections for which etiology is identified. 44, 45 Group A coxsackieviruses are the most common cause of herpangina. However, coxsackie B viruses and echoviruses have also been reported to have the same clinical manifestations. 46 Children 1-to 7-year-old are the group with the highest incidence. There is usually an abrupt onset of fever associated with sore throat, dysphagia, and malaise. Grayish white vesicles can be seen in the posterior portion of the palate, uvula, and the tonsillar pillars. The fever lasts for 1-4 days and the symptoms begin to improve in 4-5 days, and recovery is usually within 7 days of onset.

HFMD is one of the common diseases in children, especially the children under 4 years of age. The disease is usually mild, and the onset is associated with a sore throat with or without fever. Scattered vesicular lesions can be observed in the mouth, hands, feet, and hip. EV71 and coxsackievirus A 16 are closely related in genetics and both are causative agents of HFMD. However, EV71 is associated with Table II . Diseases Caused by Enteroviruses severe neurological disease, such as encephalitis, meningitis, poliomyelitis syndrome, and even fatal pulmonary edema. [48] [49] [50] [51] [52] The neurovirulence of EV71 first came to attention in 1975 in Bulgaria when 44 people died of a polio-like disease. 53 Epidemics of EV71 causing CNS disease subsequently occurred in New York, Australia, Europe, and Asia. 47, [54] [55] [56] [57] [58] An unusual epidemic of HFMD complicated with fatal myocarditis and pulmonary edema occurred in Malaysia in 1997, and EV71 was implicated as the etiology of the outbreak. 50 In 1998, there was a large scale of HFMD outbreak in Taiwan. Cox A 16 was presumed to be the cause of HFMD at the beginning of this enterovirus epidemic. However, numerous severe complications were later found to follow cases of HFMD and 78 patients died rapidly during this outbreak. EV71 proved to be the major cause of this HFMD outbreak. 51, 52, 59 

Enteroviral meningitis is the most common cause of aseptic meningitis, and occurs in 4.5-30 per 100,000 population annually with a duration of illness lasting between 7 and 14 days. 60-62 Coxsackievirus B was associated with aseptic meningitis in 62% of infants. 63 Echovirus is a very important pathogen for meningitis. 64 The onset of enteroviral meningitisis is usually sudden, with high fever of 38-40 C. The fever pattern may be biphasic. Symptoms and signs may include headache, nausea, vomiting, stiff neck, myalgia, rash, and muscle weakness. Aseptic meningitis caused by certain enterovirus serotypes is associated with particular clinical stigmata. Encephalitis due to enterovirus infection is also well documented. Unlike aseptic meningitis, enteroviral encephalitis may have more profound acute disease and long-term sequelae. 65 The illness usually begins like aseptic meningitis, with fever and other symptoms. Central nervous system (CNS) signs include confusion, weakness, lethargy, drowsiness, and irritability. Coma or seizures may also occur. Enteroviral meningitis/encephalitis usually has a good prognosis. However, as mentioned previously, EV71 meningitis/encephalitis may accompany pulmonary edema and leads to fatality. [48] [49] [50] [51] [52] 

With the great success of the poliovirus vaccination program, poliomyelitis has now been eliminated from most of the world. When polio was widespread, most of the wild-type poliovirus infections are asymptomatic and only 0.1% of poliovirus infections result in paralysis. The remaining infections caused mild flu-like illness. The paralytic manifestations of poliovirus infections reflect the regions of CNS severely affected. 66, 67 The overall mortality rate of spinal poliomyelitis is about 5%; bulbar and medullary poliomyelitis are of higher mortality rate (near 50%).

The enteroviruses are the common pathogens that cause acute myocarditis. 68 Neonates and young infants are particularly susceptible to coxsackievirus B virus-associated myocarditis. RT-PCR-based studies of endomyocardial biopsies and autopsy specimens revealed that enteroviruses were the cause of acute myocaditis. Symptoms include palpitations, chest pain, and fever. Most of the patients recover uneventfully while small percentage of patients develop congestive heart failure, chronic myocarditis, or dilated cardiomyopathy. 69, 70 F. Hemorrhagic Conjunctivitis Among enteroviruses, enterovirus 70, and coxsackievirus A 24 are the most common pathogens causing hemorrhagic conjunctivitis. Clinical manifestations caused by these two enteroviruses are indistinguishable, including eyelid swelling, lacrimation, and pain in the eyes. Recovery is usually complete within 1-2 weeks after onset while rare cases develop a poliomyelitis-like illness. [71] [72] [73] [74] 

For some viral infectious diseases, such as those infected by poliovirus, hepatitis B virus (HBV) and influenza virus types A and B, vaccination appears to be an efficient and feasible way for disease prevention. For human rhinoviruses and enteroviruses, however, this protocol may be difficult to follow due to a broad spectrum of variants. To date, at least 102 and 65 distinct serotypes, respectively, for human rhinoviruses and enteroviruses have been reported. In addition to numerous variants, the high-mutation rate during viral replication also presents a formidable challenge for the development of effective vaccines. For these reasons, effective antiviral drugs to treat diseases caused by infection of rhinoviruses and enteroviruses should not only possess high potency and low toxicity but have also a broad spectrum of activity.

In common with many other viral pathogens, several steps in the life cycle of picornaviruses, including initial attachment, RNA polymerization, and polyprotein processing, could be targeted for potential antiviral therapy. Over the past two decades, inhibiting viral attachment/uncoating by VP1 blockers and interrupting viral replication via targeting 3A coding region or 3C protease have all been attempted in order to find effective antipicornaviral agents. However, these efforts have met with little success up to this point. Despite the disappointing results, the capsid protein VP1 is considered the most promising therapeutic target due to the fact that pleconaril, a drug candidate of the Win series with potential use in fighting cold, has reached a very advanced stage (Phase III-IV) in its clinical trials. Although pleconaril was finally rejected because of safety concerns, it is believed that the underlying cause of the adverse side effects is structure-based rather than target-based in nature. The remaining sections of this review will focus on the current efforts in developing small-molecule antiviral agents, with a particular emphasis on chemical structures exerting biological activities on either VP1 capsid protein or 3C protease.

Capsid-binding molecules block viral infection by inhibiting viral uncoating and/or viral attachment to cellular receptors on host cells. The binding site for capsid-binding compounds appears to be a hydrophobic pocket inside VP1 located under the canyon floor. Between the floor and pocket is a section containing the GH loop, a region displaying the greatest changes in viral structure induced by compound binding. Two hypotheses have been proposed to explain how capsid binders mediate antiviral functions mentioned above. In terms of uncoating, insertion of a compound into the VP1 hydrophobic pocket leads to an increase in the stability of the viral particle, rendering the virus more resistant to uncoating, a process necessary for the release of viral RNA. It is believed that uncoating of viral particle requires certain degree of capsid flexibility. Interaction with capsid binders may produce a more compacted capsid structure with limited vacant space for conformational changes essential for uncoating to take place. As for attachment, binding of inhibitors to the VP1 pocket may induce a conformational change in the viral canyon floor, the binding site of cellular receptors such as ICAM-1 molecule identified as the major rhinovirus receptor, and thus prevent adsorption of the viruses to the host cells.

Among capsid-binding compounds, the Win series of compounds in Figure 2 play a remarkable role in the development of antiviral agents against both rhino-and entero-viral infections. Disoxaril, also known as Win 51711, was the first compound of this family with satisfactory biological profiles to enter the clinical trial. This compound was found to be effective in vitro against most rhino-and enteroviral serotypes tested. [75] [76] [77] [78] [79] It also showed oral efficacy in preventing poliovirus-2 and echovirus-9 induced paralysis in mice. However, its clinical studies were discontinued due to the appearance of crystallurea in healthy volunteers at high dosage. A successor, Win 54954, was subsequently evaluated in Phase II for in vivo efficacy against two rhinoviruses (RV 23 and 39) and one enterovirus (coxsackievirus A21). [80] [81] [82] The compound significantly attenuated viral titers and the severity of colds induced by coxsackievirus A21 but failed to show any efficacy against either rinhovirus. Win 54954 had a very short half-life, presumably due to the acid lability of the oxazoline ring, and was not further developed for clinical use owing to adverse effects of flushing and rash. Attempts were then made to discover more hydrolytically stable analogues with comparable antiviral potency. These studies led to the discovery of a series of 2-methyltetrazole compounds which are not only resistant to acidic conditions but maintain a broad spectrum of activity. 80, 83 Win 61605, regarded as the most promising candidate in the series, was selected for the treatment of rhino-and entero-viral infections. Unfortunately, when administered orally to beagles, this molecule caused hepatotoxic side effects. The hepatotoxicity is presumbably due to the multiple nitrogen tetrazole ring or its metabolic product(s). As a result, Win 61605 was dropped for further evaluation. In continuation of the search for structurally related bioisosteric molecules with reduced hepatotoxicity, Win 63843, also referred to as pleconaril, finally emerged as a promising new drug candidate for the treatment of human enteroviral infections. [84] [85] [86] In addition to a better metabolic stability in the monkey liver microsomal assay, the newly developed 5-methyl-oxadiazole analogue has also been shown to be more potent than its oxazoline (Win 54954) and tetrazole (Win 61605) predecessors against a variety of rhino-and entero-viruses. Pleconaril can be given by oral administration and is currently being developed by ViroPharma for the treatment of diseases associated with picornavirus infections. This drug candidate is in its late-stage clinical trials for treating viral respiratory infections and viral meningitis. Unfortunately, even though pleconaril was demonstrated to be effective in shortening the number of days patients felt sick and reducing the severity of symptoms, it was not approved by FDA for marketing due to safety concerns, making the drug's fate uncertain. Considering the tremendous synthetic efforts made as well as the two decades consumed on the development of the Win compounds, this unexpected result is extremely discouraging.

BTA-188 (Biota Scientific Management Pty. Ltd.), a lead of a new class of capsid-binding antiviral agents, has been shown to possess a broad spectrum of activity against rhinoviruses. [87] [88] [89] [90] BTA-188 (Fig. 3) inhibits 87 of 100 HRV serotypes. In the cytopathic effect reduction assay for HRV-14, BTA-188 (EC 50 ¼ 1.0 ng/mL) was found to be superior to both pleconaril (EC 50 ¼ 30 ng/mL) and pirodavir (EC 50 ¼ 3.2 ng/mL) in potency; in a virus yield reduction assay, a potent inhibition of HRV-2 with an EC 90 value of 0.73 ng/mL was also observed. Cytotoxicity of BTA-188 was detected only at much higher concentrations and in toxicopharmacokinectic studies. The high-oral bioavailability, 62-64% in rats and 21-28% in dogs, suggests that BTA-188 can be administered orally. Replacement of the oxime-substituted phenyl ring with various bicyclic heterocyclic rings led to another series of compounds, as represented by Biota Benzazole 15 (Fig. 3) , with significant activity against HRV-2 and HRV-14 even at concentrations as low as 0.006 mg/mL (IC 50 ). 91, 92 Further evaluation of these two classes are ongoing and the results have not been disclosed.

Recently, using the skeleton of Win compounds as structural templates, a structure-based drug design group at National Health Research Institutes (NHRI) in Taiwan has generated a library of virtual compounds whose minimum-energy conformations bear close similarity to the shape of VP1 pocket of human rhinoviruses and may fit into this cavity well. These studies resulted in the development of a series of imidazolidinone derivatives, such as BPR0Z 112 and 284 shown in 93, 94 The antiviral activity for EV 71 makes this series extremely significant and useful for developing potential anti-EV 71 agents. In 1998, many children in Taiwan fell victim to HFMD, aseptic meningitis/encephalitis, or acute flaccid paralysis, resulting in about 80 fatalities; EV71 was identified as a major pathogen in the etiology of these cases. Young children appear to be more susceptible to EV71 virus infection, also after infection with more severe symptoms. Unfortunately, after 1998 epidemic outbreak, EV71 has been continually isolated through the whole island all year round, and many severe cases caused by EV71 have also been reported. Pleconaril, claimed to possess broad-spectrum activity against enteroviruses, was tested for its antiviral activity against EV71. However, Pleconaril failed to neutralize the cytopathic effect (CPE) of cultured cells induced by EV71 isolated from the 1998 outbreak in Taiwan. This finding underscores the necessity of developing antiviral agents using materials isolated from local strains. Time-course studies showed that imidazolidinones effectively inhibited the early stages of EV71 viral infection, suggesting that the surface protein VP1 is highly likely to be the molecular target for this type of compounds. Currently, this class of compounds is under active investigation to evaluate their potential in therapeutic utility.

Another series of capsid-binding compounds exemplified as SCH 38057 and 47802 (Fig. 5 ) were synthesized at Schering-Plough. 95,96 SCH 38057, a phenoxyl imidazole compound in its hydrochloride salt form, is a water-soluble molecule which inhibited plaque formation of selected enteroviruses (cox B3, A21, polio 2, and echo 9) and rhinoviruses (HRV 14, 1A, 10, 28, 45, and 61) in a range of IC 50 ¼ 10.2-29.1 mM and IC 50 ¼ 20.4-29.1 mM, respectively. When administered orally (60 mg/kg, three times per day), SCH 38057 protected mice infected with either cox B3 or echo 9 from mortality for 21 days. The subsequent SCH 47802 and its derivatives SCH 48972, 48974, 49860, 49861, and 48973 (Fig. 5) exhibited potent activity against a panel of enteroviruses, including polio 2, echo 3-7, 11, and 30, cox A9, B1-3, and B5, at concentrations (IC 50 ) ranging from 0.02 to 10 mg/mL in plaque reduction assays. Cytotoxicity assays conducted on HeLa and RD cells showed that their IC 50 values were all over 50 mg/mL. SCH 47802, administered orally, protected mice with polio 2-induced encephalitis from mortality at day 21 with survival rates 57, 47, and 66% at a dose of 60, 90, and 120 mg/kg/day, respectively; its closely related analogue SCH 48973 also showed an increase in Pirodavir (R 77975) and its predecessor (R 61837), as shown in Figure 6 , were discovered at Janssen Research Foundation. Both compounds possessed significant activity in inhibiting the replication of many rhinovirus serotypes. [98] [99] [100] Compared to R 61837, pirodavir showed an improvement in potency by more than 500-fold in vitro and inhibited about 80% of rhinoviral serotypes at concentrations of 0.1 mg/mL or less. When the nasal sprays were given six times a day for 5 days to the patients, significant reductions in virus shedding occurred but no clinical benefits were observed. The lack of clinical efficacy of pirodavir could be due to the low-water solubility of this series, making it difficult to administer in an aqueous formulation compatible with respiratory secretion, and/or the labile ester functional group prone to rapid hydrolysis to form the corresponding inactive acid. Although R 61837 is much less active against most rhinovirus serotypes relative to R 77975, when given prophylactically, it was found to be effective in preventing colds in human volunteers challenged with rhinovirus 9. Given by intranasal spray sixtimes a day for 4 consecutive days (total dose, 25 mg) commencing 28 hr before virus challenge, R 61837 was able to suppress symptoms until 48 hr after medication ceased. In these studies, both compounds were formulated with 10% 2-hydroxypropyl-b-cyclodextrin to enhance their water solubility.

Independently, a class of compounds which shared the common piperazine-ring motif with R 61837 was discovered at Sandoz Forschungsinstitut. [101] [102] [103] As typified by SDZ 35682 and 880061 in Figure 7 , these novel piperazine-containing derivatives are potent and selective inhibitors against a series of human rhinovirus serotypes and some enteroviruses in vitro. The former (SDZ 35682) is active against rhinovirus serotypes such as HRV 14, 26, 35, 37, 43, and 48 with IC 50 less than 0.1 mg/ mL and echovirus 9 with IC 50 value of 0.3 mg/mL; the latter (SDZ 880061) is more selective towards human rhinoviruses with a relatively broader antiviral spectrum. Of the 89 HRV serotypes tested, SDZ 880061 inhibited 31 in 89 with IC 50 values equal to or lower than 0.0003 mg/mL and 76 in 89 with IC 50 lower than 3 mg/mL. Similar to the R series, SDZ 35682 and 880061 are also typical capsid-binding molecules, the evidence of which was individually substantiated by their co-crystallization with HRV 14. 102,103 A considerable conformational change at VP1 binding site was observed in the HRV 14/ SDZ 35682 complex in which SDZ 35682, a compound of 19 Å in length, fills the entire VP1 hydrophobic pocket including the innermost end and occupies the space more efficiently than other long antiviral agents such as Win 51711. It has been suggested that compounds fitting into the entire pocket might affect the uncoating process of the viral particles. SDZ 880061 was also found to bind at the same pocket in its HRV 14 complex structure. However, the innermost portion of the pocket is vacant, causing less alteration of the VP1 backbone conformation compared to other antiviral agents analyzed structurally. As a result, SDZ880061 only has marginal effects on viral uncoating. This may provide an explanation for the observation that, in the time-course studies, SDZ 880061 was found to primarily interfere with the HRV 14 adsorption to the cell instead of inhibiting viral uncoating. Both of these compounds showed no detectable cytotoxic effect up to 30 mg/mL. SDZ35682 at a dose of 126 mg/kg reduced echovirus 9-induced paralysis and shortened the mean time of paralysis by 70% in mice. Greater than 85% protection from Echovirus 9-induced death can be achieved by either a low dose (71 mg/kg) given for 6 days or a high dose (126 mg/kg) administered for 2 days. Although SDZ 35682 showed antiviral efficacy in mice, its clinical usefulness may be limited due to a narrow antiviral spectrum. As for SDZ 880061, its in vivo studies are not available.

Some capsid function inhibitors are synthetic derivatives or analogues based on the core structures of naturally occurring products. Rhodanine (Fig. 8) , 2-thio-4-oxothiazolidine, was synthesized and evaluated in various biological systems in the 1970s. 104, 105 The results revealed that the spectrum of the virus inhibitory activity of rhodanine was extremely narrow. At a concentration of 12.5 mg/mL, only selective inhibition against echovirus 12 was observed. However, it is non-toxic to the uninfected host cells (monkey kidney cells) at a concentration up to 150 mg/mL. Several derivatives and analogues of rhodanine were prepared and tested (Fig. 8) , but they were all considerably less potent than rhodanine itself. 104 4 0 ,6-Dichloroflavan (BW 683C) as illustrated in Figure 9 with a flavanoid-like skeleton is highly effective against some of the most prevalent rhinoviral serotypes (1A, 1B, 2, 15, 29, and 31) , with in vitro IC 50 values between 0.007 and 0.17 mM/mL. 106 Mechanistic studies indicated that BW683C blocked viral replication by inhibiting a stage immediately after the entrance of the viral RNA into host cells. In order to improve the potency as well as to broaden the antiviral spectrum, synthetic flavanoids substituted with halo, cyano, and amidino groups were prepared and tested for their in vitro activity against HRV 1B, polio 2, cox B4, echo 6, and EV71. [107] [108] [109] Among the synthetic flavanoids tested (e.g., compounds 1-4 shown in Fig. 9 ), 4 0 -chloro-6-cyanoflavan (3) was found to be not only more active against HRV 1B infection than parental BW 683C, but also showed good antienteroviral activity within micro and submicromolar range (IC 50 ¼ 0.32-1.28 mM). In contrast, BW 863C was inactive for most of the enteroviruses tested with the exception of cox B4 for which moderate activity was observed ($2.6 mM). Among the compounds listed in Figure 9 , compound 3 exhibited the most potent activity against EV71 (IC 50 ¼ 0.45 mM). However, relative to those EV71-specific imidazolidinones (Fig. 4 ) discovered at NHRI (Taiwan), this compound is much shorter in length with a decrease in activity by tenfold. The higher potency observed for the imidazolidinones could be attributed to their more efficient occupation of the VP1 pocket to produce structurally more stable virions.

4 0 -Ethoxy-2 0 -hydroxy-4,6 0 -dimethoxychalcone (Ro 09-0410) in Figure 10 is a chalcone-like synthetic compound which possesses significant activity against rhinoviruses, but shows no activity against other picornaviruses. 110,111 Among 53 rhinovirus serotypes tested, 46 were sensitive to Ro 09-0410 in HeLa cell cultures. The IC 50 value for antiviral activity is around 0.03 mg/mL while the CC 50 (50% cytotoxic concentration) is more than 30 mg/mL. Ro 09-0410 was found to be ineffective against rhinovirus infections in human volunteers, probably because of its poor water solubility and oral bioavailability. Studies on various analogues related to this antirhinovirus agent led to the identification of a novel class of chalcone amide analogues 112 (e.g., 09-0696, 09-0881 in Fig. 10) which, compared to Ro 09-0410, were 4.5-to 10-fold more active against 12 selected HRV serotypes at concentrations as low as 2-3 ng/mL and showed very low cytotoxicity (30-50 mg/mL). Labeling studies indicated that these amide compounds competitively inhibited the binding of Ro 09-0410 [ 3 H] to the viral capsid site in a manner similar to BW 863C, VP 63843, and WIN 51711. No clinical information is available for this series of compounds. The mode of action of above-mentioned capsid-binding molecules has been experimentally verified with evidence indicating that the inhibition of viral replication occurred at the early stages during viral attachment and/or uncoating. Some compounds, while their mechanisms have not been studied or reported, are also likely to function as capsid-binding molecules in light of their linear structural profiles as well as lipophilic properties. For example, a novel class of azolyalkyloxy compounds synthesized by Synphar Laboratories, Inc., Edmonton, Alberta, Canada is believed to mimic Win 51711 (disoxaril). 113 The active compounds of this series, as typified with 3methylisoxazole and 4-methylthiazole derivatives in Figure 11 , showed in vitro activity against 33-40 of a panel of 52 rhinovirus serotypes with IC 50 values ranging from 0.5 to 25 mg/mL. Some of them also showed moderate activity (IC 50 ¼ 1-25 mg/mL) against 5 to 6 serotypes of 7 enteroviruses tested. Moreover, the structure-activity relationship studies revealed that, like most of Win compounds, the optimal length of the alkyl chain between two terminal heterocyclic moieties of this series is either 6 or 7 methylene units.

In sharp contrast, some antipicornaviral agents, such as the diaryl methanes and arakylaminopyridines shown in Figure 12 and the 2-(4-pyridylaminomethyl)benzimidazole derivatives shown in Figure 13 possess a much shorter chain length in which a linker containing only one or two atoms is observed. The first two classes, diaryl methanes and arakylaminopyridines, were developed by Kenny et al. 114 Among these 26 compounds evaluated against rhinoviruses 1A, 2, and 64 as well as coxsackievires 21, several were found to exhibit moderate activity with IC 50 ranging from 0.3 to 5 mg/ mL. Based on these observations, diaryl methane 6 and arakylaminopyridine 7 (Fig. 12) were selected for further testing against a larger panel of picornaviruses and their in vivo antiviral efficacy. Both compounds exhibited similar in vitro activity, inhibiting 12-15 of the 23 picornaviruses tested at concentrations less than 5 mg/mL. In addition, the arakylaminopyridine was found to be more active in vivo in protecting cox A21-infected mice at a single oral dose of 37.5 mg/kg or at a continuous oral dose of 18.8 mg/mL per day. With these observed in vitro potency and in vivo efficacy, their potential clinical application to the treatment of picornavirus infection diseases appears to be limited. As for 2-(4-pyridylaminomethyl)benzimidazole analogues, 115 their in vitro activities against polio 2, cox B4, HRV 14 , and enterovirus 70 were tested, showing that these analogues are particularly effective against enterovirus 70 with IC 50 values as low as 0.52 mg/mL. Cytotoxicity evaluation using LLC-MK 2 and HaLa cells indicated that this series of compounds is not toxic up to concentrations more than 100 mg/mL (CC 50 ). Representative compounds 9 and 10 ( Fig. 13 ) prevented the development of Cox B4-induced hypoglycermia in mice for 2-4 days after infection when given intraperitoneally (i.p.) with an initial dose of 40 mg/kg/day followed by 80 mg/kg/day for 3 days. Results of clinical application are not available at the present time.

Some N-and O-substituted amino acid analogues were synthesized during the development of antiviral agents. Several substituted glycine analogues, as exemplified in Figure 14 , showed moderate activity against cox A13, B4, and echo 11 with IC 50 values around 1.8 mg/mL. No in vivo studies were reported.

In summary, the active capsid-binding compounds generally are characterized by a long linear methylene spacer with either an aromatic or heterocyclic ring attached to both ends, making the molecules considerably hydrophobic. From genetic point of view, these common structural features implies that the structure of ''sock-like'' VP1 coat protein are highly conserved through the evolution of piconarviruses, with the intrinsic hydrophobic property of the VP1 pocket as well as the cavity shape well preserved. Therapeutically useful antiviral drugs should have broad antiviral spectrum, high potency, and low cytotoxicity. Therefore, molecules acting at the viral VP1 pocket, where only a subtle difference in the size is observed for various serotypes in the same genus or even for different genera, could be promising therapeutic agents for treating picornaviral infection.

To date, numerous compounds with significant in vitro activity against HRVand EV have been found. However, the majority of these compounds, as shown in the previous section, bind to the viral capsid and inhibit either viral attachment/adsorption or subsequent uncoating. In addition to agents that interfere with the early stage in the picornaviral life cycle, attempts have been made recently to develop inhibitors to block the virus-coded 2A or 3C proteases at the synthetic stage of the virus replication. Antipicornaviral agents designed to target the 3C protease, which is highly conserved among different viral serotypes, have exhibited great potential in therapeutic utility. Since peptide aldehydes have been successfully used as inhibitors for cysteine and serine proteases and were shown to form reversible covalent adduct, the modified tripeptide aldehydes were designed and synthesized as inhibitors for HRV 3CP. 117 Molecular models based on the apo crystal structure of HRV-14 3CP and other trypsin-like serine proteases were constructed to approximate the binding of peptide substrate, generating transition state models of P 1 -P 1 0 amide cleavage. Since glutaminal derivatives exist predominantly in the cyclic hemiaminal form, several isosteric replacements for P 1 carboxamide side chain were designed and incorporated into the tripeptide aldehydes. The synthesized compounds were found to be potent inhibitors of purified HRV-14 3CP with K i s ranging from 0.005 to 0.65 mM. As shown in Table III , these compounds have low Table V . Inhibitory, Anti-HRV Activity, and Cytotoxicity of Compounds 14-22

The material inthistable istaken part from Ref. 119. micromolar antiviral activity, low toxicity, and reasonable therapeutic index. Along this line, structure-based design of ketone-containing tripeptidyl HRV 3CP reversible inhibitors were also reported. 118 An excellent example of such compounds (e.g., 13) displayed potent 3CP inhibition activity and in vitro antiviral property when tested against HRV serotype-14 (see Table IV ). (Table V) were also synthesized to inhibit picornaviral 3C proteases. 119 The K i for the synthesized molecules were found to be in the range of 0.0045-1.7 mM. Details of the biological screening results are summarized in Table V. HRV 3C protease was also inactivated by a series of S-nitrothiols. 120 They include SNAP, GSNO, Glucose-SNAP-2, S-nitrosocaptopril, and ELAFQCG-SNO, which exhibited inhibitory activities in a time-and concentration-dependent manner with second-order rate constants (K inact /K 1 ) ranging from 131 to 5,360/M/min (Table VI ). The inactivated enzyme was shown to be reactivated by DDT, GSH, and ascorbate, indicating that the inactivation process was through an S-transnitrosylation process.

A new class of 3CP inhibitors containing a tripeptide binding determinant as well as a Micheal acceptor moiety capable of binding irreversibly to the active site cysteine of 3C enzyme was described as agents against rhinovirus (Table VII) . 121 Indeed, analysis of the HRV-2 3CP X-ray crystal structure 122 revealed that only the trans P 1 Gln amide hydrogen atom interacted with the protease while the cis NH was found to be exposed to the solvent.

The P 1 -lactam-containing inhibitors (e.g., 24) display enhanced 3CP inhibition activity along with improved antirhinoviral properties relative to the corresponding glutamine-derived molecules (e.g., 23) (Table VIII) . Being one of the most potent inhibitors in this class, compound 24 (AG-7088), which is formulated for intranasal delivery in Phase II trial, exhibited better potency and a broader spectrum of antirhinoviral activity than pleconaril towards clinical HRV isolates. 123, 124 The median EC 50 value determined by microscopic CPE inhibition was slightly better for AG-7088 compared to Table X . 5 

The material inthistable istaken part from Ref. 127. pleconaril (P ¼ 0.02) but was indistinguishable by spectrophotometric assay (P ¼ 0.15). In the case of clinical HRV isolates, however, the median EC 50 value determined for AG-7088 either microscopically or spectrophotometrically was < 1.0 mg/mL and was found to be > 10.0 mg/mL for pleconaril.

Symptom severity in patients with HRV-induced respiratory illness is correlated with elevated levels of inflammatory cytokines interleukin-6 (IL-6) and IL-8. AG-7088 was tested for its antiviral activity and ability to inhibit the production of IL-6 and IL-8 in a human bronchial epithelial cell line, BEAS-2B. 125 Infection of BEAS-2B cells with HRV-14 resulted in the production of both infectious virus and the cytokines IL-6 and IL-8. Treatment of HRV-14 infected cells with AG-7088 resulted in a dose-dependent reduction in the levels of infectious virus as well as a reduced IL-6 and IL-8 level in the cell supernatant. AG-7088 was able to inhibit the replication of the virus in BEAS-2B cells. 126 In order to have more favorable pharmacokinetic properties and to develop orally available 3CP inhibitors, certain substituted benzamides as non-peptide inhibitors of HRV 3CP were invented. 127 a, b-Unsaturated keto benzamides (Table IX) showed good inhibitory property and moderate activity; yet 5-substituted benzamides (Table X) were found to be more active.

Evaluation of reversible, non-specific inhibitors of HRV 3C protease led to a novel series of 2,3dioxindoles (isatins) by using a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship analysis. 128 The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis. Molecular modeling using the apo crystal structure of HRV-14 3CP and a peptide substrate model provided the design template for building recognition features into P 1 and P 2 subsites, respectively, from 5-to 1-positions of isatin. The synthesized compounds (see Table XI ) were found to possess excellent inhibitory properties toward HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B.

Recently, it was claimed that compounds having quinone moiety as well as quinone analogues are useful inhibitors for cysteine proteases, in particular, caspases and 3C cysteine proteases. 129 These compounds, as exemplified in Figure 15 , have been tested against HRVs 1A, 1B, and 14 and show moderate in vitro activity with IC 50 value s around submicro to micromolar range. Mechanistically, they are assumed to act as active Michael acceptors which are prone to attack by the cysteine residue and thus disrupt the ability of cysteine protease to cleave a peptide chain.

In summary, viral proteases play an essential role in the life cycle of many viruses such as picornaviruses, herpesviruses, retroviruses, and coronaviruses, and therefore, have been selected as targets for developing antiviral drugs. Protease inhibitors including saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir are available for treating diseases caused by HIV, a species of retroviruses. Antipicornavirus compounds targeting 3C protease are currently under active investigation. Among them, AG-7088, a potential treatment against rhinovirus causing the common cold, is now in Phase II clinical trial. Most recently, AG-7088 has shown to exhibit moderate in vitro activity against the coronavirus responsible for severe acute respiratory syndrome (SARS). Modeling studies 130 also indicate that AG-7088 is a promising starting point in the search for a treatment for SARS via targeting 3CL protease, the main protease controlling the coronavirus replication.

Portraits of viruses: The picornaviruses

Enterovirus infections: Diagnosis and treatment

Picornavirus infections: A primer for the practitioner

Molecular identification of new picornaviruses and characterization of a proposed enterovirus 73 serotype

Biophysical and biochemical characterization of hepatitis V virus

Classification of hepatitis A virus as enterovirus type 72 and of hepatitis B virus as hepadnavirus type 1

Primary structure, gene organization, and polypeptide expression of poliovirus RNA

Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome

Molecular cloning and complete sequence determination of RNA genome of human rhinovirus type 14

Comparison of the complete nucleotide sequences of echovirus 7 strain UMMC and the prototype (Wallace) strain demonstrates significant genetic drift over time

Figure 15. Structures of quinone analogues

Genetic reclassification of porcine enteroviruses

Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot, and mouth disease during an epidemic in Taiwan

Echovirus 5: Infectious transcripts and complete nucleotide sequence from uncloned cDNA

Poliovirus 5 0 -terminal cloverleaf RNA is required in cis for VPg uridylation and the initiation of negative-strand RNA synthesis

Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C

Comparative sequence analysis of the 5 0 noncoding region of the enteroviruses and rhinoviruses

New model for the secondary structure of the 5 0 non-coding RNA of poliovirus is supported by biochemical and genetic data that also show that RNA secondary structure is important in neurovirulence

Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine tract-binding proteins executed by polioviral 3C(pro)

Biochemical and genetic evidence for a pseudoknot structure at the 3 0 terminus of the poliovirus RNA genome and its role in viral RNA amplification

Physical principles in the construction of regular viruses

Structure of a human common cold virus and functional relationship to other picornaviruses

Myristylation of picornavirus capsid protein VP4 and its structural significance

Antigen chimeras of poliovirus

Three-dimensional structure of poliovirus at 2.9 Å resolution

Analysis of the structure of a common cold virus, human rhinovirus 14, refined at a resolution of 3.0 Å

Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus

Viral proteinases

Proteolytic processing of poliovirus polyprotein: Elimination of 2Apro-mediated, alternative cleavage of polypeptide 3CD by in vitro mutagenesis

Cleavage specificity of coxsackievirus 3C proteinase for peptide substrate (2): Importance of the P2 and P4 residues

Proteolysis is a key process in virus replication

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex

Inhibition of translation by poliovirus: Inactivation of a specific initiation factor

Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells

Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection

Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection

Effects of poliovirus 2A(pro) on vaccinia virus gene expression

Apoptosis-inducing and apoptosis-preventing functions of poliovirus

Poliovirus protease 3C(pro) kills cells by apoptosis

Two types of death of poliovirus-infected cells: Caspase involvement in the apoptosis but not cytopathic effect

Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death

The 3C protease activity of enterovirus 71 induces human neural cell apoptosis

Respiratory illness caused by picornavirus infection: A review of clinical outcomes

Acute respiratory infection in children of developing countries: Challenge of the 1990s

Enteroviruses in human disease

Enteroviral disease in the United States, 1970-1979

Herpangina: The etiologic spectrum

Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement

Enterovirus 71 infections and neurologic disease-United States, 1977-1991

Fatal enterovirus type 71 infection: Rapid detection and diagnostic pitfalls

Fatal enterovirus 71 encephalomyelitis

Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease

An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group

Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria

New enterovirus type associated with epidemic of aseptic meningitis and/or hand, foot, and mouth disease

Enterovirus 71 infection: Report of an outbreak with two cases of paralysis and a review of the literature

Enterovirus type 71 infection in Melbourne

Virological diagnosis of enterovirus type 71 infections: Experiences gained during an epidemic of acute CNS diseases in Hungary in 1978

Monoplegia caused by Enterovirus 71: An outbreak in Hong Kong

Update on viral encephalitis

Clinical characteristics, management strategies, and cost implications of a statewide outbreak of enterovirus meningitis

Summary of notifiable diseases-United States

Enteroviral meningitis. Cost of illness and considerations for the economic evaluation of potential therapies

Group B coxsackievirus infections in infants younger than three months of age: A serious childhood illness

Profile of enterovirus disease in the first two weeks of life

An epidemic of encephalitis and meningoencephalitis in children caused by echovirus type 30 in Shanghai

A comparison of the clinical features of poliomyelitis in adults and children

Poliomyelitis in children: Experience with 956 cases in the 1955 Massachusetts epidemic

Enteroviral myocarditis and dilated cardiomyopathy: A review of clinical and experimental studies

Similar prevalence of enteroviral genome within the myocardium from patients with idiopathic dilated cardiomyopathy and controls by the polymerase chain reaction

Failure to demonstrate enterovirus aetiology in Swedish patients with dilated cardiomyopathy

An epidemic of acute haemorrhagic conjunctivitis caused by enterovirus 70 in Okinawa

Acute hemorrhagic conjunctivitis due to enterovirus 70 in India

Ten years' surveillance of viral conjunctivitis in Sapporo

Enteroviruses: Polioviruses (poliomyelitis), coxackieviruses, echoviruses, and enteroviruses

In vitro activity of WIN 51711, a new broad-spectrum antipicornavirus drug

5-Dihydro-2-oxazolyl)phenoxy]alkyl]isoxazoles. Inhibitors of picornavirus uncoating

Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug

Enantiomeric effects of homologues of disoxaril on the inhibitory activity against human rhinovirus-14

Synthesis and structure-activity studies of some disubstituted phenylisoxazoles against human picornavirus

Antipicornavirus activity of tetrazole analogues related to disoxaril

Oxadiazoles as ester bioisosteric replacements in compounds related to disoxaril. Antirhinovirus activity

In vitro and in vivo activities of WIN 54954, a new broad-spectrum antipicornavirus drug

Antirhinoviral activity of heterocyclic analogs of Win 54954

Picornavirus inhibitors: Trifluoromethyl substitution provides a global protective effect against hepatic metabolism

Treatment of enterovirus meningitis with pleconaril (VP 63843), an antipicornaviral agent, abstr. LB-27, p 14

Activity of pleconaril against enteroviruses

A new class of capsid-binder with potent inhibitory activity against rhinovirus in cell culture

In vitro anti-rhinovirus spectrum and potency of BTA 188, a novel, oral picornavirus capsid binder

Preclinical pharmacokinetics of a new rhinovirus inhibitor, BTA 188

Antiviral agents. World (PTC)

Antiviral agents. World (PTC) Patent WO-0250045

Antiviral agents. World (PTC)

Structure-based drug design of antirhinoviral compounds

Design, synthesis, structure-activity relationship of pyridyl imidazolidinones: A novel class of potent and selective human enterovirus 71 inhibitors

SCH 38057: A picornavirus capsid-binding molecule with antiviral activity after the initial stage of viral uncoating

Antipicornavirus activity of SCH47802 and analogs: In vitro and in vivo studies

SCH 48973: A potent, broad-spectrum, antienterovirus compound

Suppression of colds in human volunteers challenged with rhinovirus by a new synthetic drug (R61837)

Human rhinovirus 14 complexed with antiviral compound R 61837

Safety and efficacy of intranasal pirodavir (R77975) in experimental rhinovirus infection

In vivo efficacy of SDZ 35-682, a new picornavirus capsid-binding agent

SDZ 35-682, a picornavirus capsid-binding agent with potent antiviral activity

Synthesis and activity of piperazine-containing antirhinoviral agents and crystal structure of SDZ 880-061 bound to human rhinovirus 14

Rhodanine: A selective inhibitor of the multiplication of echovirus 12

Selective inhibition of uncoating of echovirus 12 by Rhodanine

4 0 ,6-Dichloroflavan (BW683C), a new anti-rhinovirus compound

Effect of chloro-, cyano-, and amidino-substituted flavanoids on enterovirus infection in vitro

Use of flavones, coumarins, and related compounds to treat infections. World (PTC) Patent

Anti-picornavirus activity of synthetic flavon-3-yl esters

Direct and specific inactivation of rhinovirus by chalcone Ro 09-0410

Antivirus agent, Ro 09-410, binds of rhinovirus specifically and stabilizes the virus conformation

A 'new' generation of more potent synthetic antirhinovirus compounds: Comparison of their MICs and their synergistic interactions

Novel azolyalkyloxy compounds with antipicornaviral activity

Antipicornavirus activity of some diaryl methanes and aralkylaminopyridines

2-(4-Pyridylaminomethyl)-benzimidazole derivatives having antiviral activity

Synthesis and antiviral activity of N-and O-substituted amino acids

Tripeptide aldehyde inhibitors of human rhinovirus 3C protease: Design, synthesis, biological evaluation, and cocrystal structure solution of P 1 glutamine isosteric replacements

Structure-based design of ketone-containing tripeptidyl human rhinovirus 3C protease inhibitors

Antipicornaviral compounds and compositions, their pharmaceutical uses, and materials for their synthesis

S-Nitrosothiols as novel, reversible inhibitors of human rhinovirus 3C protease

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes

Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 2. Peptide structure-activity studies

Synthetic route for the preparation of rhinovirus protease inhibitors and key intermediates

AG-7088 Pfizer

In vitro activity of pleconaril and AC7088 against selected serotypes and clinical isolates of human rhinoviruses

Inhibition of human rhinovirus-induced cytokine production by AG7088, a human rhinovirus 3C protease inhibitor

In vitro antiviral activity of AG7088, a potent inhibitor of human rhinovirus 3C protease

Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease

Cysteine protease inhibitors

Coronavirus main proteinase (3CL pro ) structure: Basis for design of anti-SARS drugs

Shih is an Associate Professor of Medical Technology at Chang Gung University and Director of Clinical Virology Laboratory at Chang Gung Memorial Hospital, Taiwan. She received her BS in Medical Technology in 1988; MS in Biochemistry in 1991 from

Chen received her PhD degree in Biochemistry and Molecular Biophysics from Virginia Commonwelth University at Richmond, VA. Her training and independent research efforts all focused on biochemical events underlying the neurological diseases. She is currently the head of high-throughput screening group at TaiGen Biotechnology

Dr. Hakimelahi was invited by National Science Council of Republic of China to join Academia Sinica in Taiwan, as a visiting specialist. Consequently, successful multidisciplinary research collaborations between the scientists of the third world countries including Iran and those based in Taiwan were started, evidenced by Dr. Hakimelahi's research publications during 1991-2003. Dr. Hakimelahi's multidisciplinary works have had a great impact on the present state of science

He received his BSc in chemistry from National Taiwan Normal University in 1964 and PhD in organic chemistry (under Professor Z. Valenta) from University of New Brunswick in 1968 and carried out postdoctoral studies at UNB (1968-1969) and Columbia University (1969-1970) under Professors Z. Valenta and G. Stork, respectively. After a short span of service (1970-1971) at UNB as a Teaching and Research Associate, he joined the faculty at U of A where he served until his recent move (1998) to NTHU. Hsing-Jang served as an Editor of the Canadian Journal of Chemistry (1995-1998) and has been an elected Fellow of the Chemical Institute of Canada since 1983

Tseng received his BSc in chemistry in 1993 and PhD degree in organic chemistry in 2001 from National Tsing-Hua University. He is currently a postdoctoral fellow at TaiGen Biotechnology with Dr. Kak-Shan Shia, performing his research on the design and synthesis of bioactive molecules targeting the chemokinemediated diseases including rheumatoid arthritis and asthma

Shia received his BSc in chemistry from National Taiwan Normal University in 1982 and MS degree from National Chiao Tung University in 1987. He undertook graduate studies in organic chemistry at the University of Alberta, where he obtained a PhD degree in 1995 under supervision of Professor Hsing

), he moved to TaiGen Biotechnology. Currently, he is the Group Leader at TaiGen Biotechnology, focusing his research on the design and synthesis of biologically active molecules targeting the chemokine-mediated diseases such as asthma and arthritis