Selective Inhibitors of Picornavirus Replication Armando M. De Palma, Inge Vliegen, Erik De Clercq, Johan Neyts Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/med.20125 ! Abstract: Picornaviruses cover a large family of pathogens that have a major impact on human but also on veterinary health. Although most infections in man subside mildly or asymptomatically, picornaviruses can also be responsible for severe, potentially life-threatening disease. To date, no therapy has been approved for the treatment of picornavirus infections. However, efforts to develop an antiviral that is effective in treating picornavirus-associated diseases are ongoing. In 2007, Schering-Plough, under license of ViroPharma, completed a phase II clinical trial with Pleconaril, a drug that was originally rejected by the FDA after a New Drug Application in 2001. Rupintrivir, a rhinovirus protease inhibitor developed at Pfizer, reached clinical trials but was recently halted from further development. Finally, Biota’s HRV drug BTA-798 is scheduled for phase II trials in 2008. Several key steps in the picornaviral replication cycle, involving structural as well as nonstructural proteins, have been identified as valuable targets for inhibition. The current review aims to highlight the most important developments during the past decades in the search for antivirals against picornaviruses. ß 2008 Wiley Periodicals, Inc. Med Res Rev Key words: picornavirus; enterovirus; antiviral 1. INTRODUCTION A. Classification and Clinical Features The picornavirus family is one of the largest virus families known, consisting of nine genera, that is, the aphtovirus, cardiovirus, enterovirus, erbovirus, hepatovirus, kobuvirus, parechovirus, rhinovirus, and teschovirus. These genera harbor several pathogens, implicated in an extensive range of clinical manifestations that affect humans as well as animals. Although often mild and self-limiting, picornaviruses may also be involved in more serious conditions, which can be lifethreatening. An overview of the most clinically (in veterinary and human medicine) relevant picornaviruses is given in Table I. The aphtoviruses contain foot-and-mouth disease virus (FMDV); major outbreaks of the virus in Taiwan (1997) and the UK (2001), were difficult to contain and resulted in direct losses of more than £3 billion.1–4 In the cardiovirus genus, Theiler’s murine Correspondence to: Johan Neyts, Rega Institute, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: [email protected] Medicinal Research Reviews ß 2008 Wiley Periodicals, Inc. 2 * DE PALMA ET AL. Table I. Overview of the Picornavirus Genera and Their Clinical Relevance encephalomyelitis virus (TMEV) is able to cause a persistent infection in the central nervous system (CNS) of susceptible mice, causing a chronic disease that resembles multiple sclerosis (MS), a common demyelating disease in humans.5 Besides TMEV, encephalomyocarditis virus (EMCV) is an important member of the cardioviruses. EMCV was isolated from several animals, as well as humans, and was shown to cause lesions in multiple organs, including heart, pancreas and CNS.6,7 The prototype of the enterovirus genus or of the picornavirus family in general, is poliovirus. This virus, causing amongst others paralytic poliomyelitis, threatened the lives of millions of people worldwide until the introduction of oral polio vaccines in 1960.8,9 Nowadays, the situation regarding polio is ‘‘controlled,’’ although the virus still remains endemic in four countries.10 The non-polio enteroviruses comprise group A and B coxsackieviruses, echoviruses, and numbered enteroviruses. The clinical manifestations associated with these viruses range from mild illnesses, such as fever, rash, hand–foot–mouth syndrome and herpangina to serious or life-threatening infections, such as meningitis, encephalitis, myocarditis, pancreatitis, acute paralysis, or neonatal sepsis.11 Rhinoviruses are responsible for more than half of all cases of the ‘‘common cold,’’ which accounts for more than 40 million days of absence from work or school.12,13 Moreover, increasing evidence is presented that rhinovirus infections account for a majority of exacerbations of both asthma and COPD.14,15 Among the hepatoviruses, hepatitis A accounts for more than 7,000 annual cases of hepatitis in the US, characterized by an acute illness with discrete onset of symptoms, jaundice, or elevated serum transaminase levels.16 The human parechoviruses generally cause mild respiratory or gastrointestinal illness, but more serious sequelae, including myocarditis and encephalitis, have been reported.17 Teschoviruses are endemic in porcine cattle, and although usually leading to nonsymptomatic infections, they can result in a neurological disorder in pigs, known as Teschen-Talfan disease.18,19 Equine rhinitis B virus is the prototype virus of the erbovirus genus, and leads to an acute respiratory tract disease in horses,20,21 while Aichi virus represents the kobuviruses, and is associated with acute gastroenteritis in humans.22,23 There is no specific therapy approved for treating Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 3 picornaviral infections. Treatment options are mainly symptomatically and as a consequence, the search for a broad-spectrum anti-picornaviral agent must go on. We here provide a non-exhaustive review of the developments in the search for inhibitors of picornavirus replication. B. Virology of Picornaviruses Picornavirions are small, non-enveloped, and spherical in shape with a diameter of about 30 nm. The icosahedrally shaped capsids are assembled from 60 protomers, each composed of four structural proteins, designated VP1 (viral protein 1), VP2, VP3, and VP4. The shell is formed by VP1, VP2, and VP3, and, hence, variations within these capsid proteins lead to different antigenic serotypes in the picornavirus family. On the contrary, VP4 lies on the inner surface and serves to anchor the capsid to the RNA genome.24 Uncoating of the virus occurs upon destabilization of VP4. The surface of the virion shows a fivefold axis of symmetry, surrounded by a deep depression or canyon.25 The structure of this canyon and implications for antiviral therapy are discussed below. The picornaviral genome consists of a single-stranded, positive sense RNA of approximately 7,500 bases in length (Fig. 1). Covalently attached to its 5 0 -end, the viral genome has a small protein called VPg, which is involved in the initiation of viral RNA replication. The genomic RNA has a Figure 1. Organization and processing pattern of the picornavirus genome. Top: Diagram of the viral genome, with the genomelinked proteinVPg at the 5 0 -end, followed by the 5 0 untranslated region (UTR), the protein coding region, the 3 0 untranslated region, and the poly(A) tail. L is a leader protein encoded in the genomes of cardioviruses and aphtoviruses but not other picornaviruses. Coding regions for the viral proteins are indicated. Bottom: Primary cleavages and processing pattern of picornavirus polyprotein. The coding region has been divided into three regions, P1, P2, and P3. The single, long open reading frames are depicted for the different picornavirus genera together with the sources of cleavage activity (curved arrows, i.e., the two viral proteinases, 2Apro and 3Cpro/3CDpro). In cells infected with enteroviruses and rhinoviruses, nascent P1 is cleaved from P2 by 2Apro. In cells infected with cardioviruses and aphtoviruses, the P1^P2 junction is cleaved by the 3Cpro. The 2A proteins of these viruses are not proteinases, but they catalyze their own release at the junction with 2B. For all genomes depicted, the P2^P3 cleavage is carried out by 3Cpro. In hepatoviruses and parechoviruses, 3Cpro is also responsible for the primary cleavage at the 2A^2B junction.The Lpro proteinase of foot-and-mouth disease virus catalyzes its release fromVP4 (adapted from Figs. 2 and10,256).The proteinase responsible for cleavage of VP0 has not been identified. Medicinal Research Reviews DOI 10.1002/med 4 * DE PALMA ET AL. highly structured 5 0 non-coding region that contains the internal ribosome entry site (IRES) and a short 3 0 non-coding region, followed by a poly(A) tract. Aphtovirus and some cardiovirus genomes contain an extended internal poly(C) tract within the 5 0 UTR. The non-coding regions at both ends are involved in replication efficiency, tissue tropism, virus infectivity and other regulatory activities during viral replication and translation (reviewed by Bedard and Semler26). The coding region of the viral genome contains non-structural and structural viral proteins divided into three primary precursor molecules (P1, P2, and P3). The structural proteins which comprise the viral capsid are derived from the P1 portion of the polyprotein, whereas the non-structural proteins are encoded by the P2 and the P3 regions. These non-structural proteins consist of two proteases (2A and 3C), one polymerase (3D), one ATPase (2C) and four other proteins that, either cleaved or as a precursor, are involved in viral replication. In addition to these proteins, the aphtoviruses and cardioviruses also code for an L protein at the N-terminus of their polyproteins. The function of this protein will be further discussed below. Upon receptor binding, the viral capsid is destabilized and VP4 is released, after which the viral RNA enters the cytoplasm of the host cell. The viral genome serves as a template for both viral protein translation and RNA replication (Fig. 2). RNA replication occurs in association with cellular membranes, and new positive-strand RNA genomes are produced through a negative-strand intermediate.26 These RNA strands are packaged into new viral structural proteins, and associate to form new viral particles, which are subsequently released from the host cell.26 Figure 2. Overview of the picornavirus replication cycle. Virus binds to a cellular receptor and the genome is uncoated. VPg is removed from the viral RNA, which is then translated.The polyprotein is cleaved nascently to produce individual viral proteins. RNA synthesis occurs on membrane vesicles.Viral (þ) strand RNA is copied by the viral RNA polymerase to form full-length () strand RNAs, which are then copied to produce additional (þ) strand RNAs. Early in infection, newly synthesized (þ) strand RNA is translated to produce additional viral proteins. Later in infection, the (þ) strands enter the morphogenetic pathway. Newly synthesized virus particles are released from the cell by lysis (adapted from Fig. 4,256). Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 5 C. Inhibiting Picornavirus Replication As discussed, the replication cycle of picornaviruses (depicted in Fig. 2) starts upon receptor binding of a virion to the host cell, and ends with the release of newly formed viral particles. Theoretically, every step during this life cycle is a potential target for blocking viral replication. In fact, antiviral drugs directed at many, but not all viral proteins have been developed during the last decades and structural, as well as non-structural proteins have been shown to be valuable targets for efficient inhibition of viral replication. In the following paragraphs, an overview is presented of compounds that selectively inhibit the replication of picornaviruses, according to their viral target. Only molecules are addressed (either synthetic or natural) that specifically interfere with viral replication. Immunomodulatory compounds, compounds acting at the cellular level, or non-small molecules that act non-specifically such as antibiotics or macromolecules will not be discussed. Some of such alternative strategies have been reviewed by Carrasco.27 2. INHIBITORS OF ATTACHMENT, ENTRY, AND UNCOATING A. Introduction The first step in viral infection is the recognition and attachment of a virus particle to receptors on the target cell. The presence or absence of a particular receptor on the cell surface determines the viral tropism for that particular cell type.28 Several cellular receptors for picornaviruses have been identified. Most receptors belong to the immunoglobulin (IgSF) superfamily. These molecules consist of tandem repeats of between two and five Ig-like domains.29 Known IgSF receptors for picornaviruses are intercellular adhesion molecule-1 (ICAM-1 or CD54) for major-group rhinoviruses, the poliovirus receptor (PVR or CD155) for polioviruses, the coxsackie-adenovirus receptor (CAR) for Coxsackie B viruses and vascular cell adhesion molecule-1 (VCAM-1) for EMCV. These receptors have their carboxy-terminal sequence anchored in the host cell’s plasma membrane, while containing their virus recognition site in the amino-terminal domain. Attachment of the virus occurs through binding of the receptors into the canyon running around each fivefold vertex of the capsid. In this ‘‘canyon hypothesis,’’ binding of the receptor triggers the uncoating process. Binding of the receptor might expel a lipid moiety (‘‘pocket factor’’) that resides in a hydrophobic pocket within VP1, which is immediately underneath the floor of the canyon, thereby destabilizing the virion and hence, initiating uncoating.30 Binding of specific molecules (‘‘pocket binders’’) in this pocket results in an increase in protein rigidity and stabilizes the particle in such a manner that the uncoating cannot proceed.31 Besides the Ig-like receptors, other known picornavirus receptors are Low-Density Lipoprotein-Receptor (LDL-R) for minor-group rhinoviruses, DecayAccelerating Factor (DAF or CD55) for echoviruses, sialic acid for Theiler’s virus, as well as several integrins.29,32 Strains of FMDV (aphtovirus genus) initiate infection by binding to any of four members of the aV subgroup of the integrin family of cellular receptors (avb1, avb3, avb6, or avb8)33–38 by binding through a highly conserved arginine–glycine–aspartic acid (RGD) tripeptide located on the GH loop of VP1.39 Other serotypes of FMDV can use heparan sulfate or oligosaccharides as a receptor.40,41 In contrast to the IgSF-like receptors, these receptors all bind outside the canyon. Hence, they do not initiate viral instability or uncoating. However, they could trigger the aggregation of other receptor molecules or they could trigger endocytosis followed by a lowering of the pH in the endosomal vesicles.29 In addition to the primary receptors for host cell binding, several ‘‘accessory factors’’ have been identified that either are indispensable for the infection to proceed or might serve to enhance the efficiency of infection. Moreover, several viruses have been reported to be able to use alternative or additional receptors. Examples are b2m, a microglobulin, which is strictly required for avb3mediated infection by CVA9,42 or integrin avb6 which has been reported to enhance CVB1 lytic Medicinal Research Reviews DOI 10.1002/med 6 * DE PALMA ET AL. infection.43 Heparan sulfates have been reported to facilitate adsorption and entry of some rhinoviruses, FMDV, CVB3, and TMEV.40,44–47 In addition to CAR, several coxsackieviruses can use DAF as an additional receptor.48–50 The use of additional receptors for infection of picornaviruses may have implications for drug therapy, considering that a virus could escape inhibition by a drug that specifically targets one particular receptor, simply by using an alternative receptor. In summary, receptor binding, uncoating and release of the viral RNA into the host cell are the primary steps in the viral life cycle. Hence, antiviral drugs that interfere with any of these processes block the subsequent steps and thus inhibit viral replication. The most extensively studied class of molecules targeting the early events of attachment, entry, and uncoating, are the so-called ‘‘WIN compounds.’’ An overview of their antiviral properties and their molecular mechanism of action are provided in the following paragraphs. B. Win Compounds Numerous studies have been performed in the past decades on a series of compounds developed by Sterling-Winthrop, commonly known as ‘‘WIN compounds.’’ Following lead optimization of an accidentally discovered compound, a drug was developed that would meet most of the criteria of a successful anti-picornavirus drug: pleconaril. An overview of the development of pleconaril is given in Figure 3. The WIN compounds were born out of a beta diketone intermediate, in a synthesis project for juvenile hormone mimetics.51–54 Several modifications to improve activity led to the development of arildone (WIN38020), an aryl diketone. This compound was shown to selectively inhibit poliovirus replication in vitro by preventing virion uncoating.55,56 In vivo, arildone prevented poliovirusinduced paralysis and death in mice.57 Further attempts to improve the activity of arildone, as well as the chemical and metabolic instability of the beta-diketone moiety, led to the development of WIN51711, also known as disoxaril.58–60 In cell culture, disoxaril proved to be a potent inhibitor of a spectrum of rhino- and enteroviruses, with minimal inhibitory concentrations (MIC’s) ranging between 0.004 and 6.2 mg/mL.61 Disoxaril, when given orally, prevented paralysis and death in mice infected with poliovirus type 2 and echovirus 9.62–64 Arildone or disoxaril in combination with other known picornavirus inhibitors mostly demonstrated synergistic activities in vitro as well as in mice.65–68 Mechanism of action studies with HRV-2 and poliovirus revealed that disoxaril and analogues prevented viral uncoating.69,70 Generation of disoxaril-resistant type 3 poliovirus, lead to the identification of 22 mutants, of which 14 proved to be drug-dependent.71 The location of the amino acid substitutions pointed to three regions of the viral capsid as important in uncoating functions: (1) the canyon wall, surrounding the fivefold vertex, (2) the lining of the drug-binding pocket in the interior of the VP1 beta barrel, and (3) the inner surface of the protein capsid.72 A more detailed overview of the mechanism of action of the WIN compounds is discussed in the next paragraph. Despite its in vivo and in vitro activity, disoxaril lacked activity against various HRV strains, was poorly bioavailable (15%), and was shown to induce crystalurea in phase I clinical studies. Consequently, the search for a more potent, highly bioavailable compound with broadspectrum of activity went on. Mono- and diaromatic substituted analogues of disoxaril were evaluated, and WIN54954 proved to be considerably more potent and bioavailable than disoxaril.73 In a murine model for infection with a diabetogenic strain of CVB4, WIN54954 when administered orally at 50 mg/kg/day reduced viral replication and had a marked effect on inflammatory lesions of the pancreas.74 In a murine CVA9induced myocarditis model, WIN54954 reduced viral replication at 50 mg/kg/day, but caused neurological toxicity at doses of >100 mg/kg/day.75 The compound was well tolerated in phase I clinical trials, and was effective in humans naturally infected with CVA21, but lacked activity against experimentally induced infections caused by rhinovirus strains 23 and 29.76 Moreover, the compound Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION O * 7 O O O • • • Beta Diketone Intermediate Cl • Ethyl side chain not necessary for activity Unsaturation not necessary for activity Replacement of carbon atom adjacent to the aromatic ring with an oxygen Further modifications O OMe O O Arildone N R O O Substituted Isoxazoles • Chemical and metabolic instability of the β-diketone moiety • • R = 2-Cl-4-OMe (1) R = 4-COOEt (2) • In vivo inactivity of the 4-carbethoxy analogue (2) due to hydrolyzation of the ester, or poor bioavailability Optimalisation of the carbon bridge length Me N R O O Me O N O Disoxaril N • Cl Me O N • O O O • • Unattractive safety profile in phase I clinical studies Not broad-spectrum Poor bioavailability N WIN 54954 Cl Me Me Me N N O O N • • • Rapidly and extensively metabolized Reversible hepatitis Three-carbon chain bridge: broader spectrum of activity • Replacement of methyl group with trifluoromethyl group in order to block metabolism at this site O Me WIN 61893 Me Me CF3 N N O O N O Me WIN 63843 (Pleconaril) Figure 3. Evolution of WIN compounds, leading to the discovery of pleconaril. was rapidly metabolized and induced reversible hepatitis, which made this analogue an uninteresting candidate for further studies.77 In a final stage for developing a successful clinical candidate in the WIN series, the oxazoline ring was substituted, since this ring appeared to be metabolically converted to several products, none of which were shown to have antiviral action. The 5-methyloxadiazole analogue (WIN61893) was shown to be a potent analogue, but did not meet all the criteria in metabolization studies. Replacement of the methyl group at the oxadiazole ring with a trifluoromethyl group, led to a compound (WIN63843 or pleconaril) whose rate of metabolism was drastically reduced.77 Pleconaril demonstrated a significant improvement in half-life and plasma clearance when administered orally Medicinal Research Reviews DOI 10.1002/med 8 * DE PALMA ET AL. to dogs at 10 mg/kg.77 Moreover, the drug was found to have a broad spectrum of antiviral activity against enteroviruses.78,79 In 1996, ViroPharma made the drug available for compassionate use in patients with potentially life-threatening enterovirus infections, including meningoencephalitis, neonatal sepsis and myocarditis. Upon pharmacokinetic studies in neonates, pleconaril at a dose of 5.0 mg/kg given every 8 hr,80 proved successful in 2 of 3 neonates who were hospitalized with severe enteroviral hepatitis.81 Follow-up data for patients with potentially life-threatening infections, including chronic meningoencephalitis, revealed the drug resulted in clinical improvement in 78% of the patients while adverse effects were minimal.82 In another double blind, placebo-controlled trial of pleconaril in infants with enterovirus meningitis, however, no significant differences in duration of positivity by culture or PCR, hospitalization or symptoms were detected between both groups, whereas twice as many adverse effects occurred per subject in the pleconaril group.83 In a phase III, double-blind, placebo-controlled clinical trial involving nearly 2,100 picornavirus-infected patients, therapy with pleconaril was shown to reduce the duration and the severity of common cold symptoms when it was administered within 24 hr of symptom onset.84 Pevear et al.85 showed that the efficacy of pleconaril was linked to the virus susceptibility to the drug. In 2002, however, the US FDA did not approve pleconaril for the treatment of the common cold, as the panel remained unconvinced about the drug’s safety profile.86 In 2003, ViroPharma licensed pleconaril to Schering-Plough. A phase II double-blind, placebo-controlled trial to study the effects of pleconaril nasal spray on common cold symptoms and asthma exacerbations following rhinovirus exposure was completed in 2007. Results of this trial have not yet been reported. C. Molecular Interactions of the Win Compounds with the Viral Capsid Following the discovery that the WIN compounds prevented receptor binding and uncoating, it was suggested that a direct compound binding site existed on the viral capsid. In 1985, Rossmann et al.25 reported the first atomic resolution structure of an animal virus, human rhinovirus 14, belonging to the major rhinovirus receptor group. This study revealed the presence of a large cleft on each icosahedral face, which was suggested to be the host cell receptor site. By hiding the receptor attachment site in a surface depression, picornaviruses were permitted to conserve residues that might be required for host cell receptor recognition without danger of attack by the host’s immune system, an idea that would later be formulated as the ‘‘canyon hypothesis.’’30,87 Structural studies of several WIN compounds complexed with human rhinovirus 14 showed that these compounds bind into an interior hydrophobic pocket, formed by the beta-barrel of VP1, underneath the floor of this canyon88–91 (Fig. 4). This binding event induces conformational changes in the floor of the HRV-14 canyon, which were shown to decrease the ability of the virions to interact with their receptor.92 Thus, in HRV-14 these drugs neutralize infectivity by inhibition of uncoating, but also by inhibition of attachment to their cellular receptor. The structure of HRV-3, complexed with WIN56291 was also determined and was found to be similar to the same compound complexed with HRV-14.93 In HRV-1A, a member of the minor receptor group, the hydrophobic pocket in VP1 is an ‘‘open’’ conformation that resembles the conformation observed in drug-bound HRV-14 and is occupied by a fatty acid, or commonly referred to as ‘‘pocket factor.’’94,95 Consequently, the pocket factor is displaced upon drug binding in HRV-1A and thus induces only very small conformational changes, in contrast to HRV-14, where much larger conformational changes occur. As a consequence, drug binding does not lead to an inhibition of viral attachment of HRV-1A. The uncoating process, however, is inhibited in HRV-1A as well as in HRV-14, due to the formation of, respectively, one and three additional interprotomer hydrogen bonds and a potential loss of flexibility of the viral capsid upon efficient packing of the pocket.94 Based on the findings that these compounds stiffen the viral capsid in HRV-1A and that the RNA is released through the pentamer channel, Vaidehi and Goddard96 postulated the Pentamer Channel Stiffness Model (PCSM): drug action on HRV-1A Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 9 Figure 4. Mechanismofactionof WIN compounds. Left:Schematicrepresentationof bindingbetween ICAM-1 (here simplifiedas a two-domain fragment) and major group HRV. ICAM-1binds to the floor of the canyon, surrounding each fivefold axis, inducing conformational changes that eventually lead to uncoating of the virus and release of the viral RNA. Right: Binding ofa‘‘WIN compound’’ occurs in an interior hydrophobic pocket, located beneath the floor of the canyon. By occupying this pocket (i) conformational changes occur in the floor of the pocket, hampering the ability of virions to interact with their receptor and (ii) virion rigidity is increased, preventing uncoating of the virus and subsequent release of viral RNA (adapted from376). constricts or stiffens the pentamer channel sufficiently that the RNA and/or VP4 cannot exit, thus preventing uncoating. Studies with HRV-14 revealed that also shorter, more hydrophilic WIN fragments (WIN 52452 and WIN 58768), consisting of only the phenyl end of the longer active WIN compounds, could bind into the pocket beneath the canyon floor. These compounds caused conformational changes similar to the longer compounds, and provided some stability to the HRV-14 capsid. It was concluded that these short compounds mimic the cellular cofactors observed in the hydrophobic pocket for some picornaviruses, but are unlikely to inhibit viral attachment.97 Another major receptor group rhinovirus serotype, HRV-16, was shown to posses a pocket factor, similar to HRV-1A.98 Although receptor attachment was prevented upon binding of WIN 56291, the presence of the drug did not cause a deformation of the pocket. The authors postulate that the receptor binding of HRV-16 can occur only when the pocket is temporarily empty, when it is possible for the canyon floor to be forced downwards into the pocket.98 The interactions of some more WIN compounds in complex with HRV-16 have been studied more in detail by Hadfield et al.99 Drug-resistant or -dependent variants have been generated for WIN compounds with human rhinovirus 14, HRV-16, poliovirus type 3 and coxsackievirus B3.71,72,100 –105 All these studies confirm the involvement of residues in the VP1 cleft regarding the interaction between WIN compounds and the viral capsid. The crucial role of amino acids L1191 and L1092 for pleconaril resistance of species B rhinoviruses and of CVB3 was demonstrated by mutational analysis and use of recombinant viruses, respectively.106,107 A systematic evaluation of several reported rhinovirus capsid binding-compounds against all serotyped rhinoviruses was performed by Andries et al.108 This study revealed the existence of two groups of rhinoviruses, which were designated antiviral group A and B. Group A rhinoviruses were preferentially inhibited by compounds with a longer chain, whereas short-chained compounds Medicinal Research Reviews DOI 10.1002/med 10 * DE PALMA ET AL. interacted more easily with rhinoviruses of group B.109 The antiviral susceptibility of both groups was shown to be highly correlated with sequence similarities, not only of amino acids lining the antiviral compound binding site, but also of amino acids of the entire VP1 protein. Another subdivision of the rhinovirus genus into two species was reported by Ledford et al.,110 who showed that all but one (HRV87) HRV serotypes can be divided in two groups, based on phylogenetic analysis of the amino acid sequence of the VP1 protein. Moreover, analysis of the amino acids constituting the hydrophobic pocket in VP1 indicated that the sequence correlates strongly with the virus susceptibility to pleconaril inhibition.110 D. R618737, Pirodavir and Related Oxime Ethers Pirodavir (R77975) (ethyl 4-[2-(1-[6-methyl-3-pyridazinyl]-4-piperidinyl)ethoxy]benzoate) and its predecessor (R61837) (Fig. 5) belong to a series of pyridazine analogues developed by the Janssen Research Foundation.111 Compared to R61837, pirodavir was 500-fold more potent as an antiviral in vitro, inhibiting 80% of 100 rhinovirus serotypes tested at concentration of 0.064 mg/mL or less.112 Moreover, pirodavir inhibited rhinoviruses of both antiviral groups, whereas R61837 was almost exclusively effective towards group B rhinovirus serotypes. As regards their mechanism of action, pirodavir (and analogues) rendered the susceptible virus serotypes non-infectious by direct contact, and the neutralized viruses became stabilized to acid and heat, strongly suggesting a direct interaction of the compound with the capsid protein.112–114 Moreover, drug-resistant mutants were shown to exhibit cross-resistance to WIN compounds and other capsid-binding agents.113 Indeed, crystallographic studies on R61837 complexed with rhinovirus 14 revealed that the compound binds to the capsid protein in the same hydrophobic pocket, underneath the canyon floor as the WIN compounds.115 Despite the same binding site, R61837 appeared to penetrate less far in the pocket, and was dependent on other atomic interactions than the WIN compounds, illustrating that considerable diversity is allowed in the nature of capsid-binding agents. In a series of double-blind placebo-controlled trials with R61837, the compound was shown to be prophylactically effective in Me N N N MeO N N N Me N N N Cl N N N Me R61837 N O COOEt O O Me N O Figure 5. Medicinal Research Reviews DOI 10.1002/med N OEt BTA-39 N OEt BTA-188 OEt O N N Pirodavir (R77975) N Compound 13 PICORNAVIRUS REPLICATION * 11 suppressing colds in human volunteers challenged with rhinovirus type 9. An intranasal treatment schedule for 4 and 6 days, with total doses of respectively 25 and 36 mg, showed substantial reductions in both mean daily clinical score and the mean daily nasal secretion weight.116 When administered shortly after virus challenge, however, the drug lacked clinical efficacy.116 In another series of clinical trials, pirodavir was shown to be clinically effective when administered 6 times a day for 5 days in experimentally induced rhinovirus infection. Administration after virus challenge led to significant reductions in virus shedding, but, as for R61837, no clinical benefits were found.117 A randomized, double-blind, placebo-controlled trial to assess the therapeutic efficacy of intranasal pirodavir in naturally occurring rhinovirus colds demonstrated no clinical benefit, and moreover, was associated with unpleasant sensations during treatment.118 The lack of clinical efficacy of pirodavir can at least partially be explained by its poor water solubility, and as a result of the rapid hydrolysis of the ester to the corresponding acid. To circumvent this problem of ester hydrolyzation, the oxime ether analogue of pirodavir was synthesized at Biota.119 This compound had good oral bioavailability (62–63% in rats and 21–28% in dogs) and illustrates that an oxime ether group can function as a metabolically stable bioisosteric for an ester functionality. This compound, which was designated BTA-188 (initially referred to as ‘‘compound 14’’119), and its analogue (BTA-39) (initially referred to as ‘‘compound 11’’119) inhibited a total of 56 HRV laboratory strains and three clinical isolates at EC50 values ranging from 0.5 to 6.7 nM.120 In a subsequent step, the effect on anti-HRVactivity was investigated after replacing the benzaldehyde oxime ether moiety in BTA-188 with a bicyclic system such as a 2-ethoxybenzoxazole.121 Over 20 analogues of these series were synthesized and evaluated for anti-HRV activity and ‘‘compound 13’’ (Fig. 5) emerged as the most potent congener. This analogue proved approximately 10 times more active than pleconaril and equipotent to both pirodavir and BTA-188, but was predicted to have a longer half life and oral bioavailability, given the greater hydrolytic stability of the 2-ethoxybenzoxazole group compared with an ethyl ester.121 Recently, a phase I clinical trial was completed with an analogue in this series (BTA-798), which was developed for the treatment and prevention of HRV infections in high risk COPD and asthma patients. Biota, which is developing the compound, is scheduling a phase II clinical trial with BTA-798 to start in 2008. E. Isoxazole Derivatives Driven from the idea that pleconaril is inactive against, for example, the cardiovirulent strain CVB3 Nancy, Makarov et al.122 synthesized a series of novel [(biphenyloxy)propyl]isoxazole derivatives of pleconaril, using a novel synthesis method. In vitro evaluation of these compounds revealed excellent activity against HRV-2 but no activity against HRV-14. Towards a pleconaril-susceptible strain of CVB3, the compounds proved moderately active. Compound VIa (Fig. 6) proved most potent in these series (EC50 against HRV-2 ¼ 9 ng/mL and EC50 against CVB3 isolate 97–927 ¼ 1.34 mg/mL) and moreover, could inhibit the pleconaril-resistant CVB3 Nancy strain.122 Recently, Kuz’min et al.123 developed a quantitative structure–activity relationship (QSAR) to predict structures of isoxazole analogues with enhanced antiviral properties. A working set of 17 pleconaril analogues was used, from which 18 new compounds were computationally designed and predicted to be antivirally active. The in vitro antiviral activity of three such compounds was evaluated (compounds 19, 20, and 21 in Fig. 6). The predicted anti-HRV-2 activity strongly correlated with the experimentally observed activity (compound 19 showed best anti-HRV-2 activity with EC50[observed] ¼ 5.01 ng/mL and EC50[predicted] ¼ 5.25 ng/mL).123 F. Pyridyl Imidazolidinones As pleconaril was not able to neutralize cytopathic effect induced by enterovirus 71, Shia et al.124 used the skeletons of pleconaril and its analogues (WIN compounds) as templates for computer-assisted drug design, synthesis, and structure–activity relationship studies, leading to the Medicinal Research Reviews DOI 10.1002/med 12 * DE PALMA ET AL. Me Me N O O F Compound VIa F Compound 19 F Compound 20 Me Me Me N O O Me F Me Me N O F O Me Me F Me Me Compound 21 N O O F Me Figure 6. development of a novel class of imidazolidinones with significant antiviral activity. SAR studies demonstrated that an aryl substituent at the para position of the phenoxyl ring and a pyridinecontaining imidazolidinone are key structural requirements for anti-EV71 activity.124 One of the molecules in this series that was selected for mechanism of action studies is BPR0Z-194 (initially referred to as ‘‘compound 11’’124) (1-[5-(4-bromophenoxy)pentyl]-3-(4-pyridyl)-2-imidazolidinone), depicted in Figure 7. This compound shows potent antiviral activity against several enteroviruses, including enterovirus 71, enterovirus 68, coxsackievirus A9 and A24, and echovirus 9.125 Time-course experiments revealed that BPR0Z-194 effectively inhibited virus replication at the early stages, namely virus attachment or uncoating. This was confirmed by sequence analysis of drug-resistant viruses, showing that a single amino acid alteration at the position 192 of VP1 can confer resistance to the inhibitory effects of BPR0Z-194.125 In terms of potency, the biphenyl analogue DBPR-103 (Fig. 7) (also previously referred to as ‘‘compound 33,’’126 ‘‘compound A2,’’127 or ‘‘compound 21’’124), with a 4-chlorophenyl moiety at the para position, is paramount with an EC50 value of 54 nM against EV71.124 To improve the spectrum and potency of these compounds, Chern and co-workers127 synthesized a series of novel pyridyl imidazolidinones, containing an oxime ether moiety. This class of compounds proved to be active as antivirals, with a high specificity for human enteroviruses, in particular for enterovirus 71. The most potent compound, ‘‘compound 8b’’ (Fig. 7) was active against various strains of coxsackievirus A and B and echovirus. The EC50 value for inhibition of EV71 was 1.0 nM, which was a marked improvement when compared to the lead DBPR-103.127 Introduction of a methyl group at the 2- or 3-position (compound 14a, Fig. 7) of the linker spacer between the imidazolidinone and biphenyl substantially improved the activity against EV71.126 The Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION N 13 Br O N * BPR0Z-194 N O Cl N O N N DBPR 103 N O O N N N OEt Compound 8b O Cl N O N Compound 14a Me N O Et N N O N Me N N O Compound 28b O Figure 7. (S)-(þ) enantiomer (EC50 ¼ 3 nM) was 10-fold more potent against EV71 than the (R)-() enantiomer.128 Furthermore, replacement of the chlorophenyl ring in this molecule (compound 14a) with the oxadiazole ring or the tetrazole ring resulted in dramatically improved inhibitory activity. The most potent analogue (compound 28b) (Fig. 7) had a selectivity index > 50.000 against EV71, and proved also activity against EV68, several coxsackieviruses A and B, echovirus 9 and 28 and rhinovirus 2 and 14.126 G. SCH 38057, SCH 47802, and SCH 48973 SCH 38057 (1-[6-(2-chloro-4-methoxyphenoxy)-hexyl]imidazole hydrochloride), developed by Schering-Plough, is a water-soluble phenoxyl imidazole compound in its hydrochloride salt form (Fig. 8). This compound was shown to inhibit plaque formation of several entero- and rhinoviruses, with EC50 values ranging between 10.2 and 29.1 mM. When administered orally (60 mg/kg) or subcutaneously (20 mg/kg), SCH 38057 protected mice infected with either CVB3 or echovirus 9 from mortality.129 Crystallographic studies, using SCH 38057 complexed with rhinovirus 14 revealed that the compound binds at the innermost end of the hydrophobic pocket within the VP1 canyon and, like previously reported capsid binders, induces conformational changes in the VP1 protein, that are even more extensive than those observed for other antivirals.130 However, the compound does not prevent attachment of the virus to the host cell, but rather exerts its activity after the initial stage of viral uncoating.129 A subsequent series of molecules, SCH 47802 (Fig. 8) and four analogues, exhibited strong antiviral properties against poliovirus, several echoviruses and coxsackieviruses, with Medicinal Research Reviews DOI 10.1002/med 14 * DE PALMA ET AL. MeO O N N.HCl Cl MeO SCH 38057 Cl O SCH 47802 MeO OMe O Cl Cl O SCH 48973 Cl O Cl Figure 8. therapeutic indices for poliovirus 2 ranging between 81 and 2,300.131 Efficacy following oral administration of SCH 47802 was demonstrated in a murine poliovirus encephalitis model, where a regimen of 60 mg/kg/day was efficient to protect mice from virus-induced mortality for 15 days, which was significantly different from placebo.131 Therapy with a close analogue, SCH 48973, increased the survival rate of infected mice when administered orally at dosages of 3–20 mg/kg/day, while the compound also reduced viral titers in brains of infected mice.132 The SCH 48974 analogue showed a moderate protective effect when given at a dosage of 90 mg/kg/day, whereas the other three analogues failed to show any in vivo efficacy at all.131 Mechanistic studies indicate that this series of molecules act at an early stage in the viral replication cycle, and specifically interact with the viral capsid.131,132 These data were confirmed with crystallographic studies, where the structure of poliovirus type 2 complexed with SCH 48973 was determined. The compound was observed to bind in a pocket within the beta-barrel of VP1, in approximately the same binding location for natural pocket factors.133 H. SDZ 35-682 and SDZ 880-061 At the Sandoz Forschungsinstitut, a class of anti-rhinoviral compounds sharing the piperazinyl moiety with R61837 was studied. Chemical modification of this series of compounds led to the identification of a new derivative, SDZ 35-682 (Fig. 9), with a limited spectrum of activity against several rhinoviruses and echovirus 9.134 Mechanistic studies revealed that SDZ 35-682 is an inhibitor of the uncoating process (of echovirus 9), although it has also some inhibitory effect on binding. Cocrystallization studies of SDZ 35-682 with HRV-14 showed that the compound fills the entire hydrophobic pocket beneath the canyon floor. In vivo efficacy for SDZ 35-682 was demonstrated in an echovirus 9 animal model. In this model, significant protection of newborn mice from paralysis and death was achieved by either a high dose (126 mg/kg) given only twice, at days 0 and 1 postinfection, or by a lower dose (102, 71, or 36 mg/kg) for the first 4 or 6 consecutive days after infection.135 Another piperazine-ring motif containing compound, that is, SDZ 880-061 (Fig. 9), inhibited 85% (76/89) of HRV serotypes tested at concentrations 3 mg/mL.136 Structural studies of SDZ 880061 bound to HRV-14 reveal that this compound binds in the same hydrophobic pocket as previously discussed, leaving the innermost portion of the pocket vacant.136 The alterations in VP1 backbone Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION N OH N N S EtOOC 15 N N O * SDZ 35-682 S N SDZ 880-061 N Figure 9. conformation are similar but less extensive, compared to other antiviral agents, such as SCH 38057 or WIN compounds. Time of drug addition/removal studies revealed that the compound primarily interferes with cellular attachment of the virus. The marginal effect of SDZ 880-061 on uncoating is most probably due to the fact that it does not completely fill the hydrophobic pocket, hence not causing enough virion stabilization.136 I. Chalcones At Roche, Ro 09-0410 (4 0 -ethoxy-2 0 -hydroxy-4,6 0 -dimethoxychalcone (Fig. 10)) was identified as a potent inhibitor of rhinoviruses, but without activity against other picornaviruses tested. The compound was discovered when studying the flavone Ro 09-0179 (which will be discussed in the following section). The concentration inhibiting 50% of the rhinovirus serotypes tested was about 30 ng/mL, whereas the 50% cytotoxic concentration was 30 mg/mL.137 Studies of various Ro 09-0410 analogues (Fig. 10) led to the identification of amide analogues (Ro 09-0535, Ro 09-0696, and Ro 090881) that were up to 10 times more active against HRV then Ro 09-0410.138 It was suggested that Ro 09-0410 binds in a specific manner to the rhinovirus capsid and stabilizes the viral capsid.139 Binding of Ro 09-0410 was competitively inhibited by other capsid binding agents (4 0 ,6-dichloroflavan and WIN-51711) and moreover, HRV-2 strains resistant to these capsid binding agents proved crossresistant to the chalcone amides.138 In a double blind clinical trial where healthy volunteers were challenged with HRV-9, Ro 09-0415, an orally bioavailable phosphorylated pro-drug of Ro 09-0410, failed to show clinical efficacy when given prophylactically.140 In another study, Ro 09-0410 was given intranasally as prophylaxis against rhinovirus infection in human volunteers, but failed again to reduce the symptoms.141 In studies where healthy volunteers were challenged with rhinovirus, resistant to or dependent on Ro 09-0410, it was shown that a drug-resistant rhinovirus was capable of infecting humans and producing disease, although its infectivity was reduced when compared to that of the wild-type. In contrast, a drug-dependent virus had lost its ability to infect humans.142 J. 4 0 ,6-Dichloroflavan (BW 683C) and Related Analogues 4 0 ,6-Dichloroflavan (BW 683C), was identified as an anti-rhinovirus compound, blocking the replication of some, but not all rhinovirus types, with EC50 values ranging between 7 and 170 nM.143,144 Mechanistic studies indicated that BW 683C (Fig. 11) binds to rhinovirus particles and stabilizes rhinovirus to heat or acid inactivation, suggesting that the compound acts as an inhibitor of viral uncoating by binding into the VP1 pocket.143–145 Double-blind, placebo-controlled clinical trials with BW 683C, administered either orally (1 mg/kg, three times daily) or intranasally (40 mg, Medicinal Research Reviews DOI 10.1002/med 16 * DE PALMA ET AL. OMe EtO OMe Ro 09-0410 OH Me O OMe O OMe Ro 09-0696 H N Me OH O OMe EtO H N OH H N Me Ro 09-0881 O Figure 10. five times daily), failed to protect healthy volunteers from experimental rhinovirus infection, despite the good oral bioavailability of the drug.146,147 Conti and co-workers148–150 synthesized a series of new flavans, isoflavans and isoflavenes substituted with halogens as well as cyano or amidino residues. Among these drugs, 4 0 , 6-dicyanoflavan (Fig. 11) proved to be more active against rhinovirus 1B than the reference molecule BW 683C, with an IC50 value of 23 nM.151 Mechanism of action studies suggested, similarly to BW 683C, interference with some early steps of virus replication.148–151 Another dichloroflavan analogue that was described by this group and reported to have antiviral activity not only against rhinovirus but also against a broader spectrum of picornaviruses, is 3(2H)isoflavene (Fig. 11).149,150 Initial studies with Sabin PV2 3(2H)-isoflavene-resistant and -dependent mutants suggested that the compound may act by interfering with viral replication at a stage between uncoating and viral RNA synthesis.149,152 Later, it was shown that 3(2H)-isoflavene exerts its action Cl 4’, 6 – Dichloroflavan (BW 683C) O CN Cl 4’, 6 – Dicyanoflavan O NC O 3(2H)-isoflavene Figure 11. Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 17 O Rhodanine HN S S Figure 12. during the uncoating step. These findings were supported by the analysis of drug-resistant viruses, that were shown to carry mutations in VP1.153 K. Rhodanine 2-thio-4-oxothiazolidine, also known as rhodanine (Fig. 12), is a selective inhibitor of echovirus 12 at a concentration of 12.5 mg/mL, showing no inhibitory effect against other viruses. The compound was demonstrated to be an uncoating inhibitor that has no effect on virus adsorption or cell entry.154,155 Virions exposed to rhodanine are protected against heat inactivation or alkaline degradation.155,156 It was revealed that the host cell shut-off that normally occurs after viral infection, was prevented by rhodanine.157 This is a logical observation given the fact that inhibition of host translation can only take place after translation of the input RNA, a step that does not take place in the presence of uncoating inhibitors.27 Mutational analysis of echovirus 12 recombinant viruses, carrying mutations of a rhodanine-resistant or -dependent variant, revealed 5 single mutations, that were capable of inducing a rhodanine-resistant or -dependent phenotype.158,159 Of these, four mutations were localized in the capsid protein VP1, whereas the fifth mutation was located in VP4. Resistant as well as dependent variants still seemed able to bind rhodanine, and apparently none of the mutations affected the putative drug binding site. Hence, the authors hypothesized that drug resistance and dependence are consequences of an increased flexibility of the virus capsid. L. 44 081 RP A synthetic compound, 2-[(1,5,10,10a-tetrahydro-3H-thiazolo[3,4b]isoquinolin-3-ylidene)amino]4-thiazoleacetic acid or 44 081 RP (Fig. 13), inhibited the replication of 39% (27/69) of tested rhinovirus strains at a concentration of 7 mg/mL.160 Mode of action studies revealed that this compound is an inhibitor of viral uncoating.161 Despite its in vitro activity, the compound had no significant clinical effect when administered intranasally as a 0.2% solution to volunteers from the day before to 5 days after inoculation with a human rhinovirus strain. M. Dibenzofuran and Dibenzosuberol Derivatives From a series of dibenzofuran and dibenzosuberol derivatives, 2-hydroxy-3-dibenzofuran carboxylic acid and dibenzosuberenone (Fig. 14) were shown to inhibit rhinovirus replication [EC50 values between 10 and 30 mM] in HRV-14- and HRV-16-induced CPE reduction assays.162 Time-of-drug S N N Figure 13. Medicinal Research Reviews DOI 10.1002/med 44 081 R.P. S N CH2COOH 18 * DE PALMA ET AL. OH 2-hydroxy-3-dibenzofuran carboxylic acid O O OH dibenzosuberenone O Figure 14. addition studies revealed that the compounds lost antiviral activity when added after the adsorption step, suggesting they act as capsid binding agents.162 N. MDL 20,610 and Related Analogues At Merrell Dow, from a class of 3,4-dihydro-2-phenyl-2H-pyrano[2,3-b]pyridines, the 6-substituted 2-(3 0 ,4 0 -dichlorophenoxy)-2H-pyrano[2,3-b]pyridines MDL 20,610, MDL 20,646, and MDL 20,957 (Fig. 15) were identified as potent inhibitors of rhinovirus replication with median plaque EC50 values of respectively 0.03, 0.006, and 0.006 mg/mL against 32 serotypes of rhinovirus tested.163,164 Mechanism of action studies with MDL 20,610 indicated that the compound binds directly to the capsid with subsequent inhibition of uncoating.165 O. Phenoxybenzenes, Phenoxypyridines and Related Analogues At Merrell Dow, a large number of phenoxybenzenes and phenoxypyridines were synthesized and evaluated for anti-picornavirus activity.166 The most active compound, 2-(3,4-dichlorophenoxy)-5nitrobenzonitrile (MDL-860) (Fig. 16), exhibited broad-spectrum anti-picornavirus activity, inhibiting 80% of the rhinovirus (72/90) and enterovirus (8/10) strains tested at 1 mg/mL.167 Moreover, MDL-860 showed in vivo efficacy while protecting mice against coxsackievirus B3induced myocarditis, as well as against lethal infection with coxsackievirus A21.166,168 The antiviral activity of another potent compound in this series, 2-(3,4-dichlorophenoxy)-5-(methylsulfonyl) pyridine or ‘‘compound 71’’ (Fig. 16), in contrast to MDL-860, appeared to be restricted to rhinoviruses.166 Using compound 71 as a scaffold, some diarylmethanes and aralkylaminopyridines were developed, replacing the oxygen linker bridge.169 Compounds (3,4-dichlorophenoxy)-(5methylsulfonyl-2-pyridinyl)-methane and (2-(3,4-dichlorobenzylamino)-5-methylsulfonylpyridine) (Fig. 16, respectively, compound 13 and 21) exhibited similar in vitro activities as compared to their parent. Mechanism of action studies with the parent compound 71 indicated that it inhibited viral uncoating, in contrast to its analogue MDL-860, that inhibited an early event in virus replication, after initial uncoating.166,170 In vivo, the aralkylaminopyridine (compound 21) was most active, and protected mice against a lethal coxsackievirus A21 challenge. Further attempts to maintain optimal structural criteria and to avoid potential toxicity associated with aromatic nitrocompounds led to the synthesis of a series of phenoxypyridinecarbonitriles.171 Based on the in vitro and in vivo properties in these series of compounds, one compound, 6-(3,4dichlorophenoxy)-3-(ethylthio)-2-pyridinecarbonitrile (DEPC) was selected for further evaluation. Preliminary mechanism of action studies with DEPC indicated that the compound inhibited picornavirus uncoating or some earlier virus-host cell-associated event.171 Additional studies, carried out by Gonzalez et al.,172 confirmed indeed that viral uncoating is the target of DEPC Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION MeSO2 N Cl O MDL 20,610 Cl Br N Cl O MDL 20,646 Cl Cl N Cl O MDL 20,957 Cl Figure 15. Structural formulae of molecules inhibiting picornavirus replication. Figure 16. Medicinal Research Reviews DOI 10.1002/med * 19 20 * DE PALMA ET AL. activity. DEPC was shown to protect mice against lethal CVA21 infection upon oral treatment at 37.5 mg/kg/day.171 In another attempt to increase the enterovirus activity, a novel series of analogues was synthesized by replacing the 3,4-dichloro group on the benzene moiety by a p-benzoyl group.173 Synthesis and evaluation of a total of 15 p-benzoylphenoxypyridines revealed that 3,4-dichloro replacement with a p-benzoyl substituent reduces anti-HRVactivity but improves in vitro and in vivo enterovirus activity.173 As for the previously discussed molecules in these series, viral uncoating was shown to be the target of their antiviral properties.173 3. REPLICATION INHIBITORS A. Guanidine Hydrochloride One of the most extensively studied picornavirus inhibitors is probably guanidine hydrochloride174–177 (Fig. 17). This molecule inhibits the replication of polioviruses, several coxsackieviruses, echoviruses, FMDV, but not the hepatitis A virus.178–182 Early studies using crude RNA replication complexes in vitro indicated that guanidine inhibited the initiation of viral RNA synthesis without inhibiting the elongation or release of completed positive-strand RNA molecules.174,183 Barton and Flanegan184 later confirmed that the compound inhibits a 2C function that is required for the initiation of negative- but not positive-strand RNA synthesis or RNA elongation. Studies with guanidine in poliovirus and FMDV revealed that resistance and/or dependence to guanidine maps to the 2C region.180,182,185 –189 In poliovirus, this resistance can be attributed mainly to two mutations in a loop adjacent to motif B, designated as ‘‘class N’’ or ‘‘class M’’ mutations.180,185 The first class involves a mutated Asn at position 179, the second class a mutated Met at residue 187. Besides these ‘‘main’’ mutations, guanidine-resistant poliovirus was shown to carry some ‘‘additional’’ mutations in 2C. With the non-structural protein 2C being identified as the antiviral target, Bienz and co-workers190 showed that guanidine prevents association of 2C/2BC with host membrane structures during viral replication, without affecting their association with viral RNA. Me N O F Cl N F N S Cl O 5-(3,4-dichlorophenyl)-methylhydantoin N CF3 H N TBZE-029 N N C N H2 NH .HCl N OH HBB H2N NH.HCl F MRL-1237 H2N Guanidine Hydrochloride Figure 17. Structural formulae of molecules targeting the picornavirus non-structural protein 2C. Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 21 Although one study showed that guanidine at millimolar concentrations inhibits ATP hydrolysis,191 other studies were not able to confirm this observation.178,192 B. HBB 2-(a-hydroxybenzyl)-benzimidazole (HBB, Fig. 17) is a selective inhibitor of picornavirus replication that blocks the replication of viral RNA.193 HBB is active against poliovirus, coxsackievirus B strains, some echoviruses and some coxsackievirus A strains, but is devoid of activity against rhinoviruses, FMDVor hepatitis Avirus.179,194 Initial mode of action studies on HBB, by Eggers and Tamm,193 indicated that the compound does not interfere with early processes such as viral entry and uncoating. The same authors confirmed these findings by showing that it was the production of poliovirus RNA that was inhibited by HBB.195 Sequence analysis of HBB-dependent and -resistant mutants variants revealed that the dependent or resistant phenotype maps to the 2C protein of echovirus 9, as observed for guanidine.178,196 The exact interaction of the compounds with 2C, and the precise role of this interaction in viral replication, however, still needs to be determined. HBB, when combined with guanidine hydrochloride proved efficient in preventing virusinduced mortality in a lethal murine model of echovirus type 9 or coxsackievirus A9 infection.197 C. MRL-1237 Another compound that, like HBB and guanidine, was shown to target the 2C protein of picornaviruses, is MRL-1237 [1-(4-fluorophenyl)-2-(4-imino-1,4-dihydropyridin-1-yl)methylbenzimidazole hydrochloride] (Fig. 17).198 Its in vitro activity was demonstrated for poliovirus and some coxsackievirus B types. In poliovirus, it was shown that mutation of a single nucleotide in the 2C coding region led to an MRL-1237-resistant mutant that appeared to be cross-resistant to guanidine hydrochloride.198 D. TBZE-029 The thiazolobenzimidazole derivative TBZE-029 ([1-(2,6-difluorophenyl)-6-trifluoromethyl1H,3H-thiazolo[3,4-a]benzimidazole]) (Fig. 17) that was initially discovered as an NNRTI active against the human immunodeficiency virus type 1 (HIV-1), was recently reported as a selective inhibitor of enteroviruses.199 The compound inhibited the replication of coxsackievirus B3 with an EC50 value of 1.2 mg/mL, and showed similar potency against coxsackievirus A9, echovirus 9 and 11 and enterovirus 68.199 Studies with coxsackievirus resistant to TBZE-029 revealed that the compound inhibits viral RNA synthesis by targeting the non-structural protein 2C. The mutations induced in resistant virus were all clustered in a short region, immediately downstream NTPase/ helicase motif C and were previously reported for other 2C inhibitors, including HBB and guanidine. Despite the fact the 2C was identified as the target of TBZE-029, the compound did not inhibit the in vitro ATPase function of 2C. E. Enviroxime and Analogues Enviroxime (2-amino-1-(isopropyl sulfonyl)-6-benzimidazole phenyl ketone oxime) (LY122771-72) (Fig. 18) is a benzimidazole derivative which proved to be highly active against the replication of rhinoand enteroviruses in vitro.200–202 For rhinoviruses, enviroxime inhibited plaque formation by 50% with EC50 values averaging 0.02 mg/mL.202 CPE formation was inhibited with 50% in 11 EV70 isolates and 15 CVA24 isolates with doses between 0.01 and 0.65 mg/mL.201 Despite this potency in vitro, the reported efficacy of this drug in preventing or treating human rhinovirus infections in clinical trials has been quite variable. Phillpotts et al. demonstrated a significant reduction in disease and virus shedding in a prophylactic study with rhinovirus-infected volunteers203 and some improvement in illness in a Medicinal Research Reviews DOI 10.1002/med 22 * DE PALMA ET AL. N NH2 N HO N Enviroxime O S O Me Me N NH2 N Enviradene O S O Me Me F Me N NH2 N Compound 12 O S O Me Me Figure 18. therapeutic trial.204 In the latter study, 21 volunteers receiving enviroxime intranasally 6 times daily, starting 44 hr after challenge with HRV9, experienced a statistically significant reduction in clinical score on day 5 p.i. The reductions in total clinical score, rhinorrhoea and virus titer in nasal washes, however, never reached statistical significance.204 This was corroborated by several other trials that failed to prove significant clinical effects of enviroxime.205–208 Moreover, subjects experienced gastrointestinal side-effects upon oral administration of enviroxime, especially vomiting and abdominal pain.203 Although factors such as irritation caused by the carrier and the uneven distribution of the drug in the respiratory tract have been suggested as causative for these varying results, the relative insolubility of enviroxime in water may be a major contributing factor. To overcome this problem, enviroxime was incorporated into liposomes, and this significantly facilitated its administration by small-particle aerosol to the respiratory tracts of mice, as well as of human volunteers.209,210 In an attempt to overcome the shortcomings of enviroxime, a series of vinylacetylene- and C2-analogues of enviroxime were synthesized.211,212 The vinylacetylene analogues were derived from enviradene (Fig. 18), which was previously shown to possess superior oral bioavailability without causing emesis in test animals.212 One analogue in this series (‘‘compound 12’’) (Fig. 18) proved to be efficacious by oral administration in the treatment of CVA21 infection in CD1 mice.212 In the C2 analogue series, the 2-amino substitution, as present in enviroxime, was the most active.211 Hamdouchi and co-workers reported on several classes of non-benzimidazole analogues of enviroxime. The compound 2-amino-6-[(E)-1-phenyl-2-(N-methylcarbamoyl)vinyl]-imidazo[1,2-a]pyridines retains the anti-rhinoviral activity of the benzimidazoles. In this new family the substitution at the 3-position was a key element for activity and both isopropyl sulfonyl and aromatic rings, as well as acyl groups were well tolerated.213,214 More recently, a series of structurally related imidazo[1,2-b]pyridazine analogues were synthesized and examined for anti-rhinovirus activity. This series was shown to have significant improvement in potency relative to the Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 23 imidazo[1,2-a]pyridines series, but did not lead to the discovery of more potent enviroxime analogues.215 Preliminary experiments by Ninomiya et al.216 suggested that enviroxime acts at the level of viral RNA replication. Subsequent studies confirmed indeed that enviroxime inhibits the initiation of plus-strand RNA synthesis by targeting the 3A protein; however, the exact molecular mechanisms regarding this inhibition remained unclear.217,218 Later studies with enviroxime-resistant virus suggested that not only the 3A protein, but possibly a complex of proteins and/or cellular factors are targeted by the drug.219 More insights into the molecular interactions that occur between enviroxime and 3A will be obtained once the crystal structure of picornavirus 3A has been solved. F. 2-FMC Thiomersal, a mercury-containing compound, was accidentally found to have antiviral properties in a placebo preparation at Johnson and Johnson.220 A subsequent screening of other mercurycontaining compounds led to the discovery of 2-FMC (2-furylmercury chloride) (Fig. 19), a compound with antiviral activity primarily against HRV serotypes belonging to the antiviral group B [IC50 values as low as 20 nM for HRV-2]. Mode of action studies indicated that 2-FMC inhibits the HRV-2 positive-strand RNA synthesis.220 G. Isothiazole Derivatives DID (5,5 0 -diphenyl-3,3 0 -diisothiazole disulfide) (Fig. 20) was selected from a series of 4 isothiazole derivatives, and proved most active (in vitro) against poliovirus type 1.221 Mode of action studies revealed that the compound exerts its antiviral activity by preventing viral RNA chain elongation via inhibition of replicase activity and/or interfering with the viral RNA polymerase complex.222 Further synthesis and antiviral evaluation of isothiazole derivatives led to the development of several 3-methylthio-5-aryl-4-isothiazolecarbonitriles.223,224 This lead optimization did, however, not result in compounds with improved antiviral activity. H. Flavonoids Flavonoids are ubiquitous in photosynthesizing cells and are commonly found in fruits, vegetables, nuts, etc. For centuries, preparations containing flavonoids have been used to treat human diseases, and many anti-microbial properties have been attributed to this class of compounds, including antiviral activity.225 Ro 09-0179 (4 0 ,5-dihydroxy-3,3 0 ,7-trimethoxyflavone) (Fig. 21), which was isolated from a Chinese medicinal herb, exhibited in vitro antiviral activity against a broad range of picornaviruses, including rhinoviruses, coxsackieviruses, and poliovirus.226 The compound was suggested to interfere with some process of viral replication that occurs between viral uncoating and the initiation of viral RNA synthesis.216,226 In a lethal CVB1 mouse model, Ro 09-0298 (an orally active derivative of Ro 09-0179) administered twice daily, increased the survival rate significantly to 30%, 40%, and 50% at doses of 10, 20, and 40 mg/kg, respectively.226 Out of a series of naturally occurring 3-methoxyflavones, isolated from plant extracts of Euphorbia grantii, 3-methylquercetin (3-MQ) proved to be the most promising agent227 with in vitro antiviral activity against polio-, coxsackie- and rhinoviruses (at concentrations as low as 10 ng/mL). O HgCl 2-Furylmercury Chloride (2-FMC) Figure 19. Medicinal Research Reviews DOI 10.1002/med 24 * DE PALMA ET AL. S S N N S S 5,5’-diphenyl-3,3’-diisothiazole disulfide (DID) Figure 20. At a dose of 20 mg/kg, the compound protected mice against lethal infection with coxsackievirus B4.227 Moreover, 3-MQ proved non-toxic to the ciliary beat frequency of cultured human nasal epithelial cells.228 Structure–activity relationship studies with 4 0 -hydroxy-3-methoxyflavones indicated that the 3-methoxyl and 4 0 -hydroxyl groups of the flavone skeleton, in addition to a substitution at the 5-position and a polysubstituted A ring, are necessary for high antiviral activity.229 OMe OH Ro 09-0179 O MeO OMe OH O OMe O O Me O MeO Ro 09-0298 OMe O O O OH Me OH 3-Methylquercetin (3-MQ) O HO OMe OH O NO2 O Compound 4h OH Cl O Figure 21. Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 25 As for Ro 09-0179, 3-MQ inhibits the synthesis of viral RNA, both in infected cells and in cellfree systems.227,230,231 The host cell shut-off of cellular proteins was shown to persist in the presence of 3-MQ.232 It was demonstrated that the presence of 3-MQ, similar to Ro 09-0179, preferentially blocks the synthesis of genomic viral RNA, but still allows the synthesis of minus strands.231,233 When added at the time of infection, however, both plus- and minus-strand viral RNA synthesis were blocked, but this might be due to the fact that there is not enough genomic RNA to serve as a template to give rise to significant amounts of negative-stranded RNA, detectable by hybridization.234 Other flavonoids isolated from medicinal plants include two flavonoids from Psiadia dentata (3-methylkaempferol and 3,4 0 -dimethylkaempferol), which are shown to inhibit the synthesis of poliovirus (þ)RNA,235 and a flavonoid from Pterocaulon sphacelatum, also exhibiting protection against poliovirus-induced CPE formation.236 Several synthetic flavonoid and isoflavonoid derivatives were evaluated for their potential antipicornavirus activity.237–241 In general, flavanones proved to be less active against picornaviruses than flavones, and 3-substitution appeared to be compulsory for potent activity.242 Among the 3-substituted analogues, the presence of a 3-hydroxy group resulted in increased activity against poliovirus, whereas a 3-methoxy group proved beneficial for the activity against HRV-1B. On the other hand, esterification of the 3-hydroxy group with bulky substituents increased toxicity without significantly improving antiviral activity.237 The synthesis of 2-styrylchromones as vinylogues of flavones, led to the identification of some analogues with antiviral activity against rhinoviruses (with EC50 values for HRV-14 of 1.33 mM for the best compound in this series).239 As expected, introduction of a methoxy- or hydroxyl group at position 3 of these 2-styrylchromones led to more potent analogues, with the best compound (‘‘compound 4h’’) (Fig. 21) showing EC50 values of 0.94 and 0.73 mM for HRV-1B and HRV-14, respectively.240 Several fluoro-, hydroxy-, methoxy-, and chloro-substituted flavonoids were weakly effective against poliovirus, while they exhibited a variable degree of activity against HRV-1B and 14.238,241,243 I. Gliotoxin Gliotoxin (Fig. 22) is a fungal metabolite with antiviral activity.244,245 Trown and Bilello246 showed that the ability of gliotoxin to inhibit poliovirus RNA synthesis was abolished when the compound was maintained in its reduced state. Indeed, the active form of gliotoxin appeared to be that containing a disulfide bridge, as present in the native oxidized state. This led the authors to suggest that gliotoxin inhibits the viral RNA-dependent RNA polymerase through the formation of a mixed disulfide with essential sulfhydryl groups on the enzyme. Rodriguez and Carrasco studied the mode of action of gliotoxin more in detail and confirmed the RdRp to be the antiviral target.247 In contrast to previously discovered inhibitors of poliovirus RNA synthesis (such as flavones), gliotoxin was shown to block (þ) strand synthesis as well as () strand synthesis. Moreover, the in vitro activity of the purified Gliotoxin N OH O O S S N Me CH2OH Figure 22. Medicinal Research Reviews DOI 10.1002/med 26 * DE PALMA ET AL. N S + S Pyrrolidine dithiocarbamate (PDTC) Figure 23. poliovirus polymerase 3D was efficiently blocked by the compound, making gliotoxin the first inhibitor of this viral enzyme.247 Gliotoxin proved to be effective in a monkey model for poliovirus infection, when administered i.m. in a total daily dose of 0.2 mg/kg, given at 8-hr intervals one day before and on each day after virus infection (until the animal developed paralysis), despite its rather high toxicity.244 J. Nucleoside Analogues The in vitro replication of FMDV was shown to be inhibited by 2 0 -C-methylcytidine with an EC50 value of 6.4 mM.248 As part of a study of lethal viral mutagens, a series of 5-substituted cytidine analogues were synthesized. The 5-nitro and 5-aminocytidine analogues were shown to be active as antivirals against poliovirus and coxsackievirus B3 by inhibiting the viral RNA-dependent RNA polymerase.249 At the highest concentration tested (2 mM), 5-nitrocytidine reduced poliovirus by more than 30-fold as compared to the non-specific RNA virus inhibitor ribavirin. K. Pyrrolidine Dithiocarbamate (PDTC) The anti-oxidant pyrrolidine dithiocarbamate (PDTC) (Fig. 23) has antiviral activity against poliovirus, coxsackievirus B3, mengovirus en human rhinoviruses.250–253 The compound was shown to interfere with viral RNA and protein synthesis, as well as with viral polyprotein processing.250–253 The antiviral activity of PDTC was attributed to its dithiocarbamate moiety and was shown to be dependent on transport of zinc ions into the cell.252 4. PROTEASE INHIBITORS: INTRODUCTION The initial step in the translation of the positive stranded genome of picornaviruses is the formation of a single large polyprotein of about 2,000 amino acids, that is, 250 kDa. This polyprotein is rapidly processed into mature viral proteins by co- and posttranslational cleavages,254 which are executed by the viral L, 2A and 3C protease (Fig. 1).255–257 The Leader (L) protein is only present in the genome of cardioviruses and aphtoviruses. In FMDV (the type species of aphtoviruses), the L protein encodes the L protease (Lpro), which cotranslationally cleaves at its own C-terminus.258,259 X-ray crystallography studies revealed a structure for Lpro, comprising a globular catalytic domain, reminiscent of that of cysteine proteases of the papain superfamily, from which a flexible C-terminal extension extrudes.260 Moreover, Lpro has been shown to cleave host translation initiation factor eIF4G, a component of the CAP-binding complex, resulting in the shut-off of host cap-dependent mRNA translation.261,262 FMDV mRNA, in contrast, is translated by a CAP-independent mechanism via an IRES and does not require intact eIF4G for viral protein production.263,264 In fact, the C-terminal cleavage fragment of eIF4G can efficiently support IRES-dependent, but not cap-dependent translation, as it retains the binding sites for IRES.265–267 Thus, as a result of FMDV infection, host cell protein synthesis is rapidly shut-off without affecting translation of viral mRNA. More recent data suggest that Lpro is implicated in antagonizing the cellular innate immune and inflammatory responses to viral infection by Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 27 degradation of nuclear factor kappa B and inhibition of beta interferon mRNA induction.268,269 In contrast to aphtoviruses, the L protein of cardioviruses is not endowed with proteolytic activity.270 In the rhinovirus and enterovirus family, a 2A protease (2Apro) function is ascribed to the 2A gene present in all Picornaviridae.271 Crystal structure determination of the 2Apro of HRV-2 identified these proteinases as members of the chymotrypsin-related protease family, with a cysteine nucleophile and comprising a four-stranded anti-parallel b-sheet as the N-terminal domain and a larger C-terminal domain containing a six-stranded anti-parallel b-barrel.272 The overall folding of the coxsackievirus B4 2Apro was shown to be similar to that of HRV-2, although the two proteins share a rather low degree of sequence similarity.273 The catalytic triad of HRV-2 2A protease contains a Cys106, His18 and Asp35 active site and cleaves the recognition site (L,I)XTX # G at its own N-terminus and the C-terminus of VP1, thereby converting the polyprotein into a capsid (P1) and a non-structural protein (P2 –P3).272,274 Similarly to the Lpro of FMDV, proteinase 2A is also implicated in the cleavage of host cell proteins, and more specifically the cleavage of eIF4G has been studied most extensively. Although cleavage does not occur at the same peptide bond as for Lpro, the effect of the cleavage is the same, that is, unefficient translation from the 5 0 -capped mRNA of the host cells.275,276 In addition, and again in parallel with the Lpro of FMDV, the 2Apro has been shown to be a trans-activator of uncapped RNA translation driven by a poliovirus IRES sequence at times when host-cell (cap-dependent) translation was not inhibited.277 In cells infected with cardiovirus and aphtoviruses, the 2A protein exerts no protease activity on host cell proteins, such as eIF4G. However, an intramolecular polyprotein processing event mediated by 2A occurs at its own C-terminus, resulting in a P1-2A or L-P1-2A protein. The P1-2A cleavage is carried out by the 3C protease.270 Although it was initially suggested that 2A acts autoproteolytically at the 2AB junction, more recent data revealed that this cleavage event is not mediated by a proteolytical reaction, but is the result of a novel translational effect, the so-called ‘‘ribosomal skip.’’278–281 During this process, the FMDV 2A protein modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, which results in release of the polypeptide from the translational complex.279,281 For hepatoviruses and parechoviruses, the 2A protein has no proteolytic activity either and the cleavage between P1 and P2 is performed by the 3C protease.282 The 20 kDa 3C protease (3Cpro) which is encoded by the 3C gene segment, excises itself from the P3 domain of the polyprotein and is responsible as such or fused to the viral RNA-dependent RNA polymerase 3D (3CDpro) for the bulk of polyprotein processing in the Picornaviridae.283,284 Although there are differences between the genomes of the different genera of picornaviruses, they all need 3Cpro to generate mature virion particles. The crystal structures of several 3C (3CD) proteases have been reported, revealing that, like Lpro and 2Apro, the 3Cpro has a cysteine nucleophile (HisGlu-Cys) but with a chymotrypsin-like serine protease folding.285–287 As well as processing viral proteins, 3Cpro can also cleave host-cell factors, for example, poly(A)-binding protein (PABP),288–290 eIF4GI,291 poly(rC)-binding protein (PCBP) isoforms 1 and 2,292 polypyrimidine tract-binding protein (PTB) isoforms 1, 2, and 4,293 TATA-binding protein294–296 and NFkBa.297,298 In addition to its proteolytic activity, 3Cpro (in the form of its precursor 3CD) can bind to the 5 0 -noncoding region of the viral genomic RNA and is involved viral replication.299–305 Given the omnipresence of the 3Cpro in the family of picornaviruses, the fact that the active site of the protease appears to be highly conserved in all rhinovirus serotypes and the fact that 3Cpro makes multiple cleavages in the polyprotein precursor and thus controls viral maturation, make this protein the most promising protease candidate for picornaviral inhibition. Inhibitors of this enzyme are expected to have broad spectrum activity. As the 3Cpro shows unique folding patterns with no known cellular homologue,283 a potential high selectivity may be expected for inhibitors of this enzyme. On the other hand, in view of the fact that the 2A protease is also highly conserved in rhinoviruses and enteroviruses and because of its important role in these two viral families, this protease may also be an interesting target for antiviral drug design. This is reflected in the effort put in the development of Medicinal Research Reviews DOI 10.1002/med 28 * DE PALMA ET AL. antiviral compounds acting predominantly against 3Cpro and 2Apro proteases, a summary of which is provided below. 5. L PROTEASE OF FMDV The thiol protease inhibitor E-64 (L-Trans-Epoxysuccinyl-Leucylamido(4-Guanidine)Butane) (Fig. 24A) is a natural product isolated from Aspergillus japonicus. The compound is an inhibitor of the cysteine protease calpain and E-64 derivates lacking charged groups inhibit intracellular proteases.306 In an in vitro translation assay, E-64 blocked the autocatalytic activity of the Lpro of FMDV and interfered with the polyprotein processing process which should lead to the release the structural protein precursor.307 In virus-infected cells, these results were confirmed, as treatment with E-64 or its membrane-permeable analogue (E-64d, Fig. 24B) caused a reduction in virus yield. E-64 and E-64d are non-toxic in animals, and doses up to 400 mg/kg body weight are well tolerated by various routes of administration.307,308 6. 3C PROTEASE INHIBITORS A. Peptidic Inhibitors The first compounds targeting the 3Cpro were designed based on the peptide substrate cleavage specificity of the enzyme. Thereafter modifications based on the 3D structure of the 3Cpro were made on the compounds to obtain higher inhibitory and antiviral activity (i.e., SAR studies in combination with rational design). 1. Peptide Aldehydes Peptide aldehydes have been broadly studied as protease inhibitors. Short 4 amino-acid peptidic aldehydes containing a P1-Gln-P1 0 -Gly bond and the aldehyde form of Gln which served as an electrophilic anchoring group, were the first 3Cpro inhibitors to be studied (Table II, compound I as a representative).309 These were designed based on the knowledge that HRV 3Cpro has a preference for cutting P1-Gln-P1 0 -Gly bonds and on the fact that cleavable substrates of 3Cpro contain at least 4 amino acids upstream of this bond.310 Furthermore, the enzyme has a preference for a non-polar A. Compound E-64 B. Compound E-64d Me O O HO Me N H O Me O O Me O Me O N H O NH Me H2N H2N Me N H Figure 24. Medicinal Research Reviews DOI 10.1002/med O NH * 29 Table II. Overview of Picornavirus Protease Inhibitors (Continued) PICORNAVIRUS REPLICATION Medicinal Research Reviews DOI 10.1002/med * DE PALMA ET AL. Table II. (Continued) 30 Medicinal Research Reviews DOI 10.1002/med * 31 (Continued) PICORNAVIRUS REPLICATION Medicinal Research Reviews DOI 10.1002/med * DE PALMA ET AL. Table II. (Continued) 32 Medicinal Research Reviews DOI 10.1002/med * 33 (Continued) PICORNAVIRUS REPLICATION Medicinal Research Reviews DOI 10.1002/med * DE PALMA ET AL. Table II. (Continued) 34 Medicinal Research Reviews DOI 10.1002/med * 35 (Continued) PICORNAVIRUS REPLICATION Medicinal Research Reviews DOI 10.1002/med * DE PALMA ET AL. Table II. (Continued) 36 Medicinal Research Reviews DOI 10.1002/med ND, notdescribed. a The methodindicatedinitalics, describesthe methodused to determinethe EC50 value. b Number in superscript indicatesthe reference showingthe data. PICORNAVIRUS REPLICATION Medicinal Research Reviews DOI 10.1002/med * 37 38 * DE PALMA ET AL. amino acid with a small side chain at position P4. Compound I inhibited HRV-14 3Cpro and demonstrated a moderate antiviral activity. Unfortunately, the aldehyde group of Gln could undergo cyclization with the unprotected side chain at P1 to form a thermodynamically more stable hemiaminal which shows diminished inhibitory activity against the enzyme (compound I, Table II).310,311 To avoid cyclization, a dipeptide aldehyde compound (compound II, Table II) containing a P1 methionine sulfone to mimic the glutamine residue, was produced as competitive inhibitor of HRV14 3Cpro.312 The compound elicited low to moderate antiviral activity. Thereafter, tripeptide aldehyde inhibitors with a relatively large substituted alkyl and aryl amide and with Kis ranging from 0.005 to 0.65 mM were produced as potent reversible HRV-14 3Cpro inhibitors.313 These compounds had low micromolar anti-HRV-14 activity, low toxicity and a reasonable therapeutic index. One of the most potent of these inhibitors is compound III (Table II) which also showed low micromolar antiviral activity against other than HRV-14 serotypes of HRV. Along this line, to prevent formation of the hemiaminal, Dragovich et al.314 also prepared potent reversible ketone-containing 3Cpro inhibitors. The most potent of these compounds (compound IV, Table II) displayed very potent levels of reversible 3Cpro inhibition along with submicromolar antiviral activity against several rhinovirus serotypes. In addition, the molecule did not exhibit significant cytotoxicity when tested in cell culture. In analogy with the development of tripeptide inhibitors against HRV-14, tetrapeptide aldehydes containing a side-chain protected P1 glutamine were designed against HAV 3Cpro. The most potent compound (compound V, Table II) contains a dimethylamide of glutamine, is a reversible slow-acting inhibitor and in addition to inhibition of HAV also shows HRV-14 3Cpro inhibition with a 50-fold less potency.315 2. Michael Acceptor-Containing Other types of inhibitors were developed with higher specific activity against 3Cpro which formed stable irreversible covalent complexes with the protease. In these inhibitors, the scissile amide carbonyl (in between Gln-Gly at positions P1 and P1 0 of the peptide inhibitor) was replaced by an electron-withdrawing group (Michael acceptor). Hanzlik and co-workers316,317 were the first to introduce this group in peptidic inhibitors for cysteine proteases and subsequently also in (tetra)peptide inhibitors for 3Cpro. These tetrapeptide Michael acceptors inhibited the HRV-14 3Cpro in the submicromolar levels (compound VI, Table II).318 In addition, a series of tripeptides with various Michael acceptor moieties as the electronwithdrawing group were reported by scientists from Agouron Pharmaceuticals (now Pfizer). A transa-b-unsaturated ethylester incorporated into a benzyloxycarbonyl-protected tripeptide showed potent irreversible antiviral activity against three HRV serotypes in cell culture (Table II, compound VII).319 Since moderate activity, no toxicity to the limit of its solubility and no inactivation by short exposure to dithiothreitol (DTT) were seen with this compound, variations based on an extensive structure–activity study were set up to enhance the activity of the Michael acceptors.319 Related a-b-unsaturated carboxylic acids were poor 3Cpro inhibitors and did not exhibit antiviral activity against the highest concentration examined (100 mM). Another group of ester-derived Michael acceptors, that is, cis-a,b-unsaturated esters or transa,b-unsaturated esters substituted at the a-position showed reduced anti-3Cpro activity when compared with compound VII. The trans-substituted esters exhibited encouraging antiviral activity, good stability in the presence of non-enzymatic thiols, low cellular toxicity, and were easy to synthesize. Amide-containing Michael acceptors displayed reduced anti-3Cpro activity, poorer or equal antiviral activity and/or increased toxicity compared to compound VII. Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 39 Aliphatic and aryl a,b-unsaturated ketones, although extremely potent inhibitors of the 3Cpro enzyme (they displayed increased anti-3Cpro activity when compared to the ester-containing inhibitors described above), had reduced antiviral activity and were toxic to cells (CC50 > 10-fold EC50). The fact that they could be inactivated by short exposure to DTT suggests that these compounds might react rapidly with biological thiols (e.g., glutathione). Vinyl sulfones, nitriles, phosphonates, oximes, and several vinyl heterocycles displayed low levels of 3Cpro inhibition and were poor antiviral agents. A compound containing an a-b-unsaturated nitro moiety demonstrated potent, irreversible 3Cpro inhibitory activity. However, this molecule was inactivated by exposure to DTT and did not display measurable antiviral activity. Although lactam, acyl oxazolidinone, and acyl urea containing Michael acceptors showed promising anti-3Cpro activity, they were inactivated by exposure to non-enzymatic thiols. In addition, the possibility of utilizing exocyclic a,b-unsaturated lactone-inhibitors was investigated. The g-lactones exhibited irreversible 3Cpro inhibitory activity similar to compound VII, while the corresponding d lactones displayed reversible 3Cpro inhibition. Inhibitors containing an N-acyl lactam derived Michael acceptor showed good, irreversible 3Cpro inhibitor activity (kobs/ [I] ¼ 155,000 M1 sec1 vs. 25,000 M1 sec1 for compound VII) and exhibited good antiviral properties (0.71 mM against HRV-14 vs. 0.54 mM for compound VII) (compound VIII, Table II). The related carbamate and N-methoxy derivates displayed reduced anti-3Cpro activity and were poor antiviral agents. The next step in the process was optimizing the protein portion of the inhibitors based on structure–activity studies.320–322 Both a single amino acid and a dipeptide inhibitor displayed significantly reduced anti-3Cpro activity and no/significantly reduced antiviral properties when compared to compound VII, indicating that at least tripeptide Michael acceptor-containing inhibitors are required for effective recognition by the 3Cpro. Since trans-a,b-unsaturated esters (like compound VII) were overall the best Michael acceptor containing compounds, these were chosen for peptidic optimization of the inhibitor. As substitution of the L-Glu at the P1 side, an S-Lactam was introduced.321 This molecule was 10 times more potent than compound VII against HRV-14 and showed fivefold better antiviral activity.321 Variations on the P2 phenylalanine moiety were also examined. From these inhibitors, only compounds containing 4-substituted phenylalanine residues exhibited increased antiviral activities. However, in some cases this increase in activity was accompanied by a decrease in the activity against some other HRV serotypes.320 Replacement of the P2 –P3 peptide bond with a ketomethylene isosteric functionality slightly reduced 3Cpro activity (kobs/[I] ¼ 17,400 M1 sec1) but significantly improved antiviral properties (EC50 ¼ 0.36 mM).322 N-methylation of the P2 –P3 amide linkage also displayed good 3Cpro inhibitory activity (kobs/[I] up to 610,000 M1 sec1) and resulted in molecules that were either equipotent or more potent against HRV-14 (EC50 approximately 0.03 mM).323 Replacement of the P2 –P3 amide linkage with an ester (‘‘depsipeptide’’) resulted in reduced HRV-14 3Cpro inhibitory activity but improved or comparable in vitro antiviral activity.324 These depsipeptide inhibitors however, may not be ideal therapeutic candidate inhibitors due to their lack of in vitro stability. A wide variety of substitutions of functionality (e.g., several other aliphatic and/or aromatic moieties) at the P3 leucine position showed improved anti-3Cpro and antiviral activity relative to compound VII in most cases.320 N-terminal (P4) inclusion of an S-alkyl thiocarbamate in the inhibitor design dramatically increased anti-3Cpro properties relative to similar carbamate-containing molecules (compound IX vs. compound VII).320 Medicinal Research Reviews DOI 10.1002/med 40 * DE PALMA ET AL. 3. AG7088/Rupintrivir Combinations of several of the above-mentioned beneficial modifications into N-terminal protected tripeptides were made by Pfizer (formerly Agouron)321–323 and finally led to AG7088/Rupintrivir, a compound with excellent irreversible inhibition capacity against 3Cpro, with strong antiviral capacity against a broad spectrum of picornaviruses and with low toxicity321,325,326 (Table II). The compound contains a five-member lactam ring at P1, a methylene group instead of the backbone amide in P2, an isoxazole group at the P4 and a Michael acceptor.327 AG7088 was shown to be highly specific for picornaviral 3Cpro and not to be active against cellular serine and cysteine proteases. Rupintrivir demonstrated potent in vitro antiviral activity against 23 of 23 HRV clinical isolates, derived from nasal lavage samples, by cell protection assays with a mean EC50 of 24 nM (range: 3–104 nM).328 In addition, potent in vitro activity was demonstrated against four HEV strains with a mean EC50 of 41 nM (range: 7–137 nM). Kaiser et al.329 demonstrated potent, broad-spectrum antiviral activity of rupintrivir against 46 untyped HRV isolates. Patick et al. investigated the efficacy of AG7088 against a panel of 48 different HRV serotypes by a cell protection assay utilizing H1-HeLa cells. Results indicated that AG7088 was active against all HRV serotypes (48 out of 48) tested with a mean EC50 of 23 nM (range: 3–81 nM).327 In addition, the efficacy of AG7088 against four related picornaviruses (CAV-21, CVB-3, EV-11, EV-70) was examined. In H1-HeLa or MRC-5 cell protection assays, AG7088 was active against all four picornaviruses tested, with EC50 values ranging from 7 to 183 nM. Overall, rupintrivir demonstrates comparable antiviral potencies against all the HRV and HEV isolates tested (n ¼ 125). In a human respiratory cell line (Beas2B), rupintrivir was shown to reduce the HRV enhanced secretion of IL-6 and IL-8 in association with its anti-HRV effect.330 Since AG7088 could be extensively hydrolyzed in the liver thereby leading to significant first pass metabolism and poor oral bioavailability,331 a clinical development program that localizes the delivery of AG7088 to the nasal cavity was preferable. The molecule was subsequently tested for safety and pharmacokinetics in healthy human volunteers in both a single-dose and multipledose study.332 In both studies, both dose levels of intranasal rupintrivir were safe and well-tolerated. Adverse effects were mild, short-lived, confined to the upper respiratory tract and similar to placebo induced adverse effects. Substantial amounts of rupintrivir were observed intranasally for at least 9 hr after multiple doses. In a human experimental HRV challenge trial rupintrivir reduced the severity of illness and viral load, providing proof of concept for the mechanism of 3C protease inhibition.333 In a subsequent natural infection study in patients, rupintrivir was not able to significantly affect virus reduction or moderate disease severity and thus was halted from further clinical development.334 Derivatives of AG7088, wherein the ethyl propenoate Michael acceptor moiety was replaced with one containing aromatic rings, possessed significantly enhanced inhibitory activity against CVB3.335 A derivative containing a naphthalene ring was the most efficient inhibitor (IC50 ¼ 93 nM) and showed CVB3 replication inhibition in a dose-dependent manner. 4. Azapeptides Azapeptides, like peptidyl bromomethylketonehydrazides containing a backbone modified glutamine, irreversibly inactivate 3Cpro by alkylating the thiol group of the active site cysteine.336 As an example compound X (Table II) is shown. Although azapeptide esters with different reactive groups at P1 were designed, these showed only moderate 3Cpro inhibition.337 Enhanced inhibitory potency was achieved by azadicarboxamides (inhibitor XI, Table II).338 These irreversible inhibitors were designed based on the substrate specificity for HAV 3C, and contain the mimicked P1 (Gln) and P2 0 (Phe) residues important for enzyme recognition. 5. Diazomethyl Ketones Diazomethyl ketones (DMK), developed on the basis of the natural cleavage site of the HRV-14 3Cpro by Murray et al.,339 were assessed for their potency. The tripeptidyl-DMK inhibitor Z-L-F-Q-CHN2 Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 41 elicited strong anti-3Cpro activity and strong antiviral efficacy against HRV-14 and HRV-16 (compound XII, Table II). The inhibitor was shown to work specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3Cpro and an accumulation of the 3CD polypeptide. 6. Peptidyl Monofluoromethylketones Peptidic aldehydes, although effective as enzyme inhibitors, are unlikely to be effective as therapeutic agents because of metabolic degradation, insufficient oral bioavailability, and transport problems. Peptidyl monofluoromethyl ketones, however, have been shown to be effective in vivo in a number of systems.340–342 A peptidyl fluoromethylketone based on the preferred HAV 3Cpro peptide substrate was therefore developed (compound XIII, Table II). This molecule irreversibly inactivated HAV 3Cpro by forming a stable complex and 50% inhibitory activity was noted at 41 mM.343 7. Keto-Glutamine Analogues Dimethyl glutamine analogues containing a ketone functionality were prepared and evaluated as potential inhibitors of HAV 3Cpro. Introduction of a phthalhydrazido group a to the ketone moiety afforded a compound which showed reversible inhibition of HAV 3Cpro with an IC50 value of 89 mM. Attaching the tripeptide (N-Ac-Leu-Ala-Ala), required for enzymatic recognition, to this compound provided compound XIV (Table II).344 The inhibitory activity of compound XIV is not affected by the presence of 100-fold excess of DTT, indicating that the phthalhydrazido system could be a specific inhibitor of HAV 3Cpro and does not react inadvertently with ubiquitous thiols (e.g., glutathione). Structural variations in these keto-glutamines, like incorporation of the 2-oxo-pyrrolidine ring moiety of AG7088 into the P1 position of compound XIV, improved inhibition against hepatitis A virus 3Cpro (IC50 ¼ 2.5 mM).345 8. S-nitrosothiols It has been demonstrated that many cysteine containing enzymes such as protein tyrosine phosphatase can be efficiently inactivated by NO or NO donors.346 Since HRV 3Cpro contains an essential cysteine residue, this enzyme should be susceptible to NO donors. S-nitrosothiols are a class of important widely used NO donors.347 The compounds studied, exhibited different inhibitory activities in a time- and concentration-dependent manner with second-order rate constants (kinact/KI) ranging from 131 to 5360 M1 min1. Inactivation of the enzyme occurred through an S-transnitrosylation process. The most potent S-nitrosothiol developed was EALFQCG-SNO (compound XV, Table II). This inhibitor which is an S-nitroso heptapeptide, was specifically designed and synthesized for HRV 3Cpro, because the peptide sequence Glu-Ala-Leu-Phe-Gln is a possible substrate for 3Cpro. 9. Peptidyl N-iodoacetamides Iodoacetamide is an irreversible cysteine protease inhibitor that inhibits the replication of HAV, HRV, and poliovirus 3Cpro.348 Displacement of iodide by the active site cysteine thiol group results in irreversible alkylation of the enzyme. Attachment of amino acid residues that mimic the P1 0 –P2 0 side chains can increase selectivity and potency. For example, ICH2CO-Val-Phe-NH2 was prepared by McKendrick et al.348 (compound XVI, Table II) and displayed 60-fold higher irreversible inhibition of HAV 3Cpro than the parent iodoacetamide. The resulting complex has an acetyl-Val-Phe-amide group covalently attached to the Sg atom of the nucleophilic Cys172 of the enzyme.349 Medicinal Research Reviews DOI 10.1002/med 42 * DE PALMA ET AL. 10. Tripeptidyl a-ketoamides A novel series of tripeptidyl a-ketoamides was synthesized by Chen et al.350 The most potent inhibitor exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14 and HRV-16 (compound XVII, Table II). B. Non-Peptidic Inhibitors 1. 2-Pyridone-Containing Peptidomimetics Because potent peptidyl 3Cpro inhibitors, previously developed by the group of Dragovich et al.351 (see Section 6.A.2: compound VII), had insufficient oral bioavailability, novel 3Cpro inhibitors were prepared with non-peptidic chemical structures. Incorporation of an appropriate P3 3-amino-2pyridone moiety into compound VII afforded a compound which displayed greatly improved anti3Cpro (i.e., about fivefold) and antiviral activity (EC50 ¼ 33 nM) relative to compound VII. Thereafter variations were made guided by previous studies of tripeptidyl 3Cpro inhibitors. This resulted in a 2-pyridone-inhibitor containing a P1 (S)-g-lactam moiety, a P2 benzyl substituent, and an N-terminal (P4) isoxazole fragment which displayed very good 3Cpro inhibition activity (50-fold increase when compared to compound VII) as well as extremely potent anti-rhinovirus activities (EC50 ¼ 2 nM for HRV-14) in cell culture. This compound also exhibited potent antiviral activity against two other HRV strains (EC50 ¼ 15 nM [HRV-1A] and EC50 ¼ 4 nM [HRV-10]). However, the compound was extensively metabolized when incubated with human liver microsomes. Introduction of an additional fluorine atom at the P2 benzyl substituent on the previous described compound displayed improved anti-3Cpro and anti-rhinovirus properties (EC50 ¼ 1 nM) relative to the mono-fluorinated molecule. Although the inhibitor displayed improved stability towards human liver microsomes, the compound was still easily metabolized in vitro. The next step was replacing the ethyl ester with a sterically larger isopropyl ester. This alteration created a compound with improved performance in the liver microsomal stability test with a simultaneous worsening of anti-3Cpro and anti-HRV potency (compound XVIII, Table II). Despite these reductions, the compound displayed good absolute anti-rhinovirus potency against 15 different HRV serotypes. Oral bioavailability studies with this compound in the dog, showed good bioavailability and pharmacokinetic properties. Unfortunately, the inhibitor was poorly bioavailable when orally delivered in solution to CMmonkeys.352 The incorporation of a bicyclic 2-pyridone moiety in the inhibitor design did not improve overall antiviral activity (compound XIX, Table II).353 Replacement of the P2 benzylic substituent in compound XVIII with an ethyl fragment led to compound XX (Table II) which exhibited plasma concentrations in the dog 7 hr after oral administration more than 14-fold greater than the EC50 value (i.e., C7h ¼ 0.682 mM vs. mean EC50 ¼ 0.047 mM).352 Similarly, oral administration of this compound to CM-monkeys afforded 7 hr plasma concentrations (i.e., C7h ¼ 0.896 mM) that far exceeded in vitro the EC50 value. Replacement of the P2 benzylic substituent in compound XVIII with a propynyl fragment furnished the irreversible inhibitor ‘‘compound XXI,’’ also referred to as ‘‘compound 1’’ (Table II).334,352 This compound demonstrated oral bioavailability in dogs and CM-monkeys with 7-hr plasma concentrations similar to or exceeding the in vitro antiviral activity against the seven HRV serotypes evaluated.352 In addition, in cell-based assays, the compound was active against 35 different HRV genotypes, against 5 clinical isolates and 8 related picornaviruses with EC50 values ranging between 7 and 249 nM (Table II).334 In vitro and in vivo non-clinical safety studies showed compound XXI to be without adverse effects at maximum achievable doses. Phase I studies showed a single oral dose of the compound to be safe and well tolerated.334 Together with rupintrivir, however, compound 1 was recently halted from further clinical development.354 Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 43 2. Substituted Benzamide Inhibitors Reich et al.355 developed non-peptidic low molecular weight inhibitors, based on the co-crystal structure of a peptide aldehyde bound to 3Cpro. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3Cpro substrate. a,b-unsaturated ketobenzamides showed good inhibitory property (compound XXII, Table II) and moderate antiviral activity, yet 5-substituted benzamides were found to be more active (compound XXIII, Table II). 3. Isatins Working on peptidic inhibitors has provided important information for the design and development of non-peptidic molecules. Based on these studies, another class of inhibitors with high affinity for the protease pocket was developed containing a reactive a-ketoamide group (Table II, compound XXIV).356 Unfortunately, although these 2,3-dioxindole(isatin)-core inhibitors showed high selectivity against HRV 3Cpro, they were devoid of antiviral activity and/or showed high cell toxicity, properties most probably attributable to their high electrophilic reactivity.356 4. Spiro-Indolinone b-Lactams Spiro-indolinone b-lactams were described for the first time by Skiles and McNeil357 as potential HRV 3Cpro inhibitors. Unfortunately they were not selective. As an example, compound XXV (Table II) inhibited in addition to HRV 3Cpro (IC50 ¼ 20 mg/mL) also human leukocyte elastase with an IC50 of 0.4 mg/mL and cathepsin G with an IC50 of 4 mg/mL. Antiviral data or toxicity data of the compound are not available. 5. Homophthalimides Following a blind screening effort, homophthalimides were found to exhibit anti-rhinovirus 3C activity.358 SAR studies resulted in homophthalimides with low micromolar IC50 (as an example compound XXVI is shown in Table II). Multiple homophthalimides displayed antiviral activities in cell culture which correlated well with the enzyme inhibition data. 6. b-Lactones b-Lactones are yet another class of non-peptidic 3Cpro inhibitors that irreversibly inactivate the HAV protease. They occur naturally in a variety of organisms and many possess potent biological activity.359 L- and D-N-Cbz-serine b-Lactones (compound XXVII) were initially studied as 3Cpro inhibitors.360–362 Despite the absence of the P1 glutamine side chain important for HAV 3C substrateinhibitor recognition, both L- and D-N-Cbz-serine were potent inhibitors of HAV 3Cpro (IC50 ¼ 35 and 6 mM, respectively). They inactivate HAV by alkylating the active site thiol group, thereby forming a covalent bound. The inhibitory activities of both compounds is not affected by the presence of 10-fold molar excess concentration of dithiothreitol (DTT), which suggests that these b-lactones are not dramatically thiophilic and could therefore be specific enzyme inhibitors that would not react randomly with free biological thiols. 7. Pseudoxazolones The unique reactivity of pseudoxazolones (i.e., they are known to add one thiol at the imine position to form thioesters) led to the proposal that these compounds could potentially be used to inhibit thiol containing enzymes. In the case of HAV 3Cpro, the enzyme active site thiolate is expected to react with the pseudoxazolone at the imine position, forming a covalent adduct. Under aqueous conditions, hydrolysis could occur to generate the covalently modified enzyme-inhibitor complex. A Medicinal Research Reviews DOI 10.1002/med 44 * DE PALMA ET AL. monophenyl pseudoxazolone inhibitor showed good time-dependent inhibition with low IC50 values against both HAV and HRV (compound XXVIII, Table II).363 8. Low Molecular Weight Non-Peptidic Inhibitors Since other non-peptidic HRV 3Cpro inhibitors, like isatins and homophthalimides, have suffered from problems such as cellular toxicity and modest antiviral activity, a new class of low molecular weight non-peptidic inhibitors was developed by Johnson et al.364 These molecules contain an ethyl ester Michael acceptor P1 lactam as a core and a P2 –P4 non-peptidic portion. From these, compound XXIX shows a rate of inactivation comparable to compound VII (i.e., the peptide lead used) but has increased antiviral activity (Table II). This may reflect the improved physical properties, leading to improved cellular permeability of the smaller molecules as compared to the larger lead compound VII. Unfortunately the activity against other HRV serotypes than HRV-14 fell off dramatically.364 C. Microbial Extracts In addition to the above described inhibitors, several other microbial fermentation extracts have also been reported as HRV 3Cpro candidate inhibitors.365–367 These include naphtoquinonelactol (compound XXX, Table II),367 a quinine-like citrinin (compound XXXI, Table II),366 radicinin (compound XXXII, Table II)366 and triterpene sulfates (compound XXXIII, Table II).365 These compounds show moderate IC50 values in the mg/mL or millimolar range. 7. 2A PROTEASE INHIBITORS A. Thiol Alkylating Agents Konig and Rosenwirth368 investigated the effect of various protease inhibitors on 2Apro activity to characterize the enzyme. From these inhibitors, iodoacetamide and N-ethylamaleimide (Fig. 25A,B) proved most potent, conferring respectively 79% and 84% inhibition of enzyme activity (at 10 mM).368 B. Classic Elastase-Specific Inhibitors Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MCPK) and elastatinal, which are both commercially available, have been reported to inhibit 2Apro of poliovirus and HRV-14, (Elastatinal: IC50 ¼ 7 mM and MPCMK: IC50 ¼ 65 mM).369 In addition, MCPK caused a reduction in the yields of the enteroviruses poliovirus type 1 and coxsackievirus A21 and of human rhinovirus 2 in infected HeLa cells but did not affect the replication of EMCV. C. Homophthalimides Wang et al.370 described several homophtalimides, which were originally designed as 3Cpro inhibitors and demonstrated their inhibitory activity against the HRV-14 2Apro, having IC50 values in A I O B NH2 O N O Et Iodoacetamide N-ethylamaleimide Figure 25. Medicinal Research Reviews DOI 10.1002/med PICORNAVIRUS REPLICATION * 45 the low-micromolar range. For example, compound XV (Table II) demonstrated an IC50 of 3.9 mM against HRV-2 2Apro. The observed antiviral activity by homophthalimides therefore might result from a dual inactivation of 3Cpro and 2Apro. D. Peptide-Based Fluoromethyl Ketones Several fluoromethyl ketone (fmk)-derivatized peptides are commercially available as inhibitors of caspases.371 Fmk acts as a trapping group which upon covalent binding to -SH of an adjacent cysteine residue causes irreversible inhibition. This stimulated Deszcz et al.372 to investigate the inhibitory capacity of the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD.fmk) and benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone (zIETD.fmk) against HRV 2Apro activity. In their methylated forms they blocked eIF4GI cleavage in vivo and in vitro and inhibited the activity of picornaviral 2Apro. The 50% inhibitory concentration values were determined as 5.6 106 M for zVAD.fmk and 7.7 106 M for zIETD.fmk, respectively. In contrast, the unmethylated form of zVAD.fmk did not inhibit the cleavage reaction. Similar results were obtained for 2Apro of CVB4. 8. ASSEMBLY AND RELEASE A. Hydantoin Hydantoin (5-(3,4-dichlorophenyl) methylhydantoin) (Fig. 17) was shown to inhibit the replication of PV1 and PV2 by more than 95% and PV3 and CVA21 by more than 99% at a concentration of 25 mg/mL.373,374 Mechanism of action studies revealed that this compound inhibits the encapsidation of viral RNA, and that the resistant phenotype to hydantoin maps to the 2C coding region of the viral genome.374 Verlinden et al.375 showed that, apart from the inhibition of viral assembly, hydantoin also blocks postsynthetic cleavage of poliovirus proteins. 9. CONCLUSIONS Picornaviruses encompass a large number of viruses with considerable clinical relevance in both human and veterinary medicine. Rhinovirus infections, although mostly mild and selflimiting, yearly account for huge economic losses in terms of lost school and working days. Moreover, recent evidence points towards rhinoviruses as a major cause in exacerbating asthma and COPD. In children and neonates, several enteroviruses are responsible for infections and epidemics, which can often be life-threatening. Poliovirus remains a threat for mankind, especially in third world countries, since the complete eradication of polio has still not reached its final stage, notwithstanding huge efforts. Despite this, there has still no therapy been approved for the treatment of picornaviruses infections, and patient care mostly remains symptomatic. Many molecules that selectively inhibit picornavirus replication and that target either structural or non-structural picornaviral proteins have been identified. The most well characterized targets are the viral capsid protein VP1 and the 3C (and 2A) protease. Targets that have been shown to be valuable, but of which the exact mechanism of action still remains to be elucidated, include the viral non-structural proteins 3A and 2C. The capsid binding compound Pleconaril was originally developed by Sanofi-Aventis and licensed to ViroPharma in 1997. A New Drug Application was submitted in 2001. The drug was not approved and ViroPharma re-licensed it to Schering-Plough in 2003. In 2007, Schering-Plough completed a phase II double-blind, placebo-controlled trial to study the effects of Pleconaril nasal spray on common cold symptoms and asthma exacerbations following rhinovirus exposure. Results Medicinal Research Reviews DOI 10.1002/med 46 * DE PALMA ET AL. of this trial are still unpublished. Biota successfully completed a double-blind phase 1 human safety trial of the HRV drug BTA-798 in 2007 and is scheduling phase II trials in 2008. This drug which, akin to pleconaril, acts as a capsid-binding agent is being developed for the prevention and treatment of HRV infections in high risk COPD and asthma patients and might be a promising future drug candidate. Pfizer developed a series of 3C protease inhibitors. Rupintrivir and its orally bioavailable analogue compound 1 were progressed to clinical development. Despite activity in a human experimental HRV challenge trial, rupintrivir proved unsatisfying in natural infection studies and, like its analogue ‘‘compound 1,’’ was halted from further clinical development (A. Patick).354 The development of highly potent and safe inhibitors of picornavirus replication remains of utmost importance. ACKNOWLEDGMENTS We thank Ward Heggermont for assistance with the drawing of the chemical structures and Dominique Brabants for excellent editorial assistance. The original work of the authors was supported by RiviGene EU FP6—Contract No SSPE-CT-2005-022639. REFERENCES 1. 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The antiviral compound 5-(3,4-dichlorophenyl) methylhydantoin inhibits the post-synthetic cleavages and the assembly of poliovirus in a cell-free system. Antiviral Res 2000;48:61–69. 376. Kolatkar PR, Bella J, Olson NH, Bator CM, Baker TS, Rossmann MG. Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor. EMBO J 1999;18:6249–6259. Armando M. De Palma (1979) graduated in 2002 as a bio-engineer in cell and gene biotechnology and is currently a PhD student at the University of Leuven (Belgium). His research project focuses on the development of novel enterovirus inhibitors, and their molecular mechanism of action. Inge Vliegen (1977) obtained her PhD degree in virology from the University of Maastricht (The Netherlands) in 2003. Until February 2005, she was a postdoctoral research fellow in the department of Medical Microbiology (University of Maastricht). Since February 2005, she is a postdoctoral fellow at the Rega Institute, University of Leuven, Belgium. Erik De Clercq (1941), M.D., Ph.D. has been Chairman of the Department of Microbiology and Immunology of the Medical School at the Katholieke Universiteit Leuven as well as Chairman of the Board of the Rega Institute for Medical Research (until September 2006). He is currently President of the Rega Foundation and a director of the Belgian (Flemish) Royal Academy of Medicine, a member of the Academia Europaea and fellow of the American Association for the Advancement of Science. He has also been the titular of the Prof. P. De Somer Chair for Microbiology. He has been teaching the courses of Cell Biology, Biochemistry and Microbiology at the K.U.Leuven (and Kortrijk) Medical School (until September 2006). Professor De Clercq received the Hoechst Marion Roussel (now called ‘‘Aventis’’) award (American Society for Microbiology), the Maisin Prize for Biomedical Sciences (National Science Foundation, Belgium), R. Descartes Prize (European Union Commission) and B. Pascal Award (European Academy of Sciences) for his pioneering efforts in the field of antiviral research. His scientific interests are in the antiviral chemotherapy field, and, in particular, the development of new antiviral agents for various viral infections, including herpes simplex virus (HSV), varicellazoster virus (VZV), cytomegalovirus (CMV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), human papilloma virus (HPV), and hepatitis C virus (HCV). He has (co)-discovered a number of antiviral drugs, currently used in the treatment of HSV infections (valaciclovir, Valtrex1, Zelitrex1), VZV infections (brivudin, Zostex1, Brivirac1, Zerpex1), CMV infections (cidofovir, Vistide1), HBV infections (adefovir dipivoxil, Hepsera1), and HIV infections (AIDS) (tenofovir disoproxil fumarate, marketed as Viread1, and, in combination with emtricitabine, as Truvada1, and, in combination with both emtricitabine and efavirenz, as Atripla1). Johan Neyts (1966) is Professor of Virology at the Rega Institute, Faculty of Medicine, University of Leuven, Belgium, where he is teaching medical virology at the school of dentistry and the school of medicine. His research is focused on the development of novel antiviral strategies against a number of viruses, including picornaviruses, flaviviruses and the hepatitis B and C virus. His team discovered the anti-HCVactivity of Debio-025, a compound which is now in phase II clinical development by DebioPharm (Lausanne, Switzerland). Together with Prof. Gerhard Puerstinger (University of Innsbruck, Austria) he also discovered a novel class of HCV inhibitors, a potent analogue of which (GS 9190) is now in phase I clinical development at Gilead Sciences (Foster City, CA). He is on the editorial board of the journals ‘‘Antiviral Research’’ and ‘‘Antiviral Chemistry and Chemotherapy,’’ ad hoc reviewer for about 30 scientific journals, member of several national and international scientific committees and on the board of directors of the International Society for Antiviral Research. He has been honoured with a number of awards including from the Royal Belgian Academy of Medicine, the Belgian Fund for Scientific Research and the International Society for Antiviral Research. Medicinal Research Reviews DOI 10.1002/med
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