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Expert Rev. Anti Infect. Ther. 9(2), 161–163 (2011)
Ben Berkhout†1 and Dirk Eggink1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, K3-110, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands † Author for correspondence: Tel.: +31 20 566 4822 Fax: +31 20 691 6531
[email protected] 1
Antivirals Congress, Celebrating 30 Years of the Journal Antiviral Research 7–9 November 2010, Amsterdam, The Netherlands The journal Antiviral Research was conceived and born in 1980, and launched in 1981, a time when very few antiviral drugs were around. This 30-year celebration meeting was convened by the publisher Elsevier and chaired by Eric de Clercq (Leuven University), who has acted as editor-in-chief for the journal for the last 30 years. Approximately 220 participants representing 36 countries attended the congress, which covered topics such as emerging viruses, virus–host interactions, new antiviral drugs and antiviral resistance. More antivirals for HIV–AIDS
Daria Hazuda (Merck, PA, USA) described the development of the novel class of HIV-1 inhibitors that target the viral integrase enzyme, which inserts the viral DNA into the host cell genome. The early drug discovery efforts were hampered by the absence of a biochemical assay that would allow high-throughput screening of drug libraries. Merck focused on the rate-limiting strand transfer and joining step of the integration process, which yielded the first drug candidates of diketo chemistry in 2000. Drugs such as raltegravir (approved in October 2007) [1] bind the enzymedonor DNA complex. This drug–enzyme complex has a very slow off‑rate, and this extended residence time may allow novel applications such as prevention strategies. What is striking is the rapid onset of the antiviral effect. This effect may relate to virus inhibition in cells where HIV-1 is arrested in the preintegration state. Details of the first co-crystal structure were published recently, and this has yielded much mechanistic insight on drug action and resistance development. Raltegravir is a Mg 2+ -binding compound and the hydrophobic groups of the inhibitor target a pocket in the active site that is created by integrase and the viral DNA. Three resistance mechanisms were reported [2] , all based on resistance mutations near to the active site of the enzyme. Single mutations give only ten- to 20-fold resistance, and more mutations usually appear over time. Integrase function is affected by these mutations, causing reduced viral fitness. Second-generation www.expert-reviews.com
10.1586/ERI.10.166
integrase inhibitors are now being developed that show an improved potency because of an even lower off-rate. Interestingly, raltegravir seems to have a broad antiretroviral spectrum, including HIV-2, human T-lymphotropic virus-I and the mouse-derived xenotropic murine leukemia virus-related retrovirus. Alex Matter (Esperanza Medicines Foundation, Singapore) discussed the goals of the ‘Universal access to antiretroviral drugs declaration’ of UNAIDS, which hopes to stop the worldwide HIV–AIDS epidemic. Some notable successes were reported, for instance a drop in new infection rates in Africa by up to 25% among the 15–24year age group. The foundation focuses on natural products with antiviral properties that should yield affordable drugs. The malaria field with quinine and artemisinin as potent drugs of natural origin was presented as a viable example of such a drug development route. Ben Berkhout (University of Amsterdam, The Netherlands) presented anti-HIV-1 fusion inhibitors, including the prototype T20 peptide and the improved versions T1249 and T2635, which were developed by Trimeris-Roche. T20 resistance is a fairly easy evolutionary process, but the selection of T1249-resistant HIV-1 strains was shown to require more dramatic amino acid substitutions in the gp41 envelope target protein. T2635 remains active against all these HIV-1 variants because of the stronger binding of the peptide and because charged residues within this peptide are not exposed for a charge repulsion mechanism [3] .
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Berkhout & Eggink
It turned out to be extremely difficult to select T2635-resistant HIV-1 variants in vitro. Many mutations are necessary to achieve a very modest tenfold resistance, and those variants exhibit a serious decrease in envelope function and viral fitness. Combined with measures to improve the stability of these peptides, these results suggest that there is a future for antiviral peptides. Sarah Palmer (Karolinska Institute, Stockholm, Sweden) described a single copy assay for HIV-1 that was used to study the persistent viremia in patients on therapy [4] . The persistent viremia was found to be independent of the potency of the treatment regimen, arguing against the involvement of ongoing virus replication and suggesting the involvement of long-lived cells that were already infected before the onset of therapy. A biphasic decline in persistent viremia was observed over 7 years of treatment, suggesting the involvement of different cellular compartments, one with a halflife of 39 weeks and another with a much longer half-life [5] . The population with a 39-week half-life are possibly resting memory T cells, which have a decreased level of the transcription factors nuclear factor of activated T cells and NF-kB. Histone deacetylase inhibitors were tested in order to influence the chromatin structure of integrated HIV-1 proviruses and their transcriptional activity, which may overcome viral latency. Drugs to halt the outbreak of serious viral infections in cattle
Johan Neyts (Leuven University, Belgium) spoke on the use of antivirals to prevent outbreaks of the classical swine fever virus (CSFV), a member of the flaviviridae family. CSFV affects domestic pigs and wild boar, and there is a continuous threat of virus immigration from Eastern Europe. Vaccines are available, but not allowed in Europe because one cannot distinguish vaccinated from infected animals. Killing all animals in the affected farms has been effective, but raises many ethical questions. As an alternative, treatment with stockpiled antiviral drugs was proposed and should prevent the further spread of virus within hours of administration. A new class of imidazopyridine drugs was identified that acts on and causes resistance mutations in the viral polymerase [6] . This 5-([4-bromophenyl] methyl)-2-phenyl-5H-imidazo(4,5-c)pyridine (BPIP) compound is active against CSFV, but can likely be improved. BPIP was subsequently tested on pigs. All drug-treated animals do eventually become infected, but a much reduced viral load and shorter viremic period was measured, combined with an absence of clinical symptoms. BPIP partially reduced transmission of the virus to untreated sentinels, which is perhaps the most important parameter for an anti-outbreak approach. Intriguingly, a related compound (tegobuvir; GS9190) was found to be active against HCV and this drug is currently being tested in clinical trials. Antivirals against emerging infections
The potential of re-emergence of the severe acute respiratory syndrome coronavirus was discussed by Dale Barnard (Utah State University, UT, USA). Severe acute respiratory syndrome induced disease was first reported in February 2003 in Asia. The infection causes severe pneumonia with a 10% mortality rate. This first epidemic was effectively contained by isolation measures. Vaccines are 162
being developed to control the viral spread in case of a new future zoonotic event or bioterrorist release. Several antivirals have been reported as potential therapeutics, including interferons, the thetadefensin antimicrobial peptide and the Griffithsin protein that binds the viral spike glycoprotein and inhibits viral entry. Mouse studies indicated that interferon is also useful in a prophylactic setting. Dengue virus is a flavivirus that is currently present through the tropics. Subhash Vasudevan (Novartis Institute for Tropical Diseases, Singapore) explained that there are four dengue serotypes and that individuals can be infected four-times because of a lack of cross-protection. An antiviral should act before or during the febrile period to prevent dengue hemorrhagic fever and dengue shock syndrome, which is an uncontrolled host response (cytokine storm). Based on the success of protease drugs against HCV – another flavivirus – the dengue nonstructural (NS)3 protease is an obvious drug target. A fluorescence-based protease assay was developed to screen more than 1 million compounds, but with disappointing results [7] . Peptidic substrate analogs were subsequently tested, and transition-state analogs showed antiviral activity. Information on the protease structure, showing an open and closed conformation [8] , was instrumental for drug optimization, yielding drugs with an IC50 in the low micromolar range. Another target is the viral helicase encoded by the full-length NS3 protein. Yet another target is the viral polymerase, and screening has yielded one drug. A potent nucleoside inhibitor (chain terminator class) was recently described by the Novartis team. Tove Bolken (SIGA Technologies Inc., OR, USA) screened a library of over 250,000 small molecule compounds in a dengue virus replication assay, yielding four lead compounds that are flavivirus-specific and active against all serotypes. Two drug candidates (ST-610 and ST-148) showed promising results in the interferondeficient mouse model, showing a 1–2 log reduction in viral load and dose-dependent reduction of disease markers. Dennis Hruby from the same SIGA company discussed anti virals against orthopox viruses. ST-246 has good antiviral activity at 3 mg/kg and this drug shows no toxicity in animals, including nonhuman primates, and no toxicity was seen in an initial Phase I clinical trial. Influenza virus & antiviral drugs
Ian Barr (WHO, Melbourne, Australia) described neuraminidase inhibitors, including zanamivir (to be inhaled), oseltamivir (oral administration) and the new drug peramivir. The seasonal H1N1 is resistant to oseltamivir, but other variants including the pandemic H1N1 influenza are sensitive. Resistance spread globally in the 2007–2008 period, but these variants have become less prominent after the spread of the pandemic H1N1 influenza. The H274Y mutation provides 1500-fold resistance to oseltamivir. Patients usually harbor a mixture of resistant and sensitive virus variants. The impact on viral fitness was tested in the ferret model, which is arguably the best small animal model for influenza. A mixture of mutant (resistant) and wild-type (sensitive) virus was quantitatively followed upon passage in the ferrets. An impact of the H274Y substitution was discussed both for the intra-host fitness (virus replication) and the inter-host fitness (transmissibility) [9] . Expert Rev. Anti Infect. Ther. 9(2), (2011)
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New pathogenic viruses & novel antiviral drugs
Larisa Gubareva (CDC, GA, USA) discussed the monitoring of globally circulating influenza A viruses with respect to drug-resistance genotypes. Oseltamivir resistance due to the H275Y mutation was reported in 72 of 8083 samples (0.9%) of the pandemic H1N1 variants. Adam Meijer (National Institute for Public Health and the Environment, Bilthoven, The Netherlands) reported a similar finding for The Netherlands. A total of 1250 clinical specimens were obtained from different sources during the H1N1 pandemic period. The oseltamivir-resistance mutation H275Y was detected in 19 samples (1.5%). These cases were not epidemiologically linked, indicating the absence of spread of the resistant virus variants in the community. Most of these patients were immune suppressed due to other reasons, which resulted in a persisting viral infection and a longer treatment window. HBV & HCV
Fabien Zoulim (INSERM, France) presented an overview of antivirals against the HBV. In fact, all available small-molecule antivirals represent nucleotide/nucleoside analogs that target the viral polymerase. Lamivudine was the first drug tested and it has shown great efficacy, but after 5 years of treatment approximately 80% of the patients developed resistance and lamivudine resistance was shown to correlate with accelerated progression of liver disease. Multiple new drugs have been developed, including tenofovir and entecavir. Cross-resistance data indicate that there is always at least one treatment option left when resistance develops, but this situation may change in the future, as there are few anti-HBV drugs in the pipeline. It remains particularly important to identify new drug targets, for example the process of HBV entry into the cell, in order to compose a true combinatorial therapy. Xiomara Thomas (University of Amsterdam, The Netherlands) described a clinical trial with the NS3 protease inhibitor narlaprevir in chronic HCV-infected individuals. A 7-day narlaprevir treatment was followed by a 4-week wash-out period and a subsequent narlaprevir plus interferon treatment period. The majority of the patients with a significant viral load at the end of the experimental treatment had acquired resistance mutations (five of seven). Among the many candidate resistance mutations, the R155K mutation was always selected. These resistance markers disappeared rapidly after References 1
Summa V, Petrocchi A, Bonelli F et al. Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV–AIDS infection. J. Med. Chem. 51(18), 5843–5855 (2008).
2
Fransen S, Gupta S, Danovich R et al. Loss of raltegravir susceptibility by human immunodeficiency virus type 1 is conferred via multiple nonoverlapping genetic pathways. J. Virol. 83(22), 11440–11446 (2009).
3
Eggink D, Langedijk JP, Bonvin AM et al. Detailed mechanistic insights into HIV-1 sensitivity to three generations of fusion inhibitors. J. Biol. Chem. 284(39), 26941–26950 (2009).
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treatment was stopped, suggesting that the resistant HCV variants have a considerably reduced viral fitness, such that they are outcompeted by the wild-type virus. Picornaviruses
Lonneke van der Linden (Nijmegen University, The Netherlands) described a cell-based high-throughput screen in a search for antivirals against Picornaviruses. The EVI-14 compound showed broadspectrum activity against coxsackievirus, enterovirus 71 and the cardioviruses mengovirus and scaffoldvirus. The subgenomic replicon system was used to demonstrate that inhibition occurs at the level of RNA replication and not mRNA translation or protein processing. Targeting the host to block virus replication
Teresa Compton (Novartis, MA, USA) introduced the important theme of virus inhibition by blocking a cellular cofactor. The theoretical advantage of targeting a host cofactor is that it would be difficult for the virus to develop resistance. An additional potential advantage is the presence of a universal cofactor that is used by many pathogenic viruses, thus increasing the chance of designing inhibitors with a broad-spectrum antiviral activity. A screen for inhibitors of the respiratory syncytial virus was performed. A library of 1.7 million compounds gave a hit rate of 1%, and additional assays, such as time-of-addition experiments, resulted in the selection of two drug candidates with isoxazole and pyrolidine cores with 2–8-nM antiviral activity. A surprisingly broad antiviral effect was scored against HCV, HIV-1, dengue virus and Yellow fever virus, but little impact was measured for DNA viruses. The host targets were identified as two components of the pyrimidine synthesis pathway, the aspartate transcarbamylase and dihydroorotase and dihydroorotate dehydrogenase enzymes. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
4
Palmer S, Wiegand AP, Maldarelli F et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 41(10), 4531–4536 (2003).
5
Palmer S, Maldarelli F, Wiegand A et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc. Natl Acad. Sci. USA 105(10), 3879–3884 (2008).
6
Vrancken R, Haegeman A, Paeshuyse J et al. Proof of concept for the reduction of classical swine fever infection in pigs by a novel viral polymerase inhibitor. J. Gen. Virol. 90(Pt 6), 1335–1342 (2009).
7
Bodenreider C, Beer D, Keller TH et al. A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease. Anal. Biochem. 395(2), 195–204 (2009).
8
Su XC, Ozawa K, Qi R, Vasudevan SG, Lim SP, Otting G. NMR analysis of the dynamic exchange of the NS2B cofactor between open and closed conformations of the West Nile virus NS2B-NS3 protease. PLoS Negl. Trop. Dis. 3(12), e561 (2009).
9
Hurt AC, Nor’e SS, McCaw JM et al. Assessing the viral fitness of oseltamivirresistant influenza viruses in ferrets, using a competitive-mixtures model. J. Virol. 84(18), 9427–9438 (2010).
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