Antiviral Research and Development Against Dengue Virus Bruno Canard, PhD. [email protected] 1 Table of Contents Part 1. Antivirals 3 A short historical view on antiviral research and therapies 3 Lessons learned from recent viral diseases and pandemies 3 The methods used to discover antivirals 4 Infected cell assays 5 Knowledge-based methods 5 The source of anti-infectious molecules 6 Why has natural product screening been neglected in antiviral research ? 8 Challenges associated with natural products in antiviral research 8 What is a validated antiviral target ? 9 Animal models 9 Patient cohorts and clinical trials 10 Frequent arguments about antiviral therapy feasibility 10 The introduction of dengue as a druggable disease 11 Diagnostics, and what does it tells us for antiviral therapy ? 11 Current treatment 11 Part 2. Dengue 13 Preamble 13 The Dengue Virus 13 The DENV targets for antiviral research 13 Overview of genome organisation 14 Overview of the DV particle and DV proteins as targets for drugs 14 The structural proteins 14 The Non-Structural proteins 15 RNA structures 17 The dengue validated targets 17 The cellular targets for antiviral research against dengue 18 siRNAs as tools and/or therapeutic agents 19 Response modifiers 20 Monoclonal antibodies 21 Mechanical devices 21 Part 3. Academic and academy-associated research centers 22 Part 4. The current industrial network of AV discovery 31 Part 5. Mapping the dengue drug design effort and needs 38 Annex 1. References 42 Annex 2. Patents 43 2 Part 1. Antivirals A short historical view on antiviral research and therapies The first significant successes of anti-infectious disease treatments originated from the discovery and use of antibiotics. The discovery of many viruses preceded largely the discovery of the first antiviral molecule, which occurred at least 40 years after that of penicillin in 1928. The first documented description of an antiviral molecule, that of 5-iodo-2'-desoxyuridine, occurred in 1959. It was discovered active against Herpes ophthalmologic infections and followed by a series of related active molecules. The fight against herpes was the perhaps the earliest and most significant driving force of antiviral research. Herpes was the only significant viral disease for which all technical elements and systems required to develop an antiviral molecule first became available (i.e., in vitro infected cell systems, animal models, chronically infected patients,…). The antiviral drug field came of age in the next decades with the first antiviral molecule finding its way to the clinic: Gertrude B. Elion discovered acyclovir(2) a scientific breakthrough for which she was later awarded the Nobel prize in 1988. The subsequent emergence of AIDS in 1981, and the following pandemics drastically changed the field of antiviral research, allowing the widening of concepts, technical developments, rules, and business. Lessons learned from recent viral diseases and pandemies HIV and HCV: chronic invaders The most important lesson comes from the following great achievement: it is possible to control a chronic infection of a very sophisticated virus, such as HIV, that hides inside the chromosomes of the infected cell. Although the victory is not total yet, it has profoundly changed the fate of the pandemic victims, at least in western countries. After being inspired by other research fields, anti-HIV research has “infected” other field of antiviral research and will continue to do so. Remarkably, after the identification of HIV, the control of HIV through antiretrovirals originated from a collective effort on a wide variety of scientific and medical fields, including efficient transfer from academia to the corporate world. More recently, hepatitis C virus (HCV) research is now boosting the antiviral chemotherapy field. Viral polymerases and proteases are targets par excellence, validated by the use of inhibitors against HIV reverse transcriptase and protease, hepatitis B polymerase, and herpes virus polymerase. Anti-HCV protease and polymerase inhibitors are in various stages of clinical trials. Novel targets and cognate inhIbitors are adding to the list, such as the HIV integrase, and the HCV NS5A. HCV (genus Hepacivirus) and DENV (genus Flavivirus) belong to the same viral family Flaviviridae sharing similar genome organization and replication strategies. Initially, research conducted on dengue virus (DENV) was the actual starting and inspiration point for HCV research, when it became known 3 that HCV had a flavivirus-like genome. Presently and conversely, knowledge and strategies gained from the successful drug discovery and design process against HCV can now be translated back to the DENV research field. SARS and Influenza (H5N1 and H1N1): “hit and run” viruses The SARS pandemic was due to a novel coronavirus which emerged in 2003 from China. The virus took the world by surprise as coronaviruses were not known to cause life threatening pathologies. Coronaviruses were clearly neglected viruses from the scientific and the medical/veterinary point-of- view. The pandemic revealed blatantly our unpreparedness to such a problem: point-of-care in hospitals crowded with contagious patients, high toll for clinicians, tracing secondary contacts of taxi drivers and plane passengers, etc…The pessimistic say that nowadays viruses travel around the world in 3 days. The optimistic say that social networks and cell phones make information travel much faster. Perhaps the true challenge is elsewhere: making people believe and adhere to an “official” information, as exemplified with the recent H1N1 crisis and the unsuccessful vaccination campaign. In any case, this crisis has been the best advocate for antivirals as a complementary strategy to prEvention and vaccination. In the case of influenza, the size of the market has been the main booster of anti-influenza drug development. This includes the availability of patients for clinical trials, and the fact that a potential devastating pandemic would undoubtedly provoke stockpiling of antivirals in the time-window into which an appropriate vaccine would available. Advice to stockpile anti-influenza drugs has been recurrently advertised, mostly after 1995 when the 1918 spanish influenza strain genome was published(8). well before the H5N1 and the H1N1 fear hit the world. These two viruses do not produce chronic infections. These types of virus produce an infection (unnoticed, mild, or acute) which resolves with virus clearance. This transient nature of the infection has long been a problem to design an efficient therapeutic answer. Indeed, there are too many unpredictable parameters to build a drug-design program based on traditional planning and funding approaches. The two biggest problems are that it is impossible to evaluate precisely the market (and invest accordingly), and that there is an unpredictable number of patients available for clinical trials. The instructive aspect of these pandemics, however, is that they greatly contributed to re-shape antiviral research at large (how can we anticipate? how money is going to be invested? ). These recent crises have shaped considerably the grand public opinion towards the necessity to have broad- spectrum anti-influenza drugs ready. The methods used to discover antivirals The original method of discovery of antivirals was partially a knowledge-based method, centered around nucleobases and nucleosides (eg., uridine derivatives mentioned above against Herpes), known to be used by viruses for their replication. The advent of AIDS and the discovery of non-nucleoside 4 reverse transcriptase inhibitors opened the era of large-scale screening, which is entirely a trial-and- error procedure, not based on previous knowledge. Millions of compounds are tested as fast as possible (using high throughput screening (HTS) techniques), and only those showing activity are selected. Infected cell assays In both cases (Herpes and AIDS), infected cell cultures provided the antiviral read out, before purified targets were available and could be used. In these assays, compounds are tested individually to see if they either cure an infected cell, or protect it from infection, pathogenic effects. The process is simple, and relies on a cell culture system able to support virus growth. Not surprisingly, the discovery of antivirals parallels the establishment of a robust infected cell based assay. When this was difficult or even not possible (eg., HCV), the use of sub-genomic replicons or surrogate viruses has nevertheless allowed drug discovery and design. There are now a wide variety of assay systems specific for each virus. Robust dengue infected cell assays are available, highly efficient in terms of characterizing the potency of a drug candidate. One significant disadvantage is the cost associated with cell culture reagents and facilities, especially in low income countries. However, this method has an impressive record of success compared to other methods. Knowledge-based methods The general trend is to reduce this trial-and-error approach and inject knowledge as much as possible in the selection process so as to reduce costs and increase efficiency. Computer-aided structure activity relationship (SAR) studies facilitate a responsive and efficient management of research results and programs. Drug-resistance must be considered as part of the drug- design process, as drug resistance mechanisms are being increasingly characterized and drug combinations optimized, in order to avoid or delay resistance.