Ten years of dengue drug discovery: Progress and prospects

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private–public funding from Novartis and the Singapore Economic Development Board. One of NITD’s missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy.     

Competitive inhibitor of cellular α-glucosidases protects mice from lethal dengue virus infection

Dengue virus infection causes diseases in people, ranging from the acute febrile illness dengue fever, to life-threatening dengue hemorrhagic fever/dengue shock syndrome. We previously reported that a host cellular α-glucosidases I and II inhibitor, imino sugar CM-10-18, potently inhibited dengue virus replication in cultured cells, and significantly reduced viremia in dengue virus infected AG129 mice. In this report we show that CM-10-18 also significantly protects mice from death and/or disease progress in two mouse models of lethal dengue virus infection. Our results thus provide a strong support for the development of CM-10-18 or its derivatives as antiviral agents to treat servere dengue virus infections.     

Anthracene-based inhibitors of dengue virus NS2B-NS3 protease

Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four "second series" analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ～2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.     

天然罗汉松二萜体外抗登革病毒活性研究

登革病毒(dengue virus,DENV)是造成热带和亚热带地区登革热和登革出血热季节性大爆发的蚊媒病原体,可导致严重威胁生命的疾病,因此亟需研发DENV疫苗和药物.本研究发现从石栗枝叶中分离的罗汉松型二萜(3α,5β,10α)-13-methoxypodocarpa-8,11,13-triene-3,12-dio...     

Combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.     

Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model

Dengue virus (DENV) infections continue to spread aggressively around the world. Here we demonstrate that celgosivir (6-O-butanoyl castanospermine), strongly inhibits all four DENV serotypes. We show by fluorescence microscopy that the antiviral mechanism of celgosivir, is in part, due to misfolding and accumulation of DENV non-structural protein 1 (NS1) in the endoplasmic reticulum. Moreover, celgosivir modulates the host’s unfolded protein response (UPR) for its antiviral action. Significantly, celgosivir is effective in lethal challenge mouse models that recapitulate primary or secondary antibody-dependent enhanced DENV infection. Celgosivir treated mice showed enhanced survival, reduced viremia and robust immune response, as reflected by serum cytokine analysis. Importantly, survival increased even after treatment was delayed till 2 days post-infection. Together the present study suggests that celgosivir, which has been clinically determined to be safe in humans, may be a valuable candidate for clinical testing in dengue patients.     

Applications of animal models of infectious arthritis in drug discovery: a focus on alphaviral disease

Animal models, which mimic human disease, are invaluable tools for understanding the mechanisms of disease pathogenesis and development of treatment strategies. In particular, animal models play important roles in the area of infectious arthritis. Alphaviruses, including Ross River virus (RRV), o'nyong-nyong virus, chikungunya virus (CHIKV), mayaro virus, Semliki Forest virus and sindbis virus, are globally distributed and cause transient illness characterized by fever, rash, myalgia, arthralgia and arthritis in humans. Severe forms of the disease result in chronic incapacitating arthralgia and arthritis. The mechanisms of how these viruses cause musculoskeletal disease are ill defined. In recent years, the use of a mouse model for RRV-induced disease has assisted in unraveling the pathobiology of infection and in discovering novel drugs to ameliorate disease. RRV as an infection model has the potential to provide key insights into such disease processes, particularly as many viruses, other than alphaviruses, are known to cause infectious arthritides. The emergence and outbreak of CHIKV in many parts of the world has necessitated the need to develop animal models of CHIKV disease. The development of non-human primate models of CHIKV disease has given insights into viral tropism and disease pathogenesis and facilitated the development of new treatment strategies. This review highlights the application of animal models of alphaviral diseases in the fundamental understanding of the mechanisms that contribute to disease and for defining the role that the immune response may have on disease pathogenesis, with the view of providing the foundation for new treatments.     

Vaccines for the prevention of dengue: development update

The dengue viruses (DENV) are mosquito-borne flaviviruses which cause a spectrum of clinical disease known as "dengue," and have emerged and re-emerged as a significant global health problem. It is estimated more than 120 countries currently have endemic DENV transmission, 55% of the world's population is at risk of infection, and there are between 70-500 million infections of which 2.1 million are clinically severe resulting in 21,000 deaths annually. By all estimates the global dengue problem will continue to worsen due to the increasing mobility of the population, ecological changes, and the inability to effectively sustain vector control. There are no licensed antivirals or vaccines to treat or prevent dengue. The development and widespread use of a safe and efficacious dengue vaccine is required to significantly reduce the global dengue burden. In this review the authors discuss dengue vaccines currently in the pre-clinical and clinical development pipeline.     

Patents related to dengue virus infection

This review discusses patents relevant to dengue virus infection. Dengue virus is a mosquito-transmitted flavivirus that usually causes an acute self-limited febrile illness, but may result in more severe life threatening disease. The lack of animal models that replicate features of the human disease has inhibited developing an understanding of the pathophysiology of dengue virus infection and consequently, limited investigation into potential therapeutics. However, there has been considerable progress in vaccine development and recent insights into the identity of the cells infected during the course of dengue virus infection and characterisation of cell-surface receptors used to mediate infectivity, have provided important information with therapeutic implications. There is no treatment for dengue virus infection and no vaccine is yet available, new approaches are urgently needed.     

The human liver-uPA-SCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV

The study of the hepatitis B virus (HBV) and the hepatitis C virus (HCV) has long been hampered by the lack of a suitable small animal model. Both viruses could only be studied in humans or in chimpanzees. Recently, a new chimeric mouse model was developed that was permissive for HBV and HCV infection. In this model, uPA+/+-SCID mice, suffering from a transgene-induced liver disease, are transplanted early after birth with primary human hepatocytes. These human hepatocytes integrate in the parenchyma and progressively repopulate the diseased mouse liver without losing their normal metabolic functions. Successfully transplanted mice can then be infected with HBV and HCV. In this review, we describe the characteristics of this chimeric mouse model in more detail and give an overview of how this model has already contributed to the development of new antiviral compounds for the treatment of viral hepatitis.     

Resistance analysis of an antibody that selectively inhibits dengue virus serotype-1

The four serotypes of dengue virus (DENV) are the causative agents of the most prevalent mosquito-borne viral disease in human. No clinically approved antiviral therapy is currently available. Therapeutic antibodies represent a viable approach for potential treatment of DENV infection. We recently isolated a human monoclonal antibody (HM14c10) that selectively neutralizes DENV serotype 1 (DENV-1), but not serotypes 2, 3, and 4. Here we report the resistance profile of DENV-1 against HM14c10 in cell culture. Escape mutant viruses readily emerged by culturing wild-type DENV-1 in the presence of the HM14c10 antibody. Sequencing of resistant viruses revealed a single T51K substitution in the domain I/II hinge region of the viral envelope protein. Residue T51 is located within the HM14c10 epitope and is highly conserved among various DENV-1 isolates. Recombinant DENV-1 containing the T51K mutation could not be neutralized by HM14c10 in vitro or in vivo. Biochemical assay revealed that the T51K mutation completely abolished the antibody binding to the DENV-1 virion. Collectively, the results demonstrate that a single amino acid change in DENV envelope protein can confer resistance to a potent antibody through abolishing the antibody-virus interaction.     

Dengue virus capsid protein interacts specifically with very low-density lipoproteins

Dengue affects millions of people worldwide. No specific treatment is currently available, in part due to an incomplete understanding of the viral components' interactions with host cellular structures. We tested dengue virus (DENV) capsid protein (C) interaction with low- and very low-density lipoproteins (LDL and VLDL, respectively) using atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR and computational analysis. Data reveal a specific DENV C interaction with VLDL, but not LDL. This binding is potassium-dependent and involves the DENV C N-terminal region, as previously observed for the DENV C-lipid droplets (LDs) interaction. A successful inhibition of DENV C-VLDL binding was achieved with a peptide drug lead. The similarities between LDs and VLDL, and between perilipin 3 (DENV C target on LDs) and ApoE, indicate ApoE as the molecular target on VLDL. We hypothesize that DENV may form lipoviroparticles, which would constitute a novel step on DENV life cycle.From the Clinical EditorUsing atomic force microscopy-based force spectroscopy, dynamic light scattering, NMR, and computational analysis, these authors demonstrate that dengue viral capsid proteins (DENV C) bind to very low density lipoprotein surfaces, but not to LDLs, in a potassium-dependent manner. This observation suggests the formation of lipo-viroparticles, which may be a novel step in its life cycle, and may offer potential therapeutic interventions directed to this step.     

In silico screening of small molecule libraries using the dengue virus envelope E protein has identified compounds with antiviral activity against multiple flaviviruses

The flaviviruses comprise a large group of related viruses, many of which pose a significant global human health threat, most notably the dengue viruses (DENV), West Nile virus (WNV) and yellow fever virus (YFV). Flaviviruses enter host cells via fusion of the viral and cellular membranes, a process mediated by the major viral envelope protein E as it undergoes a low pH induced conformational change in the endosomal compartment of the host cell. This essential entry stage in the flavivirus life cycle provides an attractive target for the development of antiviral agents. We performed an in silico docking screen of the Maybridge chemical database within a previously described ligand binding pocket in the dengue E protein structure that is thought to play a key role in the conformational transitions that lead to membrane fusion. The biological activity of selected compounds identified from this screen revealed low micromolar antiviral potency against dengue virus for two of the compounds. Our results also provide the first evidence that compounds selected to bind to this ligand binding site on the flavivirus E protein abrogate fusion activity. Interestingly, one of these compounds also has antiviral activity against both WNV (kunjin strain) and YFV.     

Sofosbuvir as treatment against dengue?

Dengvaxia^® (CTD‐TDV), the only licensed tetravalent dengue vaccine by Sanofi Pasteur, was made available since 2015. However, administration of CTD‐TDV, in general, has not received the prequalification recommendation from the World Health Organization. Having a universal antidengue agent for treatment will therefore beneficial. Accordingly, the development of nucleoside inhibitors specific to dengue viral polymerase that perturb dengue infection has been studied by many. Alternatively, we have used a marketed anti‐HCV prodrug sofosbuvir to study its in silico and in vitro effects against dengue. As a result, the active metabolite of sofosbuvir (GS‐461203) was predicted to bind to the catalytic motif (Gly‐Asp‐Asp) of dengue viral polymerase with binding affinity of −6.9 kcal/mol. Furthermore, sofosbuvir demonstrated excellent in vitro viral inhibition with an EC₉₀ of 0.4 μm . In addition, this study demonstrated the requirement of specific liver enzymes to activate the prodrug into GS‐461203 to exert its antidengue potential. All in all, sofosbuvir should be subjected to in‐depth studies to provide information of its efficacy toward dengue and its lead potential as DENV polymerase inhibitor in human subjects. In conclusion, we have expended the potential of the clinically available drug sofosbuvir as treatment for dengue.     

Naturally mutated envelope protein domain I of Chinese B dengue virus attenuated human dendritic cell maturation

Dengue virus (DENV) can infect human dendritic cells (DCs), and cause a spectrum of clinical symptoms. Envelope protein of DENV contains three distinct domains, including domain I (DI), domain II (DII) and domain III (DIII), and plays important roles in receptor binding and induction of protective antibodies. Previously, a new DENV-2 type virus (named B strain) with eight gene mutations in DI of the envelope protein was isolated from a dengue hemorrhagic fever patient. BALB/c mice infected with DENV B strain showed more prolonged viremia than mice infected with the New Guinea C (NGC) strain. However, the mechanism of prolonged viremia was not determined. In this study, DI proteins derived from B and NGC strains of DENV were expressed in Rosetta (DE3) host bacteria and purified by affinity chromatography after refolding. A flow cytometry-based binding assay and confocal microscopy indicated that both proteins could bind to human DCs induced from peripheral blood mononuclear cells (PBMCs), but DI of the B strain had a lower affinity than DI of the NGC strain, and viable B virus also show less binding efficiency with DCs. In addition, DI of the NGC strain, but not the B strain, induced IL-12 secretion and phenotypic maturation of DCs, such as up-regulated expression of CD80, CD83, CD86 and HLA-DR. NGC strain could induce more virus specific IgM/IgG. These results suggest that the naturally mutated envelope protein DI of the Chinese B strain of DENV cannot induce DC maturation as high efficiency as that of NGC strain, which may be the partial reason that DENV B strain escapes immune recognition and induce prolonged viremia. The mutated B strain envelope protein is not a good candidate for subunit vaccine target.     

登革病毒及登革热疫苗研究进展

登革病毒是引起人类疾病最重要的虫媒病毒,其感染可导致登革热、登革出血热、登革休克综合征,甚至危及患者生命。近年来,登革病毒的感染地域不断扩大,越来越多的国家和地区出现登革热流行或暴发,给公共卫生事业造成了很大的经济负担,对全球人类健康构成了极大威胁。为了加快安全有效的登革热疫苗的研发,此文就登革病毒结构、流行病学、致病机制、动物模型及疫苗的研究进展进行综述。     

Is minocycline useful for therapy of acute viral encephalitis?

Minocyline is a tetracycline derivative with anti-inflammatory, anti-apoptotic, and anti-oxidant properties. Therapy has proved useful in some experimental models of both noninfectious and infectious neurological diseases and also in clinical trials in humans, including acute traumatic cervical spinal cord injury. In models of viral encephalitis, treatment has shown both beneficial and deleterious effects. In reovirus infection in mice, minocycline delayed the disease, but did not improve either the morbidity or mortality of the disease. In neuroadapted Sindbis virus infection of mice, minocycline prevented disease, but therapy needed to be given before clinical signs were present in most of the animals. In experimental rabies in neonatal mice minocycline aggravated the disease, likely related to anti-inflammatory effects. Minocycline has also been shown to aggravate disease in a mouse model of Huntington disease, in a monkey model of Parkinson disease, and in a mouse model of hypoxic-ischemic brain injury. Hence, there is experimental evidence of benefit of minocycline in both infectious and noninfectious neurological diseases, but there is a lack of benefit and harmful effects in other diseases. This may reflect multiple mechanisms of actions that cannot be predicted in a new disease or in an infection caused by a specific viral agent. Minocycline therapy is a double-edged sword and this drug should not be given empirically to patients with acute viral encephalitis for anticipated neuroprotective effects. Much more work needs to be done in experimental models in animals as well as in clinical trials. Because patient enrollment in clinical trials on acute viral encephalitis has proven to be difficult, funding will be a challenge.     

Dengue Fever in travelers returning from southeast Asia

BACKGROUND: Dengue fever is an important illness facing travelers from nonendemic to endemic countries. METHODS: We reviewed 696 consecutive returned travelers managed at an Australian tertiary-care hospital for an illness acquired overseas. Patients with dengue fever were compared to those with a dengue-like illness, malaria, typhoid fever and rickettsial infections. RESULTS: In total, 19 cases of dengue fever and 20 cases of dengue-like illness were diagnosed, with 85% of cases acquired in Asia. The most common presenting features of dengue were fever (100%), myalgia (79%), rash (74%), headache (68%), nausea (37%), and diarrhea (37%). Compared to those with malaria, typhoid fever and rickettsial infections, patients with dengue were significantly more likely to have myalgia and a temperature <39 degrees C. Compared to all other illnesses in our returned travelers, dengue fever was 18 times more likely if fever and leukopenia were present, 71 times if fever and rash were present, and 230 times if fever, rash and leukopenia were present. All patients with dengue recovered completely. CONCLUSIONS: Dengue fever is an important health problem for travelers not only to Southeast Asia but to all endemic countries. The prevalence appears to be increasing in travelers, and health care professionals need to be familiar with its presentation in travelers. The clinical presenting features provide important guides to establishing the diagnosis.