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New Approaches to Structure-Based Discovery of Dengue Protease Inhibitors
Abstract and Figures
Dengue virus (DENV), a member of the family Flaviviridae, presents a tremendous threat to global health since an estimated 2.5 billion people worldwide are at risk for epidemic transmission. DENV infections are primarily restricted to sub-tropical and tropical regions; however, there is concern that the virus will spread into new regions including the United States. There are no approved antiviral drugs or vaccines to combat dengue infection, although DENV vaccines have entered Phase 3 clinical trials. Drug discovery and development efforts against DENV and other viral pathogens must overcome specificity, efficacy, safety, and resistance challenges before the shortage of licensed drugs to treat viral infections can be relieved. Current drug discovery methods are largely inefficient and thus relatively ineffective at tackling the growing threat to public health presented by emerging and remerging viral pathogens. This review discusses current and newly implemented structure-based computational efforts to discover antivirals that target the DENV NS3 protease, although it is clear that these computational tools can be applied to most disease targets.
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... Briefly, the first step of replication is the attachment of virus to the specific receptor present on the host cells (Tomlinson et al. 2009;Smit et al. 2011;Smith 2012). The envelope (E) glycoprotein present on DENV membrane acts as ligand that binds to receptors present on the host cell membrane. ...
... The envelope (E) glycoprotein present on DENV membrane acts as ligand that binds to receptors present on the host cell membrane. It initiates the interaction between E-glycoprotein and the host receptor(s) which leads to receptor mediated endocytosis of the virus particle (Tomlinson et al. 2009;Smith et al. 2011) ( Figure 3B). Further acidification of the endocytic vesicle; release the entrapped nucleocapsid to the cytoplasm where the virus genome is released. ...
... Further acidification of the endocytic vesicle; release the entrapped nucleocapsid to the cytoplasm where the virus genome is released. DENV is a positive sense RNA virus with single open reading frame (ORF) thus behave like messenger RNA and directly translates to produce a large single polyprotein which subsequently undergoes post translational modification mediated by viral as well as host proteases to produce seven non structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) which are necessary for virus replication and three structural proteins (capsid, pre membrane and envelope) which are involved in packaging and secretion of the DENV from infected cells (Tomlinson et al. 2009) (Table 1). Replication occurs on intercellular membranes, and assembly take place on the endoplasmic reticulum. ...
... The DENV prM is a 21 kDa protein which is the precursor of the 8 kDa M protein. Host furin proteases cleave the prM protein to separate it into the pr peptide (amino acids 1-91), ectodomain (amino acids , and M protein (amino acids [112][113][114]. The prM protein functions to prevent the conformational changes of the E protein during virus maturation in the low pH environment of the TGN [28]. ...
... This structure makes the C protein an important component in virus assembly as it facilitates encapsulation of the RNA genome for nucleocapsid formation [113]. In addition, the hydrophobic region of the C protein contains a signal sequence which anchors the C protein and partitions the prM protein to the ER membrane [114]. During virus assembly, the NS2B-NS3 protease cleaves off the C-terminal to form a mature protein [117]. ...
Dengue is a major global health threat causing 390 million dengue infections and 25,000 deaths annually. The lack of efficacy of the licensed Dengvaxia vaccine and the absence of a clinically approved antiviral against dengue virus (DENV) drive the urgent demand for the development of novel anti-DENV therapeutics. Various antiviral agents have been developed and investigated for their anti-DENV activities. This review discusses the mechanisms of action employed by various antiviral agents against DENV. The development of host-directed antivirals targeting host receptors and direct-acting antivirals targeting DENV structural and non-structural proteins are reviewed. In addition, the development of antivirals that target different stages during post-infection such as viral replication, viral maturation, and viral assembly are reviewed. Antiviral agents designed based on these molecular mechanisms of action could lead to the discovery and development of novel anti-DENV therapeutics for the treatment of dengue infections. Evaluations of combinations of antiviral drugs with different mechanisms of action could also lead to the development of synergistic drug combinations for the treatment of dengue at any stage of the infection.
... In less severe cases, patients may experience shock hypovolemia, often called dengue shock syndrome, with the level of mortality being 5% to 10% per case. 4 Although most people could recover from this disease within a period of 2 to 3 weeks, there are neither specific treatments nor available drugs for DF disease until now. ...
Dengue fever is still a major threat worldwide, approximately threatening two-fifths of the world’s population in tropical and subtropical countries. Nonstructural protein 5 (NS5) methyltransferase enzyme plays a vital role in the process of messenger RNA capping of dengue by transferring methyl groups from S-adenosyl-l-methionine to N7 atom of the guanine bases of RNA and the RNA ribose group of 2′OH, resulting in S-adenosyl-l-homocysteine (SAH). The modification of SAH compound was screened using molecular docking and molecular dynamics simulation, along with computational ADME-Tox (absorption, distribution, metabolism, excretion, and toxicity) test. The 2 simulations were performed using Molecular Operating Environment (MOE) 2008.10 software, whereas the ADME-Tox test was performed using various software. The modification of SAH compound was done using several functional groups that possess different polarities and properties, resulting in 3460 ligands to be docked. After conducting docking simulation, we earned 3 best ligands (SAH-M331, SAH-M2696, and SAH-M1356) based on ΔGbinding and molecular interactions, which show better results than the standard ligands. Moreover, the results of molecular dynamics simulation show that the best ligands are still able to maintain the active site residue interaction with the binding site until the end of the simulation. After a series of molecular docking and molecular dynamics simulation were performed, we concluded that SAH-M1356 ligand is the most potential SAH-based compound to inhibit NS5 methyltransferase enzyme for treating dengue fever.
... In addition, other characteristics have been reported, including the hyperendemicity of multiple DENV serotypes in several countries and the significant impact on human health and national economies, thus generating a global impact 3 . There is currently no specific therapy against DENV; however, palliative treatments may be used to alleviate symptoms [8][9][10] . ...
Background & objectives: Dengue is one of the most important arboviruses and public health problem associated with increasingly large outbreaks, especially in tropical countries such as Brazil. The state of Minas Gerais, in particular, has had high numbers of cases of this infection in recent years. Methods: Our study evaluated the epidemiological impact of dengue fever in the state of Minas Gerais from the National Health System (Sistema Único de Saúde, SUS) perspective between 2000 to 2015 using the Brazilian Notifiable Diseases Information System (SINAN, notification cases) and Hospital Information System (SIH)/SUS (hospitalization registers) databases. Results: The SUS database recorded 34,996 reports of dengue (International Classification of Diseases [ICD]: A90) as well as 1984 verified cases of severe dengue (ICD-A91). These hospitalizations for dengue and cases of severe dengue generally affected individuals aged 15–24 (17.74%) and 5–14 (20.86%) years, respectively. The epidemiological burden of dengue was substantial in Minas Gerais state, with the highest number of notifications nationally in 2013. Interpretation & conclusion: From retrospective data associated with dengue records, our study sought to better highlight the locations with the largest number of dengue cases in the Minas Gerais state, and contribute to direct educational and surveillance actions of these regions applied to this infection.
... DENV is categorized into four antigenically different serotypes known as DENV1, DENV2, DENV3, and DENV4. Generally, recovery from an infection by a certain serotype does not provide lifelong immunity against the other dengue serotypes (Riverts et al. 2009;Tomlinson et al. 2009). Before 1970, there were only 9 countries badly affected by severe dengue epidemics. ...
Serology-based dengue assays at times produce inaccurate results especially in the early phase of disease onset. A more precise diagnostic approach detecting dengue infections in the early phase enables better management of the disease. This helps reduce dengue-associated morbidity and mortality. Besides, an early diagnosis of dengue is also very beneficial in a dengue outbreak and in endemic regions. In this light, this study aimed to determine the potential of the dengue virus (DENV) non-structural 1 (NS1) gene as an early detection biomarker. The cytopathic effects (CPE) were monitored and the cell death of DENV serotype-2 (DENV2)-infected Vero cells was evaluated for fourteen consecutive days. Only Lemos and in-house NS1-specific primer pairs showed positive amplifications in the preliminary primer validation. Thus, both of the primer pairs were then used to amplify the NS1 gene from the infected cells. The NS1 gene was detected as early as day-2 post-infection using the in-house primers. There was no amplicon produced using the Lemos primers. This is speculated to be attributable to the relatively lower complementarity of the primer sequences with that of the template and low amount of viral mRNA in the DENV2-infected cells. Conclusively, the DENV NS1 gene is a potential early detection marker, however, the NS1-specific primers should be pre-validated to ensure a reliable dengue diagnosis.
... applied to development of dengue antivirals involving important DENV molecular target such as the three structural proteins [e.g., capsid (C), precursor membrane protein (prM) and envelope (E)] and seven nonstructural proteins (e.g., NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) 1 , [14][15][16] . In this scenario, medicinal chemistry projects using rational approaches applied to theoretical methodologies such as homology modeling [17][18] , virtual screening [19][20] , molecular docking [21][22][23][24] , molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) 17,25 have been highlighted. ...
Background & objectives: Dengue is considered one of the greatest public health challenges in the world, especially, for tropical and subtropical countries. Brazil highlights with considerable number of registers associated with this arbovirus, with emphasis on the state of Minas Gerais and its municipalities in regions such as the midwest. We aimed to evaluate the economic and epidemiological impact of dengue in the midwest region of minas Gerais, from SUS perspective.
Methods: Our study evaluated the epidemiological impact of dengue fever in the midwest region of Minas Gerais using data applied to Divinópolis city from SUS perspective between 2000 to 2015 considering SIH/SUS database (hospitalization registers) and 2007 to 2017 using the SINAN database (notification cases). We analyzed the number of notification registers and hospital services, as well as their costs, from a SUS perspective associated with this infection.
Results: There were 26,516 notifications associated with dengue, with 21,953 cases being confirmed by SEMUSA, with a median incidence of 211 cases/100,000 inhabitants of dengue notifications between 2007 and 2017. The southeast and southwest regions in the municipality are highlighted with high number of cases. Additionally, 160 hospitalizations were recorded with 33.79% in individuals between 15 and 34 years old and expenses of USD 53,737.21 for SUS between 2000 and 2015.
Interpretation & conclusion: Divinópolis is the reference (social and economic) municipality of the midwest region in Minas Gerais state. Our study is the first conducted involving a long period of follow up applied to dengue context in this locality, especially, including the costs associated with hospitalization services. We hope to contribute to the discussions regarding the coping strategies of the disease, considering the impact in its different regions, highlight- ing the need for continued efforts and initiatives aimed at combating the vector of this and other arboviruses such as Zika and chikungunya.
... It may also be possible to develop specific inhibitors of the viral protease (within the NS3 protein), which cleaves functional proteins from the viral polyprotein. 54 Finally, it may be possible to develop entry inhibitors that will prevent the virus from entering cells or inhibitors of the 5′ capping process that is required for viral replication. 52 ...
... In addition, other characteristics have been reported, including the hyperendemicity of multiple DENV serotypes in several countries and the significant impact on human health and national economies, thus generating a global impact 3 . There is currently no specific therapy against DENV; however, palliative treatments may be used to alleviate symptoms [8][9][10] . ...
Background & objectives:
Dengue is one of the most important arboviruses and public health problem associated with increasingly large outbreaks, especially in tropical countries such as Brazil. The state of Minas Gerais, in particular, has had high numbers of cases of this infection in recent years.
Methods:
Our study evaluated the epidemiological impact of dengue fever in the state of Minas Gerais from the National Health System (Sistema Único de Saúde, SUS) perspective between 2000 to 2015 using the Brazilian Notifiable Diseases Information System (SINAN, notification cases) and Hospital Information System (SIH)/SUS (hospitalization registers) databases.
Results:
The SUS database recorded 34,996 reports of dengue (International Classification of Diseases [ICD]: A90) as well as 1984 verified cases of severe dengue (ICD-A91). These hospitalizations for dengue and cases of severe dengue generally affected individuals aged 15-24 (17.74%) and 5-14 (20.86%) years, respectively. The epidemiological burden of dengue was substantial in Minas Gerais state, with the highest number of notifications nationally in 2013.
Interpretation & conclusion:
From retrospective data associated with dengue records, our study sought to better highlight the locations with the largest number of dengue cases in the Minas Gerais state, and contribute to direct educational and surveillance actions of these regions applied to this infection.
... Viral proteases are an interesting target for the development of antivirals for DENV [183]. As a viral protease complex, NS2B/NS3 cleaves various sites of the viral polyprotein to allow the conformation of both structural and nonstructural proteins; therefore, the inhibition of NS2B/NS3 leads to a clear interruption of the replicative cycle [184]. ...
Phenolic compounds have been related to multiple biological activities, and the antiviral effect of these compounds has been demonstrated in several viral models of public health concern. In this review, we show the antiviral role of phenolic compounds against dengue virus (DENV), the most widespread arbovirus globally that, after its re-emergence, has caused multiple epidemic outbreaks, especially in the last two years. Twenty phenolic compounds with anti-DENV activity are discussed, including the multiple mechanisms of action, such as those directed against viral particles or viral proteins, host proteins or pathways related to the productive replication viral cycle and the spread of the infection.
Metabolites isolated from marine invertebrates, callipeltin A, crambescidin, ptilomycalin A, celeromycalin, gymnochrome B, gymnochrome D and isogymnochrome D previously shown bioactive on either herpes simplex virus 1 or human immunodeficiency virus, were tested on a new in vitro bioassay using the dengue virus 1. Only gymnochrome D and isogymnochrome D isolated from the living fossil crinoid Gymnocrinus richeri are highly potent dengue antiviral agents.
The proteins of flaviviruses are translated as a single long polyprotein which is co- and posttranslationally processed by both cellular and viral proteinases. We have studied the processing of flavivirus polyproteins in vitro by a viral proteinase located within protein NS3 that cleaves at least three sites within the nonstructural region of the polyprotein, acting primarily autocatalytically. Recombinant polyproteins in which part of the polyprotein is derived from yellow fever virus and part from dengue virus were used. We found that polyproteins containing the yellow fever virus cleavage sites were processed efficiently by the yellow fever virus enzyme, by the dengue virus enzyme, and by various chimeric enzymes. In contrast, dengue virus cleavage sites were cleaved inefficiently by the dengue virus enzyme and not at all by the yellow fever virus enzyme. Studies with chimeric proteinases and with site-directed mutants provided evidence for a direct interaction between the cleavage sites and the proposed substrate-binding pocket of the enzyme. We also found that the efficiency and order of processing could be altered by site-directed mutagenesis of the proposed substrate-binding pocket.
We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.
A system has been developed that can automatically generate three-dimensional atomic coordinates from the constitution of a molecule as expressed by a connection table. The program, CORINA, is applicable to the entire range of organic chemistry. It can also handle structures that are beyond the scope of some other programs, e.g., macrocyclic and polymacrocyclic molecules. Computation times are short and the results compare favorably with data from X-ray crystallography and with those of molecular mechanics calculations.
A method is presented for the rapid calculation of atomic charges in σ-bonded and nonconjugated π-systems. Atoms are characterized by their orbital electronegativities. In the calculation only the connectivities of the atoms are considered. Thus only the topology of a molecule is of importance. Through an iterative procedure partial equalization of orbital electronegativity is obtained. Excellent correlations of the atomic charges with core electron binding energies and with acidity constants are observed. This establishes their value in predicting experimental data.
Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an "induced fit" mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.
Dengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 A resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.
West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.
The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.
The flavivirus envelope protein E undergoes irreversible conformational changes at a mildly acidic pH which are believed to be necessary for membrane fusion in endosomes. In this study we used a combination of chemical cross-linking and sedimentation analysis to show that the envelope proteins of the flavivirus tick-borne encephalitis virus also change their oligomeric structure when exposed to a mildly acidic environment. Under neutral or slightly alkaline conditions, protein E on the surface of native virions exists as a homodimer which can be isolated by solubilization with the nonionic detergent Triton X-100. Solubilization with the same detergent after pretreatment at an acidic pH, however, yielded homotrimers rather than homodimers, suggesting that exposure to an acidic pH had induced a simultaneous weakening of dimeric contacts and a strengthening of trimeric ones. The pH threshold for the dimer-to-trimer transition was found to be 6.5. Because the pH dependence of this transition parallels that of previously observed changes in the conformation and hydrophobicity of protein E and that of virus-induced membrane fusion, it appears likely that the mechanism of fusion with endosomal membranes involves a specific rearrangement of the proteins in the viral envelope. Immature virions in which protein E is associated with the uncleaved precursor (prM) of the membrane protein M did not undergo a low-pH-induced rearrangement. This is consistent with a protective role of protein prM for protein E during intracellular transport of immature virions through acidic compartments of the trans-Golgi network.