3D-QSAR and molecular docking studies of peptide-hybrids as dengue virus NS2B/NS3 protease inhibitors.

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R1-R2-Lys-R3-NH2) by the 3D-QSAR technique. Comparative molecular field analysis (q2 = 0.613, r2 = 0.938, r2pred = 0.820) and comparative molecular similarity indices analysis (q2 = 0.640, r2 = 0.928, r2pred = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R1 position, minor, electropositive, H-bond donor groups at the R2 position, and bulky, hydrophobic groups at the R3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease.

Investigating Lycotoxin-An1a (An1a), a defense antiviral peptide from Alopecosa nagpag venom as prospective anti-dengue agent against DENV-2 NS2B-NS3 protease.

Dengue fever is a global health concern with no effective therapy. Screening synthetic chemicals, animal-originated compounds, and phytocompounds against Dengue virus (DENV) targets has failed to find dengue antivirals. The current study examines animal drugs as antagonists against NS2B-NS3Pro, one of DENV's most promising therapeutic targets for dengue fever. Antiviral-Lycotoxin-An1a (An1a), a defence antiviral peptide isolated from the venom of Alopecosa nagpag, a toxic spider. Based on prior in vitro research, it was discovered that the venom peptide suppresses the action of DENV-2 NS2B-NS3Pro. An1a peptide with NS2B-NS3Pro wild type (WT) and two mutants (H51N and S135A) was tested for anti-dengue characteristics using in silico analysis. The WT NS2B-NS3Pro has a catalytic triad of His51, Asp75, and Ser135 in the active site, but the mutants have N51 instead of His51 and Ala135 instead of Ser135. The dynamic sites of the three proteases (WT, H51N, S135A) and the peptide toxin (An1a) were taken into account to achieve molecular docking of An1a with WT NS2B-NS3Pro in conjunction with H51N and S135A. Cluspro-2 performs rigid-flexible docking to predict peptide binding affinity, effectiveness, and inhibitory consistency. Since the ligand had a higher binding affinity, docking score, and molecular interaction network, MD simulations and MM-GBSA free energy calculations were used to investigate the stability of the three protein-peptide complexes. The computer-aided screening and manufacture of spider venom-based anti-dengue medicines yielded intriguing results in the preliminary studies. This study is significant in defining the ideal therapeutic candidate against dengue infections.

Lambda-free light chain: A serum marker of dengue disease via NS3 protease-mediated antibody cleavage.

Dengue poses a significant global public health threat, with diverse clinical manifestations due to complex interactions between the host and the pathogen. Recent reports have highlighted elevated serum-free light chain (FLC) levels in viral infectious diseases. Hence, our study aimed to investigate serum FLC levels in dengue patients. The findings revealed elevated serum λ FLCs, which were associated with the severity of dengue. Receiver operating characteristic curve (ROC) analysis demonstrated that λ FLCs may serve as a serum marker for identifying dengue disease (AUC: 0.7825, sensitivity: 80, specificity: 71.43) and classifying severe dengue (AUC: 0.8102, sensitivity: 75, specificity: 79.52). The viral protease, Dengue virus (DENV) nonstructural protein 3 (NS3), acts as a protease that cleaves viral polyproteins as well as host substrates. Therefore, we proposed that antibodies might be potential targets of NS3 protease, leading to an increase in FLCs. LC/MS-MS analysis confirmed that λ FLCs were the predominant products after antibody degradation by NS3 protease. Additionally, purified NS3 protease cleaved both human IgG and DENV2-neutralizing antibodies, resulting in the presence of λ FLCs. Moreover, NS3 protease administration in vitro led to a reduction in the neutralizing efficacy of DENV2-neutralizing antibodies. In summary, the elevated serum λ FLC levels effectively differentiate dengue patients from healthy individuals and identify severe dengue. Furthermore, the elevation of serum λ FLCs is, at least in part, mediated through NS3 protease-mediated antibody cleavage. These findings provide new insights for developing diagnostic tools and understanding the pathogenesis of DENV infection.

Discovery and structural optimization of a new series of N-acyl-2-aminobenzothiazole as inhibitors of Zika virus.

Zika virus infection is associated to severe diseases such as congenital microcephaly and Zika fever causing serious harm to humans and special concern to health systems in low-income countries. Currently, there are no approved drugs against the virus, and the development of anti-Zika virus drugs is thus urgent. The present investigation describes the discovery and hit expansion of a N-acyl-2-aminobenzothiazole series of compounds against Zika virus replication. A structure-activity relationship study was obtained with the synthesis and evaluation of anti-Zika virus activity and cytotoxicity on Vero cells of nineteen derivatives. The three optimized compounds were 2.2-fold more potent than the initial hit and 20.9, 7.7 and 6.4-fold more selective. Subsequent phenotypic and biochemical assays were performed to evidence whether non-structural proteins, such as the complex NS2B-NS3pro, are related to the mechanism of action of the most active compounds.

Characterization of pH-induced conformational changes in recombinant DENV NS2B-NS3pro.

The increasing frequency of Dengue is a cause of severe epidemics and therefore demands strategies for effective prevention, diagnosis, and treatment. DENV-protease is being investigated as a potential therapeutic target. However, due to the flat and highly charged active site of the DENV-protease, designing orthosteric medicines is very difficult. In this study, we have done a thorough analysis of pH-dependent conformational changes in recombinantly expressed DENV protease using various spectroscopic techniques. Our spectroscopic study of DENV protease (NS2B-NS3pro) at different pH conditions gives important insights into the dynamicity of structural conformation. At physiological pH, the DENV-protease exists in a random-coiled state. Lowering the pH promotes the formation of alpha-helical and beta-sheet structures i.e. gain of secondary structure as shown by Far-UV CD. The light scattering and Thioflavin T (ThT)-binding assay proved the aggregation-prone tendency of DENV-protease at pH 4.0. Further, the confocal microscopy image intensity showed the amorphous aggregate formation of DENV protease at pH 4.0. Thus, the DENV protease acquires different conformations with changes in pH conditions. Together, these results have the potential to facilitate the design of a conformation destabilizer-based therapeutic strategy for dengue fever.

Assessing the potential of NS2B/NS3 protease inhibitors biomarker in curbing dengue virus infections: In silico vs. In vitro approach.

An increase in the occurrence of viral infectious diseases is a global concern for human health. According to a WHO report, dengue virus (DENV) is one of the most common viral diseases affecting approximately 400 million people annually, with worsening symptoms in nearly 1% of cases. Both academic and industrial researchers have conducted numerous studies on viral epidemiology, virus structure and function, source and route of infection, treatment targets, vaccines, and drugs. The development of CYD-TDV or Dengvaxia® vaccine has been a major milestone in dengue treatment. However, evidence has shown that vaccines have some drawbacks and limitations. Therefore, researchers are developing dengue antivirals to curb infections. DENV NS2B/NS3 protease is a DENV enzyme essential for replication and virus assembly, making it an interesting antiviral target. For faster hit and lead recognition of DENV targets, methods to screen large number of molecules at lower costs are essential. Similarly, an integrated and multidisciplinary approach involving in silico screening and confirmation of biological activity is required. In this review, we discuss recent strategies for searching for novel DENV NS2B/NS3 protease inhibitors from the in silico and in vitro perspectives, either by applying one of the approaches or by integrating both. Therefore, we hope that our review will encourage researchers to integrate the best strategies and encourage further developments in this area.

Riboflavin, a Potent Neuroprotective Vitamin: Focus on Flavivirus and Alphavirus Proteases.

Several neurotropic viruses are members of the flavivirus and alphavirus families. Infections caused by these viruses may cause long-term neurological sequelae in humans. The continuous emergence of infections caused by viruses around the world, such as the chikungunya virus (CHIKV) (Alphavirus genus), the zika virus (ZIKV) and the yellow fever virus (YFV) (both of the Flavivirus genus), warrants the development of new strategies to combat them. Our study demonstrates the inhibitory potential of the water-soluble vitamin riboflavin against NS2B/NS3pro of ZIKV and YFV and nsP2pro of CHIKV. Riboflavin presents a competitive inhibition mode with IC50 values in the medium µM range of 79.4 ± 5.0 µM for ZIKV NS2B/NS3pro and 45.7 ± 2.9 µM for YFV NS2B/NS3pro. Against CHIKV nsP2pro, the vitamin showed a very strong effect (93 ± 5.7 nM). The determined dissociation constants (KD) are significantly below the threshold value of 30 µM. The ligand binding increases the thermal stability between 4 °C and 8 °C. Unexpectedly, riboflavin showed inhibiting activity against another viral protein; the molecule was also able to inhibit the viral entry of CHIKV. Molecular dynamics simulations indicated great stability of riboflavin in the protease active site, which validates the repurposing of riboflavin as a promising molecule in drug development against the viruses presented here.

A Computational Approach Applied to the Study of Potential Allosteric Inhibitors Protease NS2B/NS3 from Dengue Virus.

Dengue virus (DENV) is a danger to more than 400 million people in the world, and there is no specific treatment. Thus, there is an urgent need to develop an effective method to combat this pathology. NS2B/NS3 protease is an important biological target due it being necessary for viral replication and the fact that it promotes the spread of the infection. Thus, this study aimed to design DENV NS2B/NS3pro allosteric inhibitors from a matrix compound. The search was conducted using the Swiss Similarity tool. The compounds were subjected to molecular docking calculations, molecular dynamics simulations (MD) and free energy calculations. The molecular docking results showed that two compounds, ZINC000001680989 and ZINC000001679427, were promising and performed important hydrogen interactions with the Asn152, Leu149 and Ala164 residues, showing the same interactions obtained in the literature. In the MD, the results indicated that five residues, Lys74, Leu76, Asn152, Leu149 and Ala166, contribute to the stability of the ligand at the allosteric site for all of the simulated systems. Hydrophobic, electrostatic and van der Waals interactions had significant effects on binding affinity. Physicochemical properties, lipophilicity, water solubility, pharmacokinetics, druglikeness and medicinal chemistry were evaluated for four compounds that were more promising, showed negative indices for the potential penetration of the Blood Brain Barrier and expressed high human intestinal absorption, indicating a low risk of central nervous system depression or drowsiness as the the side effects. The compound ZINC000006694490 exhibited an alert with a plausible level of toxicity for the purine base chemical moiety, indicating hepatotoxicity and chromosome damage in vivo in mouse, rat and human organisms. All of the compounds selected in this study showed a synthetic accessibility (SA) score lower than 4, suggesting the ease of new syntheses. The results corroborate with other studies in the literature, and the computational approach used here can contribute to the discovery of new and potent anti-dengue agents.

Updates on Dengue Vaccine and Antiviral: Where Are We Heading?

Approximately 100-400 million people from more than 100 countries in the tropical and subtropical world are affected by dengue infections. Recent scientific breakthroughs have brought new insights into novel strategies for the production of dengue antivirals and vaccines. The search for specific dengue inhibitors is expanding, and the mechanisms for evaluating the efficacy of novel drugs are currently established, allowing for expedited translation into human trials. Furthermore, in the aftermath of the only FDA-approved vaccine, Dengvaxia, a safer and more effective dengue vaccine candidate is making its way through the clinical trials. Until an effective antiviral therapy and licensed vaccine are available, disease monitoring and vector population control will be the mainstays of dengue prevention. In this article, we highlighted recent advances made in the perspectives of efforts made recently, in dengue vaccine development and dengue antiviral drug.

A short survey of dengue protease inhibitor development in the past 6 years (2015-2020) with an emphasis on similarities between DENV and SARS-CoV-2 proteases.

Dengue remains a disease of significant concern, responsible for nearly half of all arthropod-borne disease cases across the globe. Due to the lack of potent and targeted therapeutics, palliative treatment and the adoption of preventive measures remain the only available options. Compounding the problem further, the failure of the only dengue vaccine, Dengvaxia®, also delivered a significant blow to any hopes for the treatment of dengue fever. However, the success of Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV) protease inhibitors in the past have continued to encourage researchers to investigate other viral protease targets. Dengue virus (DENV) NS2B-NS3 protease is an attractive target partly due to its role in polyprotein processing and also for being the most conserved domain in the viral genome. During the early days of the COVID-19 pandemic, a few cases of Dengue-COVID 19 co-infection were reported. In this review, we compared the substrate-peptide residue preferences and the residues lining the sub-pockets of the proteases of these two viruses and analyzed the significance of this similarity. Also, we attempted to abridge the developments in anti-dengue drug discovery in the last six years (2015-2020), focusing on critical discoveries that influenced the research.

Promising Natural Compounds against Flavivirus Proteases: Citrus Flavonoids Hesperetin and Hesperidin.

Ubiquitous in citrus plants, Hesperidin and Hesperetin flavanones possess several biological functions, including antiviral activity. Arbovirus infections pose an ever-increasing threat to global healthcare systems. Among the severe arboviral infections currently known are those caused by members of the Flavivirus genus, for example, Dengue Virus-DENV, Yellow Fever Virus-YFV, and West Nile Virus-WNV. In this study, we characterize the inhibitory effect of Hesperidin and Hesperetin against DENV2, YFV, and WNV NS2B/NS3 proteases. We report the noncompetitive inhibition of the NS2B/NS3pro by the two bioflavonoids with half maximal inhibitory concentration (IC50) values <5 µM for HST and <70 µM for HSD. The determined dissociation constants (KD) of both flavonoids is significantly below the threshold value of 30 µM. Our findings demonstrate that a new generation of anti-flavivirus drugs could be developed based on selective optimization of both molecules.

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3pro, we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3pro. Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC50 ∼ 500 µM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3pro. To identify the matching protein binding site, chemical shift perturbation studies employing 1H,15N-TROSY-HSQC experiments with uniformly 2H,15N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3pro inhibitors.

Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus.

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain-Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activity (IC50) and binding affinity (KD) of ∼5-10 µM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 µM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a "pre-open conformation", a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.

Development and utility of an in vitro, fluorescence-based assay for the discovery of novel compounds against dengue 2 viral protease.

BACKGROUND: Dengue disease is one of the most significant vector-borne illnesses in the world. The emergence and re-emergence of dengue infections in many parts of the world affect millions annually and continue to burden public health systems especially in low-income populations. Advances in dengue vaccine development showed promising results; however, protection seems to be suboptimal. There is no licensed chemotherapeutic agent against dengue to date. An ideal scenario of combinatorial vaccination of high-risk individuals and chemotherapy of the diseased during outbreaks may compensate for the meager protection offered by the vaccine. The dengue virus protease is important to viral replication and, as such, has been identified as a potential target for antivirals. It is, therefore, our objective to establish and optimize an appropriate screening method for use during the early stages of drug development for dengue. METHODS: In this study, we developed and optimized a biochemical assay system for use in screening compound libraries against dengue virus protease. We tested the selected protease inhibitors with a cell-based assay to determine inhibition of viral replication. RESULTS: We have presented direct plots of substrate kinetics data showing an apparent inhibition of the protease at excessive substrate concentrations. The most common sources of interference that may have affected the said observation were elucidated. Finally, a screen was done on an existing compound library using the developed method. The compounds selected in this study showed inhibitory activity against both the recombinant dengue protease and cell-based infectivity assays. CONCLUSIONS: Our study shows the practicality of a customized biochemical assay to find possible inhibitors of dengue viral protease during the initial stages of drug discovery.