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
Graphical abstract
Introduction
Nearly 390 million people globally are at risk of developing the arthropod-borne viral disease dengue.1 Dengue virus (DENV)2 belongs to the genus flavivirus of the Flaviviridae family.3 The genus flavivirus also includes pathogenic viruses like West Nile virus (WNV), Yellow Fever virus (YFV), and Japanese Encephalitis virus (JEV).4 There are four distinct but closely related serotypes of dengue viruses: DENV 1, DENV 2, DENV 3, and DENV 4. Further, each serotype can be sub-classified into genotypes and strains.5 The virus is primarily transmitted by the arthropod vector Aedes aegypti and, to a lesser extent, by Aedes albopictus.6 It is responsible for varying degrees of clinical manifestations in the infected individuals, including mild flu-like symptoms to severe symptoms such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Failing to attend DHF and DSS cases immediately can prove to be fatal.7 Fortunately, less than 1% of dengue patients develop DHF and DSS.8 Based on the incidence of DHF and DSS in the case of dengue infection, the severity of the four serotypes is found to be in the following order (DENV 2 > DENV 1 > DENV 3 > DENV 4). DENV 4 is rarely fatal.9 Infection with one serotype does not necessarily offer cross-immunity to other serotypes. Further, any subsequent infection can develop into severe dengue, commonly referred to as “antibody-dependent enhancement.”10, 11 Over the years, dengue has severely affected populations in the tropical and subtropical regions of the world.12 Some studies investigating the prevalence of dengue have identified 128 countries at risk of dengue13 epidemics, among which Asia alone accounts for 70% of cases. The year 2019 recorded the highest ever reported dengue cases worldwide, with Afghanistan as the new entrant, witnessing dengue infection for the first time. In the year 2020, cases were on the rise in countries like Bangladesh, Brazil, Cook Islands, Ecuador, India, Indonesia, Maldives, Mauritania, Mayotte (Fr), Nepal, Singapore, Sri Lanka, Sudan, Thailand, Timor-Leste, and Yemen.14 On the vaccine front, the current status is not highly encouraging. The currently available DENV vaccine, Dengvaxia®, launched by Sanofi Pasteur in 2015, has only been approved in 20 countries due to concerns about vaccinating seronegative patients. Seropositive and seronegative patients responded differently to the vaccine; the vaccinated seronegative patients were vulnerable to severe dengue contracting the first natural DENV infection. Currently, only individuals in the age group of 9–45 years and with at least one reported previous DENV infection are eligible for vaccination.14, 15 Hence, these limitations aggressively demand a parallel attempt at drug development. As our group has previously published the progress made in the development of DENV protease inhibitors.16 In continuation of our previous work, in this review, along with a discussion on Dengue-COVID 19 co-infection, we have compiled the progress made in the last six years (2015–2020) toward the development of DENV protease inhibitors. We have broadly categorized our discussion into peptide inhibitors, small molecule inhibitors, inhibitors identified through a rational approach, inhibitors identified through modification of previously reported inhibitors, and drug repurposing. A section on patents and information on clinical trials has also been added. Wherever possible, we have discussed the evolution of potent molecules, reported mechanism of action of the inhibitors, and their interactions with the available crystal structures of DENV protease that demonstrated inhibitory activity. Further, the emergence of COVID-19 has stirred fresh concerns in tropical regions due to anticipated threats following a dengue co-infection. While the data on this topic is relatively premature and inconclusive, we have touched upon Dengue-COVID-19 co-infections and outcomes in patients. This has also inspired us to draw two comparisons, one between the substrate-peptide residue preferences of DENV NS2B-NS3 protease (hereafter referred to as DENV NS2B-NS3 pro) and SARS-CoV 3CLpro and the other between various residues lining the sub-pockets of DENV NS2B-NS3 pro and SARS-CoV 3CLpro.
Section snippets
DENV genome and replication cycle
The DENV genome is approximately 11 kb long, single-stranded, positive-sense RNA, encoding a single polyprotein. The genome is processed into three structural proteins, namely, the capsid (C), envelope (E), and membrane (M) proteins and seven nonstructural proteins, i.e., NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 by the host proteases (Furin and signalase) and the two-component viral NS2B-NS3 protease (Figure 1A).17, 18, 19 The DENV NS2B-NS3 pro (serine protease), which belongs to the trypsin
Dengue-COVID 19 co-infection
The first case of Dengue-COVID-19 co-infection was reported by Verduyn et al. in August 2020. This has since been a significant concern in tropical areas where flaviviruses and COVID-19 infections may coexist. Since the patient showed quite severe dengue infection with no history of prior dengue infection, Verduyn et al. hypothesized that Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection might have aggravated the severity of dengue. However, this contention needs further
DENV NS2B-NS3 pro and SARS-CoV 3CLpro substrate-peptide residue preferences
The initial efforts in the search for DENV protease inhibitors were based on the residue sequences at various cleavage sites. Further, this approach was supported by the finding that NS2B-NS3 proteases of the Flavivirus family have a preference for substrates having dibasic residues (Lys or Arg) at the P1 and P2 site, and further residue preference at P1′site was a small amino acid (Gly, Ala, and Ser).29, 30 Further, Li et al. in 2005 performed functional substrate profiling of P1-P4 and P1′-P
Residues lining the sub-pockets of DENV pro and SARS-CoV 3CL pro
As mentioned above, the substrate specificities for both the proteases have been well defined. In this section, the corresponding sub-pocket occupancies of the residues involved have been discussed. Table 2 summarizes the residues lining the corresponding S4, S3, S2, S1, and S1′ pockets of both DENVpro 31 and SARS-CoV-2 3CLPro.46, 47, 48, 49 The overlap of residues within the pockets is due to the common wall between the pockets. It is evident from the comparison that there is no exact overlap
Design and discovery of DENV NS2B-NS3 pro inhibitors
This section summarizes the research on inhibitors reported in the last six years (2015–20) and summarizes the predicted interactions of a few key residues. Most interactions were common among reported peptide inhibitors. Inhibitors other than peptides were also critically examined for their predicted interactions with the proteases. Besides, any common attributes from different chemical structures were also examined. Since we have already compared the substrate-peptide residue preferences and
Conclusion
Despite best efforts by various research groups over the years, none of the DENV protease inhibitors has entered clinical trials. The failure of the only vaccine developed has further increased the demand for the search for a potent drug. The structure and function of DENV protease is well understood. Based on the efforts made by researchers in the industry and academia, it is well understood that aiming only to develop inhibitors with sub-micromolar or nanomolar affinities will not serve the
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors would like to thank the funding agency, DST-SERB, Govt. of India, for providing financial support through their project (EMR /2016/005711) dated 7th August 2017 and Birla Institute of Technology, Mesra, Ranchi, India for providing the necessary infrastructural facilities.
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