Allosteric Inhibition of the NS2B-NS3 Protease from Dengue Virus

Abstract

Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

“…As virus-encoded proteases have been shown to be essential for the replication and infectivity of many viruses, consequently they become important targets for design of anti-viral drugs [42,43]. Indeed, drugs have been successfully developed to treat HIV and HCV infections by targeting their proteases [44,45]. The Dengue NS3 protease has its catalytic machinery hosted by a chymotrypsin fold, which has been extensively shared by a variety of proteases.…”

“…It has been proposed that the flat and charged nature of its active site may at least partly contribute to the current failure in developing effective inhibitors [7,45]. To overcome these difficulties, alternative strategies are requested to targets sites other than the active sites of the viral proteases, such as to inhibit the dimerization required for activity [46], to trigger allosteric inhibition [45], or even to block the folding of the protease [54]. In this regard, our present study successfully implies potential strategies to perturb the NS2B-NS3pro interface for future developing effective and specific inhibitors for the dengue protease.…”

Policresulen, a novel NS2B/NS3 protease inhibitor, effectively inhibits the replication of DENV2 virus in BHK-21 cells

“…For example, one cyclopentapeptide was proposed as an inhibitor of the DENV2 NS2B/NS3 protease [21] , and MrIA, a type of conotoxin, was also determined to be an inhibitor for this protease [22] . Recently, varied types of potential inhibitors against the DENV2 NS2B/NS3 protease have been reported [11,[23][24][25][26][27][28] , but few inhibitors exhibited antiviral activities. Therefore, it is urgent to discover novel NS2B/NS3 protease inhibitors with high activities against dengue virus.…”

Reaching beyond HIV/HCV: nelfinavir as a potential starting point for broad-spectrum protease inhibitors against dengue and chikungunya virus

“…(55). ; C. one of the promising NS2B-NS3 inhibitors reported by Yildiz et al (57). ; C. one of the promising NS2B-NS3 inhibitors reported by Yildiz et al (57).…”

“…Antiviral activity of HIV/HCV protease inhibitors against DENV and CHIKV Table 3. ; C. one of the promising NS2B-NS3 inhibitors reported by Yildiz et al (57). (55).…”