Discovery of Zika Virus NS2B/NS3 Inhibitors That Prevent Mice from Life-Threatening Infection and Brain Damage.
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ABSTRACT:
SUBMITTER: Coluccia A
PROVIDER: S-EPMC7549096 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zika virus is a mosquito-transmitted flavivirus that causes devastating fetal outcomes in the context of maternal infection during pregnancy. An important target for drugs combatting Zika virus pathogenicity is NS2B-NS3 protease, which plays an essential role in hydrolysis and maturation of the flavivirus polyprotein. We identify hydroxychloroquine, a drug that already has approved uses in pregnancy, as a possible inhibitor of NS2B-NS3 protease by using a Food and Drug Administration-approved drug library, molecular docking, and molecular dynamics simulations. Further, to gain insight into its inhibitory potential toward NS2B-NS3 protease, we performed enzyme kinetic studies, which revealed that hydroxychloroquine inhibits protease activity with an inhibition constant (K i) of 92.34 ± 11.91 μM. Additionally, hydroxychloroquine significantly decreases Zika virus infection in placental cells.
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Project description:The Zika virus presents a major public health concern due to severe fetal neurological disorders associated with infections in pregnant women. In addition to vaccine development, the discovery of selective antiviral drugs is essential to combat future epidemic Zika virus outbreaks. The Zika virus NS2B-NS3 protease, which performs replication-critical cleavages of the viral polyprotein, is a promising drug target. We report the first macrocyclic peptide-based inhibitors of the NS2B-NS3 protease, discovered de novo through in vitro display screening of a genetically reprogrammed library including noncanonical residues. Six compounds were selected, resynthesized, and isolated, all of which displayed affinities in the low nanomolar concentration range. Five compounds showed significant protease inhibition. Two of these were validated as hits with submicromolar inhibition constants and selectivity toward Zika over the related proteases from dengue and West Nile viruses. The compounds were characterized as noncompetitive inhibitors, suggesting allosteric inhibition.
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Project description:Zika virus (ZIKV) serine protease, indispensable for viral polyprotein processing and replication, is composed of the membrane-anchored NS2B polypeptide and the N-terminal domain of the NS3 polypeptide (NS3pro). The C-terminal domain of the NS3 polypeptide (NS3hel) is necessary for helicase activity and contains an ATP-binding site. We discovered that ZIKV NS2B-NS3pro binds single-stranded RNA with a Kd of ~0.3 μM, suggesting a novel function. We tested various structural modifications of NS2B-NS3pro and observed that constructs stabilized in the recently discovered "super-open" conformation do not bind RNA. Likewise, stabilizing NS2B-NS3pro in the "closed" (proteolytically active) conformation using substrate inhibitors abolished RNA binding. We posit that RNA binding occurs when ZIKV NS2B-NS3pro adopts the "open" conformation, which we modeled using highly homologous dengue NS2B-NS3pro crystallized in the open conformation. We identified two positively charged fork-like structures present only in the open conformation of NS3pro. These forks are conserved across Flaviviridae family and could be aligned with the positively charged grove on NS3hel, providing a contiguous binding surface for the negative RNA strand exiting helicase. We propose a "reverse inchworm" model for a tightly intertwined NS2B-NS3 helicase-protease machinery, which suggests that NS2B-NS3pro cycles between open and super-open conformations to bind and release RNA enabling long-range NS3hel processivity. The transition to the closed conformation, likely induced by the substrate, enables the classical protease activity of NS2B-NS3pro.
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Project description: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 ?M 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 ?M. 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.
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Project description:The Zika virus (ZIKV), a member of the Flaviviridae family, is considered a major health threat causing multiple cases of microcephaly in newborns and Guillain-Barré syndrome in adults. In this study, we targeted a transient, deep, and hydrophobic pocket of the "super-open" conformation of ZIKV NS2B-NS3 protease to overcome the limitations of the active site pocket. After virtual docking screening of approximately seven million compounds against the novel allosteric site, we selected the top six candidates and assessed them in enzymatic assays. Six candidates inhibited ZIKV NS2B-NS3 protease proteolytic activity at low micromolar concentrations. These six compounds, targeting the selected protease pocket conserved in ZIKV, serve as unique drug candidates and open new opportunities for possible treatment against several flavivirus infections.
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Project description:Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.
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Project description:Dengue virus (DENV) infection causes serious health problems in humans for which no drug is currently available. Recently, DENV NS2B-NS3 protease has been proposed as a primary target for anti-dengue drug discovery due to its important role in new virus particle formation by conducting DENV polyprotein cleavage. Triterpenoids from the medicinal fungus Ganoderma lucidum have been suggested as pharmacologically bioactive compounds and tested as anti-viral agents against various viral pathogens including human immunodeficiency virus. However, no reports are available concerning the anti-viral activity of triterpenoids from Ganoderma lucidum against DENV. Therefore, we employed a virtual screening approach to predict the functional triterpenoids from Ganoderma lucidum as potential inhibitors of DENV NS2B-NS3 protease, followed by an in vitro assay. From in silico analysis of twenty-two triterpenoids of Ganoderma lucidum, four triterpenoids, viz. Ganodermanontriol (-6.291 kcal/mol), Lucidumol A (-5.993 kcal/mol), Ganoderic acid C2 (-5.948 kcal/mol) and Ganosporeric acid A (-5.983 kcal/mol) were predicted to be viral protease inhibitors by comparison to reference inhibitor 1,8-Dihydroxy-4,5-dinitroanthraquinone (-5.377 kcal/mol). These results were further studied for binding affinity and stability using the molecular mechanics/generalized Born surface area method and Molecular Dynamics simulations, respectively. Also, in vitro viral infection inhibition suggested that Ganodermanontriol is a potent bioactive triterpenoid.
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Project description:The pivotal role of viral proteases in virus replication has already been successfully exploited in several antiviral drug design campaigns. However, no efficient antivirals are currently available against flaviviral infections. In this study, we present lead-like small molecule inhibitors of the Zika Virus (ZIKV) NS2B-NS3 protease. Since only few nonpeptide competitive ligands are known, we take advantage of the high structural similarity with the West Nile Virus (WNV) NS2B-NS3 protease. A comparative modeling approach involving our in-house software PyRod was employed to systematically analyze the binding sites and develop molecular dynamics-based 3D pharmacophores for virtual screening. The identified compounds were biochemically characterized revealing low micromolar affinity for both ZIKV and WNV proteases. Their lead-like properties together with rationalized binding modes represent valuable starting points for future lead optimization. Since the NS2B-NS3 protease is highly conserved among flaviviruses, these compounds may also drive the development of pan-flaviviral antiviral drugs.
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Project description:The Zika virus (ZIKV) protease is an attractive drug target for the design of novel inhibitors to control the ZIKV infection. As the protease substrate-binding site contains acidic residues, inhibitors with basic residues can be beneficial for the inhibition of protease activities. Molecular dynamics (MD) simulation and molecular mechanics with generalized Born and surface area solvation (MM/GBSA) techniques are employed herein to design potent peptide inhibitors and to understand the nature of the basic residues that can potentially stabilize the acidic residues of the protease substrate-binding site. It is found that the inclusion of K, R, and K at P1, P2, and P3 positions, respectively, and Y at the P4 position (YKRK) would generate a highly stable tetrapeptide-protease complex with a ΔGbind of ~ -80 kcal/mol. We have also shown that the C-terminal extension of this and the second most stable tetrapeptide (YRRR) with small polar residues, such as S and T would generate even more stable hexapeptide-protease complexes. The modes of interactions of these inhibitors are discussed in detail, which are in agreement with earlier experimental studies. Thus, this study is expected to aid in the design of novel antiviral drugs against the ZIKV.
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