High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

Balasubramanian, Anuradha; Manzano, Mark; Teramoto, Tadahisa; Pilankatta, Rajendra; Padmanabhan, Radhakrishnan

doi:10.1016/j.antiviral.2016.08.014

Cited by 30 publications

(22 citation statements)

References 60 publications

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“…DENV-2 Protease Inhibitors used for Assay Correlations Chu, et al 16, Tomlinson, et al 17 a different procedures for virus titer reduction assay, for detailed information see 16,18…”

Section: S7mentioning

confidence: 99%

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays

et al. 2020

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Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure–activity relationship study provides a perspective for further drug development against flaviviral infections.

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“…DENV-2 Protease Inhibitors used for Assay Correlations Chu, et al 16, Tomlinson, et al 17 a different procedures for virus titer reduction assay, for detailed information see 16,18…”

Section: S7mentioning

confidence: 99%

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays

et al. 2020

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“…RNA encodes a single polyprotein that is cleaved by viral and host proteases to produce three structural proteins (C, prM and E) and eight nonstructural proteins including the cleavage product of NS4A (NS1, NS2A, NS2B, NS3, NS4A, NS4A-2K, NS4B and NS5) (for a review, (Padmanabhan and Strongin, 2010)). Previous HTS campaign using purified DENV2 NS2B-NS3pro as a target and subsequent cherry picks for validation by secondary assays identified curcumin as a potential hit that inhibited the purified viral protease NS2B-NS3pro (unpublished results; see also (Balasubramanian et al, 2016)). Subsequently, four curcuminoids were synthesized and characterized for their antiviral activity.…”

Section: Introductionmentioning

confidence: 94%

“…Plaques were visually examined after fixing the cells with 4% paraformaldehyde and staining with 1% crystal violet for 30 min. The number of plaque forming units (PFU) per ml was determined (Balasubramanian et al, 2016;Boonyasuppayakorn et al, 2014). The EC50 (PFU) was calculated from two independent experiments.…”

Section: Plaque Assaysmentioning

confidence: 99%

Inhibition of dengue virus by curcuminoids

et al. 2019

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The dengue virus is considered to be a globally important human pathogen prevalent in tropical and subtropical regions of the world. According to a recent estimate, the disease burden due to DENV infections is ~390 million infections per year globally in ~100 countries including the southern US, Puerto Rico and Hawaii, resulting in nearly ~25,000 deaths mostly among children. Despite the significant morbidity and mortality that results from DENV infections, there is currently no effective chemotherapeutic treatment for DENV infections. We identified curcumin as an inhibitor of DENV2 NS2B/NS3protease in a previous high-throughput screening (HTS) campaign. We synthesized four analogues of curcumin (curcuminoids) and tested the in vitro protease activity and inhibition of replication by cell-based assays. The results revealed that curcumin is a weak inhibitor of the viral protease. However, the analogues exhibited more potent inhibition of DENV infectivity in plaque assays suggesting that the cellular pathway(s) required for viral replication and/or assembly are targeted by these compounds. Further analysis shows that inhibition of genes involved in lipid biosynthesis, and of actin polymerization by curcuminoids, are likely to be involved as their mode of action in DENV2-infected cells. Three of the curcumin derivatives possess good selectivity indices (SI) (>10) when compared to the parent curcumin.

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“…Tolcapone, tannic acid and suramin have been reported as hits from a high-throughput screening with K i values significantly below 1 μM [1]. Tannic acid (K i = 0.34 μM) showed also exceptional activity in a viral plaque assay with an EC 50 value of 0.084 μM and only limited cytotoxic effects.…”

Section: Small-molecular Dengue Virus Protease Inhibitorsmentioning

confidence: 99%

Strategies Towards Protease Inhibitors for Emerging Flaviviruses

Nitsche

2018

Advances in Experimental Medicine and Biology

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Infections with flaviviruses are a continuing public health threat. In addition to vaccine development and vector control, the search for antiviral agents that alleviate symptoms in patients are of considerable interest. Among others, the flaviviral protease NS2B-NS3 is a promising drug target to inhibit viral replication. Flaviviral proteases share a high degree of structural similarity and substrate-recognition profile, which may facilitate a strategy towards development of pan-flaviviral protease inhibitors. However, the success of various drug discovery attempts during the last decade has been limited by the nature of the viral enzyme as well as a lack of robust structural templates. Small-molecular, structurally diverse protease inhibitors have been reported to reach affinities in the lower micromolar range. Peptide-based, substrate-derived compounds are often nanomolar inhibitors, however, with highly compromised drug-likeness. With some exceptions, the antiviral cellular activity of most of the reported compounds have been patchy and insufficient for further development. Recent progress has been made in the elucidation of inhibitor binding using different structural methods. This will hopefully lead to more rational attempts for the identification of various lead compounds that may be successful in cellular assays, animal models and ultimately translated to patients.

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High-throughput screening for the identification of small-molecule inhibitors of the flaviviral protease

Cited by 30 publications

References 60 publications

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays

Inhibition of dengue virus by curcuminoids

Strategies Towards Protease Inhibitors for Emerging Flaviviruses

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