Discovery of Dengue Virus NS4B Inhibitors

Abstract

The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC 50 ], 10 to 80 nM) but not DENV-1 and -4 (EC 50 , >20 M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial "hit" (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2-and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions Emerging infectious pathogens represent a major threat to public health. Vaccines and therapeutics are two key countermeasures against these pathogens. Many viruses of the genus Flavivirus within the family Flaviviridae are arthropod-borne human pathogens, among which the four serotypes of dengue virus (DENV) alone cause 390 million human infections each year (1). Several promising DENV vaccines are currently in clinical development (2). The most advanced vaccine (CYD-TDV) exhibited good efficacy against DENV-1, -3, and -4 but weak protection against DENV-2 (3-5). For antiviral development, four compounds have been tested in dengue clinical trials, including balapiravir (a nucleoside inhibitor) (6), celgosivir (a cellular ␣-glucosidase inhibitor) (7), chloroquine (a malaria drug with antiviral and immunomodulatory activities) (8), and prednisolone (a corticosteroid drug) (9). None of them showed any antiviral activity or clinical benefits in dengue patients. Notably, all these compounds were repurposed from existing drugs or compounds previously developed for other viruses. Bona fide inhibitors specifically designed for DENV have never advanced to clinical trials (10).In this paper, we report the identification of a novel class of small-molecule anti-DENV agents, the spiropyrazolopyridones, using phenotypic screening. These inhibitors block DENV replication by targeting nonstructural protein 4B (NS4B), a nonenzymatic transmembrane protein functioning as an essential component of the viral r...

Therapeutic Potential of Spirooxindoles as Antiviral Agents

“…Resistance analysis and direct ligand-protein binding confirmed that variations at amino acid 63 of DENV nonstructural protein 4B (NS4B), a nonenzymatic transmembrane protein functioning as an essential component of the viral replication complex, were responsible for the observed resistance in DENV-2 and -3 (Figure 6B). 72 Thus, it confirms that ( R )- 43 specifically targets DENV NS4B to block viral RNA synthesis or accumulation. Although ( R )- 43 was found to be poorly soluble in aqueous media (11 μ M) and highly lipophilic with a LogP value of 4.9, ( R )- 43 remains a promising starting point for lead optimization efforts in an attempt to improve the solubility while retaining or improving its potency.…”

“…(B) Membrane topology and sequence alignment of the DENV NS4B protein. Reprinted with permission from ref 72. Copyright 2015 American Society for Microbiology Limited.…”

Synthesis of Hexahydrospiro[pyrazolo[3,4‐b]quinoline‐4,1′‐pyrrolo[3,2,1‐ij]quinoline‐2′,5(1H,4′H)‐diones] from 5,6‐dihydro‐4H‐pyrrolo[3,2,1‐ij]quinoline‐1,2‐dione Using Fe3O4@Cu(OH)x as a Nanocatalyst

“…Such structures have prompted cutting‐edge total syntheses, for example, of structurally complex alkaloids (e.g., horsfiline and welwitindolinone A ). Compounds with diverse medicinal interests are other examples containing spiro[3,3′‐oxindoles] moieties: broad‐spectrum anticancer potency , relief of inflammatory pain , antimicrobial activity , inhibitors of the dengue virus , and of the influenza virus . Natural products, too, show medicinal potential, for example, thiopyran‐spirooxindoles isolated from Isatis indigotica have activity against the influenza virus .…”

Escape Mutations in NS4B Render Dengue Virus Insensitive to the Antiviral Activity of the Paracetamol Metabolite AM404

“…All of these compounds inhibit viral RNA accumulation, with EC 50 s ranging from 42 nM for compound 14a to 0.4 to 1.9 M for the other compounds (33). A direct interaction between compound 14a and NS4B has been demonstrated (18); the NS4B-targeting activities of the other compounds have been deduced from the emergence of resistance mutations in NS4B (11,22,32). The pleiotropic effects of AM404 and the relatively high EC 50 will be hurdles for further drug development.…”

“…Several other flavivirus NS4B inhibitors have recently been reported, including SDM25N (11), NITD-618 (22), spiropyrazolopyridone compound 14a (18), and CCG-3394 and CCG-4088 (32). All of these compounds inhibit viral RNA accumulation, with EC 50 s ranging from 42 nM for compound 14a to 0.4 to 1.9 M for the other compounds (33).…”