We performed a focused siRNA screen in an A549 dengue type 2 New Guinea C subgenomic replicon cell line (Rluc-replicon) that contains a Renilla luciferase cassette. We found that siRNA mediated knock down of mevalonate diphospho decarboxylase (MVD) inhibited viral replication of the Rluc-replicon and DEN-2 NGC live virus replication in A549 cells. When the Rluc-replicon A459 cells were grown in delipidated media the replicon expression was suppressed and MVD knock down could further sensitize Renilla expression. Hymeglusin and zaragozic acid A could inhibit DEN-2 NGC live virus replication in K562 cells, while lovastatin could inhibit DEN-2 NGC live virus replication in human peripheral blood mononuclear cells. Renilla expression could be rescued in fluvastatin treated A549 Rluc-replicon cells after the addition of mevalonate, and partially restored with geranylgeranyl pyrophosphate, or farnesyl pyrophosphate. Our data suggest genetic and pharmacological modulation of cholesterol biosynthesis can regulate dengue virus replication.

Free radical-induced lipid peroxidation has been implicated in a number of human diseases including atherosclerosis, cancer, and neurodegenerative diseases. F2-Isoprostanes (IsoPs) are isomers of prostaglandin PGF2α that are generated in vivo from the free radical-initiated peroxidation of arachidonic acid independent of cyclooxygenase enzymes. Since the discovery of the IsoPs in the early 1990s, a large body of evidence has been accumulated to indicate that quantification of these F2-IsoPs represents the most reliable biomarker to assess oxidative stress in vivo. A variety of analytical approaches have been developed for the quantification of these novel compounds; these methods include mass spectrometry (MS) detection coupled to gas chromatography (GC) or liquid chromatography (LC) separation, and detection using immunological approaches. This article summarizes our current methodology to quantify F2-IsoPs in biological fluids and tissues using GC-MS. This method includes solid phase extraction (SPE), thin layer chromatography (TLC) purification, chemical derivatization, and MS detection using negative ion chemical ionization (NICI) coupled with GC. The protocol described herein has been optimized and validated to provide the best sensitivity and selectivity for quantification of F2-IsoPs from a variety of biological sources.

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Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model. KEYWORDS: Dengue virus (DENV), spiropyrazolopyridones, structure−activity relationship, lead optimization D engue fever is a febrile disease caused by dengue virus (DENV), which is transmitted by Aedes aegypti, a mosquito that feeds on humans.1 DENV threatens up to 2.5 billion people in more than 100 endemic countries. According to a World Health Organization (WHO) report, 2 there are 50− 100 million infections annually (and yet this number could be far underestimated), 3 with approximately 500,000 cases of dengue hemorrhagic fever and 22,000 deaths globally. 4,5 Despite the clear unmet medical need, currently there is no clinically approved vaccine or antiviral therapy available for treatment of dengue.6−10 Therefore, it is urgent to develop safe and effective therapeutics.High-throughput phenotypic screening is a powerful tool to identify compounds that are active against DENV by inhibiting either host pathways and/or viral proteins, which are essential for viral replication.11−16 As a result of DENV-2 screening on Novartis compound library, a new chemical class spiropyrazolopyridone 1 was identified to be an interesting starting point for further optimization (Figure 1). As compound 1 was a racemate, two enantiomers were subsequently separated by chiral high-performance purification chromatography (HPLC). The structure of R enantiomer 1a was unambiguously determined by X-ray crystallography (Figure 2). Interestingly, when tested in the DENV-2 in vitro assay, these two individual enantiomers displayed remarkably different potency where R enantiomer 1a was greater than 200-fold more potent than the S enantiomer 1b (Figure 1). Very recently, genetic analysis demonstrated that mutations in dengue viral NS4B conferred the resistance of compound 1a, suggesting that this class of compounds inhibited DENV replication by targeting NS4B protein. 11,17−19 In this report, lead optimization efforts to explore the structure−activity relationships (SARs) and improve the physicochemical properties are described.Following the identification of compound 1, which was subsequently found to be poorly soluble in aqueous media, we initiated a lead optimization effort in an attempt to improve the solubility while maintaining the potency. The general synthetic route to the spiropyrazolopyridone derivatives is outlined in Scheme 1. The synthesis, featuring a three component...

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Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

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