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Identification of Broad-Spectrum Antiviral Compounds and Assessment of the Druggability of Their Target for Efficacy Against

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Identification of Broad-Spectrum Antiviral Compounds and Assessment of the Druggability of Their Target for Efficacy Against Identification of broad-spectrum antiviral compounds SPECIAL FEATURE and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV) Aurelio Bonaviaa, Michael Frantia, Erin Pusateri Keaneya, Kelli Kuhena, Mohindra Seepersauda, Branko Radeticha, Jian Shaoa, Ayako Hondaa, Janetta Dewhursta, Kara Balabanisa, James Monroea, Karen Wolffa, Colin Osbornea, Leanne Lanieria, Keith Hoffmastera, Jakal Amina, Judit Markovitsa, Michelle Broomea, Elizabeth Skubaa, Ivan Cornella-Taracidoa, Gerard Jobertyb, Tewis Bouwmeestera, Lawrence Hamanna, John A. Tallaricoa, Ruben Tommasia, Teresa Comptona, and Simon M. Bushella,1 aNovartis Institutes for Biomedical Research, Inc., 250 Massachusetts Avenue, Cambridge, MA 02139; and bCellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved February 25, 2011 (received for review November 17, 2010) The search for novel therapeutic interventions for viral disease is a confounded many conventional antiviral products. Targeting a challenging pursuit, hallmarked by the paucity of antiviral agents cellular protein might afford antiviral compounds with a broader currently prescribed. Targeting of viral proteins has the inextricable spectrum of activity and have the attractive property to lessen challenge of rise of resistance. Safe and effective vaccines are not the chance of resistance development. However, targeting the possible for many viral pathogens. New approaches are required host could result in toxicity, especially if the protein or pathway to address the unmet medical need in this area. We undertook a used is crucial for cell survival. This risk is mitigated if the virus cell-based high-throughput screen to identify leads for develop- uses the host target in a substantially different manner from the ment of drugs to treat respiratory syncytial virus (RSV), a serious obligate cell process or the biological process is not essential. pediatric pathogen. We identified compounds that are potent Therefore, an up-front understanding of antiviral mechanism of CHEMISTRY (nanomolar) inhibitors of RSV in vitro in HEp-2 cells and in primary action (MoA) is required to mitigate such risks. Given the inher- human bronchial epithelial cells and were shown to act postentry. ent challenges, we took an unbiased approach and sought chemi- Interestingly, two scaffolds exhibited broad-spectrum activity cal starting points that exert an antiviral phenotype in whole cells among multiple RNA viruses. Using the chemical matter as a probe, that could potentially reveal inhibitors of virtually any facet of we identified the targets and identified a common cellular path- the viral life cycle. By not restricting the screen to a biochemical way: the de novo pyrimidine biosynthesis pathway. Both targets assay for virally encoded protein, we did not preclude identifying were validated in vitro and showed no significant cell cytotoxicity inhibitors of such. Rather, we opened the additional possibility to except for activity against proliferative B- and T-type lymphoid identify host factors critical to propagation of the viral life cycle. cells. Corollary to this finding was to understand the consequences Cognizant of the potential toxicity caveats associated with inhibi- of inhibition of the target to the host. An in vivo assessment for tion of host factors, we prioritized target identification to facil- antiviral efficacy failed to demonstrate reduced viral load, but re- itate an assessment of druggability (12–14) of any such targets vealed microscopic changes and a trend toward reduced pyrimidine should they arise from our screen. We present here a small- pools and findings in histopathology. We present here a discovery molecule–driven paradigm to identify, and subsequently interro- program that includes screen, target identification, validation, and gate, the druggability of specific host factors for antiviral efficacy. druggability that can be broadly applied to identify and interro- gate other host factors for antiviral effect starting from chemical Results matter of unknown target/mechanism of action. Screening. A whole cell screen in HEp-2 cells infected with RSV-A (Long) was performed using approximately 1.7 M compounds espiratory syncytial virus (RSV) (1) is an increasingly impor- that identified >16;000 primary hits (approximately 0.95% hit Rtant pediatric pathogen (2). RSV affects all age groups (3), rate) with >50% cell protection (denoted as CPE assay for cell with symptoms ranging from mild rhinorrhea, cough and fever cytopathic effect) at a single concentration (10 μM) (Fig. 1A). (4) to more serious bronchiolitis or pneumonia (5), and it is the Hit confirmation (CPE, 10 μM, triplicate) delivered approxi- single most common cause of childhood hospitalization (6). With mately 7,000 validated candidates (48% confirmation rate) that the exception of the prophylactic antibody Synagis® (palivizu- were further characterized to determine their purity/integrity mab) (7), for high-risk populations, there is no specific treatment (liquid chromatography–UV-MS), antiviral efficacy (EC50), and for RSV,and the prospects for a safe and effective vaccine remain cell cytotoxicity (CC50) (eight-point dose response curve, half-log remote at this time (8). Overall, the effort to develop effective serial dilution) to deliver a working set of approximately 5,000 therapies for pathogenic viral infections is one of the most chal- compounds (one-third of validated candidate list) from which lenging in public health (9). The large-scale screening for natural two distinct chemical series were selected, pyrazole-isoxazole products able to kill bacteria in vitro, which was the basis for the (6b) and proline (14b) (Fig. 1B). boom of antibiotics in the 1950s, was not successful for antivirals (10). Because of an increased understanding of the molecular Author contributions: A.B., M.F., E.P.K., K.K., C.O., K.H., I.C.-T., T.B., L.H., J.A.T., R.T., T.C., mechanisms of viral life cycles, many viral proteins have emerged and S.M.B. designed research; A.B., M.F., E.P.K., K.K., M.S., B.R., J.S., A.H., J.D., K.B., as targets for therapeutic intervention. However, antiviral drug J. Monroe, K.W., C.O., L.L., K.H., J.A., J. Markovits, M.B., E.S., I.C.-T., G.J., T.B., and discovery can be targeted at either viral proteins or cellular pro- S.M.B. performed research; and A.B. and S.M.B. wrote the paper. teins as in the case of Selzentry® (maraviroc, targeting CCR5) for The authors declare no conflict of interest. human immunodeficiency virus (HIV) (11). Although the former This article is a PNAS Direct Submission. is likely to yield compounds with a narrow spectrum of antiviral 1To whom correspondence should be addressed. E-mail: [email protected]. activity, few effective and safe agents have emerged, and these This article contains supporting information online at www.pnas.org/lookup/suppl/ face the inextricable challenge of high mutation rates that have doi:10.1073/pnas.1017142108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1017142108 PNAS Early Edition ∣ 6739 of 6744 Downloaded by guest on September 30, 2021 Fig. 1. Identification of 2 series with anti-RSV antiviral effect in a whole-cell high-throughput screening (HTS). (A, Right) Flow chart of the HTS for RSV in HEp-2 cells using cell protection as efficacy determination. (A, Left) Images of healthy HEp-2 cells protected by RSV604 or killed by RSV infection. (B) The isoxazole- pyrazole (6b) and the proline (14b) series identified. (C) Effect of compounds (10 μM) in HEp-2 cells at 96 h postinoculation at an MOI 0.1 measured by plaque assay. (D) Time of addition experiments. Compounds (10 μM) were added at time of addition and 4 h postinoculation to HEp-2 cells at MOI 1. Virus was quantified by qRT-PCR from total cell RNA isolated from infected cells at 12 h postinoculation. (E) Efficacy in HBEC polarized. Virus was measured by qRT-PCR from the apical surface supernatant at days 2, 3, 4, and 5 postinoculation after 5 μM compound addition. In Vitro Antiviral Activity. Addition of 6b or 14b to HEp-2 cells 1 h natants collected at 2, 3, 4, and 5 d postinoculation (Fig.1E),con- prior to inoculation with RSV-A-Long at multiplicity of infection firming activity in primary polarized cells. (MOI) 0.1 resulted in a reduction of 4 logs of virus production compared to DMSO control as measured by plaque assay at 96 h Broad-Spectrum Activity. To test the specificity of the compounds postinoculation. This strong reduction was similar to the effect against RSV, both 6b and 14b were screened against four differ- by the replication inhibitor RSV604 (Fig. 1C). Time of addition ent RNA virus families (Paramyxoviridae, Orthomyxoviridae, studies were performed to determine at which step of the viral Flaviviridae, and Retroviridae). Potent EC50 values in the range μ cycle the compounds were acting (15). The compound was added 0.002 to 0.3 M are observed for RSV, influenza virus, hepatitis C 4 h postinoculation at MOI 1 (Fig. 1D). Both 6b and 14b signifi- virus (HCV), dengue virus, yellow fever virus, and HIV (Table 1). cantly reduced the viral copies measured by quantitative RT-PCR The broad-spectrum activity was not only observed among the (qRT-PCR) from the whole cell RNA compared to DMSO con- different viral families but within a family as well. The compounds trol, similar to inhibitions observed with replication inhibitors are active against strains of RSV groups A and B, influenza virus A/PR/8/34, A/Udorn/72 and B/Lee, HCV infectious virus group RSV604 and RI. RSV604 targets the replication complex, 2a and replicon 1a, dengue virus 2 and replicon, yellow fever virus whereas RI targets the L-polymerase. In contrast, FI, a fusion 17D, and the multidrug-resistant strain MDR769. In all assays the inhibitor that targets the F protein, could not suppress viral CC50 was >10 μM.
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