Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis Hans-Heinrich Hoffmanna, Andrea Kunza,b,1, Viviana A. Simona,b,c, Peter Palesea,b,2, and Megan L. Shawa,2 aDepartment of Microbiology, bDepartment of Medicine, and cGlobal Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, NY 10029 Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved February 25, 2011 (received for review February 4, 2011) Compound A3 was identified in a high-throughput screen for Favipiravir (T-705), which is currently in Phase III trials in inhibitors of influenza virus replication. It displays broad-spectrum Japan, also displays broad-spectrum antiviral activity, but only antiviral activity, and at noncytotoxic concentrations it is shown to among RNA viruses. In vivo efficacy has been demonstrated for inhibit the replication of negative-sense RNA viruses (influenza influenza viruses (A, B, and C), flaviviruses (West Nile virus, viruses A and B, Newcastle disease virus, and vesicular stomatitis Yellow Fever virus), bunyaviruses (Punta Toro virus, Rift Valley virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, fever), arenaviruses (Pichinde virus), and picornaviruses (foot- West Nile virus, and dengue virus), DNA viruses (vaccinia virus and and-mouth disease virus) (11, 12). T-705 is converted to the human adenovirus), and retroviruses (HIV). In contrast to mamma- ribofuranosyl triphosphate form by host cell enzymes, and it was lian cells, inhibition of viral replication by A3 is absent in chicken shown that the antiviral activity of T-705 can be reversed by an cells, which suggests species-specific activity of A3. Correspond- excess of purines (13). Nevertheless, the mechanism of T-705 is ingly, the antiviral activity of A3 can be linked to a cellular protein, not fully understood. It does not affect the synthesis of cellular dihydroorotate dehydrogenase (DHODH), which is an enzyme in DNA or RNA (13, 14), and may therefore target viral RNA- the de novo pyrimidine biosynthesis pathway. Viral replication of dependent RNA polymerases directly or may be preferentially both RNA and DNA viruses can be restored in the presence of excess incorporated into viral RNA, thereby causing hypermutations. uracil, which promotes pyrimidine salvage, or excess orotic acid, Brequinar was described as a broad-spectrum antiviral agent ca- which is the product of DHODH in the de novo pyrimidine bio- pable of inhibiting both negative- and positive-strand RNA viruses synthesis pathway. Based on these findings, it is proposed that A3 (15), and leflunomide and a derivative, FK778, have been repor- acts by depleting pyrimidine pools, which are crucial for efficient ted to inhibit human cytomegalovirus (HCMV) and herpes sim- virus replication. plex virus 1 (HSV-1) (16–19). This group of compounds all target the cellular enzyme dihydroorotate dehydrogenase (DHODH) (20–22), although leflunomide shows the weakest activity against mall-molecular-weight compounds with antiviral activity can this enzyme (23). DHODH is a key player in the pyrimidine de act by inhibiting viral proteins or host cell proteins that are S novo biosynthesis pathway and converts dihydroorotate (DHO) required for virus replication. Although drugs directed at viral into orotate (24). Both the pyrimidine de novo biosynthesis proteins are more virus specific, they can easily lead to the se- pathway and the uracil salvage pathway channel into the pro- lection of resistant mutants. For example, the use of adamant- duction of uridine monophosphate (UMP) (25), which is the anes which target the M2 ion channel of influenza A viruses is precursor for all pyrimidine nucleotides needed for RNA (UTP, now precluded due to wide-spread resistance (1, 2). By targeting CTP) and DNA (dTTP, dCTP) synthesis. Leflunomide (Arava) is host cell proteins, resistance is less likely to occur, and if that a Food and Drug Administration (FDA)-approved therapy for protein is necessary for replication of a variety of different vi- rheumatoid arthritis, and its immunosuppressive properties are ruses, broad-spectrum antiviral activity can be achieved. Such related to inhibition of T-cell proliferation, which is heavily reliant compounds are presumed to be more prone to toxicity, and it is on pyrimidine pools (26). Virus replication is also dependent on important to identify targets that are not critical for cell growth large nucleotide pools, and therefore the antiviral activity of these but that are absolutely essential for the virus. compounds is likely due to pyrimidine depletion. Recently, two small molecules were reported, LJ-001 (3) and Here we report that the small-molecular-weight compound dUY11 (4), that demonstrate broad antiviral activity against all A3, which was identified in a previously reported influenza virus enveloped viruses. These so-called rigid amphiphiles resemble high-throughput screen (27), possesses broad-spectrum activity phospholipids and are incorporated into viral membranes, where against RNA-, DNA-, and retroviruses and acts by targeting they modulate the membrane curvature needed for the mem- pyrimidine metabolism. brane fusion event and they therefore inhibit viral entry. The fi virus speci city of these compounds takes advantage of struc- Results tural differences in virion membranes versus cellular membranes Antiviral Activity of Compound A3. Approximately 61,600 com- and also the lack of repair mechanisms for viral membranes. To mercial, small-molecular-weight compounds were screened in date, the only approved broad-spectrum antiviral that is effective duplicate in a high-throughput screen (HTS) assay described against both RNA and DNA viruses is ribavirin (5), which is currently used in combination with IFN for hepatitis C therapy (6). Ribavirin is a ribosyl purine analog, the carboxamide group of which can resemble adenosine or guanosine, depending on its Author contributions: H.-H.H., P.P., and M.L.S. designed research; H.-H.H. and A.K. per- formed research; A.K. and V.A.S. contributed new reagents/analytic tools; H.-H.H., A.K., rotation. As a prodrug, it is sequentially phosphorylated by cel- V.A.S., P.P., and M.L.S. analyzed data; and H.-H.H., V.A.S., and M.L.S. wrote the paper. MICROBIOLOGY lular kinases, and all its intermediates such as ribavirin mono- Conflict of interest statement: A provisional patent application has been filed by Mount (RMP), di-(RDP) and triphosphate (RTP) are inhibitors of certain Sinai School of Medicine covering the A3 compound. viral RNA-dependent RNA polymerases (7). RTP is incorporated This article is a PNAS Direct Submission. into RNA and pairs equally well with uracil and cytosine, inducing 1Present address: Institute of Tropical Medicine and International Health, Charité, Univer- lethal hypermutations (8), and RMP has been shown to target sity Medicine Berlin, Berlin 14050, Germany. the cellular inosine monophosphate dehydrogenase (IMPDH) and 2To whom correspondence may be addressed. E-mail: [email protected] or peter. thereby depletes intracellular pools of GTP (9, 10). This lack of [email protected]. GTP may explain the inhibition of DNA viruses, as well as the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. general cytotoxicity of ribavirin. 1073/pnas.1101143108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1101143108 PNAS | April 5, 2011 | vol. 108 | no. 14 | 5777–5782 Downloaded by guest on September 28, 2021 A B 1E+8 120 100 1E+7 Fig. 1. Compound A3 and its antiviral activity against influenza A/WSN/33 virus. (A) Chemical 80 structure of compound A3 and its molecular 1E+6 IC50=0.178μM CC50=268μM weight (MW). (B) A549 cells were infected with 60 influenza A/WSN/33 virus (MOI = 0.01) in the 1E+5 ter [pfu/ml] ter presence of increasing concentrations of com- 40 cell viability [%] pound A3. Viral titers were determined at 24 h MW: 356 g/mol viral 1E+4 20 postinfection and the IC50 calculated (left-hand scale, blue curve). Mean of three replicates ± SD 1E+3 0 are shown. Cell viability (CC50) was determined 0.001 0.01 0.1 1 10 100 independently for a 24-h incubation period (right- hand scale, red curve). Mean of five replicates ± compound concentraon [μM] SD are shown. previously (27). Briefly, a compound was defined to be a strong the viral life cycle, leading to the hypothesis that replication and inhibitor of influenza virus replication if the luminescence signal transcription may be targeted. of an influenza virus inducible firefly luciferase reporter was We addressed this question by performing an influenza virus decreased by at least 90% compared with that in untreated cells. minigenome assay to determine whether influenza virus poly- Compound A3 (Fig. 1A) from the Asinex1 library displayed a merase activity is affected by A3. A549 cells were transfected very strong effect on viral replication, such that there was no with expression plasmids for the influenza virus polymerase luminescence detectable. The compound was further evaluated proteins (PB1, PB2, and PA), the nucleoprotein (NP), and the for cytotoxity, and its CC50 in A549 cells was determined to be previously described influenza virus-specific firefly luciferase re- 268 μM over a 24-h incubation period (Fig. 1B). To confirm the porter (27). To normalize for transfection efficiency, a Renilla results of the primary screen, A3 was tested at noncytotoxic luciferase plasmid was cotransfected. Compounds were added at concentrations in viral replication assays performed at an MOI 4 h before transfection and were present for the duration of the of 0.01. A reduction in viral titers of 4 logs was detected at a assay. A3 strongly inhibits influenza virus polymerase function by concentration of 2 μM, and the IC50 over a 24-h replication 98% compared with the DMSO control, without affecting cel- period was determined as 0.178 μM (Fig. 1B). This resulted in lular gene expression as monitored by Renilla luciferase activity a selective index (SI = CC50/IC50) of 1,505, which indicates (Fig.