Sulfated Small Molecules Targeting EBV in Burkitt Lymphoma: From In Silico Screening to the Evidence of In Vitro Effect on Viral Episomal DNA Raquel T. Lima 1,2,† , Hugo Seca 1,3,† , Andreia Palmeira 1,2,4 , Miguel X. Fernandes 5 , Felipe Castro 2,4 , Marta Correia-da-Silva 2,4 , Maria S. J. Nascimento 2,3 , Em  ılia Sousa 2,4 , Madalena Pinto 2,4 and M. Helena Vasconcelos 1,3, * 1 Cancer Drug Resistance Group, IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal 2 CEQUIMED-UP, Centre of Medicinal Chemistry – University of Porto, Porto, Portugal 3 Department of Biological Sciences, Laboratory of Microbiology, Faculty of Pharmacy, University of Porto, Porto, Portugal 4 Departamento de Ci^ encias Qu  ımicas, Laborat orio de Qu  ımica Org^ anica e Farmac^ eutica, Faculdade de Farm acia, Universidade do Porto, Porto, Portugal 5 Centro de Qu  ımica da Madeira, Centro de Compet^ encia de Ci^ encias Exactas e da Engenharia, University of Madeira, Funchal, Portugal *Corresponding authors: M. Helena Vasconcelos, hvasconcelos@ipatimup.pt; E. Sousa, esousa@ff.up.pt † These authors equally contributed to this work. Epstein–Barr virus (EBV) infects more than 90% of the world population. Following primary infection, Epstein– Barr virus persists in an asymptomatic latent state. Occasionally, it may switch to lytic infection. Latent EBV infection has been associated with several diseases, such as Burkitt lymphoma (BL). To date, there are no available drugs to target latent EBV, and the existing broad-spec- trum antiviral drugs are mainly active against lytic viral infection. Thus, using computational molecular docking, a virtual screen of a library of small molecules, including xanthones and flavonoids (described with potential for antiviral activity against EBV), was carried out targeting EBV proteins. The more interesting molecules were selected for further computational analysis, and sub- sequently, the compounds were tested in the Raji (BL) cell line, to evaluate their activity against latent EBV. This work identified three novel sulfated small molecules capable of decreasing EBV levels in a BL. Therefore, the in silico screening presents a good approach for the development of new anti-EBV agents. Key words: antivirals, Burkitt lymphoma, Epstein–Barr virus, sulfated small molecules, virtual screening Received 24 May 2012, revised 13 November 2012 and accepted for publication 8 January 2013 Epstein–Barr virus (EBV) infection has been associated with several human malignancies, including Burkitt lym- phoma (BL), as EBV is responsible for transforming B cells and contributing to their malignant phenotype. Epstein–Barr virus is a 184-kbp double-stranded DNA lin- ear genome virus which, once in the nucleus of infected cells, circularizes adopting an episomal shape (1). Epstein –Barr virus episome presents two origins of replication, oriP and oriLyt, which are used to replicate the virus gen- ome during its latent or lytic phase, respectively. Epstein– Barr virus may adopt different types of latency (mainly type I, II, and III) during the latent phase of infection, depending on the proteins expressed (2). In type I latency, a very restricted number of latent proteins are expressed, whereas in type III latency, all set of latent proteins are expressed. Epstein–Barr virus nuclear antigen 1 (EBNA1) is the only latent protein that is expressed in all types of latencies. Importantly, the association of this protein with the oriP sequence is required for both initia- tion and maintenance of replication of EBV episomes in infected cells (3–5). Moreover, EBNA1 is also able to reg- ulate its own expression (6) and, by binding to oriP, to enhance transcription from several viral promoters, such as the LMP1 promoter, leading to the regulated expres- sion of other latent proteins (7). The replication of EBV episomal DNA also requires cellular proteins, such as DNA polymerase, which is not sensitive to antiviral drugs (8). The switch from latency to lytic phase may occur following different stimulus (9–13). The expression of the immediate- early protein Zta was found to trigger viral lytic replication by activating several promoters, therefore leading to the expression of early lytic genes such as the early-antigen- diffuse component (EA-D), the EBV DNA polymerase (involved in viral DNA replication), and the BamHI fragment H rightward open reading frame 1 (BHRF1, an anti-apop- totic molecule homologue of cellular Bcl-2). Following this stage, the expression of late EBV proteins involved in the structure of the virus will occur, and viral DNA replication will not depend on the cellular machinery anymore. Com- pletion of the viral lytic cycle results in the production of ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12109 631 Chem Biol Drug Des 2013; 81: 631–644 Research Article 17470285, 2013, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cbdd.12109 by Universidade De Madeira, Wiley Online Library on [09/02/2023]. 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