In Vitro Assays

The discovery of antiviral agents is currently an active field of research in academia and in the biotechnology and pharmaceutical industries [27]. More than 20 new chemical and biological agents have received U.S. Food and Drug Administration approval for treatment of human viral diseases in the last 10 years. Numerous compounds, recombinant proteins, and monoclonal antibodies are currently under active investigation as antiviral or immune-boosting agents. Some but not all antiviral agents have been tested for their ability to prevent variola virus infection of cultured cells in the Biological Safety Level 4 (BSL-4) biological containment facilities at the Centers for Disease Control and Prevention (CDC) in Atlanta or the State Center of Virology and Biotechnology (VECTOR) in Kotsovo.*

Cidofovir, for example, is an antiviral, initially developed as a DNA polymerase inhibitor for the treatment of cytomegalovirus (CMV) retinitis, then found to be active in preventing variola infection of cultured cells (see also Chapter 5). While cidofovir's low oral bioavailability and potential for severe renal toxicity limit its clinical utility for the treatment of variola infection, numerous advances in drug discovery technology—such as combinatorial chemistry, molecular modeling, and high-throughput screening—are providing many new chemical entities that could be tested for their safety and efficacy against variola [27, 28]. Moreover, successful strategies for blocking the infectivity of other types of viruses may suggest new approaches for combating variola. If a large outbreak of smallpox is a credible threat, the infrastructure for testing the antivariola activity of existing and future antiviral agents must be retained by CDC on an ongoing basis. Should a backlog of promising agents develop, expansion of the infrastructure might well be considered.

Discovery of a new antiviral agent is a complex and costly process, typically requiring evaluation of many tens of thousands of candidates in several assays. The primary screening assay typically tests the ability of an agent to bind and inactivate a recombinant, cell-free target protein of viral or human origin, or inhibit replication of the virus itself. Intrinsic potency is determined at this stage in titration experiments, while measurement of potency against closely related proteins provides an indication of specificity. Since single amino acid changes can have dramatic effects on the potency and specificity of antiviral agents, it is imperative that authentic target proteins be tested. Authentication of the target protein includes sequence analysis of the corresponding DNA from several clinical isolates.

The potency and efficacy of an antiviral agent are influenced by many factors in addition to the agent's ability to bind to the target protein. These factors include

*  

John W. Huggins, Personal communication, December 1998.



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