Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly

Abstract

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.Poxviruses have been at the forefront of advances in medicine for 200 years, from Jenner's paper on the efficacy of vaccination against smallpox in 1798 to the successful use of vaccinia virus to eradicate human smallpox disease, as proclaimed by the World Health Organization in 1977. Throughout the 1990s, the scientific community debated the proposed destruction of the last known stocks of smallpox virus (24, 31). Hanging over this discussion was the fear that undeclared stocks of smallpox could be used as a bioterror weapon against an unvaccinated population. The urgency of this threat was amplified by the terrorist attacks of 2001, followed by the dissemination of anthrax via the postal service. The outbreak of human monkeypox infections in the United States in 2003 further highlighted the risks of reemergence of human poxvirus disease.Public health and research efforts have been mobilized accordingly to (i) exploit a modified live smallpox vaccine that maintains efficacy while minimizing complications, (ii) pursue alternatives to live vaccination for smallpox prophylaxis, and (iii) discover and bring forward for FDA approval new antipoxviral drugs. Two very different clinical scenarios present different challenges for drug therapy. Prophylaxis of a low-risk population in the event of a threat or actual outbreak mandates an orally available drug with minimal side effects. In contrast, the treatment of confirmed cases of smallpox (which can have a Ն30% fatality rate) need not be hindered by concerns about route of administration and non-life-threatening side effects. The goal is to have at least two approved antipoxviral drugs that act on different molecular targets.Although many inhibitors of poxvirus replication in culture or animal models have been described previously (50), the initial efforts post-2001 focused on the nucleoside analog cidofovir, an inhibitor of the viral DNA polymerase (28), which was already FDA approved for treatment of cytomegalovirus retinitis. Cidofovir was found to be effective in animal models of orthopoxvirus infection (37,50,53). However,...

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

“…Nicks would provide binding sites for the two enzymes on newly synthesized DNA (where the topoisomerase might serve to unknot and decatenate the genome), and the loss of nicks through their ligation would cause the simultaneous dissociation of both enzymes. Adding etoposide or mitoxantrone to this environment would cause the conversion of a presumably beneficial enzymatic association into a toxic adduct that inhibits virus replication and/or maturation (10). An interesting feature of what poxviruses are doing is that ligase I and topoisomerase II are both also recruited to cellular sites of replication.…”

“…2) (27). A peculiar property of these mutant strains is that ligase mutations also create resistance to etoposide and mitoxantrone, even though etoposide has no known effect on the activity of purified poxvirus DNA ligases (9,10,33). Both drugs are highly specific topoisomerase poisons, which work by trapping topoisomerase cleavage complexes on DNA and thus blocking processes like DNA replication and repair (3).…”

“…Targeted gene deletions create similar, although not identical, phenotypes (9,27). Interestingly Deng et al have recently shown that mitoxantrone, a structurally unrelated topoisomerase poison, is also an antipoxviral agent and independently isolated mito-xantrone-resistant virus encoding some of the same A50R mutations conferring etoposide resistance (10). The genetics is not explained by targeting of these drugs to the vaccinia ligase, because its activity is not inhibited by etoposide in vitro (33).…”

“…The N terminus of vaccinia ligase also plays some role in mediating etoposide and mitoxantrone resistance, since drug resistance can be conferred by a single N-terminal C11Y mutation. Mitoxantrone-resistant virus encoding a nearby in-frame indel (adding an isoleucine at position 14) and a mutation in what homologous structures suggest could be the cysteine-11 disulfide partner at cysteine-145 have also been isolated (10). In this communication, we show that this complex phenotype is explained by the fact that cellular topoisomerases II␣ and ␤ are recruited to virus "factories" and that the N terminus of vaccinia DNA ligase plays an important role in stabilizing these interactions.…”

Validation of a High-Content Screening Assay Using Whole-Well Imaging of Transformed Phenotypes

“…[25][26][27][28][29][30][31][32] Aligning a highcontent assay with the correct imaging platform is critical for its development and execution in HTS. The choice of an imaging platform would impact the overall quality of image acquisition for the recognition and quantification of objects by the analysis protocols.…”