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Antimicrobial Agents and Chemotherapy, September 2009, p. 3860-3870, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00503-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Target Analysis of the Experimental Measles Therapeutic AS-136A{triangledown}

Jeong-Joong Yoon,1,{dagger} Stefanie A. Krumm,1,{dagger} J. Maina Ndungu,2 Vanessa Hoffman,1 Bettina Bankamp,3 Paul A. Rota,3 Aiming Sun,3 James P. Snyder,3 and Richard K. Plemper1,4*

Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, Georgia 30322,1 Department of Chemistry, Emory University, Atlanta, Georgia 30322,2 Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,3 Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 303224

Received 16 April 2009/ Returned for modification 19 May 2009/ Accepted 8 June 2009

No effective therapeutic is currently in place for improved case management of severe measles or the rapid control of outbreaks. Through high-throughput screening, we recently identified a novel small-molecule class that potently blocks activity of the measles virus (MeV) RNA-dependent RNA polymerase (RdRp) complex in transient replicon assays. However, the nature of the block in RdRp activity and the physical target of the compound remained elusive. Through real-time reverse transcription-PCR analysis, we demonstrate that the lead compound AS-136A blocks viral RNA synthesis in the context of an infection. Adaptation of different MeV strains to growth in the presence of the compound identified three candidate hot spots for resistance that are located in conserved domains of the viral polymerase (L protein) subunit of the RdRp complex. Rebuilding of individual mutations in RdRp-driven reporter assays and recombinant MeV traced the molecular basis for resistance to specific mutations in L. Mutations responsible for resistance cluster in the immediate vicinity of the proposed catalytic center for phosphodiester bond formation and neighboring conserved domains of L, providing support for effective inhibition of a paramyxovirus RdRp complex through interaction of a nonnucleoside small-molecule inhibitor with the L protein. Resistance mutations are located in regions of L that are fully conserved among viral isolates, and recombinant MeV harboring individual resistance mutations show some delay in the onset of viral growth in vitro. Taken together, these data support the hypothesis that acquiring mutations in these L domains may reduce virus fitness.

* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Pediatrics, 520 Children's Center, 2015 Uppergate Drive, Emory University School of Medicine, Atlanta, GA 30322. Phone: (404) 727-1605. Fax: (404) 727-9223. E-mail: rplempe{at}emory.edu

{triangledown} Published ahead of print on 15 June 2009.

{dagger} These authors contributed equally to the study.

Antimicrobial Agents and Chemotherapy, September 2009, p. 3860-3870, Vol. 53, No. 9
0066-4804/09/$08.00+0     doi:10.1128/AAC.00503-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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