Hinnuliquinone, a C 2 -symmetric dimeric non-peptide fungal metabolite inhibitor of HIV-1 protease Sheo B. Singh a, * , John G. Ondeyka a , Nasios Tsipouras a , Carolyn Ruby a , Vinod Sardana b , Marvin Schulman a , Manuel Sanchez c , Fernando Pelaez c , Mark W. Stahlhut b , Sanjeev Munshi b , David B. Olsen b , Russell B. Lingham a a Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA b Merck Research Laboratories, West Point, PA 19486, USA c CIBE, Merck Sharp & Dohme de Espan ˆa, S. A. Josefa Valca ´ rcel 38, 28027 Madrid, Spain Received 25 August 2004 Available online 21 September 2004 Abstract HIV-1 protease is one of several key enzymes required for the replication and maturation of HIV-1 virus. An almost two-decade research effort by academic and pharmaceutical institutions resulted in the successful commercialization of seven drugs that are potent inhibitors of HIV-1 protease activity and which, if used correctly, are highly effective in managing viral load. However, iden- tification of clinical viral isolates that are resistant to these drugs indicates that this is a significant problem and that new classes of inhibitors are continually needed. Screening of microbial extracts followed by bioassay-guided isolation led to the discovery of a natural hinnuliquinone, a C 2 -symmetric bis-indolyl quinone natural product that inhibited the wild-type and a clinically resistant (A44) strain of HIV-1 protease with K i values of 0.97 and 1.25 lM, respectively. Crystallographic analysis of the inhibitor-bound HIV-1 protease helped explain the importance of the C 2 -symmetry of hinnuliquinone for activity. Details of the isolation, biological activity, and crystallographic analysis of the inhibitor-bound protease are herein described. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Hinnuliquinone; Fungal metabolite; HIV-1 protease inhibitor; Inhibitor-bound X-ray structure of protease HIV-1 encodes for an aspartic protease that is essen- tial for maturation of infectious virus. This is one of two key biological targets (HIV-1 reverse transcriptase is the other) to which therapeutic drugs have been developed and approved by the FDA. Currently, at least seven HIV-1 protease inhibitors are commercially available and include saquinavir, nelfinavir, ritonavir, indinavir, amprenavir, lopinavir, and atazanavir [1]. The clinical use of HIV-1 protease inhibitors as monotherapy, in the absence of other anti-viral agents, is contraindicated due to the rapid emergence of drug-resistant strains of HIV. The current drug treatment for HIV-1 usually con- sists of a cocktail of anti-viral and other drugs including anti-fungal and/or anti-bacterial drugs. The identifica- tion and approval of novel HIV-1 protease inhibitors would add significantly to the management of the dis- ease in this and other countries. Currently approved protease inhibitors are peptidomi- metics and are one of the first examples of structure-based drug design that utilized structural information of inhib- itor binding to the active site of HIV-1 protease as well as inhibitors of other aspartic proteases (e.g., renin) [1]. This approach successfully identified chemical leads for this target and accelerated the clinical availability of therapeu- tically useful drugs. The unresolved issue of viral resis- tance to current therapy is a daunting challenge and continues to hamper efforts to halt the progression and 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.234 * Corresponding author. Fax: +1 732 594 6880. E-mail address: sheo_singh@merck.com (S.B. Singh). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 324 (2004) 108–113 BBRC