HTV-1 protease is one of several key enzymes required for the replication and maturation of HTV-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, identification 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 muM, 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. (C) 2004 Elsevier Inc. All rights reserved.