Inhibitors of the HIV-1 aspartyl protease mayprovide novel therapeutic agents in the treatment of AIDS1 2). Rational design based on structural data of this viral protease has led to the synthesis of molecules with specific inhibitory activity3 ~ 7). There are, however, few reports of HIV-1 protease in- hibitors from natural sources8'9*. Production of protease inhibitors, in the wider sense, by microorganisms is well known10). We have recorded in literature 188 published compounds; three quarters being produced by actinomycetes, the remainder evenly divided between other bacteria and fungi. Consequently, we decided to develop a solid-phase immunoassay designed to detect thepresence of HIV-1 protease inhibitors in fermentation broths11*. The assay is based on a recombinant protein consisting of Escherichia coli β-galactosidase fused to a portion of the HIV-1 gag polyprotein containing the pl7~p24 cleavage site. This fusion protein acts as a specific substrate for HIV-1 protease. The proteolytic activity is detected using a very specific monoclonal antibody immobilized on standard 96-well microtiter plates. This antibody binds the fusion protein in the gag region only ifthis has not been cleaved by the HIV-1 protease. More than 12,000 strains have been screened using this method, 70%of which were actinomycetes, the remainder being fungi. After retesting and eliminating replicate strains, 3 1 positive activities have been found. 24 activities were found in the broths of strains belonging to the genus Streptomyces; 4 Micromonospora, 2 Nocardia and 1 fungal producer were also found.The activity from strain GE16457 was isolated and characterized. The producer strain after morphological and chemotaxonomic evaluation was found to be a member of the genus Streptomyces. Production of the inhibitor was obtained by growing the producer strain for 72 hours in a medium composed of: glucose 2%, soybean meal 0.8%, yeast extract 0.2%, calcium carbonate 0.4%, sodium chloride 0.1%, pH 7.4. The protease inhibitor was adsorbed from the filtered broth on the polystyrenic resin SI12 (Dow Chemical Co.) and was subsequently extracted from the concentrated acetonic eluates with butanol. This crude preparation was purified by normal pressure reverse-phase chromatography and subsequently preparative HPLCon a 250 x 25 mmcolumn packed with Lichrosorb Merck RP-18 (7/im). The separation was accomplished with a linear gradient of acetonitrile (25 to 50%)and phosphate buffer (0.02m, pH 6.0) with UV detection at 220nm. The purified inhibitor was found structurally belonging to the MAPIcomplex of a and /? epimers which was previously described as microbial protease inhibitor12 ~ 14). The identification was reached as a result of FAB-MSanalysis (Fig. 1). ¹H and ¹³C NMRof the isolated product confirmed the attribution.The GE16457 product was treated with KMnO4 and the oxidation product was hydrolyzed (6 n HC1 with 1 % phenol; 105°C for 24 hours) and derivatized with pentafluoropropionic anhydride and with 2-propanol-HC1. The analysis by HR-GCusing a Heliflex Chirasil-Val (Alltech) capillary column1 5) found L-phenylalanine along with L-valine and L-arginine. This is consistent with the structure of the a-MAPI epimer, as the hydrolysis derivative of a-MAPI contains two L-phenylalanine residues13). /?-MAPI is the epimer at the carbon atom adjacent to the aldehydic function and would be characterized by the presence of D-phenylalanine14). /?-MAPI was also isolated from the crude butanol extract, as D-phenylalanine was found in the hydrolysates after oxidation. This product showed no activity against HIV- 1 protease. The oxidation product of the active a-MAPI was also found to be inactive. These data indicate that the aldehydic function and the configuration of its adjacent atom is crucial for theactivity of these molecules on the HIV-1 protease. Pepstatin A is a known inhibitor of aspartic proteases including the HIV-1 protease8'9). a-MAPI has been previously characterized as an inhibitor of alkaline proteases, with no activity on pepsin or other aspartic proteases12). Nevertheless, in our HIV-1 protease assay (Fig. 2) a-MAPI isolated from strain GE16457 and pepstatin A show similarinhibition profiles. A high sensitivity HPLCmethod was set up for the rapid detection of the MAPImolecules in the positive broths (Fig. 3). Of the 24 positive activities detected from strains belonging to the genus Streptomyces, the MAPIcomplex was found ten times, thus appearing a commonsecondary metabolite of the Streptomyces strains isolated for