From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 M when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively. NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion.We prepared ϳ9,000 different extracts from solitary colonies obtained by incubation of bacterial samples from various sources, including soil specimens collected in Japan. The methanol extract from each bacterial colony was tested for antihuman immunodeficiency virus type 1 (HIV-1) activity by using a colorimetric assay employing a tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide [MTT]) and MT4 cells (9). A crude extract of strain SKH-2344 (FERMP-14239), a Streptomyces species strain isolated from soil collected in Yamaguchi, was found to be active against HIV-1 (data not shown). The strain was then grown in culture medium and subjected to fermentation. On day 5 of fermentation, 20 liters of culture broth medium was centrifuged, and the supernatant was concentrated in vacuo to yield a paste, which was resuspended in 1 liter of methanol for 12 h. The methanol extract was then filtered and concentrated to yield a pale yellow oily specimen (fraction A). The pellet (the mycelium) was homogenized in 10 liters of methanol with agitation for 12 h, and the filtered solution was concentrated in vacuo to yield a yellow oily specimen (fraction B). Fractions A and B were combined, dissolved in water-saturated butanol, and concentrated in vacuo. The extract was column chromatographed on silica gel with ethyl acetate-methanol-water (10:5:0.25) and purified (Ͼ99%) by high-performance liquid chromatography with CH 3 CN-H 2 O-CF 3 COOH (38:62:0.1) to yield a white powder (100 mg), designated NP-06.Amino acid analysis of the NP-06 hydrolysate revealed the presence of one residue each of Ser, Ile, Leu, and Tyr; two residues each of Phe, Ala, Val, and Asp; and four residues each of Cys and Gly, although whether the two Asp residues represented Asn was to be confirmed. Thin-layer chromatography analysis of the hydrazine hydrolysate of NP-06 revealed that the C terminus was Trp. NP-06 could not be sequenced by the Edman degradation method, although the physical properties of NP-06 showed that it was a peptide. Therefore, nuclear magnetic resonance (NMR) spectroscopy was used to determine the amino acid sequence of NP-06. Inspection of the relayed signals arising from the amide resonances in a homonuclear Hartmann-Hahn spectroscopy experiment revealed the presence of 21-amino-acid spin systems. One Leu, one Ile, one Ser, two Val, two Ala,...