NP-06: a novel anti-human immunodeficiency virus polypeptide produced by a Streptomyces species

Abstract

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,...

Siamycin Attenuates fsr Quorum Sensing Mediated by a Gelatinase Biosynthesis-Activating Pheromone in Enterococcus faecalis

“…The target molecule of siamycins in anti-HIV activity has been proposed to be HIV envelope glycoprotein gp41 because siamycins have a strong inhibitory effect on syncytium formation and exhibit sequence similarity to part of gp41 (7,11,18). However, unlike the sequence similarity between siamycin and gp41, there is no sequence similarity between siamycin I and GBAP.…”

“…Mass spectrometry suggested that the molecular mass of this compound was 2,164, which is identical to the molecular masses of siamycin I (14, 61), NP-06 (7), and RP-71955 (18). These compounds were previously screened for anti-human immunodeficiency virus (HIV) activity and are known to have antimicrobial activity against gram-positive bacteria as well (7,18,30,61). They were found to be tricyclic peptides consisting of 21 amino acids.…”

Inhibition of Human Immunodeficiency Virus Type 1 Entry by a Binding Domain of Porphyromonas gingivalis Gingipain

“…Chokekijchai et al (9) screened 9,000 extracts of bacterial samples from various sources for anti-HIV activity. A crude extract of strain SKH-2344, a Streptomyces strain isolated from soil, showed high anti-HIV activity.…”

Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection

“…7), from Streptomyces have been described: siamycin II (RP 71955, BMY 29303), 22±24 NP-06 (FR 901724) 25,26 and siamycin I (BMY-29304). 24,27 They differ from one another only at position 4 (Val or Ile) or 17 (again, Val or Ile), which should not make much of a difference in terms of conformational or functional properties.…”

“…The siamycins have been found to inhibit HIV infection in vitro; they exert a strong inhibitory effect on syncytium formation while only weakly inhibiting virus-cell binding. 25,26 The most likely target for the mode of action of siamycins is the HIV envelope glycoprotein gp41. 24 In fact, 33% sequence identity was observed between siamycin II and residues 608±628 of gp41 (amino acid numbering for the gp160 precursor).…”