Inhibition of Human Immunodeficiency Virus Type 1 Replication in ...

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Eisai Company, Tsukuba, Ibaraki 300-2635,2 Japan. Received 11 January 1999/Returned for modification 16 April 1999/Accepted 15 July 1999. In a search for ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1999, p. 2350–2355 0066-4804/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 10

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Acutely and Chronically Infected Cells by EM2487, a Novel Substance Produced by a Streptomyces Species MASANORI BABA,1* MIKA OKAMOTO,1

AND

HITOSHI TAKEUCHI2

Division of Human Retroviruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, Kagoshima 890-8520,1 and Department of Exploratory Drug Research, Eisai Company, Tsukuba, Ibaraki 300-2635,2 Japan Received 11 January 1999/Returned for modification 16 April 1999/Accepted 15 July 1999

In a search for effective HIV-1 transcription inhibitors, we have evaluated more than 75,000 compounds for their inhibitory effects on Tat-induced human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-driven reporter gene expression and found that EM2487, a novel small-molecule substance produced by a Streptomyces species, is a potent and selective inhibitor of HIV-1 replication in both acutely and chronically infected cells. Its 50% effective concentration for acute HIV-1 infection was 0.27 ␮M in peripheral blood mononuclear cells (PBMCs), while the 50% cytotoxic concentration for mock-infected PBMCs was 13.3 ␮M. EM2487 proved inhibitory to a variety of HIV-1 strains and HIV-2 in acutely infected T-cell lines (MOLT-4 and MT-4). The compound could suppress tumor necrosis factor alpha (TNF-␣)-induced HIV-1 production in latently infected cells (OM-10.1 and ACH-2) as well as constitutive viral production in chronically infected cells (MOLT4/IIIB and U937/IIIB) without showing any cytotoxicity. EM2487 did not affect early events of the HIV-1 replication cycle, as determined by proviral DNA synthesis in acutely infected MOLT-4 cells. In contrast, the compound selectively prevented viral mRNA synthesis in OM-10.1 cells, suggesting that HIV-1 inhibition occurs at the transcriptional level. Furthermore, EM2487 did not inhibit TNF-␣-induced HIV-1 LTR-driven reporter gene expression but did inhibit that induced by Tat, irrespective of the presence or absence of the nuclear factor ␬B binding sites in the LTR. These results suggest that the mechanism of action is attributable in part to the inhibition of Tat function. inhibitor of HIV-1 replication in acutely and chronically infected cells.

The progress of combination chemotherapy with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease inhibitors has achieved long-sustained suppression of viral replication in HIV-1-infected individuals (8, 17). However, considering the high cost and low patient compliance of long-term combination chemotherapy (12), discovery of novel anti-HIV-1 agents with different mechanisms of action is still highly desirable. In addition, recent studies have revealed that replication-competent virus can be recovered from resting CD4⫹ T cells even in patients with prolonged suppression of plasma viremia (more than 100 weeks) by combination chemotherapy (13, 32). Therefore, it is clear that the current chemotherapy cannot be terminated unless such reservoir cells have been eradicated or viral recovery from these cells can be completely suppressed. In this regard, inhibitors that selectively prevent HIV-1 gene expression have the potential of inhibiting the recovery of latent virus from resting CD4⫹ T cells as well as infected macrophages, which are also considered to be a long-surviving chronically infected cell population in HIV-1-infected patients (26). In our extensive search program for HIV-1 transcription inhibitors, we have evaluated more than 75,000 compounds for their inhibitory effects on Tat-induced reporter gene expression in cell cultures and found that EM2487 (Fig. 1), a novel small-molecule substance produced by a Streptomyces species, is a potent and selective

MATERIALS AND METHODS Compounds. Preparation and purification of EM2487 (Mr, 829) carried out in collaboration with Mercian Corporation (Tokyo, Japan) will be described elsewhere (27a). The purity of the final preparation was more than 99.9% (data not shown). The Tat antagonist Ro24-7429 (18) was synthesized by Eisai Co. (Tsukuba, Japan). The HIV-1 transcription inhibitor K-12 (3) and the protease inhibitor nelfinavir were kindly provided by Daiichi Pharmaceutical Co. (Tokyo, Japan) and Japan Tobacco Co. (Takatsuki, Japan), respectively. The HIV-1 RT inhibitors lamivudine and MKC-442 (4) were supplied from Mitsubishi Chemical Corporation (Yokohama, Japan). Dextran sulfate was purchased from Sigma Chemical Co. (St. Louis, Mo.). Except for dextran sulfate, all compounds were dissolved in dimethyl sulfoxide at 20 mM or higher concentration to exclude any antiviral or cytotoxic effect of dimethyl sulfoxide. Dextran sulfate was dissolved in distilled water. Cells and viruses. MOLT-4 cells (20), MT-4 cells (21), peripheral blood mononuclear cells (PBMCs), OM-10.1 cells (7), ACH-2 cells (9), MOLT-4/IIIB cells, and U937/IIIB cells were used in the antiviral assays. OM-10.1 and ACH-2 cells are clones of HL-60 and CEM cells latently infected with HIV-1, respectively. MOLT-4/IIIB and U937/IIIB cells are MOLT-4 and U937 cells chronically infected with HIV-1 (IIIB strain), respectively. PBMCs were obtained from healthy donors and stimulated with phytohemagglutinin. Establishment of W-3 and KM-3 cells and their reporter gene constructs will be reported elsewhere (27a). These cells are clones of CEM cells that stably integrate a HIV-1 long terminal repeat (LTR)-driven secreted alkaline phosphatase gene (14). The integrated HIV-1 LTR contains two intact NF-␬B-binding sites in W-3 cells, whereas both of the sites are mutated in KM-3 cells. Three strains of HIV-1 (IIIB, HE, and Ba-L) and one strain of HIV-2 (EHO) were used in the antiviral assays. HE and Ba-L are a T-cell line-tropic clinical isolate (24) and a macrophage-tropic strain, respectively. Antiviral assays. The activities of the compounds against acute HIV-1 and HIV-2 infections were based on the inhibition of virus-induced cytopathicity in MOLT-4 and MT-4 cells and p24 antigen production in PBMCs, as previously described (1). MOLT-4 and MT-4 cells (105/ml) were infected with the virus at a multiplicity of infection of 0.1 and 0.02, respectively, and cultured in the presence of various concentrations of the test compounds. After a 4-day incubation at 37°C, the MOLT-4 cells were subcultured at a ratio of 1:5 with fresh

* Corresponding author. Mailing address: Division of Human Retroviruses, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Phone: (81) 99-275-5930. Fax: (81) 99-275-5932. E-mail: baba @med3.kufm.kagoshima-u.ac.jp. 2350

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Reporter gene assays. W-3 and KM-3 cells (3 ⫻ 106/ml) were either treated with 10 ng of TNF-␣/ml or transfected with 10 ␮g of a plasmid expressing HIV-1 Tat, containing the second exon, under the control of the simian virus 40 promoter (modification of pSV2tat72) by electroporation (300 V; 1,000 ␮F). The cells were cultured in the presence of various concentrations of the test compounds. After a 2-day incubation at 37°C, the culture supernatants were collected, incubated at 65°C for 30 min to inactivate the alkaline phosphatase activity of fetal calf serum, and examined for secreted alkaline phosphatase levels. At the same time, the number of viable cells was determined by the MTT method. FIG. 1. Structure of EM2487.

RESULTS culture medium containing appropriate concentrations of the test compounds and further cultured. The number of viable cells was measured by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method on day 7 (MOLT-4 cells) and day 4 (MT-4 cells) after virus infection (25). For the assays in PBMCs, the cells (105/ml) were infected with HIV-1 at a multiplicity of infection of 0.1. After virus adsorption for 2 h, the cells were extensively washed to remove unadsorbed virus particles and cultured in the presence of various concentrations of the test compounds. After a 6-day incubation at 37°C, the culture supernatants were collected and their p24 antigen levels were determined with a sandwich enzyme-linked immunosorbent assay kit (Cellular Products, Buffalo, N.Y.). The cytotoxicities of the test compounds were evaluated in parallel with their antiviral activities. They were based on the viability of mockinfected cells, as determined by the MTT method. The activities of the compounds against chronic HIV-1 infection were based on the inhibition of p24 antigen. OM-10.1 and ACH-2 cells (105/ml) were incubated in the absence or presence of the test compounds for 2 h, stimulated with 1 ng of tumor necrosis factor alpha (TNF-␣) (Boehringer-Mannheim, Mannheim, Germany)/ml, and further incubated. On the other hand, MOLT-4/IIIB and U937/IIIB cells (105/ml) were cultured in the absence or presence of the test compounds without any stimulation. After a 3-day incubation at 37°C, the culture supernatants were collected and examined for their p24 antigen levels. The cytotoxicities of the test compounds for the chronically infected cells were also determined by the MTT method. PCR analysis. The effects of the compounds on HIV-1 proviral DNA synthesis were analyzed by PCR (23). MOLT-4 cells (2.5 ⫻ 105) were infected with HIV-1 at a multiplicity of infection of 1.0 and cultured in the absence or presence of the test compounds. After a 24-h incubation at 37°C, total DNA was extracted from the cells with a DNA extraction kit (Wako, Osaka, Japan). The extracted DNA was subjected to PCR amplification with the HIV-1 gag-specific primer pair SK38 and SK39 (23) and the control primer pair GH20 and PC04 for ␤-globin (5). The amplification was carried out for 35 cycles (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min). The amplified products were electrophoresed in an agarose gel and visualized by ethidium bromide staining. Northern blot analysis. OM-10.1 cells (106/ml) were incubated in the absence or presence of EM2487 for 2 h, stimulated with 10 ng of TNF-␣/ml, and further incubated. After a 24-h incubation at 37°C, total RNA was extracted from the cells with an RNA extraction kit (RNAzol B; Tel-Test, Friendswood, Tex.). The extracted RNA (20 ␮g) was electrophoresed and transferred to Hybond-N⫹ membrane (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). The blot was hybridized with a 32P-labeled full-length HIV-1 molecular clone, HXB2 (27).

Antiviral activity in acutely infected cells. When we evaluated EM2487 for its inhibitory effects on HIV-1 (IIIB strain) replication in PBMCs, it completely inhibited p24 antigen production in culture supernatants at a concentration of 4 ␮M (Fig. 2A). EM2487 did not reduce the proliferation and viability of mock-infected PBMCs at this concentration. Its 50% effective concentration (EC50) was 0.27 ␮M, while the 50% cytotoxic concentration (CC50) was 13.3 ␮M (Table 1). Thus, the selectivity index (SI), based on the ratio of its CC50 to its EC50, was 49. EM2487 was also active against the macrophagetropic strain Ba-L and a macrophage-tropic clinical isolate (KK strain) in PBMCs. The EC50s for Ba-L and KK were 2.1 and 0.072 ␮M, respectively (Table 1 and data not shown). EM2487

TABLE 1. Inhibitory effects of EM2487 on HIV-1 replication in acutely infected cellsa Compound

EM2487

Cell

Virus

EC50b (␮M)

CC50c (␮M)

SId

MOLT-4

IIIB HE EHO IIIB IIIB Ba-L IIIB IIIB IIIB IIIB IIIB IIIB

0.093 ⫾ 0.005 0.25 ⫾ 0.11 0.27 ⫾ 0.02 0.88 ⫾ 0.08 0.27 ⫾ 0.12 2.1 ⫾ 0.2 0.23 ⫾ 0.09 ⬎2.7 0.082 ⫾ 0.010 0.51 ⫾ 0.04 0.038 ⫾ 0.013 0.61 ⫾ 0.35

9.0 ⫾ 0.2 9.0 ⫾ 0.2 9.0 ⫾ 0.2 4.3 ⫾ 1.2 13.3 ⫾ 1.0 13.3 ⫾ 1.0 2.8 ⫾ 0.8 2.7 ⫾ 0.1 8.0 ⫾ 1.2 3.1 ⫾ 1.3 ⬎20 ⬎20

97 36 33 4.9 49 6.3 12 ⬍1 98 6.1 ⬎526 ⬎33

MT-4 PBMC Ro24-7429 K-12 Lamivudine a

MOLT-4 MT-4 MOLT-4 MT-4 MOLT-4 MT-4

All data are means ⫾ standard deviations for three separate experiments. Concentration required for 50% inhibition of viral cytopathicity in MOLT-4 and MT-4 cells or p24 antigen production in PBMCs. c Concentration required for 50% inhibition of proliferation and viability of mock-infected cells. d Selectivity index (ratio of CC50 to EC50). b

FIG. 2. Inhibitory effects of EM2487 on HIV-1 replication in acutely infected PBMCs (A) and TNF-␣-stimulated OM-10.1 cells (B). In acute infection, phytohemagglutinin-stimulated PBMCs were infected with HIV-1 (IIIB strain) and cultured in the presence of various concentrations of the test compound. In chronic infection, OM-10.1 cells were incubated in the absence or presence of the test compounds for 2 h, stimulated with TNF-␣ (1 ng/ml), and further incubated. After a 6-day incubation for PBMCs and a 3-day incubation for OM-10.1 cells, the p24 antigen levels of the culture supernatants (■) were measured by antigen capture ELISA. At the same time, the number of viable cells (䊐) was determined by the MTT method. The p24 antigen levels of the control culture supernatants (in the absence of EM2487) were 70 ⫾ 15 and 174 ⫾ 11 ng/ml in PBMCs and OM-10.1 cells, respectively. The experiments were repeated three times, and representative results are shown.

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TABLE 2. Inhibitory effects of EM2487 on HIV-1 replication in chronically infected cellsa Compound

EM2487

Ro24-7429 K-12 Lamivudine

Cell

EC50b (␮M)

CC50c (␮M)

SId

OM-10.1 ACH-2 MOLT-4/IIIB U937/IIIB OM-10.1 MOLT-4/IIIB OM-10.1 MOLT-4/IIIB OM-10.1 MOLT-4/IIIB

0.075 ⫾ 0.032 0.52 ⫾ 0.23 0.31 ⫾ 0.07 0.24 ⫾ 0.03 2.6 ⫾ 0.8 0.34 ⫾ 0.08 0.034 ⫾ 0.003 0.069 ⫾ 0.010 ⬎20 ⬎20

12.5 ⫾ 4.1 ⬎20 14.6 ⫾ 5.0 6.7 ⫾ 2.0 ⬎20 ⬎20 9.6 ⫾ 1.0 7.0 ⫾ 0.4 ⬎20 ⬎20

167 ⬎38 47 28 ⬎7.7 ⬎58 282 101

a

All data are means ⫾ standard deviations for three separate experiments. Concentration required for 50% inhibition of p24 antigen production in TNF-␣-stimulated OM-10.1 or ACH-2 cells and unstimulated MOLT-4/IIIB or U937/IIIB cells. c Concentration required for 50% inhibition of proliferation and viability of the cells. d Selectivity index (ratio of CC50 to EC50). b

proved effective against HIV-1 replication in MOLT-4 and MT-4 cells (Table 1). Like other HIV-1 transcription inhibitors, such as Ro24-7429 and K-12, EM2487 appeared to be a less potent inhibitor of HIV-1 in MT-4 cells than in MOLT-4 cells. EM2487 also inhibited the replication of HE (an HIV-1 clinical isolate) and HIV-2 (EHO strain) in MOLT-4 cells (Table 1). Furthermore, the compound was equally inhibitory to AZT (zidovudine)-sensitive and AZT-resistant strains (data not shown). EM2487 was active against simian immunodeficiency virus but inactive against human T-cell lymphotropic virus type I (data not shown). Antiviral activity in chronically infected cells. In the next experiment, we examined whether EM2487 could inhibit HIV1 replication in chronically infected cells. OM-10.1 cells produce little or no HIV-1 under basal conditions but do produce a significant level of virus after stimulation with TNF-␣ or phorbol 12-myristate 13-acetate (7). In fact, the level of HIV-1 p24 antigen in culture supernatants was 0.2 to 0.5 ng/ml in the absence of TNF-␣, yet it increased more than 200-fold after stimulation with 1 ng of TNF-␣/ml (data not shown). As shown in Fig. 2B, EM2487 suppressed p24 antigen production in TNF-␣-stimulated OM-10.1 cells in a dose-dependent manner. The compound completely prevented antigen production at a concentration of 0.8 ␮M. However, it did not reduce the viability and proliferation of OM-10.1 cells at concentrations up to 4 ␮M. The EC50 and CC50 were 0.075 and 12.5 ␮M, respectively (Table 2), indicating that EM2487 is a potent and selective inhibitor of HIV-1 replication in chronically infected cells. EM2487 was also inhibitory to HIV-1 replication in TNF-␣stimulated ACH-2 cells as well as MOLT-4/IIIB and U937/IIIB cells, both of which constitutively produce a large amount of virus without stimulation (data not shown). When the antiHIV-1 activity of EM2487 was compared with those of Ro247429, K-12, and lamivudine, K-12 was found to be a slightly more potent inhibitor of HIV-1 than EM2487 in OM-10.1 and MOLT-4/IIIB cells. Ro24-7429 was less active, and the RT inhibitor lamivudine was totally inactive in these cell lines (Table 2). Effect on HIV-1 proviral DNA synthesis. To gain insight into its mechanism of action, we examined whether EM2487 could inhibit HIV-1 proviral DNA synthesis in acutely infected cells. The PCR analysis revealed that, like the protease inhibitor nelfinavir, EM2487 did not affect the synthesis of HIV-1 proviral DNA even at a concentration of 5 ␮M, which was more than 50-fold higher than its EC50 in MOLT-4 cells (Fig. 3A). In

contrast, apparent suppression of HIV-1 proviral DNA synthesis was observed in the presence of the adsorption inhibitor dextran sulfate (5 ␮M) and the nonnucleoside RT inhibitor MKC-442 (1 ␮M). These results indicate that EM2487 does not interfere with early events of the viral replication cycle. Inhibitory effect on HIV-1 transcription. Since EM2487 was selected through screening in a Tat-induced reporter gene expression system, the compound was expected to be an HIV-1 transcription inhibitor. Therefore, Northern blot analysis was conducted to determine whether EM2487 could prevent HIV-1 mRNA synthesis in TNF-␣-stimulated OM-10.1 cells. As shown in Fig. 3B, EM2487 selectively suppressed TNF-␣induced HIV-1 mRNA synthesis at concentrations nontoxic to the host cells, indicating that EM2487 inhibits HIV-1 replication at the transcriptional level. To elucidate whether EM2487 primarily inhibits Tat or the cellular transcriptional factor NF␬B, experiments involving transfection of the Tat expression plasmid into W-3 and KM-3 cells were conducted. Transfection with the Tat expression plasmid or treatment with 10 ng of TNF-␣/ml induced approximately 450- or 5.6-fold increase of alkaline phosphatase production in W-3 cells, respectively (Fig. 4A). Under such conditions, EM2487 could reduce the Tatinduced alkaline phosphatase production in a dose-dependent fashion (Fig. 4B). Interestingly, EM2487 appeared to enhance the TNF-␣-induced alkaline phosphatase production in W-3 cells. On the other hand, KM-3 cells, in which the HIV-1 LTR contained two mutated NF-␬B binding sites, did not respond to TNF-␣ stimulation but did strongly respond to Tat (Fig. 4C). EM2487 also reduced the Tat-induced alkaline phosphatase production in KM-3 cells (Fig. 4D). In both cell systems, the compound did not affect basal alkaline phosphatase produc-

FIG. 3. (A) Effect of EM2487 on HIV-1 proviral DNA synthesis in MOLT-4 cells. The cells were mock infected (lane 1) or infected with HIV-1 (lanes 2 to 6) and cultured in the absence (lane 2) or presence of either 5 ␮M EM2487 (lane 3), 5 ␮M dextran sulfate (lane 4), 1 ␮M MKC-442 (lane 5), or 1 ␮M nelfinavir (lane 6). After a 24-h incubation, total DNA was extracted and subjected to PCR amplification with the HIV-1 gag-specific primer pair SK38 and SK39 (a) and the control primer pair GH20 and PC04 (b) for ␤-globin. The amplified products were electrophoresed and visualized by ethidium bromide staining. (B) Inhibitory effect of EM2487 on HIV-1 mRNA synthesis in OM-10.1 cells. The cells were incubated with the compound for 2 h, stimulated (⫹) with TNF-␣ (10 ng/ml), and further incubated. After a 24-h incubation, total RNA was extracted from the cells, blotted, and hybridized with a 32P-labeled full-length HIV-1 molecular clone, HXB2.

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FIG. 4. Characterization of W-3 and KM-3 cells and effects of EM2487 on Tat- or TNF-␣-induced transactivation in these cells. W-3 (A and B) and KM-3 (C and D) cells were either transfected with the Tat expression plasmid (10 ng) or treated with TNF-␣ (10 ng/ml). The cells were cultured in the presence of various concentrations of the compound. After a 2-day incubation, the culture supernatants were collected and examined for their alkaline phosphatase levels. At the same time, the number of viable cells was determined by the MTT method. (A and C) Alkaline phosphatase (AP) activities of culture supernatants in the absence of EM2487. (B and D) Effects of EM2487 on Tat-induced (■) or TNF-␣-induced (Œ) transactivation and viable cell number (䊐).

tion (data not shown). Furthermore, the inhibitory effect of EM2487 on Tat-induced gene expression was confirmed by a cotransfection experiment with an HIV-1 LTR-driven chloramphenicol acetyltransferase-expression plasmid and the Tatexpression plasmid in HeLa cells (data not shown). These results suggest EM2487 is inhibitory to HIV-1 Tat rather than NF-␬B. DISCUSSION Control of HIV-1 gene expression is an attractive approach to chemotherapy of AIDS. Although the cellular transcription factor NF-␬B is a potent activator of HIV-1 gene expression (15, 22), the viral transactivator protein Tat seems to play a more important role in sustaining a high level of viral replication in acutely infected cells. Since several lines of evidence suggest that repetitive acute infection accounts for most plasma viruses in HIV-1-infected individuals (26), a selective Tat inhibitor may have great potential as a candidate for the treatment of HIV-1 infection. In addition, TNF-␣-triggered

activation of NF-␬B leads to rapid production of Tat, which is necessary for continuous HIV-1 gene expression in latently infected cells, such as OM-10.1 and ACH-2. Therefore, an effective Tat inhibitor suppresses TNF-␣-induced HIV-1 gene expression in these cells, assuming that it is also able to suppress the recovery of latent virus from resting CD4⫹ T cells as well as infected macrophages in vivo. The Tat inhibitors first described in the literature are Ro53335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepin-2(H)-one] and its congener Ro24-7429 (18, 19). These compounds were shown to be active against HIV-1 in both acute and chronic infections. In fact, we also confirmed that Ro24-7429 was a selective inhibitor of HIV-1 replication in acutely infected MOLT-4 cells and chronically infected cells (OM-10.1 and MOLT-4/IIIB) (Tables 1 and 2). However, as previously reported, Ro24-7429 did not display selective inhibition of HIV-1 replication in MT-4 cells (31) (Table 1). Ro24-7429 is assumed to target a host cellular factor that binds to the transactivating response element (TAR) (6). Clinical trials of Ro247429 were halted due to lack of efficacy and some side effects

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in patients (11). More recently, it was reported that CGP64222, a hybrid peptoid-peptide oligomer of nine residues, inhibited HIV-1 replication in peripheral blood lymphocytes by blocking the formation of the Tat-TAR RNA complex (16). GCP64222 was discovered from a pool of 3.2 ⫻ 106 individual chemical entities, suggesting the extreme difficulty in discovering this class of compounds by random screening. In this study, we have identified EM2487, a novel substance produced by a Streptomyces species and a potent and selective inhibitor of HIV-1 replication in acutely and chronically infected cell cultures. Among 75,000 compounds examined for their inhibitory effects on Tat-induced HIV-1-driven reporter gene expression, less than 10 compounds were found to be active (data not shown). The active compounds were further evaluated for their inhibitory effects on HIV-1 replication in acutely infected MOLT-4 cells. EM2487 was the only compound that displayed selective inhibition of HIV-1 replication. The chemical structure of EM2487 is unique (Fig. 1), which hampers its modification and structure-activity relationship studies. EM2487 totally differs in chemical structure from CGP64222 or the fluoroquinoline derivative K-12. The latter has recently been reported as a potent and selective inhibitor of HIV-1 transcription (3). K-12, a representative of the fluoroquinoline derivatives, inhibited HIV-1 replication in both acutely and chronically infected cells, and its anti-HIV-1 activity appeared to be similar to that of EM2487 (Tables 1 and 2). Unlike Ro24-7429, both EM2487 and K-12 displayed selective inhibition of HIV-1 replication in acutely infected MT-4 cells, although their SIs were smaller than those in MOLT-4 cells (Table 1). Since K-12 is able to suppress Tat-induced transactivation, it is possible that EM2487 and K-12 share the same target molecule for Tat inhibition. However, as shown by recent studies of its mechanism of action, K-12 inhibits the Tat function in a TAR-independent fashion (unpublished data). In addition, K-12 was also inhibitory to murine retroviruses, which are devoid of accessory genes such as tat and rev, and some herpes viruses (30). K-12 reduced the production of TNF-␣ and interleukin-6 in mitogen-stimulated PBMCs (2). Thus, K-12 does not seem to directly block Tat itself or the Tat-TAR interaction but may prevent the interaction of some cellular factors with Tat. Although we have not completed extensive studies of the EM2487 mechanism of action, a recent preliminary study revealed that neither EM2487 nor K-12 interfered with Tatinduced transactivation in a cell-free assay system (data not shown), suggesting that EM2487 does not interrupt the TatTAR interaction. In conclusion, EM2487 appears to be a selective inhibitor of Tat, yet we cannot exclude the possibility that EM2487 also interacts with known and unknown cellular transcriptional factors involved in the Tat-induced transactivation (10, 28, 29, 33–35). ACKNOWLEDGMENTS OM-10.1 cells and pSV2tat72 were obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, Bethesda, Md. (contributors were S. Butera [OM-10.1 cells] and A. Frankel [pSV2tat72]). This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture. REFERENCES 1. Baba, M., E. De Clercq, H. Tanaka, M. Ubasawa, H. Takashima, K. Sekiya, I. Nitta, K. Umezu, H. Nakashima, S. Mori, S. Shigeta, R. T. Walker, and T. Miyasaka. 1991. Potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) by 5-ethyl-6-phenylthiouracil derivatives through their interaction with the HIV-1 reverse transcriptase. Proc. Natl. Acad. Sci. USA 88:2356–2360.

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