Human immunodeficiency virus gene regulation as a target for ...

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transcripts (Hammarskjold et al., 1989). Second, Rev may decrease the effect of instability elements (INS) on the viral transcripts (Rosen et al., 1988; Felber et al.
Antiviral Chemistry & Chemotherapy 10: 1–14

Review

Human immunodeficiency virus gene regulation as a target for antiviral chemotherapy Dirk Daelemans*, Anne-Mieke Vandamme and Erik De Clercq Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium *Corresponding author: Tel: +32 16 33 21 76; Fax: +32 16 33 73 40; E-mail: [email protected]

Inhibitors interfering with human immunodeficiency virus (HIV) gene regulation may have great potential in anti-HIV drug (combination) therapy. They act against different targets to currently used anti-HIV drugs, reduce virus production from acute and chronically infected cells and are anticipated to elicit less virus drug resistance. Several agents have already proven to inhibit HIV gene regulation in vitro. A first class of compounds interacts with cellular factors that bind to the long terminal repeat (LTR) promoter and that are needed for basal level transcription, such as NF-κB and Sp1 inhibitors. A second class of compounds specifically inhibits the transactivation of the HIV LTR promoter by the viral Tat protein, such as the peptoid CGP64222. A third class of compounds

prevents the accumulation of single and unspliced mRNAs through inhibition of the viral regulator protein Rev, such as the aminoglycosidic antibiotics. Most of these compounds have been tested in specific transactivation assays. Whether they are active at the postulated target in virus replication assays has, for many of them, not been ascertained. Toxicity data are often lacking or insufficient. Yet these data are crucial in view of the toxicity that may be expected for compounds that primarily interact with cellular factors. Although a promising lead, considerable research is still required before gene regulation inhibitors may come of age as clinically useful agents. Keywords: HIV; gene regulation; inhibition

Introduction During the last decade, numerous compounds have been reported to inhibit the replication of human immunodeficiency virus (HIV), the causative agent of AIDS (De Clercq, 1995). One of the major problems in HIV chemotherapy is the development of virus drug resistance (for review, see Vandamme et al., 1998). This is because HIV has a very variable genome, owing to the low fidelity of its reverse transcriptase and the high replication rate. Any treatment that is not able to completely suppress viral replication is bound to select for drug-resistant variants. Although triple-drug combination therapy can lead to a sustained suppression of viral replication, its efficiency in preventing resistance remains to be proven. A second problem in HIV chemotherapy is that even when viral replication is virtually shut down, latently infected cells can be stimulated by cytokines so as to produce virus that very rapidly repopulates the body once therapy is interrupted (Chun et al., 1998). Therefore, both free virions and latently infected cells have to be kept under

©1999 International Medical Press 0956-3202/99/$17.00

control. The replicative cycle of HIV comprises a number of steps that could be considered adequate targets for chemotherapeutic intervention (De Clercq, 1997). It can be postulated that intervention at more than a single step in the HIV replication cycle will be more efficient in suppressing viral replication and avoiding resistance. At present, 12 drugs have been approved by the US Food and Drug Administration (FDA). They are targeted at only two events in the HIV replication cycle; reverse transcription and processing by the viral protease. Therefore, the search for drugs interacting at additional steps in the viral replication cycle remains essential. HIV gene regulation inhibitors may have great potential in HIV drug combination therapy. They act against a different target of the HIV replication cycle than current clinical drugs and they may force the virus into its dormant state. Indeed, it has been demonstrated that impairment of Tat function causes post-integration latency (Emaliani et

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Figure 1. Recognition sites for cellular factors known to interact with the HIV LTR Br H O

Br

NH2 C

O + Cl

P

Cl

Et3N/Et2O

C

OCH3

CH3

O Br

O

P

O

Cl

Cl

OH 2

Cl

P

O 3

4

Cl

O

O H3C

5

H 6 N HO

5'

H3C

O

N-methylimidazole +

4

Br

O

O

NH

H3C Br H3CO O P

O

P

O

O

5'

O

NH H3CO2C

O

O

H 6 N

O

THF

Br

5

NH

N3 5 (AZT)

Br2 /MeOH

CO2CH3

N H

H

5

6

N3 6

NH N

O

5'

O

NH H3CO2C

N3

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TAR, transactivation responsive element; InR, initiator; IST, inducer of short transcripts; LBP-1, leader binding protein-1; TCF-1α, T cell factor-1α; ILF-1, interleukin factor-1; NFAT, nuclear factor of activiated T lymphocytes; AP-1, activation protein-1; COUP, chicken ovalbumin upstream promoter element; USF, upstream stimulating factor; LEF, in lymphocytes enriched factor.

region. This region includes the E-box and Ets binding sites for activators that are highly enriched in T cells: LEF, a lymphocyte-specific high mobility group (HMG) protein; the Ets-1 protein, which is a T-cell-specific factor, and the E-box binding protein USF. Recently USF has been identified as a direct interaction partner of Ets-1 and this interaction is required for full transcriptional activity of the HIV LTR in T cells (Sieweke et al., 1998). It is presumed that these cellular factors produce a complex that maintains the promoter in an open configuration ( Jones & Peterlin, 1994). The 5′-end region of the LTR contains a number of sequences that are similar to well-characterized response elements in other promoters. These include AP-1 bindingsites, a steroid-responsive sequence present in the chicken ovalbumin promoter (COUP), a sequence found in the IL2 promoter (ILF-1), and sequences implicated in the activation of T cells (NFAT). However, the functional importance of these factors for HIV replication and transcription is unclear (reviewed in Gaynor, 1995).

Transactivation by Tat al., 1998). Moreover, Tat antagonists have proven to inhibit both acute and chronic HIV infection (Hsu et al., 1991). Furthermore, it may be argued that antivirals targeted at HIV gene expression would develop less resistance as HIV gene regulation requires the interplay of both viral and cellular components.

HIV promoter elements The promoter of the HIV LTR is situated in the 5′ LTR and is similar to promoters of many cellular genes in that it contains the key regulatory elements required for transcription. The core promoter contains three tandem Sp1-binding sites, a TATA box and an initiator element (InR) (Figure 1). These three elements are essential for minimal promoter activity in vitro and in vivo ( Jones et al., 1986; Garcia et al., 1989). Viral transcripts are initiated 22 bp downstream of the TATA box. Sequences downstream of the viral RNA start site contain three different elements: LBP-1 (loop-binding protein 1), IST (inducer of short transcripts) and TAR (transactivation responsive element). The IST is a DNA element, located between –5 and +82, that stimulates the production of short abortive RNA transcripts which accumulate in cells in the absence of Tat (Sheldon et al., 1993; Pessler & Hernandez, 1998). TAR, located between +1 and +80, is an enhancer that stimulates the synthesis of productive transcripts in the presence of Tat. The role of LBP-1 in virus transcription is not yet clearly defined. Upstream of the Sp1 sites, the HIV LTR contains two NF-κB binding sites that are the focus of particular study because binding of NF-κB results in a dramatic stimulation of HIV transcription. The upstream region of the NF-κB binding sites is a less well-defined

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HIV is a complex retrovirus because control of HIV RNA synthesis is mediated via the interplay of viral and multiple cellular factors. In a first phase of HIV expression, cellular stimulation is sufficient to allow a low transcription level, resulting in only multiply spliced mRNAs (Figure 2a). These spliced mRNAs encode the HIV regulator proteins Tat and Rev. Tat is responsible for high-level HIV transcription directed from the HIV LTR promoter and is essential for viral replication (Dayton et al., 1986; Fisher et al., 1986; Feinberg et al., 1991; reviewed in Cullen, 1991, 1995). High levels of Rev result in the nucleocytoplasmatic export of unspliced and partially spliced mRNAs. This regulation is essential, as HIV relies upon differential splicing to generate the full range of viral proteins (Figure 2b) (Kim et al., 1989, Pomerantz et al., 1992). Tat functions through a specific interaction with the cisacting transactivation responsive element (TAR) of the nascent RNA that is present at the 5′-terminus of all viral mRNAs (reviewed in Gait & Karn, 1993). This 86 amino acid Tat protein has two functional domains. These are a cofactor-binding domain extending from the N terminus to residue 48 and an arginine-rich RNA-binding motif, from residue 49 to 58, that also acts as a nuclear localization signal. TAR is a secondary RNA stem structure that is unique in terms of eukaryotic transcription control because it is functional as an RNA element. In the absence of Tat protein only short, prematurely terminated transcripts from the HIV LTR promoter are initiated (Kao et al., 1987). There is a block to transcription elongation, and the polymerase complex is said to be poorly processive (Figure 2a). In contrast, in the presence of Tat this processivity block is bypassed, suggesting that Tat promotes the processivity of

©1999 International Medical Press

HIV gene regulation as an antiviral target

Figure 2. Stages in HIV gene expression (a) Early regulatory gene expression

(b) Late structural gene expression

TAR RNA Pol II

LTR TAR

P

CTD

RNA Pol II

Elongation block

P Low transcription level

CDK9/cyclin T

LTR

P P P CTD P P

High transcription level

Tat RRE Splicing and transport

Splicing and transport

Gag–Pol Rev Nef Tat

Env

+ Gag–Pol

+ Tat Nef Rev

Env RRE

(a) In a first phase only multiply spliced mRNAs encoding the regulatory proteins are expressed. Tat, through its interaction with the TAR RNA element, functions as a potent amplifier of viral gene expression, leading to a high level of regulator gene products. In the abscence of Tat, transcription elongation is inefficient because of the hypophosphorylation of the C terminal domain (CTD) of RNA polymerase II (RNA Pol II). Poorly processive RNA Pol II is able to synthesize the bulged stem loop structure TAR, which consists of the first 80 nucleotides of the transcript. The viral Tat protein in a complex with CDK9/cyclin T binds to TAR near the stalled RNA Pol II. CDK9 then hyperphosphorylates the CTD, which stimulates efficient transcriptional elongation of the nascent viral RNA. (b) Once Rev reaches its threshold level, it activates the expression of the HIV structural genes while simultanously inhibiting the production of regulatory proteins. This is mediated through interaction of Rev with its Rev responsive element (RRE). Rev is thus important in governing the transition from expression of regulatory genes to expression of structural genes.

initiated RNA polymerase II molecules (reviewed in Kingsman & Kingsman, 1996). Regulation of transcription elongation is known to be modulated by phosphorylation of the C terminal domain (CTD) of RNA polymerase II (Reines et al., 1996; Hermann & Rice, 1995; Parada & Roeder, 1996). For HIV transcriptional regulation, this hyperphosphorylation is accomplished by a cellular protein kinase called CDK9 which specifically binds to cyclin T (Wei et al., 1998), which in turn interacts directly with the activation domain of Tat (Zhu et al., 1997). Therefore, the role of Tat appears to be to recruit CDK9 kinase to the HIV LTR promoter by binding cyclin T, resulting in a hyperphosphorylation of the CTD of the RNA polymerase II complex, leading to a transition from non-processive to processive transcription (Figure 2b) (Bieniasz et al., 1998; reviewed in Cullen, 1998; Emerman & Malim, 1998). A number of studies have also shown increases in transcriptional initiation in addition to effects on elongation (Laspia et al., 1989; Bohan et al., 1992; Veschambre et al., 1995). Although Tat appears to be essential for HIV replication, it is possible, experimentally, to bypass or minimize the Tat requirement (Kaufman et al., 1987). If the level of transcription initiation is increased by the action of cytokines or viral transactivators then the probability of full-length transcripts being produced is increased. One such viral transactivator is the Tax protein present in, for

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example, the human T-lymphotropic virus (HTLV)-transformed MT-4 cell line. This is the major reason why it is very difficult to investigate the activity of Tat in MT-4, cells which are routinely used for anti-HIV drug evaluation.

Transactivation by Rev In the infected cell, Rev binds, like Tat, to a secondary RNA stem structure called the Rev responsive element (RRE) situated in the Env open reading frame (reviewed in Gait & Karn, 1993). The 116 amino acid Rev protein contains an arginine-rich stretch located toward the N terminus and serves as both an RNA-binding motif and as a nuclear localization signal. This stretch is flanked by residues that mediate Rev multimerization. Binding of Rev to the viral transcripts containing the RRE, shifts the balance from multiple spliced transcripts (encoding Tat, Rev and Nef in the early stage of virus replication) to both singly spliced and unspliced transcripts (encoding viral structural proteins in the late stage of replication) (Figure 2b) (reviewed in Luciw, 1996). The Rev post-transcriptional transactivator is hypothesized to play a critical role in viral latency and activation (Pomerantz et al., 1992). One of the difficulties involved in unravelling the Rev function is the fact that this viral regulatory protein functions through several cellular post-transcriptional mechanisms which are complex and not well understood. Rev binds to RRE-con-

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Table 1. HIV transcription inhibitors targeted at NF-κ κ B activation Compound Antioxidants N-acetylcysteine

Anti-transactivation* Activity Toxicity

Acute

IC50‡: 7–15 mM

CC50§: >50 mM

90% at 1 mM

Yes

α-Lipoic acid

IC50:1 mM

CC50:3 mM

Complete at 1 mM



3-hydroxykynurenine

Complete at 1 mM







Not up Staal et al. (1993); to 30 mM Lee et al. (1997); Mihm et al. (1991); Raju et al. (1994); Shoji et al. (1994) – Suzuki et al. (1992); Shoji et al. (1994); Baur et al. (1991); Merin et al. (1996) – Sekkai et al. (1997)

PKC inhibitors Pentoxifylline (PTX)

IC50: 0.3–1 mM

Less than activity¶

Yes

71% at 1 mM

26% at 1 mM

IC50: 2 µM 70% at 30 µM 62.3% at 300 nM

Not at 10 µM Yes >1000 nM

– Yes –

– – 50–100 nM



90% at 50 µM 40% at 2.5 µM 70% at 50 µg/mM 80% at 2mM

98% at 50 µM 76% at 2.5 µM 37% at 50 µg/mM –







Fabbri et al. (1993)













Fabbri et al. (1993); Jiang et al. (1996) Jiang et al. (1996)







Kopp & Ghosh (1994)









Kopp & Ghosh (1994)

>1 µM







Yasui et al. (1997)

ET-18-OCH3 Curcumin Gö-6976

Others Nitrogen mustard Quinacrine mustard Chloroquine

Sodium salicylate Acetylsalicylic 60% at 2 mM acid (Aspirin) Carboxyamidotriazole 90% at 1 µM

Anti-HIV activity† Chronic

Toxicity

References

Biswas et al. (1993a,b); Biswas et al. (1994); Fazely et al. (1991); Mhashilkar et al. (1997) Daniel et al. (1995)

>5000nM Mhashilkar et al. (1997); Qatsha et al. (1993)

*Activity and toxicity values obtained in a cellular bioassay based on the expression of a reporter gene under the control of the HIV LTR and stimulated with HIV Tat or mitogens. †Activity and toxicity values obtained in a cellular HIV replication assay. ‡Concentration of inhibitor required for 50% inhibition of reporter gene activity in a cellular transactivation bioassay. §Concentration of inhibitor required for 50% cytotoxicity. ¶No real toxicity measurement was performed but inhibitory activity was measured using equal amounts of protein –, Data are not known.

taining viral transcripts in the nucleus and increases their half-life. This could be achieved in several ways. First, Rev may diminish the efficiency of splicing of RRE-containing transcripts (Hammarskjold et al., 1989). Second, Rev may decrease the effect of instability elements (INS) on the viral transcripts (Rosen et al., 1988; Felber et al., 1989). The INS are found in the transcripts of gag, pol and env and promote degradation of these transcripts (Cochrane et al., 1991; Schwartz et al., 1992). The interaction of Rev with RRE may overcome the destabilizing effects of these sequences. Third, Rev may facilitate transport of RRE-containing HIV mRNAs from the nucleus to the cytoplasm (reviewed in Cullen, 1998). Although data are available that support these three mechanisms, their relative importance has not been entirely established.

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Since both the Tat and Rev functions are essential for viral replication and no cellular counterparts exist, it is clear that both Tat and Rev and the processes that they engender may serve as excellent targets for antiviral therapy.

HIV-1 Transcription Inhibitors Transcription of the HIV provirus is governed by the viral LTR promoter. This promoter’s activity is determined by a number of positive and negative transcriptional regulators. Transactivation of the HIV LTR is mediated by the viral Tat protein and by cellular factors. Most of the cellular factors known to interact with the LTR promoter are described in Figure 1. They can be considered as potential targets for HIV regulation inhibition.

©1999 International Medical Press

HIV gene regulation as an antiviral target

Almost all the HIV transactivation inhibitors described here were found to be active in a bioassay based on the expression of a reporter gene under the control of the HIV LTR. In such an assay, the reporter gene is expressed upon stimulation of the LTR. Inhibition of HIV gene expression can be monitored by a decrease in reporter gene activity. For several compounds, the antiviral activity of the inhibitors was confirmed in viral replication-based assays, which can measure either inhibition of acute infection or inhibition of chronic infection. In some cases, specific binding assays have been used to evaluate the effect of the compounds on Tat/TAR or Rev/RRE binding. Inhibitory concentrations can differ in these assays, since they are performed under different conditions.

Inhibitors of NF-κB activation Within the modulatory element of the HIV LTR, the most influential positive regulatory elements are the two tandemly repeated NF-κB elements (Figure 1). Cytoplasmatic NF-κB is detected as a complex with the inhibitory protein IκB (Baeuerle & Baltimore, 1988). Phosphorylation of IκB releases NF-κB, which is then translocated into the nucleus, where it interacts with its response elements and stimulates transciption from numerous promoters including the HIV LTR. A variety of stimuli have been found to induce this NF-κB activation, including protein kinase activators, inflammatory cytokines such as TNF-α or IL-1, viral infection, UV irradiation, oxidative stress, B- or T cell activation and other physiological and non-physiological stimuli. Several inhibitors of cellular NF-κB activation have been described to block the HIV LTR transcription (Table 1). One class of inhibitors that blocks HIV transcription through inhibition of NF-κB activation is the antioxidants. It has been shown that oxidative stress can induce the expression and replication of HIV-1 (Schreck et al., 1991). N-acetylcysteine (NAC), a well-characterized antioxidant that counteracts the effects of reactive oxygen intermediates in living cells, prevents the activation of NF-κB through inhibition of IκB degradation (Schreck et al., 1991). NAC effectively inhibits the stimulatory effect of TNF-α or phorbol 12-myristate 13-acetate (PMA) on LTR expression and the replication of HIV in T cells at a concentration that is well below the cytoxic concentration (Table 1) (Roederer et al., 1990; Staal et al., 1993; Merin et al., 1996). However, in monocyte-derived macro-phages, NAC upregulates HIV-1 replication through stimulation of viral gene expression mediated by NF-κB (Nottet et al., 1997). Administration of NAC has historically been used as a mucolytic agent in a variety of respiratory diseases and clinical studies are underway to evaluate the efficacy of NAC in HIV-infected patients. Functional protein kinase C (PKC) has been shown to

Antiviral Chemistry & Chemotherapy 10:1

be required for the activation of HIV-1 transcription ( Jakobovits et al., 1990). Futhermore, induction by phorbol esters, such as PMA (Kaufman et al., 1987), has been reported to enhance binding of NF-κB to the HIV LTR, presumably as result of the PKC-mediated dissociation of inhibitory IκB protein and NF-κB (Baeuerle & Baltimore, 1988). Protein kinase C inhibitors have been shown to inhibit HIV LTR-driven gene expression and replication through interference with NF-κB activation (Table 1). Pentoxifylline (PTX), a specific PKC inhibitor, inhibits HIV-1 LTR-driven gene expression at submillimolar concentrations (Biswas et al., 1993a, 1994). No direct toxicity measurements were carried out, inhibition of reporter gene activity driven by the HIV LTR was measured using equal amounts of protein at each concentration of PTX tested. There was a dose-dependent inhibition of reporter gene activity. The antiretroviral activity of PTX appears to be associated, at least in part, with decreased NF-κB binding to the LTR (Biswas et al., 1993b). PTX could be an interesting compound since clinical studies have shown that PTX is safe in patients. Patients in the later stages of AIDS have increased levels of serum TNF-α. Clinical studies with PTX revealed a decreased expression of TNF-α in HIV-infected patients, but these levels never fell below normal values (Dezube & Lederman, 1995). Additionally, no reduction in viral load could be demonstrated (Mole et al., 1994). Yet other PKC inhibitors may be useful in down-regulating transcription of HIV-1 provirus and thereby reducing virus replication in HIV-infected patients (Table 1).

HIV gene expression inhibitors interfering with cellular factors other than NF-κB Factors other than NF-κB that also interact with the HIV LTR promoter are suitable targets for interference with HIV gene expression. Several of these factors are illustrated in Figure 1. Interference with these cellular factors, such as interference with Sp1 binding, may lead to inhibition of HIV LTR gene expression (Gnabre et al., 1995) (Plant lignan Mal4, Table 2). Other cellular factors and enzymatic or metabolic pathways have recently been identified to interfere with HIV gene expression (Table 2), since inhibitors of these pathways seem to inhibit HIV gene expression. For example, various analogues of adenosine have been described as inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase and some of these have been reported to inhibit the replication of HIV-1 (Mayers et al., 1995). Inhibition of AdoHcy hydrolase results in an accumulation of AdoHcy, a product inhibitor of methylation reactions that use AdoMet as a methyldonor. AdoHcy hydrolase catalyses the reversible hydrolysis of AdoHcy to adenosine and L-homocysteine. Methylations of DNA, RNA, proteins and phospholipids play a crucial role in numerous

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Table 2. HIV transactivation inhibitors interfering with cellular factors other than NF-κB Anti-transactivation (µM)*

Anti-HIV activty (µM)†

Compound

Activity‡

Toxicity§

Acute

Toxicity

Specific target

References

Plant lignan Mal4

25



21

Chronic –

Little toxicity

Interferes with the binding of Sp1

Gnabre et al. (1995)

AdoHcy hydrolase inhibitors

0.26–182

>30–252

0.29–248



37–>191

Inhibits AdoHcy hydrolase

Daelemans et al. (1997); Mayers et al. (1995)

CT-2576

8–10

>32

1

1

No toxicity

Inhibits phosphatidic acid metabolism but not NF-κB activation

Leung et al. (1995)

SB203580

0.1–1









Inhibits p38 MAPkinase

Kumar et al. (1996)

*Activity and toxicity values obtained in a cellular bioassay based on the expression of a reporter gene under the control of the HIV LTR and stimulated with HIV Tat or mitogens. †Activity and toxicity values obtained in a cellular HIV replication assay. ‡Concentration of inhibitor required for 50% inhibition of reporter gene activity in a cellular transactivation bioassay. §Concentration of inhibitor required for 50% cytotoxicity. –, Data are not known.

biological processes. For a series of adenosine analogues we recently found a close correlation among their inhibitory effect on AdoHcy hydrolase activity, their inhibitory effect on HIV-1 replication and their inhibitory effect on the HIV-1 Tat-dependent and -independent transactivation of the LTR (Daelemans et al., 1997) (Table 2). These results suggest that AdoHcy hydrolase and the associated S-

adenosylmethionine-dependent methylations play a role in LTR transactivation. Recently, Turpin et al. (1998) identified a bistriazoloacridone derivative that inhibited acute HIV-1 infections and suppressed the production of virus from chronically infected cells. They showed that this bistriazoloacridone derivative exerted its mechanism of antiviral

Table 3. Inhibitors of the Tat/TAR interaction Anti-transactivation* Activity§ Toxicity¶

Anti-HIV activity† Acute Toxicity

Reference







Lapidot et al. (1995)

Yes



IC50: 10 nM– 5 µM

No toxicity

Sumner-Smith et al. (1995); O’Brien et al. (1996)

12 nM

3–5 µM

>100 µM

Completely at 30 µM

Not up to 100 µM

Hamy et al. (1997)

Tat10biotin

45 nM

15 µM

>150 µM

Strongly at 100 µM

Not at 100 µM

Choudhury et al. (1998)

CGP 40336

22 nM

1.2 µM

30% at 10 µM





Hamy et al. (1998)

Neomycin

1 µM









Wang et al. (1998)

Compound

CD50‡

THP(A)

Ki = – 50–100 nM

ALX40-4C

Yes

CGP64222

*Activity and toxicity values obtained in a cellular bioassay based on the expression of a reporter gene under the control of the HIV LTR and stimulated with HIV Tat or mitogens. †Activity and toxicity values obtained in a cellular HIV replication assay. ‡Concentration of inhibitor required for 50% dissociation of Tat/TAR complex in a gel mobility shift assay in vitro. §Concentration of inhibitor required for 50% inhibition of reporter gene activity in a cellular anti-transactivation bioassay. ¶Concentration of inhibitor required for 50% cytotoxicity. –, Data are not known.

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©1999 International Medical Press

HIV gene regulation as an antiviral target

Table 4. HIV transactivation inhibitors with an unknown molecular mechanism of action Anti-transactivation*

Anti-HIV activity†

Compound Ro5-3335

Activity IC50: 0.1– 0.5 µM

Toxicity 105 µM

Acute IC50: 0.4– 2.6 µM

Chronic IC50: 0.1– 1 µM

Toxicity CC50: 2.6– 120 µM

Reference Hsu et al. (1991); Hsu et al. (1992); Witvrouw et al. (1992)

Ro24-7429

80% at 25 µM

Not at 25 µM

IC50: 0.2– 0.7 µM

IC50: 0.1– 1 µM

CC50: >50 µM

Hsu et al. (1993)

D-Penicillamine

90% at 40 µg/ml

Not up to 200 µg/ml







Chandra et al. (1988)

Thiamine disulphide 64% at 500 µM



99% at 500 µM

80–99 % at 500 µM

Not up to 1000 µM

Shoji et al. (1994)

Oncostatin M

IC50: 9.5 ng/ml

CC50: >100 ng/ml§

IC50: >400 ng/ml



CC50: >400 ng/ml

Esté et al. (1995)

Keto/enol epoxy steroids

IC50: 2.6– >130 µM

130



SI >3‡

GCPK

IC50: 2.24 µM

SI: 13‡

IC50: 0.13– 0.62 µg/ml

IC50: 1 µg/ml

CC50: Kira et al. (1996) 3–7.9 µg/ml

Doxorubicin

Completely at >1 µM 1 µM IC50: 14 µM >60 µM

49% at 0.01 µg/ml –

– –

Not at 0.01 µg/ml –

Nakashima & Yamamoto (1987); Jeyaseelan et al. (1996) Prochaska et al. (1996)

Fluoroquinolone derivative (K-12)

IC50: 7.1 µM

CC50: >50 µM

IC50: 0.2–0.6 µM

IC50: 0.05 µM

CC50: 2.3–24 µM

Baba et al. (1998); Witvrouw et al. (1998)

Topotecan

IC50: 0.05 µM

Not at 2 µM

IC50: 0.003– 0.009 µM

IC50: 0.008 µM

CC50:0.037– 0.072 µM

Li et al. (1993)

β-lapachone

IC50: 0.3 µM

Not toxic

Yes

60% at 2.5 µM