VIROLOGY

190,

269-277

(1992)

Prevention

of the Spread of HIV-1 Infection with Nonnucleoside Reverse Transcriptase Inhibitors

M. B. VASUDEVACHARI,* C. BATTISTA,* H. C. LANE,t M. C. PSALLIDOPOULOS,” B. ZHAO,” J. COOK,* J. R. PALMER,+ D. L. ROMERO,+ W. G. TARPLEYJ AND N. P. SALZMAN*r’ *Laboratory of Molecular Retrovirology, Department of Microbiology, Georgetown University Medical Center, Washington, DC 20007; tClinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; *Medicinal Chemistry Research, Upjohn Laboratories, Kalamazoo, Michigan 4900 1; §Cancer and Infectious Diseases Research, Upjohn Laboratories, Kalamazoo, Michigan 49001 Received

March

30,

1992;

accepted

May

22,

1992

Certain bisheteroarylpiperazines (BHAPs) directly inhibit the replication of human immunodeficiency virus type 1 (HIV-l) and block the spread of infection to susceptible populations of cells. At a 1 pM concentration three analogs, U-87201, U-88204, and U-89674, inhibited the replication of HIV-l in MT-2 cells by 83, 100, and 93%, respectively. At the same concentration, U-88204 completely inhibited replication of primary HIV-l isolates in peripheral blood mononuclear cells. Replication of 3’-azido-2’,3’-dideoxythymidine (AZT)-resistant strains of HIV-1 was also inhibited by U-88204. When MT-2 cells that were lytically infected with HIV-I were mixed with uninfected MT-2 cells, U-88204 provided complete protection to the uninfected cells. Integrated proviral DNA sequences were not detected by the polymerase chain reaction technique in this culture after 15 days in the presence of drug. The resultant healthy cell culture was subsequently maintained without drug with no evidence of latent proviral DNA. Serial passage of a laboratory strain and a primary isolate of HIV-l in cell culture in the presence of increasing concentrations of U-88204 yielded virus populations which were at least 1 OO-fold resistant to the drug. These resistant viruses also showed cross-resistance to the pyridinone class of nonnucleoside inhibitors but were sensitive to AZT. Analysis of the nucleotide sequence of resistant viruses revealed mutations at conserved regions of the reverse transcriptase (RT) gene. The results presented here suggest the therapeutic potential of U-88204 in the combination therapy for HIV-l infection. o 1992 Academic

Press, Inc.

INTRODUCTION

verse transcriptase activity, although they do not all share the same mechanism of action. Recently, several nonnucleoside compounds that are derivatives of BHAPs were shown to be potent inhibitors of HIV-1 (Romero et al., 1991). One of these compounds, U-87201, at a dose of 200 mg per kg per day, was further shown to reduce the viral burden in HIV-l-infected severe combined immune deficiency mice that had been reconstituted with a human immune system (SCID-hu mice). In this report we describe the antiviral properties of three such BHAPs, U-87201, U-88204, and U-89674, against both laboratory strains and primary isolates of HIV-l. U-88204, the most active of these three, offers complete protection to HIV-susceptible cells when mixed with cells that are lytically infected with HIV-l. As an approach to understanding the mechanism of action of these compounds, we have isolated and characterized two drug-resistant mutants of HIV-l.

In recent years, there has been an extensive search for drugs that selectively block HIV-l replication. Among the various steps in the life cycle of HIV-l, the process of reverse transcription has been widely targeted in the design of antiviral compounds (Gilboa et a/., 1979; Mitsuya and Broder, 1987; Varmus, 1988). Inhibitors of reverse transcriptase cannot protect infected cells containing proviral DNA but they are able to prevent infection of cells. AZT, which effectively blocks chain elongation by reverse transcriptase, has been approved for treatment of HIV-1 infection (Fischl et al., 1987; Yarchoan et al., 1986) and offers significant protection to HIV-seropositive individuals. However, long-term use of AZT can be complicated by toxic side effects (Richman et al., 1987) and is associated with the emergence of AZT-resistant virus strains (Land et al,, 1990; Larder et al., 1989; Larder and Kemp, 1989). A variety of other nucleoside (Yarchoan et a/., 1989a,b) and nonnucleoside (Goldman et al., 1991; Merluzzi et al., 1990; Pauwels et a/., 1990) compounds inhibit HIV-l replication by interfering with re’ To whom

reprint

requests

should

MATERIALS

AND

METHODS

Drugs Compounds U-87201, U-88204, obtained from Upjohn Laboratories

be addressed. 269

0042-6822192

and U-89674 were (Upjohn Company, $5.00

Copyrcght 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed.

270

VASUDEVACHARI

loo”

0 b

Inhibition of virus replication

2 0

u 0

”

. ..~.Y.~

I

1o-3 1o-2 10-l loo 10’ lo2 Drug concentration (PM)

1

lo3

FIG. 1. Effect of BHAPs on HIV-1 N,, replication in MT-2 cells. Stock solutions (2 mg/ml) of the drugs were appropriately diluted in RPMI1640 medium and 100 ~1 of each dilution was added to three replicate wells of a 96.well flat-bottomed microtiter plate. MT-2 cells were inoculated with 1 TCID (tissue culture infectious dose) of HIV1 LA,, in a T-25 flask and incubated for 2 hr at 37”. Infected cells (2 X 1 04) were distributed to each of the wells. In cell controls and drug toxicity studies, uninfected cells were added to wells containing the required concentration of the compounds. After incubation at 37” for 7 days (in 5% CO* and humidified conditions), cell viability was determined in each well by the MTT assay. Some control wells also received vehicle ethanol without added compound. Solid lines represent inhibitory effect of BHAPs on cell killing by HIV-l; cell killing by virus in the virus control was taken as 100%. Dotted lines represent the cytotoxic activity of the compounds. Viability of cells in the wells that did not receive any compound was taken as 0% killing or 100% viability. (0) U-87201 ; (A) U-88204; and (W) U-89674.

Kalamazoo, Ml). The chemical synthesis and structures of U-87201 and U-88204 have been described (Romero et al., 1991); these drugs were dissolved in ethanol at concentrations of 2 mg/ml. U-89674 is a modified form of U-88204 in which the 5 position of the indole is substituted with a dimethylamidino group and the drug was prepared by treating the five-amino analog with /V,IV-dimethylformamide dimethylacetal in dimethylformamide. U-89674 and AZT (Sigma) were dissolved in phosphate-buffered saline at 2 mg/ml. Pyridinone compounds L-697,639 and L-697,661 (Goldman et al., 1991) were kindly provided by Emilio Emini (Merck Sharp & Dohme Research Laboratories, West Point, PA). Viruses HIV-1 (LAV.04/A3.01, catalog no. 235) was obtained from Malcom Martin (Barre-Sinoussi et a/., 1983), two AZT-resistant strains of HIV-1 (catalog nos. 629 and 964) and MT-2 cells (catalog no. 237) from Douglas Richman (Richman et a/., 1987; Harada et a/., 1985), and the HS/HTLV-III, cell line from Robert Gallo (Popo-

ET AL.

vie et al., 1984) through the AIDS Research and Reference Reagent Program, NIAID, NIH (Bethesda, MD). Primary HIV-1 isolates were obtained by culturing peripheral blood mononuclearcells(PBMC)fromseropositive individuals who are participating in clinical programs at the NIAID, NIH (Bethesda, MD) with normal phytohemagglutinin-stimulated PBMC [maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 10% interleukin-2, 1.5 mM HEPES, and gentamicin (60 pg/ml)]. Cell-free supernatant fluid collected when the cultures showed peak reverse transcriptase activity was used as the virus stock. Quantitation

of virus

Release of infectious virus was measured by collecting aliquots of supernatant fluid at several time points and measuring p24 antigen levels using the Coulter p24 antigen assay (Coulter Immunology, Hialeah, FL). Cytotoxicity

assay

The microtiter cytotoxicity assay used was based on the ability of living cells to reduce the tetrazolium salt MTT (3-4, 5-dimethylthiazol-2-yl-2-5-diphenyl tetrazolium bromide) and form a blue formazan product (Tada et a/., 1986). Polymerase

chain reaction

(PCR)

Cellular DNA isolated at the indicated time points was assayed using a PCR amplification method (Saiki et al., 1988; Psallidopoulos et a/., 1989) with SK 68/69 and SK 145/l 50 primers from the HIV-1 env and gag

TABLE

1

EFFECTOFBHAPSONTHEREPLICATIONOFPRIMARYISOLATESOF Inhibition Drug U-87201 U-88204 U-89674 AZT

of p24 antigen

HIV-1 synthesis

(%)

A”

B

C

D

E

87’ 100 100 100

47 100 100 100

63 100 100 100

48 100 100 100

64 100 99 100

a PBMC were infected with the virus stocks from five HIV-1 seropositive patients (A, B, C, D, and E) at 5 TCID. Virus was allowed to adsorb for 2 hr at 37”, then the cells were washed and resuspended at a final concentration of 1 05cells per milliliter in medium containing the indicated drugs at a concentration of 1 PM. The cell cultures were maintained for 7 days at 37” in 5% CO, and humidified conditions. b Values represent percentage inhibition of p24 antigen synthesis compared to control (HIV-l-infected cultures without any added drugs).

NONNUCLEOSIDE

regions, 1988).

respectively

(Krone et al., 1990;

Ou et al.,

Radioimmunoprecipitation On Day 8 after exposure to virus, cells were incubated in medium lacking methionine for 1 hr and then labeled with [35S]methionine and [35S]cysteine (total radioactive isotope concentration: 0.075 mCi/ml) for 6 hr. The labeled cells and supernatants were separated by centrifugation. The cells were lysed with RIPA buffer [20 mMTris-HCI (pH 7.5), 2 mM EDTA, 150 mM NaCI, l%Triton X-l 00,0.25% SDS, and 1 mM phenylmethylsulfonyl fluoride] and mixed with pooled human serum containing high-titer antibodies to HIV-1 and with protein A-Sepharose beads, and the mixture was rotated at 4” overnight. The beads were washed twice with RIPA buffer and bound proteins were eluted from the beads. Eluted proteins were subjected to electrophoresis on 12.5% SDS-polyacrylamide gels, and the gels subjected to fluorography. Isolation

and analysis of the drug-resistant

viruses

Two isolates of HIV-l (LAV and a primary patient isolate, F890) were serially passaged in MT-2 cells in the presence of increasing concentrations of U-88204 (0.15,0.3,0.6, 1.0,3.0,and lOj&‘)ateachpassageof 1O-l 4 days. Supernatants from cultures treated with 3 pM U-88204 were collected and used as drug-resistant viral stock (LAV’ and F890’). A cDNA copy of viral RNA was synthesized using synthetic oligonucleotide primer 3’-GTCCATTTATCAGGATGG (nucleotides 3365-3381). PCR amplification of the cDNA was carried out using the primer pair 5’-CCAGTCAACATAATTGGA (2606-2623) and 3’-AGTTCATAACCCATCCAAAG (3344-3363). The PCR-amplified segments were ligated with a TA cloning vector (pCR1000 from lnvitrogen Corp., San Diego, CA) and used to transform Escherichia co/i. Bacterial colonies containing the cloned sequences were identified by colony hybridization and the DNAs purified from positive clones were sequenced using the Sanger chain termination method (Sanger et al., 1977) and Sequenase T7 DNA polymerase (U.S. Biochemical Corp., Cleveland, OH). RESULTS Inhibition

of HIV-1 replication

by BHAPs

We investigated the effect of three BHAPs on infection of MT-2 cells with the LAV strain of HIV-1 (HIV1LAv)in vitro. The cultures were maintained for 7 days in the presence of drug and cell viability was determined

INHIBITORS

OF

271

HIV-1

by the MTTassay. All three compounds inhibited HIV-1 infection in a dose-dependent manner, but U-88204 showed an antiviral effect at lower concentrations than U-87201 and U-89674 (Fig. 1). The IC,, values (concentration required for 50% inhibition of viral ~24 antigen synthesis) for U-87201, U-88204, and U-89674 were 0.39, 0.07, and 0.22 pM, respectively. The cytotoxic effect of BHAPs on MT-2 cells is also shown in Fig. 1. All three compounds at a concentration of 10 pM or lower had no cytotoxic effect during 7 days as judged by MTT uptake by cells. The TD,, values (concentration at which 50% of the cells are killed) of the three compounds were in the range of 28 to 35 pM. No changes in cell morphology were observed when cultures were incubated with the drugs at concentrations of 10 pM or lower. Two AZT-resistant strains of HIV-l were as susceptible to U-88204 as the AZT-sensitive viruses (data not shown). The ability of BH,APs at a concentration of 1 pM to inhibit the replication of primary isolates of HIV-1 in PBMC was also studied. AZT (1 pM) was also included as a control. Growth of all five primary isolates tested was completely inhibited by U-88204, U-89674, and AZT. The drug U-8720 1, however, inhibited replication of the isolates by 47 to 87% (Table 1). The relative effectiveness of these three drugs was the same in PBMC infected with various primary isolates as in HIV1,-infected MT-2 cells (Fig. 1).

Blocking the spread of HIV-1 infection

by U-88204

In the patients with HIV-l infection, only a fraction of peripheral blood lymphocytes are infected. To more closely approximate those conditions that exist in viva we wanted to determine if these antiviral compounds would protect uninfected cells in vitro by preventing the spread of HIV-1 from infected cells. For this purpose, HIV-1 -infected MT-2 cells (2 days after infection) were cultured together with uninfected MT-2 cells at a ratio of 1: 1000 in the presence or absence of BHAPs or AZT. These conditions differ significantly from those of the previous experiments in that cell-to-cell transmission of virus can occur with much higher frequency. Cultures were diluted every 2 to 3 days to maintain cell densities in a suitable range for exponential growth. Using replicate cultures on days 12, 15, 18, 21, and 24, drug treatment was terminated by total replacement of the medium with fresh drug-free medium, and the cultures were continued for up to 47 days. Viable cell number and viral p24 antigen in the supernatant were used as measures of HIV-l spread. In the absence of drug treatment, most of the virus-infected cells were killed by Day 6 and no viable cells were

272

VASUDEVACHARI

-

I?

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ET AL

2

1

0

" g 4

lo4

g

lo2 loo

0

10:

5

15

C. Protochl

20

25

for Drug

30 35 Days

U-88204

40

and

45

50

55

60

PCR Analyses

Culture Terminated INFECTION DAYS 'OSTSAMPLE

NUMBER

3

6

9

12

15

15

18

40

A

A

A

A

A

A

Lb hi

Days

P:??&J

142bp+ &?a&!

FIG. 2. Effect of BHAPs on the spread of HIV-1 infection. MT-2 cells were infected with 5 TCID of HIV-l. After 2 days the cells were washed and mixed with uninfected MT-2 cells at a ratio of 1: 1000, and the mixed cell population was placed in 24-well tissue culture plates at a final cell density of 2 X 1 O5 cells per well. Individual BHAPs or AZTwere added to selected wells to a final concentration of 1 PM. On Day 3 and every 2 to 3 days thereafter, the cultures were diluted approximately threefold by removing two-thirds of the cell suspension and replacing the volume with fresh medium containing the corresponding drugs, in order to avoid overcrowding of cells. On Days 12 (vertical dotted line) and 15 (vertical dotted-dashed line), U-88204.treated cells were separated by centrifugation and resuspended in medium without drug. Surviving cells were reinfected with HIV-1 on Day 47 (solid vertical line). (A) Relative viable cell numbers were determined by the MTT procedure and are expressed as absorbance units at a wavelength of 570 nm (A&. (B) The concentration of p24 antigen in the culture supernatant was quantitated by p24

NONNUCLEOSIDE

present by Day 9 (Fig. 2A). The increases in the concentrations of p24 antigen were, as expected, directly proportional to the extent of cell killing (Fig. 2B). All three BHAPs and AZT delayed killing of the cells by virus, but only cells cultured in the presence of U-88204 survived until Day 15. Cells treated with U-88204 grew as well as uninfected cells and the amount of p24 antigen produced at Day 9 was 0.01% of that produced in cultures where no drug was added. In the culture in which compound U-88204 was discontinued on Day 12, cell viability gradually decreased, with a simultaneous increase in p24 antigen, and the cells were completely killed by Day 21. However, when the drug was removed on Day 15, cell viability was comparable to uninfected controls and p24 antigen concentrations remained undetectable for the entire time the culture was maintained (47 days). Similar data were obtained when U-88204 was removed on Days 18, 21, and 24 (data not shown). Prior treatment with U-88204 did not alter the susceptibility of the cells to HIV-l infection, and surviving cells were readily infected on Day 47 when fresh virus was added in the absence of the drug (Fig. 2). To determine whether these surviving cells in the U-88204-treated culture contained integrated proviral DNA, we performed polymerase chain reaction (PCR) amplification of the DNA isolated from cells (Fig. 2C). HIV-1 DNA was detected for up to 12 days after mixing infected and uninfected cells, but was absent on Day 15 in cultures maintained in the presence of the drug. When U-88204 was removed on Day 12 and the culture was incubated for another 3 days in the absence of drug, there was a strong PCR signal, indicating renewed virus replication. Surprisingly, in cultures in which the drug was removed on Day 15, at all times during the subsequent 32 days the cells remained negative by PCR performed with primers selected to amplify either the gag or the env genes, even when the autoradiographs were exposed for as long as 48 hr.

INHIBITORS

OF

HIV-1

Effect of U-88204

273

on HIV-I viral protein

synthesis

Viral proteins in cells that had been exposed to HIV1 ,for 8 days in the presence or absence of drug were labeled with [35S]methionine and [35S]cysteine. Cell lysates obtained in the absence of drug revealed all the major HIV-l structural proteins when subjected to immunoprecipitation with antiserum to HIV-1 (Fig. 3A, lane 1). However, no viral structural proteins were synthesized in the presence of 1 @III U-88204 (Fig. 3A, lane 2). Viral proteins were also absent in the supernatants of the culture treated with U-88204 (data not shown). Lysates from both untreated and drug-treated cells were subjected to electrophoresis without immunoprecipitation to monitor the effect of the drug on cellular protein synthesis. Treatment of cells with drug did not seem to affect host cell protein synthesis as indicated by the similar protein band patterns in both treated and untreated samples (Fig. 3B, lanes 1 and 2). Isolation viruses

and characterization

of drug-resistant

Passaging of LAV and F890 at increasing concentrations of U-88204 resulted in the selection of drug-resistant mutants (LAV’and F890’, respectively). Replication of mutants was observed in the presence of up to 10 PM U-88204. We tested the cross-resistance of the two mutant viruses to AZT and pyridinone inhibitors. Replication of both LAV’ and F890’ was inhibited by AZT at 1 PM concentration similar to their parent virus isolates LAV and F890 (data not shown). F890’ showed cross-resistance to two pyridinone class inhibitors (L-697,661 and L-697,639) and was able to replicate at concentrations as high as 3 PM. LAV’ showed cross-resistance, but was able to replicate only at drug concentrations of pyridinone inhibitors of 0.03-0.1 pM (Table 2). cDNAs prepared from the viral RNA of both U-88204susceptible (LAV and F890) and -resistant (LAV’and F890’) viruses were cloned into plasmid vec-

antigen enzyme immunoassay. (0) U-87201 ; (A) U-88204; (m) U-89674; (v) AZT; and (+) no drug added. (a) Cells after removal of U-88204. (C) PCR analysis of the culture performed in the presence of U-88204. Pos and Neg correspond, respectively, to infected and uninfected MT-2 cell controls. DNA from cell lysates of 5 X 1 O5 MT-2 cells (collected on the indicated days) was amplified with HIV-1 -specific primers from either the gag (SK i 451150) or the env genes (SK 68/69). The specific amplified sequences were detected by liquid hybridization with 3ZP-labeled SK 102 or SK 70 probes for gag- and env-specific PCR products, respectively. The heteroduplex product representing part of the PCR material was analyzed on 10% polyacrylamide gels, and autoradiography was performed at -70” for 18 hr. Bands corresponding to the PCR-specific products of 142.bp length are indicated in the figure. The protocol for U-88204 is illustrated schematically in the middle of the figure, Shaded areas indicate presence of U-88204 in the culture medium. Aliquots of cells were obtained on Days 3, 6, 9, and 12 (samples 1, 2, 3, and 4 respectively). On Day 12 the culture was divided. One culture was maintained free of U-88204. Sample 5 was harvested on Day 15 from the culture that had been free of drug for 3 days. The second culture was maintained in the presence of U-88204 until Day 15 when a fraction of the cells were collected for PCR analysis (sample 6). The remainder of the cells were collected by centrifugation and resuspended in drug-free medium. Cells obtained on Days 18 (sample 7) and 41 (sample 8) were also used for PCR analysis. In all PCR analyses, an HIV-l-positive control (ACH2 cells), an HIV-l-negative control (A3.01 cell line), and a buffer control containing all reagents except DNA were included. With the gag primers used, some DNA is retained at the top of the gel and its concentration is related to the quantity of the specific 142-bp PCR product, We have not further characterized the retained DNA.

274

VASUDEVACHARI

B

A 1 200 -

TABLE 2 +

COMPARISON

-U-88204

gP160 HIV-1 ~85 P55 P51 clP41

6843 -

~32 ~24

18-

(b) F890r

FIG. 3. Effect of U-88204 on HIV-1 and cellular protein synthesis. (A) lmmunoprecipitated proteins from infected cells and (B) total labeled cellular proteins from uninfected cells without immunoprecipitation. Cultures in lanes 1 were incubated in the absence of U-88204 and cultures in lanes 2 were incubated with drug. Molecular mass standards in kDa are indicated on the left. The positions of HIV-1 specific proteins are also indicated. The intense bands at approximately 200 and 43 kDa are cellular proteins seen both in the presence and absence of the drug.

the DNAs from positive clones were seThe clones correspond to amino acid (aa) between 80 and 200 of the HIV-l RT (which 560 aa). The deduced aa sequences from

TABLE

2

ABILITYOF U-88204~XTANTHIV-1 VARIANTSTOGROW IN THE PRESENCE OF NONNUCLEOSIDE INHIBITORS

Virus? LAV’

F890’

resistant

to

Pyridinone RT inhibitor (L-693,593) Nevirapine (BI-RG-587) TIBO (R-8291 3) BHAP (U-88204) (a) LAVr

29 -

Drug concn (WV

Extent

of virus

replication

(%)”

U-88204

L-697,661

L-697,639

0.03 0.1 0.3 1.0 3.0 0.03 0.1 0.3 1.0 3.0

100 100 100 100 43 100 100 98 78 32

68 10 1 0 0 100 100 94 63 56

40 4 0 0 0 100 100 100 68 43

a Values represent percentage of control replication (virus without added drug). Virus replication was determined by p24 antigen levels in the culture supernatant on Day 6 after infection. b Prior to selection, replication of LAV and F890 was completely inhibited (00/o growth) by 0.03 and 0.1 PM concentration of the drugs, respectively.

3

OF AMINO ACID CHANGES OBSERVED IN NONNUCLEOSIDE HIV-1 RT DRUG-RESISTANT STRAINS

clP120

97-

tors and quenced. positions contains

ET AL.

Mutations in HIV-1 RT at aa position K-103 Y-181 Y-181 Y-l 88

to to to to

N C I or V I or L

L-100 to I (all three clones) V-106 to A (clone 1) Y-181 to C (all three clones)

Reference Nunberg et a/., 1991 Shih et al., 1991

Present communication

three positive clones in each category were compared with the corresponding aa sequences of HXB2 (Myers et a/., 1991). The three LAV’ clones each showed an L-l change at aa position 100 in the RT gene (one also showed a V-A change at aa position 106). The F890’ clones showed a Y-C change at aa position 181 of the RT gene (Table 3). In addition one LAV’ clone had V-l 89 to I change and one F890’ clone had Y-l 44 to H and N-147 to K changes. DISCUSSION One of the unusual characteristics of the HIV disease process is the variability from individual to individual in the rate of disease development from the time of seroconversion to the development of acquired immune deficiency syndrome (AIDS). There is evidence that suggests that infectious virus is present throughout the course of the disease (Jackson et al., 1990; Michael et al,, 1992), and the presence of infectious virus in plasma provides direct evidence of a chronic viremic state. When patients with AIDS and AlDS-related complex (ARC) are compared to asymptomatic seropositive individuals, there is a pronounced increase in the extent of plasma viremia associated with disease progression (Coombs et a/., 1989; Ho et al., 1989). The extended periods of time during which low virus levels persist in the absence of clinically adverse effects may reflect containment of infectious virus by the immune system (Sei et a/., 1989). Although factors that trigger an accelerated rate of virus replication are not understood, disease progression is associated with elevated levels of virus in plasma. High levels of newly synthesized viruses result in lytic infection of CD4+ T lymphocytes, with a concomitant decrease in

NONNUCLEOSIDE

their number and a rapid progression of the disease (Schnittman er al., 1989, 1990). Agents that suppress virus replication can thus be expected to extend the quiescent stage of the disease. Our results demonstrate that BHAPs and, in particular, U-88204, are potent anti-HIV-l agents, capable of suppressing in vitro infection by extracellular virus as well as blocking the spread of virus from infected to uninfected cells. These drugs have been shown to inhibit HIV-l reverse transcriptase activity by binding to the enzyme in a noncompetitive manner with respect to the substrate and template:primer (Romero et al., 1991). Nonnucleoside inhibitors of reverse transcriptase, such as 6-substituted acyclouridine derivatives (Baba et al., 1989) and benzodiazepines (TIBO derivatives) (Merluzzi et al., 1990; Pauwels et al., 1990), have been described recentlywhich, like BHAPs, also specifically block the replication of HIV-l. All these inhibitors show a wide separation of their antiviral concentrations and their cytotoxic concentrations. U-88204 inhibited HIV-l replication at a concentration that was almost one-hundredth of that at which cytotoxic effects were apparent. Although the three BHAPs and AZT when tested at 1 PAI effectively inhibited HIV-1 replication in MT-2 cells, except for U-88204 they could only delay the virus-induced cell killing when uninfected MT-2 cells were cultured with infected cells. U-88204, however, at 1 pLM, was able to completely block the spread of infection between infected and uninfected cells. In most instances, the inhibition of viral growth by U-88204 was more dramatic than that by AZT. The absence of HIV-1 proviral DNA sequences in the cells cultured with HIV1 -infected cells in the presence of drug for 15 days indicates that U-88204 completely blocked a new cycle of infection. In addition to protection of uninfected cells and the complete exclusion of proviral DNA sequences, U-88204 treatment resulted in healthy cell cultures that could be maintained indefinitely without evidence of latent virus and also remained susceptible to fresh virus challenge. The conditions under which U-88204 was effective parallel conditions that exist in individuals with AIDS, in whom a fraction of the susceptible cell population (1 in 100 to 1000 CD4+ T lymphocytes) is actively infected (Schnittman et a/., 1989). A single infectious unit of HIV-l has been shown to kill a population of A3.01 cells within 14 to 21 days (Ashorn et a/., 1990). The absence of p24 antigen and proviral DNA in our cultures even 32 days after removal of U-88204 indicates that the drug completely inhibited virus spread and that the infected cells harboring HIV-1 had died during that time period. Similar results have been observed when HIV-l-infected cultures were

INHIBITORS

OF

HIV-l

275

treated with both AZT and human CD4 linked to Pseudomonas exotoxin (Ashorn et a/., 1990). The drug U-88204 at a concentration of 2 @I did not inhibit the replication of HIV-l in chronically infected H9 cells (HS/HTLV-Ill,). However, when these chronically infected cells were cultivated with uninfected H9 cells at a ratio of 1 :lOOO, U-88204 completely inhibited new rounds of HIV-1 infection on Days 4 and 7 as evidenced by undetectable concentrations of p24 antigen (unpublished results). The concentration of U-88204 required to inhibit p24 antigen synthesis by 50% in such a culture system was found to be 60 nM, a value comparable to that obtained with acutely infected MT2 cells. Such results are typical of drugs that effectively block the synthesis of proviral DNA. We have identified mutations in a portion of the RT gene in drug resistant isolates. They are located in regions previously reported as responsible for resistance to other nonnucleoside compounds, such as pyridinone inhibitors, nevirapine, and TIBO compounds (Nunberg et a/., 1991; Shih et a/., 1991). The region that surrounds aa position 100-l 15 is involved in PPi exchange (Larder et al., 1987; Nunberg eta/., 1991). The YG/MDD motif (around aa position 181) is conserved in many DNA and RNA polymerases (Argos, 1988; Barber et a/., 1990; Johnson et al., 1986; Smith et al., 1990; Nunberg eta/., 1991). While aa changes in either of these domains generate drug-resistant virus variants, changes at Y-l 81 or Y-l 88 confer a higher level of drug resistance than changes that occur around aa position 100 of RT (Nunberg et al., 1991; Shih et a/., 1991). In agreement with these findings, LAV’ that contains mutations in the domain encompassing aa 100-l 06 was resistant to lower concentrations of pyridinone inhibitors than F890’ which had a mutation at the YG/MDD motif. However, LAV’ and F890’ both show resistance to the same concentration (33 PM) of U-88204. It is possible that the two physically distinct regions (PPi exchange and YG/MDD) may spatially interact in the folded RT molecule contributing to the comparable resistance to U-88204 observed with both mutants. The variation in the pattern of resistance to U-88204 and pyridinone inhibitors may reflect differences in their enzyme-binding properties. It has been shown that pyridinones bind only to the enzyme-template-primer complexes and not to the enzyme alone (Goldman et al., 1991) while BHAPs bind to the free enzyme. The interpretation of the sequence data, while consistent with other published data, needs further experimental verification by transferring the restriction fragments containing the mutations into the HXB2 genetic background to confirm that the observed changes indeed confer drug resistance. We cannot exclude the possibility that our limited analysis may not

276

VASUDEVACHARI

reflect all the possible changes that could be associated with drug resistance. Selection of resistant variants in PBMC or other types of primary cell culture might reveal even more variation among the drug-resistant viral strains. The emergence of drug-resistant strains of HIV-l and the toxicity of several antiviral nucleoside analogs has led to the use of combination antiretroviral regimens for the treatment of HIV-l infection (Groopman, 1991). In this context, U-88204 is a good candidate for combination therapy. Combination therapy approaches may also benefit from potential synergism in the antiviral effects of mechanistically distinct inhibitors of reverse transcriptase while minimizing the selection of drug-resistant strains. ACKNOWLEDGMENTS We thank Kathy Uffelman for providing the initial stocks of primary viral isolates and Robin Dewar and V. Natarajan for reviewing the manuscript and Ann Stephens for help in reading the nucleotide sequence. This work was supported by funds from the Department of Health and Human Service under contract NOI-Al-05058 with Program Resources, Inc.

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