Proc. Natl. Acad. Sci.
USA
Vol. 90, pp. 1711-1715, March 1993
Medical Sciences
Potent and highly selective human immunodeficiency virus type 1
(HIV-1) inhibition by a series of a-anilinophenylacetamide
derivatives targeted at HIV-1 reverse transcriptase
RUDI PAUWELS*t, KOEN ANDRIES*, ZEGER DEBYSER*, PAUL VAN DAELE*, DOMINIQUE SCHOLS*,
PAUL STOFFELS*, KAREN DE VREESE*, ROBERT WOESTENBORGHSI, ANNE-MIEKE VANDAMME*,
CORNELUS G. M. JANSSENt, JEF ANNE*, GEERT CAUWENBERGHI, JAN DESMYTER*, JOZEF HEYKANTS*,
MARCEL A. C. JANSSENt, ERIK DE CLERCQ*, AND PAUL A. J. JANSSEN*
*Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and *Janssen Research Foundation,. Turnhoutseweg 30,
B-2340 Beerse, Belgium
Communicated by Baruch S. Blumberg, September 8, 1992
ABSTRACT In vitro evaluation of a large chemical library
of pharmacologically acceptable prototpe compounds in a
high-capacity, cellular-based screening system has led to the
discovery of another famlly of human m virus
type 1 (HIV-1) inhibitors. Through optimiation of a lead
compound, several a-anilMnphenylacetamide (a-APA) deriv-
atives have been identified that inhibit the repUcation of several
HIV-1 strains (1B/LAI, RF, NDK, MN, HE) in a variety of
host cell ypes at concentrations that are 10,000- to 100,000-
fold lower than their cytotoxic concentrations. The IC5. of the
a-APA derivative R 89439 for HIV-1 cytopathicity in MT-4
cells was 13 nM. The median 90% inhibitory concentration
(IC5o) in a variety of host cells was 50-100 uM. Although these
a-APA derivatves are active against a tetrahydrohmkao
[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-reslstant
HIV-1 strain, they do not inhibit replication of HIV-2 (strains
ROD and EHO) or simian immunodeficency virus (strins
Mac251, mndGB1, and agm3). An HIV-1 strain containing the
Tyrl1 -) Cys mutation in the reverse transcriptase region
displayed reduced sensitivity. a-APA derivative R 89439 in-
hibited virion and recombiant reverse transciptase of HIV-1
but did not inhibit that of HIV-2. Reverse transcriptase inhi-
bition depended upon the template/primer used. The rela-
tively uncomplicated synthesis of R 89439, its potent anti-
HIV-1 activity, and its favorable pharmacokinetic profie make
R 89439 a good candidate for clinical studies.
As of 1992, the World Health Organisation (WHO) estimates
that worldwide -10 million people are infected with the
human immunodeficiency virus (HIV). In particular, Africa;
Southeast Asia, and South America are now faced with a
rapidly growing number of HIV infections. In addition to
preventive campaigns and treatment of opportunistic dis-
eases, managing and counteracting the HIV/AIDS pandemic
also requires effective antiviral therapy. Ideally, anti-HIV
drugs should be highly selective, have good oral availability
and favorable pharmacokinetics, and allow large-scale pro-
duction at reasonable costs to bring them within reach of
more of the world population.
We described previously a family of highly potent and
selective reverse transcriptase (RT) inhibitors, the tetrahy-
droimidazo[4,5,1-jkl[1,4]benzodiazepin-2(1H)-one and
-thione (TIBO) derivatives that exhibit distinctive specificity
for HIV-1 (1, 2) and are devoid of serious side effects upon
administration in vivo (3). Other compounds have been
reported, which, despite their different chemical structures,
exhibit a TIBO-like antiviral profile. These include the [1-(2-
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
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1711
hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) de-
rivatives (4), the dipyridodiazepinones (5), the pyridinones
(6), and the bis(heteroaryl)piperazines (7). All of these com-
pounds interact allosterically with the RT-template/primer
complex (1, 2, 5-9).
Here we describe another chemical family of HIV-1-
specific RT inhibitors, the a-anilinophenylacetamide (a-
APA) derivatives, which inhibit HIV-1 replication in vitro at
nanomolar concentrations 10,000- to 100,000-fold lower than
their cytotoxic concentrations. One of them, R 89439, has
good oral availability and favorable pharmacokinetics and
can be easily synthesized on a large scale.
MATERIALS AND METHODS
Compounds. The a-APA derivatives were prepared from
2,6-dichlorobenzaldehyde, a substituted aniline, and sodium
cyanide in acetic acid (10), followed by hydrolysis of the
intermediate aminonitrile in sulfuric acid (11). The radiola-
beled a-APA derivative R 18893 and its two enantiomers R
87231 and R 87232 were synthesized by tritiation of 3-bromo-
2,6-dichlorobenzylalcohol to 2,6-dichloro-3-tritiobenzylalco-
hol. Oxidation to the benzaldehyde, aminonitrile synthesis
with 2-nitroaniline, and hydrolysis gave the 3H-labeled R
18893 in a 40% radiochemical yield. Resolution by means of
HPLC over a chiral column (ChiralCel O.J.) afforded both
enantiomers with enantiomeric excess >99%o (261 GBq/
nmol, radiochemical purity >99.0%6).
Viruses. The origin of the virus strains was as follows: IIIB
(LAI) and RF (R. C. Gallo, National Institutes of Health);
NDK (12); MN (Medical Research Council AIDS Directed
Programme Reagent Project and the National Institute of
Allergy and Infectious Diseases AIDS Research and Refer-
ence Reagent Program); HE, isolated in our laboratory from
a Belgian AIDS patient; ROD (L. Montagnier, Pasteur In-
stitute, Paris); EHO (13); simian immunodeficiency virus
(SIV)M=,251 (14); SIV., (15); -)SIV,mndGBl (16). The HIV-1
mutant strains 13MB1 (Leu00 Ile) and 13CN1 (Tyrl81-*
Cys) were isolated in our laboratory after serial passage of the
IIIB/LAI strain (MT-4 cells) and the NDK strain (CEM) cells,
respectively, in the presence of TIBO R 82913 at 0.5 ,tg/ml
(unpublished data).
Antiviral Assays. These assays have been described in
more detail elsewhere (1, 17). Exponentially growing MT-4
Abbreviations: HIV-1, human immunodeficiency virus type 1;
a-APA, a-anilinophenylacetamide; ddI, dideoxyinosine; TIBO, tet-
rahydroimidazo[4,5,1-jkI[1,4]benzodiazepin-2(1H)-one and -thione;
HEPT, [142-hydroxyethoxy)methyl]-6(phenylthio)thymine; RT, re-
verse transcriptase; p.i., postinfection; MTT, 3-(4,5-dimethylthia-
zol-2-yl)-2,5-oliphenyltetrazolium bromide.
tTo whom reprint requests should be addressed.
1712 Medical Sciences: Pauwels et al. Proc. Natl. Acad Sci. USA 90 (1993)

cells were either infected with 100-500 CCID50 (1 CCID50
being the viral dose infective for 50% of the cell cultures) or
mock-infected. Five (MT-4 cells) to 6 (MOLT-4) days after
infection the viability of mock- and HIV-infected cells was
examined either microscopically by trypan blue exclusion
(MOLT-4) or spectrophotometrically (MT-4, MOLT-4) by a
tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT)-based method (17). Phytohe-
magglutinin-stimulated peripheral blood lymphocytes were
infected with a concentrated HIV-1 [strain IIIB(LAI)J stock
solution. Fresh medium without compound was added at day
4 after infection. HIV-1 p24 core protein in the cell culture
medium was quantified at 8 days after infection by an
antigen-capture assay (DuPont). The inhibitory activity of the
to parallel cultures in microtiter plates at different time points
post infection (p.i.) at concentrations -100-fold higher than
their IC50 values in the MT-4/MTT assay. HIV-1 p24 core
protein (p24) was determined 29 hr p.i. by a sandwich ELISA
(DuPont).
RT Assays. In the exogenous RT assay (8), the reaction
mixture (50 ,ld) contained 50 mM Tris HCl (pH 8.4), 10 mM
MgCl2, 100 mM KCI, 2.2 mM dithiothreitol, 2.5 ,uM dGTP,
and 0.05% (wt/vol) Triton X-100. The templates poly(C) or
poly(dC) and the primer (dG)12 18 (Pharmacia) were used at 40
and 6 ,ug/ml, respectively. Recombinant HIV-1 RT p66/p66
(P. J. Barr, Chiron) and HIV-2 p68/p68 RT (S. Hughes,
Frederick Cancer Research Facility) were used at concen-
trations of 1.1 nM. Recombinant RT Tyr'81 --
Cys, Tyr181 -_

compounds on expression of HIV-1 proteins in CEM cells
was determined at day 4 after infection by an indirect
immunofluorescence-laser cytofluorometric (FACS) method
(18). All experiments were conducted at least twice, and final
data are expressed as the median of the IC50 values.
Time of Addition Experiment. Delineation of the drug-
sensitive phase was determined in MT-4 cells infected at a
high multiplicity of infection (0.1-1) with HIV-1 (IIIB/LAI).
After 60-min incubation at 37°C, unadsorbed virus was re-
moved by three washing steps. Compounds were then added
Ile, and Tyr188 Leu mutants were constructed by site-
directed mutagenesis as described (19) and were used at 20
nM. The vector pKRT2 containing the HIV-1 RT-encoding
fragment of HIV-1 under control of trc promoter was ob-
tained through the AIDS Research and Reference Reagent
Program, Division of AIDS (National Institute of Allergy and
Infectious Diseases) (20). For kinetic studies, recombinant
p66/pSi (R. Bhikhabhai and B. Strandberg, University of
Uppsala) was used. In the endogenous RT assay, the reaction
mixture (50 ,ul) consisted of 50 mM Tris-HCl (pH 8.4), 2.5 mM

H R2

0 NH

H R

Compound Mr Isomer R1 R2 IC50,t nM CC50,t AM SOi RT IC50,1 p&M

Test
R 15345 325.2 (+) OCH3 H 610 15 25 9
R 18893 340.2 (±) NO2 H 88 80 909 1
R 87231 340.2 (+) NO2 H 1,700 82 48 67
R 87232 340.2 (-) NO2 H 33 66 2,000 0.4
R 88703 337.2 (+) C(O)CH3 H 26 130 5,000
R 88976 337.2 (+) C(O)CH3 H 6,500 120 18
R 88977 337.2 (-) C(O)CH3 H 19 52 2,737
R 89439 351.2 (+) C(O)CH3 CH3 13 710 54,615 0.2
R 90384 351.2 (+) C(O)CH3 CH3 2,100 270 129
R 90385 351.2 (-) C(O)CH3 CH3 5 420 84,000
Reference
AZT 267.2 0.5 3.5 7,000
ddC 211.2 190 86 453
ddI 236.2 4,800 1,000 208
PMEA 273.2 15,300 380 25
R 82150 287.4 44 550 12,500
R 82913 321.9 33 34 1,030
R 86183 321.9 5 140 28,000
E-EPU 306.4 48 200 4,167
L-697,661 352.2 22 320 14,545
Ro 318959 670.9 14 17 1,214
FIG. 1 Structure-activity relationship for inhibition of HIV-1 cytopathicity and cytotoxicity in MT-4 cells by a-APA derivatives. Reference
compounds include the following: PMEA, 9-(2-phosphonylmethoxyethyl)adenine; R 82150, TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-
methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jkl[l,4]benzodiazepin-2(1H)-thione; R 82913, TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-9-chloro-
5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione; R 86183, TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-8-
chloro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione; E-EPU, HEPT derivative 5-ethyl-1-ethoxymethyl-6-
(phenylthio)uracil; L-697,661, pyridinone derivative 3-1[(4,7-dichloro-1,3-benzoxazol-2-yl)methyl]amino)-5-ethyl-6-methylpyridin-2(1H)-one
(Merck Sharp & Dohme), provided by M. Goldman; Ro 318959, HIV protease inhibitor QC-AsnPhe[CH(OH)CH2N]DIQ-NHtBu [where QC,
quinoline-2-carbonyl; DIQ, (4as, 8as)-decahydro-3(S)-isoquinolinecarbonyl], provided by N. Roberts (Roche Products, Welwyn Garden City,
U.K.)
tIC50 in MT-4 cells 5 days p.i. (MTT procedure).
tFifty percent cytotoxic concentration in MT-4 cells (MTT procedure).
§Selectivity index or ratio of CC50 to IC50.
lIC50 of HIV-1 virion RT activity with poly(C)-(dG)j2j8 and dGTP as the template/primer and substrate, respectively.
 Medical Sciences: Pauwels et al. Proc. Natl. Acad. Sci. USA 90 (1993) 1713
MgCl2, 100 mM KCI, 4 mM dithiothreitol, 30 mg of bovine assay conditions, dideoxyinosine (ddI) and 3'-azido-3'-
serum albumin per ml, 0.5 mM EGTA, and 0.01% (wt/vol) deoxythymidine (AZT) had an IC50 of 4800 nM and 0.5 nM,
Triton X-100. Of the four deoxynucleotides (dNTPs), three respectively (Fig. 1).
were used at a saturating concentration of 100 AM, whereas Antiretroviral Activity Profile of a-APA Derivative R 89439.
the tritium-labeled dGTP (Amersham) was used at a concen- Next we examined the possible role of different host cell
tration of 2.5 ,uM. systems and the susceptibility of different retroviruses to R
89439, ddI, and AZT (Table 1). In phytohemagglutinin-
RESULTS stimulated peripheral blood lymphocyte cultures, HIV-1 in-
fection was monitored by measuring the viral p24 core
Discovery of the Lead Compound. From a chemical library production in the cell supernatant at 8 days p.i. Under these
of 90,000 entities we selected 2000 compounds that belonged conditions R 89439 achieved a median 50%1o inhibition at 14
to different chemical classes and that were without effect in nM (Table 1). R 89439 and R 18893 proved >90%o protective
standard toxicological and pharmacological assays. These at "'100 and 600 nM, respectively. In the non-human T-lym-
compounds were evaluated in a high-flux screening system photropic virus type I-transformed CD4+ T-cell line CEM, R
for their cellular cytotoxicity and capacity to inhibit HIV-1 89439 inhibited HIV-1 antigen expression, as measured by
[IIIg(LAI)] replication in MT-4 cells. This screening program FACS analysis at an IC50 of 4 nM (Table 1).
led to the discovery of the a-APA derivative R 15345, which We subsequently investigated the sensitivity of a number
inhibited HIV-1 replication by 50%o at a concentration of 0.6 of HIV-1 strains to R 89439 inhibition in MT-4 cells (Table 1).
,uM (Fig. 1). Cytotoxicity was seen at a 25-fold higher The MN strain, which has a V3 loop typical for strains
concentration. Through evaluation of structurally related circulating in North America and Europe, proved the most
compounds we identified R 18893 as a more potent inhibitor sensitive; its IC50 was 6 nM. The Haitian strain RF, the HE
of HIV-1 replication with an IC50 of 88 nM (Fig. 1). This strain (Belgium), and the Zairian strain NDK exhibited IC50
increased potency was not paralleled by higher cytotoxicity, values of 15, 30, and 22 nM, respectively. An HIV-1 strain
as the selectivity index [ratio of the 50% cytotoxic concen- that was totally resistant to the TIBO derivative R 82913
tration (CCso) to IC50] was 909. A program of synthesis was (IC5o, 18 pM; strain 13MB1) was still sensitive to R 89439
subsequently initiated, aimed at enhancing the anti-HIV (IC5o, 44 nM). On the other hand, an HIV-1 strain carrying a
potency. The HIV-1 iihibition of R 18893 appeared to be Tyr-181 --
Cys mutation (strain 13CN1) was nearly 500-fold
stereospecific because the newly synthesized (-)-isomer R less sensitive than the prototype IIIB/LAI strain. No activity
87232 was 50-fold more potent (IC50, 33 nM) as compared was found against the HIV-2 strains ROD and EHO and the
with the (+)-isomer R 87231 (IC50, 1700 nM) (Fig. 1). When SIV strains MAC251, mndGB1, and agm3. The latter strain is
the nitro-substituent in the aniline moiety of the a-APA inhibited by TIBO compound R 86183 at an IC5o of 2 ,M (21).
structure was replaced by an acetyl group (R 88703), the The combined anti-HIV-1 activity of a-APA derivative R
anti-HIV-1 activity increased (IC50, 26 nM). The potency was 89439 with either AZT, ddI, and the protease inhibitor Ro
further optimized by introducing an additional methyl group 318959 proved to be synergistic (data not shown).
in the 5-position of the aniline moiety. This a-APA deriva- Mechanism of Action Studies. The drug-sensitive phase in
tive, R 89439, had an IC5o of 13 nM and a selectivity index of the HIV-1 replicative cycle was determined by a time of
54,614. The stereospecificity seen for the enantiomers of R addition experiment in which equipotent drug concentrations
18893 extended to these derivatives. The (-)-isomer of R (i.e., 100-fold IC50 concentrations) were added at different
89439, R 90385, was the most potent of the series; it had an times p.i. to MT4 cells (Fig. 2). Analysis of p24 antigen levels
IC50 of S nM and a selectivity index of 84,000. Under similar shortly after the end of the first replicative cycle indicated
Table 1. Antiretroviral spectrum of a-APA derivative R 89439, ddI, and AZT
R 89439 ddI AZT
Cell IC50, CC50, ICso, CCso, ICso, CCso,
Virus Strain line nM ,M SI nM AM SI nM ,M SI
HIV-1 IIIB(LAI) MT-4t 13 710 54,615 4,800 1000 208 0.5 3.5 7,000
IIIB(LAI) PBL* 14 >300 >21,429 210 >250 >1,190 2.1 52 24,762
IIIB(LAI) CEM§ 4 >500 >125,000 1,400 760 543 1.1 38 34,545
RF MT-4 15 710 47,333 4,000 1000 250 5.2 3.5 673
NDK MT-4 22 710 32,273 14,000 1000 71 1.7 3.5 2,059
MN MT-4 6 710 118,333 1,300 1000 769 0.4 3.5 8,750
HE MT-4 30 710 23,667 9,400 1000 106 0.9 3.5 3,889
13MB1 MT-4 44 710 16,136 1,800 1000 556 0.6 3.5 5,833
13CN1 MT-4 5,900 710 120 6,800 1000 147 1.1 3.5 3,182
HIV-2 ROD MT-4 >710,000 710 <1 18,000 1000 56 2.6 3.5 1,346
EHO MT-4 >710,000 710 <1 14,000 1000 71 2.2 3.5 1,591
SIV mac2si MT-4 >710,000 710 <1 5,300 1000 189 1.8 3.5 1,944
mndGB MOLT41 >250,000 >250 <1 12,000 >250 >21 1.1 >4 >3,636
agm3 MOLT411 >250,000 >250 <1 11,000 >250 >23 1.1 >4 >3,636
Data for IC5o represent median values of at least two separate experiments. CC50, cytotoxic concentration; SI, selectivity index or ratio of
CC5o to IC5o. Strains were as follows: RF, Haitian strain; NDK, Zairian strain; MN, HIV-1 strain with V3-loop typical for Europe/USA; HE,
Belgian strain; 13MB1, HIV-1 strain resistant to TIBO R 82913, obtained after serial passage of IIIB/LAI in MT-4 cells and contains a Leul(X
Ile mutation in RT; 13CN1, HIV-1 strain resistant to TIBO R 82913, obtained after serial passage of NDK in CEM cells and contains a Tyr181
Cys mutation in RT; mndGB, derived from mandrills; agm3, derived from African green monkeys.
tDetermined 5 days p.i. by the MTT procedure.
tDetermined by measuring HIV-1 p24 core protein production 7 days p.i. Cytotoxicity was determined by the MTT procedure.
§HIV-1 antigen expression was determined by FACS analysis.
lData determined by the MTT procedure.
'iData determined by the trypan blue dye-exclusion method.
1714 Medical Sciences: Pauwels et al. Proc. Natl. Acad Sci. USA 90 (1993)
rium dialysis experiments, the same stereospecificity as well
as the template-dependent pattern became apparent (data not
shown). Also within a series of a-APA derivatives, a corre-
lation was found when the IC50 values for inhibition of HIV-1
04 replication were compared with the IC50 values for RT
C., inhibition (Fig. 1). Enzyme kinetic analysis of RT inhibition
by R 89439 indicated that the effects were noncompetitive
with respect to the substrate dGTP (Ki, 0.34 uM) and the
template/primer poly(C) (dG)i2_j8 (data not shown). HIV-1
._

Cts
RT mutants obtained by site-directed mutagenesis in which
Tyr'81 was replaced by either cysteine or isoleucine, dis-
played reduced sensitivity by, at least, a factor of 250 and
1250, respectively. Similarly, the Tyr88 Leu mutation also
led to >1250-fold reduction in sensitivity (Table 2).
0 5 10 15 20 25 Pharmacokinetic Profile of a-APA Derivatives. To select a
Time of compound addition, hr suitable candidate for clinical evaluation, four a-APA deriv-
FIG. 2. Variation of p24 antigen levels in acutely infected MT-4 atives were tested in healthy male volunteers. The plasma
cells (29 hr) with time of addition of compounds dextran sulfate (Mr levels of the active (-) enantiomers were determined by
5000) at 50 ,g/ml (-*-), aurintricarboxylic acid at 50 ,ug/ml enantioselective HPLC. The ratio of plasma levels, deter-
(- -*- -), AZT at 0.05 ,g/ml (o), dideoxycytidine ddC at 5 ,ug/ml (o), mined at 8 hr after oral intake of 100 mg in solution, over the
ddl at 100 &g/ml (A), TIBO R 82913 at 0.05 ,ug/ml (o), a-APA R 87303 IC50 values was the highest for R 89439 (Cp, 50 ng/ml; ratio,
at 1 pg/ml (m), and HIV protease inhibitor Ro 318959 at 0.05 ug/ml
().
30). Previous studies have indicated that for the TIBO
derivative R 82913 this ratio was =5 when a 120- to 200-mg
that the a-APA derivative R 87303 (5-methyl-substituted dose was given i.v. (3).
derivative of R 87231, IC50 in MT-4 cells (IIIB, MTT method,
20 nM) lost its protective capacity during a time frame that DISCUSSION
coincided with that of TIBO compound R 82913-i.e., 6-10 The a-APA derivatives represent another chemical family of
hr p.i. This period fell shortly after that ofother RT inhibitors, anti-HIV-1 agents with selectivity indexes up to 4 to 5 orders
such as AZT and ddl, and clearly differed from those periods of magnitude and with antiviral potencies, demonstrated in
sensitive to the adsorption inhibitors dextran sulfate and different host cell types, in the nanomolar range. These
aurintricarboxylic acid and the protease inhibitor Ro 318959. agents exhibit stereospecificity toward inhibition of HIV-1
Treatment of acutely infected MT cells resulted in a dose- replication and RT activity. That this class of drugs is
dependent inhibition of HIV-1 DNA formation as determined targeted at the RT was further shown through (i) identifica-
by PCR analysis (data not shown). In addition R 89439 did not tion of the drug-sensitive phase of the replicative cycle,
mask the CD4 receptor on MT-4 cells, it did not prevent which corresponded to that of other RT inhibitors; (ii) the
binding of HIV-1 particles to the host cells, and it did not concentration-dependent inhibition of HIV-1 DNA formation
affect syncytium formation between uninfected MOLT-4 in acutely infected target cells; (iii) the correlation between
cells and persistently HIV-1-infected HuT-78 cells. In the cellular IC50 values and IC50 values for RT inhibition; and (iv)
latter, HIV-1 progeny formation, as measured by p24 pro- the HIV-1 specificity found both in cell culture and at the RT
duction, was not affected by 4 days of incubation with 30 ,uM level. All tested HIV-1 strains, originating from different
R 89439. geographical regions, proved sensitive to a-APA inhibition;
RT Inhibition by a-APA Derivatives. R 89439 inhibited a all tested HIV-2 or SIV strains proved insensitive to a-APA
poly(A)-(dT)12_18- and a poly(C) (dG)12.18-driven RT reaction inhibition. This specific spectrum is reminiscent of the spec-
by 50% at 3.1 and 0.1 AM, respectively (Table 2). A trum of the nonnucleoside RT inhibitors with TIBO-like
poly(dC) (dG)12_18-directed polymerase reaction was inhib- properties [TIBO, HEPT, dipyridodiazepinones, pyridino-
ited by 50% at 1.3 ,uM. The RT inhibition by a-APA deriv- nes, and bis(heteroaryl)piperazines]. The most potent
atives was stereospecific because the (+)-isomer of R 18893 a-APAs also act as noncompetitive inhibitors of HIV-1 RT,
(R 87231) was >150-fold less active (IC50, 67 ,AM) than the exhibit a similar template preference for poly(C), and seem to
(-)-isomer R 87232 (IC50, 0.4 ,uM) (Fig. 1). When a tritium preferentially inhibit RNA-directed DNA polymerase activ-
label was introduced in the latter three a-APA derivatives ity. Photoaffinity labeling studies (22), characterization of
and their RT-binding properties were investigated in equilib- drug-resistant HIV-1 isolates obtained in cell culture (23),
Table 2. Inhibition of HIV RT
IC50, AM
Template/primer RT R 89439 R 82150 R 86183 ddGTP AZT-TP
Endogenous
(viral RNA)t HIV-1 virion 0.2 0.2 0.05 0.01 0.02
Poly(A)-(dT)12_18 HIV-1 virion 3.1 10 >5.0 0.05
Poly(C)-(dG)12_1s HIV-1 virion 0.1 0.2 0.06 0.02
Poly(C)-(dG)12-18 HIV-1 p66/p66 0.2 0.3 0.06 0.04
Poly(dC)-(dG)12-18 HIV-1 p66/p66 1.3 12 0.5 0.004
Poly(C)'(dG)1218 HIV-1 p66/pSi Y181 - Ct >50 45 0.01
Poly(C)-(dG)12.18 HIV-1 p66/pSi yl81 I§ >250 >250 0.03
Poly(C).(dG)12_18 HIV-1 p66/pSi yl88 - LI >250 >250 0.01
Poly(C)-(dG)12_18 HIV-2 p68/p68 >250 >250 >250 0.06
AZT-TP, AZT-5'-triphosphate.
tTritium-labeled dGTP (2.5 ,M) was used as the substrate. The other dNTPs were used at saturating concentrations (100
,uM). Recombinant mutagenized HIV-1 RT with a Tyr181 -) Cys (t) or -* Ile (§) mutation or a Tyr188 -. Leu mutation (¶).
 Medical Sciences: Pauwels et al.
construction of chimeric HIV-1/HIV-2 RTs (24), and site-
directed mutagenesis of RT (19, 24) all point to the important
role of amino acids Tyr"8' and Tyr'88 for interaction with the
TIBO-like compounds. These residues flank the highly con-
served "polymerase signature sequence" (YXDD), of which
the first aspartic residue may well participate in catalysis, as
shown for the analogous Asp882 in Escherichia coli Klenow
fragment (25). Here we demonstrate, at the RT level as well
as with an HIV-1 strain containing the Tyr"8" -3 Cys muta-
tion, that Tyr181 and Tyrt88 are also crucial for interaction
with a-APA derivatives. All TIBO-like compounds, includ-
ing a-APA derivatives, seem to have a common target on RT.
On the other hand, the a-APA derivative R 89439 proved
inactive against agm3, the first SIV strain shown to be
susceptible to TIBO and HEPT inhibition (21, 26). Further-
more, an HIV-1 strain resistant to TIBO, R 82913, that
contains a Lys00 -- Ile mutation was still sensitive to R
89439, indicating that resistance to a particular TIBO-like
compound does not automatically lead to cross-resistance to
other nonnucleoside RT inhibitors. These observations sug-
gest that inside the binding site for the TIBO-like compounds
("TIBO-pocket"), which we previously indicated to be func-
tionally and possibly spatially related to the substrate-binding
site (9), the nonnucleoside RT inhibitors, depending on their
chemical structure, display quantitative and/or qualitative
differences in their interaction with the amino acids that
constitute this putative pocket and/or may induce different
conformational changes. This hypothesis could be confirmed
when the high-resolution structure of RT becomes available,
and these findings can be placed in a three-dimensional
context. During preparation of this manuscript two groups
reported significant advances in the elucidation of the struc-
ture of RT p66/pSi heterodimer (27, 28). One of these
crystallographic studies showed that the nonnucleoside RT
inhibitor Nevirapine binds in a pocket of the p66 subunit on
top of a f-hairpin motif containing Asp185 and Asp186, two
residues that are just underneath the active site for polymer-
ization (28). The side chains of Tyr181 and Tyr'" also con-
tacted this inhibitor (28), explaining why their mutation led to
resistance. Other amino acids, such as Lys'03 and the above-
mentioned Leu'00, identified by characterization of HIV
strains resistant to TIBO-like compounds, all seem to be part
of this pocket on p66. Having a totally different chemical
structure, the a-APA derivatives may, therefore, contribute
to a better understanding of the mechanism of action of the
TIBO-like compounds, which seems to involve a highly
vulnerable site on the RT that is now inhibited by at least six
different chemical families. The a-APA derivatives rank
among the most potent and selective agents of these different
families. The relatively easy chemical synthesis of R 89439,
its good oral availability, and its favorable pharmacokinetics
make R 89439 a valid candidate for clinical studies.
We thank Hilde Azijn, Barbara Van Remoortel, and Sonia Van
Dooren for excellent technical assistance. Work at the Rega Institute
was funded by the Janssen Research Foundation and also supported
by the Belgian Fonds voor Geneeskundig Wetenschappelijk Onder-
zoek, the Belgian Geconcerteerde Onderzoeksacties, and the AIDS
Basic Research Programme of the European Community.
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