Frank, K. B., Noll, G. J., Connell, E. V. & Sim, I. S. (1991)J. Biol. Chem. 266 ... J. M., Andries, K., Pauwels, R., Janssen, P. A. J., Shannon,. W. M. & Chirigos, M. A. ...
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6952-6956, August 1993 Medical Sciences
Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tertbutyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"dioxide)]-f8-D-pentofuranosyl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors J. BALZARINI*t, A. KARLSSONt, A.-M. VANDAMME*, M.-J. PEREZ-PEREZ§, H. ZHANG1, L. VRANG1, B. OBERG1, K. BACKBROII, T. UNGEII, A. SAN-FELIX§, S. VELAZQUEZ§, M.-J. CAMARASA§, AND E. DE CLERCQ* *Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; tKarolinska Institute, S-104 01 Stockholm, Sweden; 'Medivir AB, S-141 44 Huddinge, Sweden; lnstituto de Quimica Medica, 28006 Madrid, Spain; and I'Department of Molecular Biology, Biomedical Centre, University of Uppsala, S-751 24 Uppsala, Sweden Communicated by Gertrude B. Elion, March 3, 1993 (received for review October 6, 1992)
nucleoside derivatives nevirapine and pyridinone are still sensitive to AZT (22). In fact, HIV-1 strains resistant to AZT or ddI show amino acid substitutions in the HIV-1 RT (23-25) that are clearly distinct from those seen in nevirapine- and pyridinone-resistant HIV-1 strains (26). We have discovered an entirely different class of nucleoside analogues, the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'spiro-5"-(4"-amino- 1", 2"-oxathiole-2", 2"-dioxide)] -/3-Dpentofuranosyl derivatives of pyrimidines and purines (designated TSAO) that are endowed with potent and selective activity against HIV-1 (27-31) and that are targeted at HIV-1 RT (27, 28, 32). The prototype TSAO compound is the thymine derivative (designated TSAO-T). For TSAO-T to be
We recently reported that a newly discovered ABSTRACT class of nucleoside analogues-[2',5'-bis-O-(tert-butyldimethylsilyl)-3' -spiro-5"-(4"-amino-1",2"-oxathiole-2r,2r-dioxide)]-13D-pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)-are highly specific inhibitors of human inmunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of IIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jkl[1,4]benzodiazepin-2(lH)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu --+ Lys) in the RT of all TSAO-resistant HIV-1 strains. IIV-1 RT in which the Glu-138 -* Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia col could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138 -* Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4'-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT.
0
0
CH
HN
O
HN JSCH3
N
O
N
esio H2N,
soS°os0 /~~~~~~~IlS.-0
S
(A) (ribo) TSAO-T
A number of different structural classes of nonnucleoside
analogues-i.e., 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine (HEPT) (1-5), tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO) (6, 7), nevirapine (8, 9), pyridinones (10, 11), bis(heteroaryl)piperazine (12), and a-anilinophenylacetamide (13)-have recently been identified as potent and highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication. It has been demonstrated for the HIV-1-specific nonnucleoside derivatives that they interact with a site of HIV-1 reverse transcriptase (RT) that is clearly distinct from the substrate binding site (8, 10, 12-20). HIV-1 strains that are resistant to 3'-azido-3'-deoxythymidine (AZT) or ddI are still sensitive to the HIV-1-specific nonnucleoside inhibitors (21), and, vice versa, HIV-1 strains selected for resistance against the non-
o*
osi$
NH2
X
(S) (xylo) TSAO-T
active against HIV-1, the 3'-spiro moiety must be in the R (ribo) configuration. TSAO derivatives with the 3'-spiro moiety in the S (xylo) configuration are devoid of anti-HIV-1 activity (27). Here we report that HIV-1 strains resistant to the TSAO nucleoside derivatives are still highly sensitive to other HIV-1-specific nonnucleoside analogues. We identified the 3'-spiro 4"-amino group as the pharmacophore of the TSAO molecules being responsible for direct interaction with the glutamic acid residue at position 138 of the HIV-1 RT. Abbreviations: HEPT, 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine; TIBO, tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin2(1H)-one and -thione; HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; AZT, 3'-azido-3'-deoxythymidine; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; FPMPA, (S)-9-(3fluoro-2-phosphonylmethoxypropyl)adenine. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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MATERIALS AND METHODS Cells. MT-4 cells were provided by N. Yamamoto (Tokyo Medical School and Dental University School of Medicine, Tokyo) and CEM cells were obtained from the American Type Culture Collection. The cells were grown in RPMI 1640 medium supplemented with 10% (vol/vol) inactivated fetal calf serum (GIBCO), 2 mM L-glutamine (Flow Laboratories), and 0.075% (vol/vol) NaHCO3 (Flow Laboratories). Virus. The HIV-1 strain HTLV-IIIB was obtained from R. C. Gallo and M. Popovic (National Institutes of Health, Bethesda, MD) (33). Test Compounds. Synthesis of the TSAO derivatives of thymine (TSAO-T), N3-methylthymine (TSAO-m3T), N3ethylthymine (TSAO-e3T), N3-allylthymine (TSAO-a3T), uracil (TSAO-U), cytosine (TSAO-C), 5-methylcytosine (TSAO-m5C), and adenine (TSAO-A) has been described (29-31). TIBO R82150 and TIBO R82913 were kindly provided by Zhang Hao (National Institutes of Health). Nevirapine (BIRG-587) and pyridinone L-697,661 were kindly provided by P. Ganong (Boehringer Ingelheim) and M. Goldman (Merck, Sharp & Dohme), respectively. The HEPT derivatives E-EBU-dM and I-HEPU-SdM were provided by M. Baba (Fukushima Medical College, Fukushima, Japan). ddI was from Bristol-Myers Squibb (Syracuse, NY); ddC and ddG were provided by David G. Johns (National Institutes of Health). AZT was from Sigma and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) were synthesized by A. Holy and J. Jindrich (Prague). Antiviral Activity of TSAO Nucleoside Derivatives. CEM cells were suspended at 250,000 cells per ml of culture medium and infected with 100 CCID5o (50% cell culture infective dose) HIV-1(IIIB). Then, 100 ,ul of the infected cell suspensions was added to 200-,ul microtiter plate wells containing 100 ul of an appropriate dilution of the test compounds. The inhibitory effect of the test compounds against HIV-1-induced syncytium formation in CEM cells was examined at day 4 postinfection as described (34). Selection of TSAO-Resistant HIV-1 Strains. HIV-1(IIIB) was subjected to two passages in 6-ml CEM cell cultures (4 x 105 cells per ml) in the presence of 2-3 times the EC50 of TSAO-T, TSAO-m3T, or TSAO-e3T in 25-cm2 culture flasks (Falcon, Becton Dickinson). Passages were performed every 4-5 days by adding 0.5-1.0 ml of the infected cultures to a fresh culture volume of 5 ml containing 4 x i0s uninfected CEM cells per ml and increasing amounts of inhibitor (=5fold). The remaining supematants were frozen in aliquots at -700C. Testing of Sensitivity of TSAO-Resistant HIV-1 Strains Against HIV-1-Specific Test Compounds in CEM Cell Cultures. The assay procedure was essentially as described (35). Determination of the Amino Acid Sequence of the RTs of TSAO-Resistant HIV-1 Strains. The procedure of MT-4 cell infection with different HIV-1 strains, preparation for PCR assay, amplification of proviral DNA, and sequencing of the 727-bp fragment covering amino acids 50-270 have been described (35). RT Assay. The culture supematants of HIV-1(IIIB)- and HIV-1/TSAO-m3T-infected CEM cells were ultracentrifuged for 2 h at 105,000 x g. Then the pellets were suspended in 5 mM Tris HCl, pH 7.8/1 mM dithiothreitol/20 pg of bovine serum albumin per ml/0.1% Triton X-100/0.5 M KCl and stored at -20°C until use. The HIV-1 RT reaction mixtures (50 ul) contained 50 mM Tris HCl (pH 7.8), 5 mM dithiothreitol, 300 uM glutathione, 500 AM EDTA, 150 mM KCl, 5 mM MgCl2, 1.25 ug of bovine serum albumin, 3.5 ;kM dGTP (2 uCi per assay mixture; 1 Ci = 37 GBq), a fixed concentration of the template/primer poly(C)-oligo(dG) (0.1 mM),
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0.05% Triton X-100, 10 til of inhibitor solution (containing various concentrations of the test compounds), and 5 ,ul of the HIV-1(IIIB) and HIV-1/TSAO-m3T RT preparations. The reaction mixtures were incubated at 37°C for 30 min, at which time 150 ,ul of yeast RNA (2 mg/ml) and 0.75 ml of 5% (vol/vol) trichloroacetic acid in saturated Na4P207 solution were added. The solutions were kept on ice for 20 min, after which the acid-insoluble material was washed and analyzed for radioactivity. For cloned HIV-1 RT, the assay was as described (36). Using extract from Escherichia coli without RT expression, incorporation of [3H]dGTP was =2000 cpm as trichloroacetic acid-precipitable material per 50 ,ul of assay mixture. Using the E. coli extracts that expressed wild-type HIV-1 RT and mutant HIV-1 RT, containing the Glu-138 -* Arg substitution, incorporation was -150,000 cpm in the absence of inhibitors. Expression and Preparation of HIV-1 RT. Single-stranded DNA was isolated from the plasmid pRT.BS (36), containing the HIV-1(IIIB) RT sequence. Oligonucleoside-directed sitespecific mutagenesis was done according to the method by Sayers et al. (37). Glu-138 (GAG) was mutated to Lys (AAG) and to Arg (AGG). The expression vectors for the two mutant forms of RT (Glu-138 -- Lys) and RT (Glu-138 -* Arg) were constructed by transfer of the mutated RT sequences to the plasmid pETlia (38). Expression was performed in the E. coli strain BL21(DE3). Transformed cells were grown to OD = 1.0 before induction with isopropyl /3D-thiogalactopyranoside. Cells were harvested after 3 h of induction. E. coli was lysed in a French press giving a concentration of -0.1 ,ug/,ul. The lysate was then diluted 1:100 before use in the RT assay. One of the mutant enzymes, RT (Glu-138 -3 Lys), could not be isolated or used as a lysate because of instability even in the presence of protease inhibitors. E. coli cell lysates of HIV-1 RT (Glu-138 - Arg) and wild-type HIV-1 RT were used without further purification. Wild-type RT was also purified with Q-Sepharose, heparin-Sepharose, and S-Sepharose (K.B., unpublished data).
RESULTS Antiviral Actity of TSAO Nucleoside Derivatives. Among the most active TSAO derivatives are TSAO-T, TSAO-m3T, TSAO-e3T, the uracil derivative TSAO-U, and the adenine derivative TSAO-A (Table 1). The 50% antivirally effective concentrations of these compounds range from 0.05 to 0.13 ,uM in CEM cells. TSAO-T is cytotoxic at a 50%o cytotoxic concentration (CC50) of 12 AM (data not shown). However, TSAO-m3T and TSAO-e3T are antivirally effective in HIVl(IIIB)-infected CEM cells at concentrations that are 1000- to 4000-fold lower than their cytotoxic concentration (CC50 2 150 ,uM). The antiviral potencies of the TSAO nucleoside derivatives in MT-4 and C8166 cells rank to the same order of magnitude as those recorded in CEM cells (data not shown). Sensitivities of TSAO-Resistant HIV-1 Strains Against HIV1-Specific Test Compounds. Drug-resistant HIV-1 variants could be obtained after three passages of the virus in the presence of increasing concentrations of the test compounds. These virus strains were 100-fold (TSAO-T) or >1000-fold (TSAO-m3T and TSAO-e3T) less susceptible to the inhibitory action of the TSAO congeners in which they were raised (Table 1). Moreover, the three TSAO-resistant HIV-1(IIIB) strains also proved cross-resistant to other TSAO analogues. As a rule, full cross-resistance was observed with all TSAOresistant HIV-1 strains against all TSAO derivatives, except for TSAO-T, which still proved slightly active against these HIV-1 strains (EC50, 3.1-6.2 ,uM). However, this concentration was close to the toxicity threshold (CC50, 12 uM). Also, the virus that emerged after the third subcultivation in the presence of test compound did not prove phenotypically
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Table 1. Inhibitory effects of test compounds on different HIV-1(IIIB) mutant strains in CEM cells EC50,* ,M HIV-1/TSAO-e3T HIV-1/TSAO-m3T HIV-1/TSAO-T Compound HIV-1(IIIB) >8 >8 >8 0.13 ± 0.06 TSAO-U 3.1 ± 0.0 4.7 ± 2.6 6.2 ± 2.2 0.05 ± 0.008 TSAO-T >80 46 ± 26 >80 0.05 ± 0.008 TSAO-m3T >80 >80 >80 0.09 ± 0.08 TSAO-e3T >8 >8 >8 0.08 ± 0.06 TSAO-A 0.07 ± 0.0 0.07 ± 0.02 0.31 ± 0.10 0.09 ± 0.02 TIBO R82150 0.63 ± 0.29 0.16 ± 0.16 0.31 ± 0.22 0.06 ± 0.03 TIBO R82913 0.09 ± 0.09 0.42 ± 0.24 0.27 ± 0.24 0.009 ± 0.003 E-EBU-dM 0.04 ± 0.018 0.01 ± 0.013 0.003 ± 0.0005 I-HEPU-SdM 0.07 ± 0.0 0.11 ± 0.07 0.07 ± 0.0 0.12 ± 0.11 Nevirapine 0.07 ± 0.0 0.57 ± 0.21 0.25 ± 0.18 0.02 ± 0.01 Pyridinone L697,661 0.002 ± 0.001 0.002 ± 0.0009 0.009 ± 0.009 AZT 0.003 ± 0.001 0.03 ± 0.04 0.03 ± 0.01 0.09 ± 0.09 0.035 ± 0.007 ddC ± 7.1 ± 0.0 10 5.3 ± 2.9 15 4.6 ± 2.6 ddI 3.0 ± 1.4 3.5 ± 2.9 0.77 ± 0.32 1.1 ± 0.28 ddG 7.5 ± 3.5 ± 7.1 7.0 ± 3.0 15 8.5 ± 2.1 PMEA ± 0.0 ± 0.0 5.4 ± 1.1 12 30 9.0 ± 0.0 FPMPA *Effective concentration or compound concentration required to inhibit HIV-1-induced syncytium formation in CEM cells by 50%. Data are means (±SD) of at least two to six independent experiments.
different from the virus recovered after further passages with respect to its lack of sensitivity to the TSAO derivatives (data not shown). All three TSAO-resistant HIV-1(IIIB) strains retained high sensitivity to TIBO R82150 and TIBO R82913. However, in some cases there was a 2.5- to 10-fold decrease in sensitivity to TIBO derivatives, depending on the particular resistant virus strain (i.e., HIV-1/TSAO-T, HIV-1/TSAO-m3T, or HIV-1/TSAO-e3T) and the nature of the compound (i.e., TIBO R82150 or TIBO R82913) (Table 1). The dipyridodiazepinone derivative nevirapine was equally active against the wild-type virus and the HIV-1/TSAO mutants. However, the HEPT derivative E-EBU-dM and the pyridinone derivative L697,661 had considerably reduced activity against HIV-1/ TSAO-m3T and, to a lesser extent, reduced activity toward HIV-1/TSAO-T and HIV-1/TSAO-e3T. However, it should be stressed that all TSAO-resistant HIV-1 strains retained sensitivity to the test compounds at concentrations that were far below 0.5 ,uM; this concentration is easily attainable in plasma from humans. In addition, the TSAO-resistant HIV-1 strains retained full sensitivity to the 2',3'-dideoxynucleoside analogues AZT, ddI, ddC, and ddG, and the acyclic nucleoside phosphonates PMEA and FPMPA (Table 1). Determination of the Amino Acid Sequence of the RT of TSAO-Resistant HIV-1 Strains. We found that all three TSAOresistant HIV-1 strains contained a single amino acid change from Glu to Lys at position 138 of the RT. This resulted from the transition mutation of the first purine (G) of the Glu codon (GAG) to another purine (A), thus converting the Glu codon into a Lys codon (AAG). No other amino acid changes were observed in the TSAO-resistant mutant RTs, as compared to wild-type (IIIB) RTs within the domain of amino acids 50-270. In contrast with the nevirapine- and pyridinone-resistant HIV-1 strains, the TSAO-resistant HIV-1 strains did not reveal any amino acid changes at positions 103 and 181. Thus, a
single amino acid change (Glu-138
-+
Lys) in the HIV-1
RT
could be held responsible for the >1000-fold decreased sensitivity that HIV-1 required upon passage in cell culture in the presence of the TSAO derivatives. In fact, such fully resistant virus already emerged after the first passage of the virus in the presence of TSAO-m3T at a concentration that was only 3-fold higher than the EC5o (data not shown). Sensitivity of RT of HIV-1/TSAO-m3T to HIV-i-Specific Test Compounds. The RT derived from the HIV-1/TSAO-
m3T mutant proved fully resistant to the inhibitory effect of TSAO-T (IC50, >155 ,uM) but still sensitive to nevirapine, TIBO R82150, and TIBO R82913, and the HEPT derivative I-HEPU-SdM (5) (Table 2). The data obtained for RT activity are in full agreement with our observations that in cell culture the TSAO-resistant HIV-1 mutant strains remain sensitive to the HIV-1-specific nonnucleoside analogues (Table 1). These data also provide additional proof for RT being the principal target for the HIV-1-specific activity of the TSAO class of nucleoside analogues. Properties of Recombinant HIV-1 RT Containing Lys or Arg at Position 138. The HIV-1 RT containing the Glu-138 -- Lys substitution was expressed in E. coli but could not be purified or used as a crude extract because of very rapid degradation (data not shown). The mutant HIV-1 RT containing Arg-138 proved more stable in the bacterial extract than the Lys-138 mutant RT and could be used to determine the inhibitory effects of TSAO-T, TSAO-m3T, TIBO R82913, and pyridinone L697,661 (Table 3). While the mutant HIV-1 RT (containing Arg instead of Glu at position 138) was only 2.8- to 4.0-fold less sensitive to the inhibitory effect of pyridinone and TIBO R82913, it had lost virtually all affinity for TSAO-T and TSAO-m3T (Table 3). The sensitivity of highly purified wild-type HIV-1 RT to the HIV-1-specific inhibitors was similar to that of the wild-type HIV-1 RT expressed in the bacterial extract.
DISCUSSION Recently, Nunberg (26) reported that mutant HIV-1 strains selected for resistance against the pyridinone inhibitor Table 2. Inhibitory effects of test compounds on RTs derived from HIV-1(IIIB) and HIV-1/TSAO-m3T virions ICso,* ,uM HIV-1/TSAO-m3T Compound HIV-1(IIIB) >155 TSAO-T 20 0.75 0.94 Nevirapine 1.1 4.8 TIBO R82150 5.3 TIBO R82913 4.0 I-HEPU-SdM 16.8 29.9 *Inhibitory concentration or compound concentration required to inhibit RT by 50%.
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Table 3. Inhibition of wild-type and mutant (Glu-138 -. Arg) HIV-1 RT by HIV-1-specific inhibitors
IC5o,* AM Wild-type HIV-1 RT Wild-type HIV-1 RT Mutant HIV-1 RT (Glu-138 -- Arg) Mutant HIV-1 RT/wild-type Test compound (purified) (bacterial extract) (bacterial extract) HIV-1 RTt Pyridinone L697,661 0.08 0.12 0.36 3 0.20 0.31 1.24 4 TIBO R82913 >161 TSAO-T 0.62 0.93 >150 (30o)t TSAO-m3T 1.1 1.52 >150 (23%)t >99 *RT reaction was carried out in the presence of poly(C)-oligo(dG) and [3H]dGTP as the template/primer and radiolabeled substrate, respectively. tRatio of IC50 for mutant HIV-1 RT (Glu-138 -+ Arg) to IC5o for wild-type HIV-1 RT. *Percentage of inhibitory activity of TSAO derivatives against mutant RT enzyme at 150 ,uM is indicated in parentheses.
L-697,639 are cross-resistant to the structurally unrelated RT inhibitors TIBO R82150 and BI-RG-587 (nevirapine). Also, Richman et al. (22), Mellors et al. (39), and Balzarini et al. (35) reported that HIV-1 mutants selected for resistance against the dipyridodiazepinone BI-RG-587 are cross-resistant to TIBO R82913 (22, 35), the HEPT derivatives E-EPU and E-BPU-S (22), and TIBO R82150 (39). These data suggest a common binding site at the HIV-1 RT for all these nonnucleoside HIV-1 inhibitors. Resistance to nevirapine, TIBO, HEPT, and the pyridinones has been ascribed mainly to the amino acid change Tyr-181 -+ Cys and, in some instances, to the amino acid changes Leu-100 -+ Ile and Lys-103 -* Asn in the RT enzyme (26, 35, 39). It is of particular interest to note that the virus that emerged after the third passage in cell culture in the presence of relatively low concentrations of the TSAO derivatives (i.e., '=0.2 ,uM) already proved fully resistant to TSAO-m3T and TSAO-e3T. This observation is strongly suggestive for the preexistence of fully resistant virus particles in the initial virus load. This situation is completely different from the mechanism of resistance of HIV against AZT, since it has been clearly demonstrated by Larder and Kemp (23) and St. Clair et al. (24) that the degree of resistance of HIV to AZT depends on the number (and nature) of accumulating mutations in the RT gene of the particular resistant HIV-1 strains. Our studies revealed that TSAO-resistant HIV-1 strains retain marked sensitivity to other HIV-1-specific compounds. Also, the TSAO-resistant HIV-1 strains were invariably characterized as having a mutation at position 138 (Glu -- Lys) of the RT, which has never been reported to occur in nevirapine-, pyridinone-, or TIBO-resistant HIV-1 strains. Recently, these observations were confirmed for five other TSAO-resistant strains that had been selected in independent experiments (data not shown). In all cases, Glu-138 -* Lys was the only amino acid change detected in the RT of these TSAO-resistant HIV-1 strains. Structure modeling studies and space-filling models of TSAO-T derivatives showed that the 4"-amino group on the 3'-spiro moiety of the (antivirally inactive) S (xylo) derivative of TSAO-T is sterically more crowded than the 4"-amino group on the 3'-spiro moiety of the (antivirally active) R (ribo) isomer of TSAO-T. This makes the amino group on the 3'-spiro moiety of the S derivative of TSAO-T less accessible to interaction with other molecules, whereas the amino group on the 3'-spiro moiety of the R (ribo) derivative seems more accessible to interaction with another molecular entity. Therefore, it is postulated that this amino group is most likely responsible for a specific interaction of the TSAO molecule with the RT. The carboxylic acid group of the glutamic acid residue at position 138 of the HIV-1 RT is a likely site of interaction since a replacement of this acidic Glu by a basic Lys residue results in TSAO-resistant HIV-1 strains. Attempts to express and purify the mutant HIV-1 RT (containing the Glu-138 -+ Lys substitution) have so far failed because of very rapid degradation of this enzyme in E. coli extracts, despite the presence of protease inhibitors. However, the
mutant HIV-1 RT (containing the Glu-138 -* Arg substitution) was stable and showed a marked resistance to both TSAO-T and TSAO-m3T (Table 3). These observations add further proof of the importance of Glu-138 for the interaction of HIV-1 RT with the TSAO derivatives. The fact that the structure-activity relationship of the TSAO derivatives is very stringent with respect to the sugar part [requiring a 3'-spiro moiety in the R (ribo) configuration] but not with respect to the base part (Table 1; ref. 28) further corroborates this hypothesis. Also, analysis ofthe amino acid sequences of the RTs of HIV-2 and simian immunodeficiency virus revealed the presence of Ala at position 138 (40), which may thus explain why these viruses are not sensitive to the antiviral action of the TSAO derivatives. Moreover, HIV-1 RT that had been mutated (by site-directed mutagenesis) such that the Glu residue at position 138 was replaced by the basic amino acid Arg lost virtually all affinity for the TSAO derivatives but not for other HIV-1-specific inhibitors such as TIBO R82913 and pyridinone (Table 3). The latter observation strongly suggests that TSAO derivatives interact with Glu-138 of HIV-1 RT in a highly specific manner. Recently, the crystal structure at 3.5 A resolution of HIV-1 RT complexes with nevirapine has been reported (41). The polymerase domain of p66 is anatomically analogous to a right hand, containing a finger domain, a palm domain, a thumb domain, and a connection domain. It is striking that all amino acid mutations that have been reported so far to be responsible for HIV-1 resistance to the nonnucleoside inhibitors of HIV-1 (i.e., amino acids 100, 103, 108, 181, and 188) (22,26,38, **) are all clustered in the palm domain. In striking contrast, the amino acid change (Glu-138 -- Lys) that confers resistance to TSAO is situated at the top of the finger domain (,(8 finger), which is quite distinct from the palm domain. In conclusion, our findings that HIV-1 strains resistant to the HIV-1-specific TSAO nucleoside derivatives are still sufficiently sensitive to other HIV-1-specific nonnucleoside analogues point to the potential usefulness of simultaneous or alternating combination modalities of two or more HIV-1specific RT inhibitors belonging to different structural classes (TSAO, TIBO, etc.). Subtle differences seem to exist among different classes of compounds with regard to the exact mode of interaction of these compounds with HIV-1 RT. As to the TSAO class, the 3'-spiro 4"-amino pharmacophore of these molecules and the Glu residue at position 138 of the HIV-1 RT appear crucial for the interaction of these molecules with the
enzyme. **Emini, E. A., Schleif, W. A., Bymes, V. W., Condra, J. H., Sardanal, V. V., Kappes, J. C., Saag, M. & Shaw, G., Abstracts of the HIV Drug-Resistance Workshop, July 16-18, 1992, Noordwijk, The Netherlands, p. 14. This research was supported in part by the AIDS Basic Research Programme of the European Community; by grants from the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (Krediet 3.0026.91 and 3.0097.87), the Belgian Nationaal Fonds voor Weten-
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Proc. Natl. Acad Sci. USA 90 (1993)
Medical Sciences: Balzarini et al.
schappelijk Onderzoek (Krediet 3.3010.91), and the Belgian Geconcerteerde Onderzoeksacties (Project 90/94-2); by North Atlantic Treaty Organization Collaborative Research Grant CRG920777; by the Spanish Plan Nacional de I+D Farmaceuticas (Project FAR 880160/1) and the Ministerio de Educacion y Ciencia of Spain (grants to A.S.-F. and M.-J.P.-P.); and by the Medical Faculty of the Karolinska Institute, the Swedish Society of Medicine, and the Swedish Board of Technical Development (Projects 623-90-02526 and 623-90-2581). 1. Baba, M., Tanaka, H., De Clercq, E., Pauwels, R., Balzarini, J., Schols, D., Nakashima, H., Pemo, C.-F., Walker, R. T. & Miyasaka, T. (1989) Biochem. Biophys. Res. Commun. 165, 1375-1381. 2. Miyasaka, T., Tanaka, H., Baba, M., Hayakawa, H., Walker, R. T., Balzarini, J. & De Clercq, E. (1989) J. Med. Chem. 32, 2507-2509. 3. Tanaka, H., Baba, M., Hayakawa, H., Sakamaki, T., Miyasaka, T., Ubasawa, M., Takashima, H., Sekiya, K., Nitta, I., Shigeta, S., Walker, R. T., Balzarini, J. & De Clercq, E. (1991) J. Med. Chem. 34, 349-357. 4. Baba, M., De Clercq, E., Tanaka, H., Ubasawa, M., Takashima, H., Sekiya, K., Nitta, I., Umezu, K., Walker, R. T., Mon, S., Ito, M., Shigeta, S. & Miyasaka, T. (1991) Mol. Pharmacol. 39, 805-810. 5. Tanaka, H., Takashima, H., Ubasawa, M., Sekiya, K., Nitta, I., Baba, M., Shigeta, S., Walker, R. T., De Clercq, E. & Miyasaka, T. (1992) J. Med. Chem. 35, 337-345. 6. Pauwels, R., Andries, K., Desmyter, J., Schols, D., Kukla, M. J., Breslin, H. J., Raeymaeckers, A., Van Gelder, J., Woestenborghs, R., Heykants, J., Schellekens, K., Janssen, M. A. C., De Clercq, E. & Janssen, P. A. J. (1990) Nature (London) 343, 470-474. 7. Kukla, M. J., Breslin, H. J., Pauwels, R., Fedde, C. L., Miranda, M., Scott, M. K., Sherrill, R. G., Raeymaekers, A., Van Gelder, J., Andries, K., Janssen, M. A. C., De Clercq, E. & Janssen, P. A. J. (1991) J. Med. Chem. 34, 746-751. 8. Merluzzi, V. J., Hargrave, K. D., Labodia, M., Grozinger, K., Skoog, M., Wu, J. C., Shih, C.-K., Eckner, K., Hattox, S., Adams, J., Rosenthal, A. S., Faanes, R., Eckner, R. J., Koup, R. A. & Sullivan, J. L. (1990) Science 250, 1411-1413. 9. Koup, R. A., Merluzzi, V. J., Hargrave, K. D., Adams, J., Grozinger, K., Eckner, R. J. & Sullivan, J. L. (1991) J. Infect. Dis. 163, 966-970. 10. Goldman, M. E., Nunberg, J. H., O'Brien, J. A., Quintero, J. C., Schleif, W. A., Freund, K. F., Gaul, S. L., Saari, W. S., Wai, J. S., Hoffman, J. M., Anderson, P. S., Hupe, D. J., Emini, E. A. & Stem, A. M. (1991) Proc. Natl. Acad. Sci. USA 88, 6863-6867. 11. Saari, W. S., Hoffman, J. M., Wai, J. S., Fisher, T. E., Rooney, C. S., Smith, A. M., Thomas, C. M., Goldman, M. E., O'Brien, J. A., Nunberg, J. N., Quintero, J. C., Schleif, W. A., Emini, E. A., Stem, A. M. & Anderson, P. S. (1991) J. Med. Chem. 34, 2922-2925. 12. Romero, D. L., Busso, M., Tan, C.-K., Reusser, F., Palmer, J. R., Poppe, S. M., Aristoff, P. A., Downey, K. M., So, A. G., Resnick, L. & Tarpley, W. G. (1991) Proc. Natl. Acad. Sci. USA 88, 8806-8810. 13. Pauwels, R., Andries, K., Debyser, Z., Van Daele, P., Schols, D., Vandamme, A.-M., Stoffels, P., De Vreese, K., Woestenborghs, R., Janssen, C. G. M., Annd, J., Cauwenbergh, G., Desmyter, J., Heykants, J., Janssen, M. A. C., De Clercq, E. & Janssen, P. A. J. (1993) Proc. Natl. Acad. Sci. USA 90, 1711-1715. 14. Debyser, Z., Pauwels, R., Andries, K., Desmyter, J., Kukla, M., Janssen, P. A. J. & De Clercq, E. (1991) Proc. Natl. Acad. Sci. USA 88, 1451-1455. 15. Cohen, K. A., Hopkins, J., Ingraham, R. H., Pargellis, C., Wu,
16. 17. 18.
19. 20. 21.
22. 23. 24.
25. 26. 27.
28. 29.
30.
31. 32. 33.
34. 35. 36.
37. 38. 39. 40. 41.
J. C., Palladino, D. E. H., Kinkade, P., Warren, T. C., Rogers, S., Adams, J., Farina, P. R. & Grob, P. M. (1991) J. Biol. Chem. 266, 14670-14674. Wu, J. C., Warren, T. C., Adams, J., Proudfoot, J., Skiles, J., Raghavan, P., Perry, C., Potocki, I., Farina, P. R. & Grob, P. M. (1991) Biochemistry 30, 2022-2026. Frank, K. B., Noll, G. J., Connell, E. V. & Sim, I. S. (1991)J. Biol. Chem. 266, 14232-14236. Baba, M., De Clercq, E., Tanaka, H., Ubasawa, M., Takashima, H., Sekiya, K., Nitta, I., Umezu, K., Nakashima, H., Mori, S., Shigeta, S., Walker, R. T. & Miyasaka, T. (1991) Proc. Natl. Acad. Sci. USA 88, 2356-2360. White, E. L., Buckheit, R. W., Jr., Ross, L. J., Germany, J. M., Andries, K., Pauwels, R., Janssen, P. A. J., Shannon, W. M. & Chirigos, M. A. (1991) Antiviral Res. 16, 257-266. Dueweke, T. J., Kezdy, F. J., Waszak, G. A., Deibel, M. R., Jr., & Tarpley, W. G. (1992) J. Biol. Chem. 267, 27-30. Richman, D., Rosenthal, A. S., Skoog, M., Eckner, R. J., Chou, T.-C., Sabo, J. P. & Merluzzi, V. J. (1991) Antimicrob. Agents Chemother. 35, 305-308. Richman, D., Shih, C.-K., Lowy, I., Rose, J., Prodanovich, P., Goff, S. & Griffin, J. (1991) Proc. Natl. Acad. Sci. USA 88, 11241-11245. Larder, B. A. & Kemp, S. D. (1989) Science 246, 1155-1158. St. Clair, M. H., Martin, J. L., Tudor-Williams, G., Bach, M. C., Vavro, C. L., King, D. M., Kellam, P., Kemp, S. D. & Larder, B. A. (1991) Science 253, 1557-1559. Gao, Q., Gu, Z., Parniak, M. A., Li, X. & Wainberg, M. A. (1992) J. Virol. 66, 12-19. Nunberg, J. H., Schleif, W. A., Boots, E. J., O'Brien, J. A., Quintero, J. C., Hoffman, J. M., Emini, E. A. & Goldman, M. E. (1991) J. Virol. 65, 4887-4892. Balzarini, J., Perez-Perez, M.-J., San-Felix, A., Schols, D., Pemo, C.-F., Vandamme, A.-M., Camarasa, M.-J. & De Clercq, E. (1992) Proc. Natl. Acad. Sci. USA 89, 4392-4396. Balzarini, J., Perez-Perez, M.-J., San-Felix, A., Velazquez, S., Camarasa, M.-J. & De Clercq, E. (1992) Antimicrob. Agents Chemother. 36, 1073-1080. Camarasa, M.-J., Perez-Perez, M.-J., San-Felix, A., Balzarini, J. & De Clercq, E. (1992) J. Med. Chem. 35, 2721-2727. Perez-Perez, M. J., San-Felix, A., Balzarini, J., De Clercq, E. & Camarasa, M. J. (1992) J. Med. Chem. 35, 2988-2995. Perez-Perez, M.-J., San-Felix, A., Camarasa, M.-J., Balzarini, J. & De Clercq, E. (1992) Tetrahedron Lett. 33, 3029-3032. Balzarini, J., Perez-Perez, M.-J., San-Fdlix, A., Camarasa, M.-J., Barr, P. J. & De Clercq, E. (1992) J. Biol. Chem. 267, 11831-11838. Popovic, M., Sarngadharan, M. G., Read, E. & Gallo, R. C. (1984) Science 224, 497-500. Balzarini, J., Naesens, L., Slachmuylders, J., Niphuis, H., Rosenberg, I., Holy, A., Schellekens, H. & De Clercq, E. (1991) AIDS 5, 21-28. Balzarini, J., Karlsson, A., Perez-Perez, M.-J., Vrang, L., Walbers, J., Zhang, H., Oberg, B., Vandamme, A.-M., Camarasa, M.-J. & De Clercq, E. (1993) Virology 192, 246-253. Zhang, H., Vrang, L., Unge, T. & Oberg, B. (1993) Antiviral Chem. Chemother., in press. Sayers, J. R., Schmidt, W. & Eckstein, F. (1988) Nucleic Acids Res. 16, 791-802. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990) Methods Enzymol. 185, 60-89. Mellors, J. W., Dutschman, G. E., Im, G.-J., Tramontano, E., Winkler, S. R. & Cheng, Y.-C. (1992) Mol. Pharmacol. 41, 446-451. Barber, A. H., Hizi, A., Maizel, J. V. & Hughes, S. H. (1990) AIDS Res. Hum. Retroviruses 6, 1061-1072. Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790.
