characterization of the binding site for nevirapine (bi-rg-587), a

THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No . 22, Issue of ’ August 5, pp, 14670-14674, 1991 Printed in U. S. A.

Characterization of the Binding Site for Nevirapine (BI-RG-587), a Nonnucleoside Inhibitor of Human Immunodeficiency Virus Type- 1 Reverse Transcriptase*

(Received for publication, March 26, 1991)

Kenneth A. CohenSg, Jerry HopkinsS, Richard H. Ingrahamll, Chris Pargellisll, Joe C. WuII, Deborah E. H. PalladinoS, Peter Kinkadell, Thomas C. WarrenII, Sheri Rogersll, Julian AdamsY, Peter R. Farinall, and Peter M. Grob 11 From the Departments of $Analytical Sciences, llMedicina1 Chemistry, and 11 Biochemistry, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877

Nevirapine (BI-RG-587) is a potent and specific nonnucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is noncompetitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues.

Human immunodeficiency virus type-1 (HIV-1)’ is the causative agent of acquired immune deficiency syndrome (AIDS). Since the infectivity as well as the replication of this pathogen requires reverse transcription (RT), the inhibition of HIV-1 R T is an important therapeutic target (Fauci, 1988). The dipyridodiazepinone, nevirapine (BI-RG-587), has re- cently been reported as a potent and specific inhibitor of HIV- 1 R T pol activity with an ICbo value of 84 nM. The inhibition is noncompetitive with respect to template, primer, and sub- strate nucleoside triphosphates. BI-RG-587 inhibits p24 protein production, syncitia and infectious center formation in T-cell cultures, and exhibits an EC50 value of 40 nM (Merluzzi, et al., 1990).

BI-RJ-70, an azido photoaffinity analogue of BI-RG-587, binds to a noncatalytic modulatory site (RT,MS) on HIV-1 RT. Significantly, a thiobenzimidazolone derivative which inhibits HIV-1 R T also binds to this site as shown by competitive binding experiments. Only the p66 subunit of the p66/p51 heterodimeric enzyme is labeled by BI-RJ-70 (Wu et

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should he addressed Boehringer In- gelheim Pharmaceuticals, Inc., Dept. of Analytical Sciences, 90 E. Ridge, P. 0. Box 368, Ridgefield, CT 06877. Tel.: 203-798-5178; Fax:

The abbreviations used are: HIV-1, human immunodeficiency virus type-1; HIV-2, human immunodeficiency virus type-2; RT, reverse transcriptase; RP, reversed-phase; SEC, size exclusion chromatography; RTIMS, HIV-1 reverse transcriptase modulatory site; TLCK, W-p-tosyl-L-lysine chloromethyl ketone; HPLC, high per- formance liquid chromatography; SDS-PAGE, sodium dodecyl sul- fate-polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin.

203-790-6815.

al., 1991). Upon UV irradiation, the azide moiety on the photoaffinity probe is transformed to a highly reactive nitrene capable of insertion into proximal bonds of the protein back- bone and/or side chains of amino acids. The covalent bond formed between the photoaffinity probe and protein results in irreversible inactivation of the enzyme with a 1:l stoichi- ometry of probe:RT heterodimer (Wu et al., 1991).

Experiments described in the present paper determine where this novel inhibitor binds to RT. We now report that the use of a photoaffinity label, BI-RJ-70, has allowed us to show that both Tyr-181 and Tyr-188 are located at the binding site of BI-RG-587.

MATERIALS AND METHODS

Escherichia coli (strain J M 109) transformed with pKRT-2 was obtained from Dr. W. Summers, Yale University (D’Aquila and Summers, 1989) and is also available from the National Institutes of Health AIDS Research and Reference Reagent Program Catalog (cat. 393). Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone- treated) was purchased from Worthington and endoproteinase Lys- C and TLCK were purchased from Boehringer Mannheim. The SEC- HPLC column (7.75- X 300-mm TSK G2000 SW) was purchased from Phenomenex (Torrance, CA). The CM RP column (150 X 2.1 mm) was purchased from Millipore-Waters (Milford, MA), and the CR RP column (4.6 mm X 250 mm) was purchased from The Nest Group (Southborough, MA). Ready Safe scintillation fluid was obtained from Beckman (Fullerton, CA), Amplify was obtained from Amersham (Arlington Heights, IL), Kodak XAR-5 x-ray film from Sigma, nitrocellulose membrane from Schleicher & Schuell (Keene, NH), and all reagents for electrophoresis from Bio-Rad. Solvents were HPLC grade and all other chemicals were sequence or reagent grade.

Synthesis of Photoaffinity Label-A detailed description of the synthesis of the azido photoaffinity tritiated probe, [3H]BI-RJ-70, is described by Wu et al. (1991) and Hargrave et al. (1991). The final product was >98% pure (2.02 Ci/mmol). Storage was a t -20 “C in the dark.

Preparation of rHZV-1 RT-Recombinant HIV-1 R T was produced by E. coli transfected with the pKRT-2 plasmid encoding for a 66- kDa R T gene product and purified as previously described (Wu et al., 1991).’ SDS-PAGE analysis of this enzyme preparation shows 66- and 51-kDa bands in approximate equimolar proportion and greater _ _ . . than 95% purity.

Photoaffinitv Labeling of HIV-1 RT-The photolabelinn of HIV-1 R T with [’H]BI-FLJ-70 was conducted as described (Wu e ta l . , 1991). In a typical experiment, a sample of R T with a specific activity of 32.6 units/mg (1 unit = 1 nmol of dGTP hydrolyzed per min) was photolabeled with [3H]BI-RJ-70 (2.02 Ci/mmol) under weak UV irradiation (15 pW/cm2) until less than 30% of the initial RT pol

~~

T. C . Warren, J. J. Miglietta, A. Shrutkowski, J. M. Rose, S. L. Rogers, K. Lubbe, C.-K. Shih, G. 0. Caviness, R. Ingraham, D. E. H. Palladino, G. C. Chow, E. B. Kopp, K. A. Cohen, J. A. Glinski, P. R. Farina, and P. M. Grob, submitted for publication.

14670

Binding Site of Nevirapine (BI-RG-587) on HIV-1 Reverse Transcriptase 14671

activity remained. The initial molar ratio of RT to [3H]BI-RJ-70 was 1 to 0.8. The unbound photoaffinity label was removed by Sephadex G-50 gel filtration. Isolated product [3H]BI-FLJ-70-RT exhibited a specific activity of 9.6 units/mg with n = 0.73 and r = 0.29 where n represents the average number of covalent labels per mol of enzyme and r represents the ratio of specific activity of the labeled RT to that of unlabeled control enzyme.

Tryptic Mapping-RT covalently labeled with [3H]BI-RJ70 or endoproteinase lys-C-derived fragments were subjected to trypsin digestion under denaturing conditions (Stone et al., 1986). Briefly, 40-1000 fig of labeled RT or polypeptide (0.34-8.5 nmol) in 35-250 pl were treated with approximately an equal volume of 8 M urea in 400 mM ammonium bicarbonate, pH 7.8, and vortexed. Typically two volumes of water were added and then 10-25 p1 of trypsin in water so that the trypsin:RT ratio was 1:25 by mass. Following a 2.5-h incubation at 37 "C, the reaction was arrested by injecting the digest onto the ClX RP-HPLC column. Typically, 70-100% of the radioactivity of the digested protein was recovered following chromatographic separation.

Size Exclusion HPLC-Peptides purified by RP-HPLC were subjected to SEC-HPLC using a mobile phase of 0.1% trifluoroacetic acid in water/acetonitrile (50/50, v/v) and a 1-ml/min flow rate at ambient temperature. Fractions of 0.25 ml were collected, and radioactivity was measured by mixing the fractions with 5.5 ml of liquid scintillation mixture. The samples were counted for 1 min on a Beckman model LS 5000 TA scintillation counter equipped with automatic quench control.

Peptide Sequencing-Labeled peptides were sequenced by auto- mated Edman degradation on an Applied Biosystems, Inc. (ABI; Foster City, CA) model 477A pulsed-liquid phase sequenator. At the completion of each Edman cycle, 40% of the PTH-amino acid sample was transferred on-line to an AB1 model 120A microbore HPLC and amino acid analyzer. The remaining 60% was collected by a fraction collector for subsequent liquid scintillation counting.

Peptide Synthesis-Peptide synthesis was performed with an AB1 120A synthesizer using t-butoxycarhonyl chemistry, followed by hy- drofluoric acid cleavage and HPLC purification.

Partial Proteolysis with Endoproteinase Lys-C-HIV-1 RT (-11.5 nmol) treated with ["H]BI-RJ-70 was diluted to a protein concentration of 1.0 mg/ml. Endoproteinase Lys-C (0.1 mg/ml) was added at a mass ratio of 1:270 and incubated at room temperature for 24 h. Previous work had established these conditions as optimal for the production of the desired radiolabeled peptide. The reaction was stopped by the addition of TLCK to a final concentration of 10 fig/ ml.

RP-HPLC and SDS-PAGE of Endoproteinase Lys-C Digest-Pep- tides of [:'H]BI-RJ-70-RT photoadduct were separated on a C, RP- HPLC column as described in the legend to Fig. 5. Aliquots of HPLC fractions exhibiting radioactivity were concentrated to dryness by rotoevaporation. The residue was subjected to tryptic mapping as described above or alternatively, the residue was dissolved in buffer containing 8 M urea and 1% SDS for analysis by SDS-PAGE (Laem- mli, 1970). These latter samples were combined with a standard loading solution containing 2-mercaptoethanol and boiled for 3 min prior to loading onto 15% SDS-PAGE. The gel was stained with Coomassie Brilliant Blue-R250 followed by treatment with Amplify enhancing fluid for 30 min. The gel was dried, exposed to Kodak XAR-5 x-ray film for 4 days at -80 "C and developed with an X- Omat developer (AFP Imaging, Elmsford, NY).

RESULTS AND DISCUSSION

The structures of BI-RG-587 and [3H]BI-RJ-70 are shown in Fig. 1. These compounds inhibit HIV-1 RT with ICbo values of 84 and 160 nM, respectively. Upon irradiation with UV light, RT was covalently labeled by [3H]BI-RJ-70. The photoadduct, [3H]BI-RJ-70-RT, was subsequently purified by gel filtration, denatured with urea, and digested with trypsin. The resulting mixture of tryptic peptides was resolved by RP- HPLC. As shown in the chromatogram in Fig. 2 A , approximately 70 peaks or shoulders were observed which is consistent with a prediction of 65 unique peptides based on complete tryptic digestion. At 210 nm the differences between the chromatographic profile in Fig. 2A and that obtained from a digest of unlabeled RT were comparatively minor (not shown). Some of these differences were ascribed to the presence or

n hi,

BI -RG-587 [3H]-B1-RJ-70

FIG. 1. Structure of BI-RG-587 and r3H]B1-RJ-70. BI-RG- 587 is ll-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-~2',3'- e][l,4]diazepin-6-one. BI-RJ-70 is 9-azido-6,11-dihydro-ll-ethyl-6- methyl-5H-pyrido[2,3-b][1,5]benzodia~epin-5-one-3-t and is tritiated at position 3 on the C ring as shown.

h

140

E 7

0

N v

2 E

0 100 I ' 1

I I

0 25 50 75 100 125 150

Retention Time ( Min )

FIG. 2. Separation of tryptic peptides of [3H]BI-RJ-70-RT photoadduct by RP-HPLC. A 375-pmol sample of [3H]BI-RJ-70- RT, which had been subjected to tryptic digestion, was applied to a C,, RP 2.1- X 150-mm column and developed as follows. Solvent A was 0.06% trifluoroacetic acid in water and solvent B was 0.052% trifluoroacetic acid in 3070 water:acetontrile. A gradient of 0-40% B in 90 min, 40-70% B in 40 min, 70-100% B in 20 min, 100% B for 5 min, 100-0% B in 15 min, and 0% B for 10 min was programmed with a 0.2-ml/min flow rate. A, detection was by UV absorbance at 210 nm. B, radioactivity was measured by liquid scintillation counting of column fractions of 400 pl. C, detection was by UV absorbance at 335 nm.

absence of BI-RJ-70-labeled peptides. Both the radioactivity of [3H]BI-RJ-70, and the UV ab-

sorbance of this photoaffinity probe at 335 nm, were exploited to locate [3H]BI-RJ-70-labeled peptides in the tryptic map. As shown in Fig. 2B, the overwhelming majority of eluted radioactivity (i.e. 91%) was recovered in the retention window of 95-120 min. Moreover, 40% of this recovered radioactivity was present in a single fraction eluting between 110 and 112 min. To eliminate the possibility that the radioactivity was due to breakdown products of [3H]BI-RJ-70, a mock digestion was performed in which trypsin was omitted. No radioactivity was found between 95 and 120 min. Instead, nearly all ( i e . 87%) of the radioactivity of this mock digest coeluted with undigested photolabeled RT between 125 and 140 min (data not shown). The absence of radioactivity in the retention window of 95-120 min in this control chromatogram strongly suggested that the tritiated analytes in Fig. 2B were of peptide character.

14672 Binding Site of Nevirapine (BI-RG-587) on HIV-1 Reverse Transcriptase

The precise peak that corresponded to the major [3H]BI- RJ-70 labeled peptide eluted at 111 min as indicated by absorption at 335 nm (Fig. 2C). Absorbance at 335 nm is indicative of the presence of BI-RJ-70 since peptides exhibit negligible UV absorbance at this wavelength. Zero and second order derivative UV absorbance spectra taken of this peak suggest the presence of tyrosine but not tryptophan or phen- ylalanine (methodology of Palladino et al., 1991).

When the 110-112-min fraction was analyzed by SEC- HPLC, 75% of the radioactivity applied to the column eluted at a retention consistent with that of a peptide of ~ 2 2 0 0 Da (Fig. 3). The size exclusion separation employed an acidic/ organic mobile phase which was denaturing and afforded a size-dependent separation of small peptides (Taneja et al., 1985). The remainder of the recovered radioactivity is prob- ably due to labeled degradation products since it coeluted with photolysed [3H]BI-W-70 at 600 Da. These results, together with the UV spectral data, confirm that the labeled component is a peptide and not a degradation product of the photoaffinity probe.

To purify the major labeled component in Fig. 2C for Edman degradation sequence analysis, reversed-phase chromatographic conditions were modified to include an isocratic segment in the retention vicinity of the major BI-RJ-70- labeled peptide. Digested [3H]BI-RJ-70-RT was separated by this methodology and amino acid sequencing was performed on 10-200-pmol samples of labeled peptide. The peptide, as shown in Fig. 4, begins with Gln-174 and does not appear to extend past the next putative tryptic cleavage site, Arg-199. Unlabeled peptide, lacking the hydrophobic BI-RJ-70 probe, eluted earlier (107 min in Fig. 2 A ) than the labeled peptide as demonstrated by amino acid and sequence analysis, and coelution with synthetic 174-199 peptide (not shown).

Approximately 200 pmol of labeled peptide were measured in Fig. 4, compared to 164 pmol of radiolabel applied to the sequenator, indicating essentially stoichiometric labeling (i.e. 0.82). The major labeled residue within the peptide is Tyr- 181 which appeared in cycle 8, and accounts for 18% of the applied radioactivity. The PTH-Tyr signal in cycle 8 was substantially smaller than that of cycle 10. This was expected

4 1 0 \ 1 30

6 8 10 la 14

retention volume (ml)

FIG. 3. Size exclusion HPLC of photoadduct peptide purified by RP-HPLC. The standards (e) ribonuclease A (13,000 Da), insulin (5780 Da), ribonuclease S peptide (2160 Da), bradykinin (1240 Da), leucine enkephalin (556 Da), tryptophan (204 Da), and sodium azide (65 Da) and sample (0) were chromatographed on a TSK G2000SW column. Least squares, curve fit for elution of standards, were calculated (Rz = 0.98) and plotted (dashed line). A 0.2-ml aliquot of fraction 110-112 min was directly injected, fractions of 0.25-ml were collected, and radioactivity was measured by liquid scintillation counting. Retention volumes for sample fractions were adjusted to account for delay volume (0.3 ml) between detector flow cell and fraction tube.

- DPM

2 5 0 m o l o f PTH-amlno acld D P j j o o o o

Resldues 174-199

FIG. 4. Amino acid sequence of primary labeled peptide. Approximately 200 pmol of the major labeled peptide peak was subjected to Edman degradation. Of the PTH-amino acids released during each Edman cycle, 40% was injected on-line to a PTH-amino acid analyzer for identification and quantification. The quantities of PTH-amino acids shown have been corrected for total sample volume. The level of background signal was 2-5 pmol. The remaining 60% of sample volumes from each cycle were collected and subjected to liquid scintillation counting. DPM values have been corrected for background.

since tyrosine covalently labeled with BI-RJ-70 probe would not be identified as PTH-Tyr. The majority of radioactivity observed in cycles 9-12 was attributed to incomplete Edman degradation. Only 78% of Tyr-181 was cleaved from the peptide during cycle 8, leaving the remaining 22% of this residue to be cleaved in cycle 9. Similarly, approximately 22% of this carry-over Tyr-181 in cycle 9 will not cleave until cycle 10. Taking this carry-over into account, it appears that Tyr- 181, but not the succeeding 4 residues, was labeled in the 111- min-labeled peptide. Significantly, carry-over does not explain the radioactivity associated with Tyr-188 in cycle 15 suggesting that the Ill-min fraction is a mixture of two forms of modified peptide 174-199, with one species covalently labeled at Tyr-181 and the other a t Tyr-188.

As shown by Fig. 2B, other labeled peptides are present in the digest and elute predominantly between 95-110 and 112- 120 min. Sequence analysis, of 10 fractions spanning these retention windows and accounting for approximately 50% of the initial radioactivity, indicated the presence of various identifiable peptide components. Although not always the major component, peptides containing the sequence 174-199 were identified in nine fractions. The highest amount of radioactivity was always found in Edman cycle 8 from all 10 fractions. In addition, cycle 15 usually contained significantly more radioactivity compared to adjacent cycles. Relatively minor amounts of radioactivity, not attributable to carry- over, were found in cycles 9-16 suggesting the presence of additional labeled residues.

These data were confirmed when photoaffinity-labeled RT was subjected to partial proteolysis with endoproteinase Lys- C under nondenaturing conditions. When the digest was chromatographed on a Cs RP column, several radiolabeled peptides were resolved with approximately 48% of applied radioactivity being recovered in fractions 123-129 (Fig. 5). Analysis of these fractions by SDS-PAGE indicates a major peptide of approximately 30 kDa which is also the major site of radioactivity as shown by fluorographic analysis (Fig. 6). Amino acid sequence analysis of this 30-kDa peptide indicates that it begins with the expected N terminus of recombinant HIV-1 RT and presumably extends up through approximately residue 230. A further 21% of applied radioactivity was re-

Binding Site of Nevirapine (BI-RG-587) on HIV-1 Reverse Transcriptase 14673

0 25 50 75 100 125 150

Retention Time ( Min )

FIG. 5. Separation of endoproteinase Lys-C peptides of [3H] BI-RJ-70-RT photoadduct by RP-HPLC. An 8.5-nmol sample of [:'H]BI-RJ-70-RT photoadduct which had been subjected to partial proteolysis with endoproteinase lys-C was applied to a CR R P (4.6 X 250 mm) column as follows. Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% triflouroacetic acid in 10:90 watexacetonitrile. A gradient of 0-10% B in 10 min, 10-55% B in 140 min, and 55-100% B in 10 min was carried out a t a flow rate of 1 ml/min. Fractions of 1 ml were collected every 1.0 min from 0 to 160 min. A , detection by UV absorbance a t 214 nm. B, radioactivity of 50-pl aliquots was determined by liquid scintillation counting. C, detection by UV absorbance a t 335 nm.

n B Mr I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 I 2 3 4 5 6 78910l1l21314 1 0 6 80 50 ,

33 . 2a .

FIG. 6. Analysis of C8 R P fractions by SDS-PAGE. Column fractions were applied to a 15% SDS-PAGE gel using an SE-280 Tall Mighty Small gel apparatus purchased from Hoeffer Scientific (San Francisco, CA). Numbered lanes contain the following materials: 1 , [:'H]BI-RJ-70-RT photoadduct; 2, endoproteinase Lys-C digest of [:'H]BI-RJ-70-RT photoadduct; 3-14, C, R P fractions 123-130 and 142-145, respectively. A, protein detection by Coomassie Brilliant Blue staining. Numbers, molecular mass in kDa; arrow, position of the p66 subunit from HIV-1 RT. B, detection of 'H radioactivity by fluorography on X-Omat AR film.

covered in fractions 142-145. SDS-PAGE analysis indicates that the major radiolabeled polypeptide is undigested p66. Several other peptides containing minor amounts of radioactivity are apparent, including one of approximately 45 kDa (fraction 145) and another of approximately 33 kDa (fraction 130). However, due to the low amounts of radioactivity, these peptides were not analyzed further.

Tryptic mapping of the 30-kDa peptide (Fig. 7) yielded profiles highly analogous to those obtained with ['HJBI-RJ- 70-RT photoadduct (Fig. 2). Not surprising, due to the smaller size of the 30-kDa peptide relative to intact RT, fewer peptides were evident a t 210 nm in Fig. 7A as compared to Fig. 2A. As is the case for digested [:'H]BI-RJ-70-RT in Fig. 2, B and C, the most highly labeled peptide of the digested 30-kDa frag- ment elutes a t precisely 111 min in Fig. 7, B and C. Indeed, sequence analysis of this I l l-min fraction identified amino acids 174-199. Again, the largest amount of radioactivity, approximately 22% of that applied to the sequenator, was

0 25 50 75 100 125 150 Retention Time ( Min )

FIG. 7. Separation of tryptic peptides of ["HJBI-RJ-70 (30- kDa polypeptide) by RP-HPLC. Approximately 1.5 nmol of labeled, endoproteinase lys-C-derived 30-kDa polypeptide was analyzed as described in the legend of Fig. 2.

associated with cycle 8. After correcting for sequenator carry- over, the second largest amount of radioactivity was associated with cycle 15. These data corroborate the results shown in Fig. 4. The radioactivity eluting a t approximately 30 min in Fig. 7B is attributable to degraded ['H]BI-RJ-70, as radioactivity at the same retention is observed in the mock digestion in which trypsin is omitted (see above).

Our interpretation of these data is that the primary labeled peptide includes residues 174-199 and accounts for 75% of the radioactivity initially present in the tryptic digest of intact ["H]BI-RJ-70-RT. The most highly labeled individual residues are Tyr-181 and Tyr-188. The different RP-HPLC retention characteristics exhibited by the minor radiolabeled species may result from several factors including variations in peptide length due to incomplete tryptic digestion, reaction of ['HJBI-RJ-70 with other residues, and bond formation with different atoms on tyrosines 181 and 188. Since a single affinity probe was utilized for these studies, the possibility exists that additional regions of the RT molecule may directly participate in BI-RG-587 binding but were not affinity labeled by BI-RJ-70 for chemical or steric reasons. Thus, mapping studies are planned which will employ additional photoaffinity probes.

The possibility that Tyr-181 or Tyr-188 were initially more highly labeled, or that other residues within the peptide may also have reacted with the probe cannot be discounted since 30% of the applied radioactivity was found in the sequenator waste. This radioactivity may no longer be covalently bound to peptide since the relative instability of photoadducts gen- erated using the azido moiety is well documented (Bayley and Knowles, 1977; Bayley, 1983; Guillory, 1989; Rush and Kon- igsberg, 1990; Liu, 1990). Although adducts involving C-H insertion are the most stable and are likely to form upon reaction with hydrophobic residues such as tyrosine (Guillory, 1989), cleavage of radioprobe to liberate the free amine analogue of BI-RJ-70 is likely to occur under the reductive acidic conditions and elevated temperatures utilized in protein sequencing.

The observation that Tyr-181 and Tyr-188 are present in

14674 Binding Site of Nevirapine (BI-RG-587) on HIV-1 Reverse Transcriptase

the HIV-1 RT binding site of nonnucleoside inhibitors of the dipyridodiazepinone and thiobenzimidazolone classes is in- triguing. The region (residues 174-199) contains the most highly conserved block of amino acids present in RT from other retroviruses and is also found in viral RNA polymerases (Argos et al., 1988; Webster et al., 1989). This structural block consists of an Asp-Asp dipeptide flanked predominantly by hydrophobic residues. This conserved sequence apparently plays an important functional role since site-directed muta- genesis demonstrated that conversion of Asp-185 to His re- sulted in a complete loss of HIV-1 RT pol activity, and conversion of Tyr-183 to Ser yielded enzyme with only 1% of wild-type activity (Larder et al., 1987).

HIV-1 RT contains Tyr-181 and Tyr-188, whereas RT from most other characterized retroviruses does not contain tyrosine at both of these positions. Interestingly, reverse transcriptase from three retroviruses which do not contain tyrosine at positions 181 and 188 were tested (simian immunodeficiency virus, feline leukemia virus, and HIV-2), and these enzymes were not significantly inhibited by BI-RG-587 (Mer- luzzi et al., 1990; Wu et al., 1991). We suggest that Tyr-181 and Tyr-188 play a critical role in the binding of these classes of inhibitors of HIV-1 RT. Additional evidence to support this conclusion is provided by amino acid substitutions from HIV-2 RT into HIV-1 RT at these two positions which result in RTs that retain pol activity but are not sensitive to inhibition by the BI-RG-587 series of compound^.^

Acknowledgments-[3H]BI-RJ-70 was provided by John Proud- foot, Jerry Skiles, Palayakotai Raghavan, Ian Potocki, and Clark Perry. Recombinant HIV-1 RT was produced and purified by Klaus Lubbe, Gary Caviness, Julie Mikell, and Jan Glinski. Mass spectros- copy and amino acid analysis were performed by Walter Davidson, Keith McKellop, and Robin House. We thank Steve J. Benkovic,

"C.-K. Shih, J. M. Rose, G. L. Hansen, J. C. Wu, A. Bacolla, and J. A. Griffin, submitted for publication.

Cheng-Kon Shih, Jim Stevenson, Karl Hargrave, Christine Grygon, Gordon Hansen, Peter Pallai, Frank Hatch, Richard Shansky, and Mark Skoog for helpful discussions. We also thank Fritz Hess for his support of this project.

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