AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 8, Number 2, 1992 Mary Ann Liebert, Inc., Publishers

'>

Nonnucleoside Inhibitors of HIV-1 Reverse

Transcriptase: Nevirapine as a Prototype Drug

PETER M.

GROB, JOE C. WU, KENNETH A. COHEN, RICHARD H. INGRAHAM, CHENG-KON SHIH, KARL D. HARGRAVE, TARI L. MCTAGUE, and VINCENT J. MERLUZZI

ABSTRACT

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an ICS0 84nM in enzyme assays and ICS0 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic. =

=

INTRODUCTION IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) encodes at least three enzymes (reverse transcriptase, integrase, protease) that are required for virus replication. Reverse tran-

HUMAN

scriptase (RT) is responsible for an RNA-dependent DNA polymerase activity, an RNase H activity, and a DNA-dependent DNA polymerase activity. Early in the infection, all three of the catalytic activities of RT are essential for the production of proviral DNA which then integrates into the host cell genome. Inhibition of any one of these RT enzymatic activities should block viral replication and prevent the spread of HIV-1 infection (for review see Ref. 1). The first drug approved for HIV-1 infection (AZT, zidovudine) inhibits RT via a mechanism involving chain termination.2 This

occurs

because zidovudine is

a

nucleoside

analog

Boehringer Ingelheim Pharmaceuticals Inc., 90 East Ridge,

which

Box 368,

lacks the 3'-hydroxyl group necessary for phosphodiester bond formation with the succeeding nucleotide in the nascent DNA.3'4 While AZT and other nucleoside analogs are effective RT inhibitors, their use results in significant clinical side effects such as bone marrow suppression and peripheral neuropathy.5-7 Our initial studies began with a random search for HIV-1 RT inhibitors. The first active compound, a pyrido[2,3-è][l,4) benzodiazepinone was an analog of the M,-selective muscarinic receptor antagonist, pirenzepine.8 Synthesis of additional analogues and selection based on potency, specificity, bioavailability, metabolism, and physicochemical properties provided a dipyrido[3,2-b:2',3'-e][l,4]diazepinone (nevirapine, BI-RG587) (Fig. 1,/) as a candidate for development. We present here a review of the enzyme specificity, virology, structure-activity relation (SAR), mechanism of nevirapine action, and nevirapine resistance/sensitivity resulting from site-directed mutagenesis.

Ridgefield, CT 06877.

145

GROB ET AL.

homopolymer template primer or a heteropolymer template primer10 show that this compound is not a competitive inhibitor of HIV-1 RT. Nevirapine decreases the rate of the enzymatic catalysis (kcal) but does not affect the binding of the substrates (ks).9'I0 This observation indicates that nevirapine binds to both the binary (RT:template primer) and ternary (RT:template primendGTP) complexes of this enzyme.10

1, Nevirapine

VIROLOGY

Nevirapine inhibits HIV-1 replication in c8166 T-cell cultures having an IC5() against HIV-1,,,,, of 40 nM as determined by inhibition of cytopathic effect (CPE) and 10 nM as determined of p24 production; (Imax 100% for both asby inhibition ' ' was determined by in this cell culture system says).9, Viability =

of a tetrazolium salt (MTT) metabolic assay. This assay showed 50% cytotoxicity of nevirapine at 321,000 nM providing a selectivity ratio in vitro of >8000.9 Other isolates have been tested including the HIV-1RF strain and isolates from patients. In all cases, nevirapine was effective in reducing CPE and p24 production with similar IC50s.9 Maximum inhibition was also achieved against all isolates and strains. In situ hybridization experiments using peripheral blood mononuclear cells (PBMC) and a clinical isolate of HIV-1 have shown that nevirapine inhibits the accumulation of HIV-1 RNA. In the absence of nevirapine, 1 of 12 cells were positive for HIV-1 RNA whereas in the presence of nevirapine, 1 of 18,600 cells were positive.9 These results were significant in that these isolates were never adapted to grow in cell culture other than fresh PBMC and they were tested after only three passages.9 The effect of nevirapine on HIV-2 was tested in a plaque reduction assay using CD4-transfected HeLa cells and a syncytia assay using c8166 T-lymphoblastoid cells. In both cases, this compound was inactive against HIV-2.9' " '2 These results were not surprising because nevirapine did not inhibit SIV RT and HIV-2 RT.9 Since the target for RT inhibition by nevirapine is different from that of nucleoside analogs known to act as terminaors, we hypothesized that nevirapine would be active against AZTresistant strains of HIV-1 and may synergize with AZT for inhibition of HIV-1 replication in culture. In fact, both hypotheses are correct. Nevirapine is effective against AZT-resistant isolates. Viral isolates obtained from patients before and after AZT treatment differ severalfold in their sensitivity to AZT. Nevirapine was found to be equally effective on AZT-sensitive and AZT-resistant isolates.I2 Nevirapine is also synergistic with AZT for inhibiting HIV-1 replication in CD4-transfected HeLa cells.12 Additionally, no antagonism was observed between '2 nevirapine and AZT at high concentrations. Additional studies have shown that nevirapine inhibits HIV-1 p24 production in CEM T-lymphoblastoid cells and in primary human monocyte cultures. '2 This compound did not inhibit CPE in HeLa cells infected with rhinovirus 54 or poliovirus I, bovine kidney cells infected with influenza A virus, or human lymphoid cells infected with vaccinia virus."12 These results demonstrate further specificity of nevirapine in its inability to inhibit RNA and DNA viruses not dependent upon reverse transcriptase in their life cycle. A summary of the effects of nevirapine on virus replication is shown in Table 2. means

3, BI-RJ-70

FIG. 1. Structure of nevirapine (I); 1 l-cyclopropyl-5,11dihydro-4-methyl-6//-dipyrido[3,2-£:2',3'-e][ 1,4]diazepin-6one; general structure of dipyrido[3,2-¿>:2',3'-e][l,4]diazepinone (2); and structure of photoaffinity probe BI-RJ-70 (3).

ENZYME SPECIFICITY AND KINETICS

Nevirapine is a highly specific inhibitor of HIV-1 RT RNAand DNA-dependent polymerase activities. As shown in Table 1, this compound inhibits HIV-1 RT but has no effect on any other RT tested and is inactive against all four human DNA polymerases.9 Steady-state kinetics of nevirapine using either a Table 1. Enzyme Specificity of Nevirapine

Enzyme HIV-1 reverse transcriptase HIV-2 reverse transcriptase FLV reverse transcriptase SIV reverse transcriptase Human DNA polyerase alpha Human DNA polymerase beta Human DNA polymerase delta Human DNA polymerase

Inhibition +

by nevirapine*

(IC50

=

84 nM)

gamma HIV-1 protease

a(+): indicates positive inhibition by nevirapine; (—) indicates no inhibition by nevirapine. Abbreviations: FLV = feline leukemia virus; SIV simian immunodeficiency virus. =

NEVIRAPINE

147

Table 2. Effect of Nevirapine on Virus Replication

Target cell

Virus

HIV-l,IIb

Measurement

c8166T cells c8166T cells Human PBMC c8166T cells c8166 T cells Human PBMC CD4+ HeLa CEM T cells Human monocytes CD4+ HeLa CD4+ HeLa c8166 T cells HeLa HeLa MDBK HeLa

HTV-lnn, HlV-lmb HW-Irf HIV-1UMGL

HIV-1 isolates

LAV-Ibru LAV-1BRU HTLV-IIIBal.85

HIV-1 AZTR isolates

HIV-2R0D HIV-2rod

Rhinovirus 54 Coxsackie A13 Influenza A Poliovirus type I

Nevirapine* + + + + + + + + + +

Syncytia p24 p24 Syncytia Syncytia p24 Plaque p24 p24 Plaque Plaque Syncytia CPE CPE CPE CPE

a(+) Inhibition of virus replication; (—): no inhibition of virus replication. Abbreviations: AZTR AZT-resistant clinical isolates; PBMC peripheral blood mononuclear cells; MDBK bovine kidney cells; CPE = cytopathogenic effect. =

=

=

IMMUNOLOGY, PHARMACOLOGY, AND PHARMACOKINETICS

Preliminary studies indicate that nevirapine is not immunosuppressive and is not toxic to bone marrow progenitors.913

Furthermore, initial studies of metabolism and tissue distribution were carried out in rodents and primates. Such tissue distribution studies after oral administration indicate a plasma:brain ratio of 0.8:1,0.9 These results are encouraging for the development of nevirapine and its analogs as antiviral agents for the treatment of HIV-1 infection in humans which frequently exhibits CNS involvement. The nonnucleoside nature of this compound may circumvent the toxicities normally associated with nucleoside inhibitors of RT.

STRUCTURE-ACTIVITY RELATIONSHIPS

Nevirapine belongs to a class of novel tricyclic dipyridodiazepinones (Fig. 1,2), the synthesis and detailed SAR of which recently have been reported.14 These compounds were initially evaluated in an HIV-1 RT enzyme assay, and subsequent testing for their ability to block HIV-1 replication in cell culture demonstrated a direct correlation in potency between the two assays (Hargrave KD, Koup RA, Eckner RJ, Merluzzi VJ, Brewster FE, and Sullivan JL, unpublished data). Substituent effects were explored at the two diazepinone nitrogen atoms and at each of the six available aromatic positions. Several thiocarbonyl analogs were also prepared. Although generally more potent than the corresponding carbonyl compounds, these sulfur derivatives are significantly less soluble and are rapidly metabolized in vivo.14 At the lactam nitrogen (position 5), substituent effects are altered by the presence or absence of a substituent on the 4-position of the pyridine ring. If a hydrogen is present on position 4, then small alkyl or acyl groups on the lactam nitrogen confer greater potency, with

methyl generally being optimum for activity. On the other hand, unsubstituted lactam nitrogen is preferred if a substituent is present on position 4. At the other diazepinone nitrogen (N-l 1), a small alkyl group such as ethyl or cyclopropyl is optimum, with ethyl usually being the most potent. In addition, although the ethyl analogs are more water soluble, the corresponding cyclopropyl derivatives are generally more bioavailable in the an

rat

(Riska P, Erickson D, Dinallo R, Hanson G, and Hattox S,

unpublished data). Improved potency is observed with proper substitution on the A-ring (Fig. 1,2). At position 2, a variety of groups (e.g., methyl, nitro, amino) with diverse physicochemical properties are tolerated. In contrast, the same substituents at position 3 reduce significantly the potency relative to the unsubstituted compound. Small lipophilic groups such as methyl, ethyl, and chloro are preferred at position 4. Methyl, however, provides maximum potency when coupled with an unsubstituted lactam nitrogen, and this combination is found in all of the most active compounds. In the C-ring, substituents on positions 7, 8, or 9 generally have a detrimental effect on potency, although an amino group at position 8 is tolerated. MECHANISM OF ACTION The inhibitory mechanism and the primary structure of the nevirapine binding site in HIV-1 RT were delineated by the use of a photoaffinity analog. This azido-pyrido[2,3-b][ 1,5]-benzodiazepinone analogue, BI-RJ-70 (Fig. 1, 3 ), exhibits many of the characteristics, especially its inhibitory specificity and potency against HIV-1 RT. BI-RJ-70 has an IC50 of 160 nM for inhibiting HIV-1 RT but exhibits no activity against other DNA polymerases including HIV-2 RT.9'5 Upon ultraviolet (UV) light illumination, the azido moiety on BI-RJ-70 is rapidly transformed into a nitrene intermediate, which presumably is capable of reacting with proximal amino acid residues at or near

148

GROB ET AL.

the inhibitor binding site. Consequently, the enzyme is covalently labeled and inactivated. The resulting labeled HIV-1 RT can thus be used to determine the stoichiometry of binding and the location of the inhibitor binding site in the enzyme. During weak UV illumination, the rate of HIV-1 RT photoinactivation can be conveniently monitored by assaying the enzyme activity after the removal of free and noncovalently bound labels at different illumination periods. It was found that the HIV-1 RT polymerase activity decreased with time t following the pseudo-first-order equation -d[ln(A/A0)]/dt k', where A and A0 denote the activities of the labeled and unlabeled HIV-1 RT, respectively.15 When photoinactivation was conducted in the presence of nevirapine, the compound protected the enzyme from inactivation by the photoaffinity probe in a competitive manner. This suggested that the photoaffinity probe BI-RJ-70 and nevirapine bind to HIV-1 RT at the same site. The results presented in Figure 2 demonstrate the stoichiometry of the photoaffinity labeling of HIV-1 RT by [3H]BI-RJ-70. Clearly, HIV-1 RT heterodimer can be inactivated by binding with one molecule of [3H]BI-RJ-70. Subsequent analysis of the labeled HIV-1 RT by sodium dodecyl sulfide-polyacrylic gel electrophoresis (SDS-PAGE) followed by autoradiography indicated that the probe was preferentially associated with the p66 subunit. The photoaffinity labeling of HIV-1 RT by BI-RJ-70 appeared to be very selective, since even in the presence of a 500-fold excess of cytosolic proteins from human peripheral blood leukocytes only the p66 subunit was labeled. Evidence has been presented that the polymerase and RNAse '6 H active sites of HIV-1 RT are located on the p66 subunit and are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex.1720 The nevirapine binding site apparently is distinct from either of these two catalytic sites because the enzyme substrates dGTP, poly(rC)/ oligo(dG), and t-RNA provided no protection against photoaffinity labeling of HIV-1 RT by BI-RJ-70.15 This assertion is further supported by steady-state kinetics data showing noncompetitive inhibition9 and the observation that nevirapine failed to inhibit other DNA polymerases.9 It should be noted that nevirapine and its analogues are not the only compounds that can bind to the nevirapine site. At least three other structurally distinct classes of nonnucleoside HIV-1 RT inhibitors, namely, TIBO,21 HEPT,22 and L-697,63923 were also found to compete with nevirapine for the same binding site in HIV-1 RT. It appears that HIV-1 RT possesses a modulatory site (RT,MS) that can be occupied by compounds with a high degree of structural diversity which inhibit the polymerase activity of the enzyme. However, it is not clear why only HIV-1 RT, but not HIV-2 RT or other DNA-polymerases, has this modulatory site. =

IDENTIFICATION OF THE NEVIRAPINE BINDING SITE ON HIV-1 RT The amino acid residues labeled by [3H] BI-RJ-70 at the nevirapine binding site were identified by tryptic mapping and peptide sequencing.24 The photoadduct [3H]BI-RJ-70 RT was digested with trypsin and the resulting mixture was resolved by

n

5000 5

4000 +

Ho. o

ZZ]pd6

12

25

28 43

55 min

.I

"L^r>.

—

3000

2000

1000

0

FIG. 2.

Selective

2.5

0

?

's

?

i_g, 5 12

25

28

y

y

43

55

B

Time of Illumination

(min)

photoaffinity labeling

of HIV-1 RT

by

[,H]BI-RJ-70. (A) Linear relationship between the inhibition of HIV-1 RT and the average number of BI-RJ-70-label covalently attached to the enzyme, r ratio of the specific activity of the =

labeled HIV-1 RT to that of the unlabeled enzyme; n = the number of covalently attached [3H]BI-RJ-70 label per enzyme. The linear r vs. n plot has a slope of drldn 1, showing that each label completely inactivates one HIV-1 RT heterodimer. (B) SDS-PAGE gel of the labeled HIV-1 RT was autoradiographed, showing that the p66 subunit was preferentially labeled.17 (Reproduced with permission by Biochemistry.) =

—

high-performance liquid chromatographs (HPLC) (Fig. 3). Both the radioactivity of [3H]BI-RJ-70 (Fig. 3B) and its absorbance at 335 nm (Fig. 3C) were exploited to locate (3H]BI-RJ-70-labeled peptides in the tryptic map. The primary labeled peptide, which eluted at 111 min (Fig. 3C), corresponded to residues 174-199 as determined by peptide sequencing (Fig. 4) and amino acid

analysis.

In the primary labeled peptide the major labeled residue which appeared in cycle 8 (Fig. 4) was identified as Tyr,si. Significantly, radioactivity also appeared in cycle 15 which corresponds to Tyr188. This result suggests that the 111-min fraction contained two isomers of labeled peptide, with one isomer labeled at Tyrm and the other at Tyr,88. The peptide region containing amino acids 181-188 of HIV-1 RT is therefore implicated in the binding of dipyridodiazepinone

and TIBO classes of nonnucleoside inhibitors. This region, which consists of an Asp-Asp dipeptide flanked predominantly by hydrophobic residues, is the most highly con-

NEVIRAPINE

149 The sensitivity of these chimeric RTs to nevirapine, as well as representative TIBO derivative, were quite varied (Table 3). Chimeric enzymes in the HIV-1 RT background with single amino acid substitutions from corresponding residues in HIV-2 RT exhibited dramatically reduced sensitivity to these nonnucleoside inhibitors. These results demonstrate that Tyr,8, and Tyr]88 of HIV-1 RT are necessary for sensitivity to these nonnucleoside inhibitors. These results also raised the possibility that resistant variants of HIV-1 could arise by mutations of the binding site of the nonnucleoside inhibitors. In fact, cell culture variants of HIV-1 exhibiting resistance to nevirapine, TIBO derivative, and L-693,593 have been reported recently with mutations at amino acids 181 and 103.34 Similar observations were made where an HIV-1 isolate with a single change at residue 181 from tyrosine to cysteine results in 200-fold reduced to a

sensitivity to nevirapine.35

O

25

50

75

Retention Time

100

(

Min

125

150

)

FIG. 3.

Separation of tryptic peptides of 3H-BI-RJ-70-RT photoadduct by reversed-phase high-performance liquid chromatography. A 375-pmol sample of 3H-BI-RJ-70-RT digested by trypsin was separated by a C18 narrow bore column and a gradient of acetonitrile in water containing 0.052-0.060% trifluoroacetic acid. (A) detection was by UV absorbance at 210 nm. (B) radioactivity was measured by liquid scintillation counting of column fractions of 400 (xl. (C) detection was by UV absorbance at 335 nm. (Reproduced with permission by Journal of Biological Chemistry.) served sequence motif in RT and viral polymerases.25-27 It is functionally important, as conversion of TyrI83 to Ser, or Asp,85 to Asn results in dramatic loss of HIV-1 RT polymerase

activity.28

SITE-DIRECTED MUTAGENESIS

Although structurally and functionally similar to HIV-1 RT,29~31 HIV-2 RT is completely insensitive to nevirapine (IC50 > 200 u,M). This differential sensitivity to nevirapine prompted us to construct chimeric HIV-l/HIV-2 reverse transcriptases in order to identify domains that confer sensitivity/ resistance to this inhibitor. Structural characterization of the

photoadduct [3H]BI-RJ-70-RT identified two labeled residues, Tyr,8, and Tyr188 (Fig. 4).24Tyrosines at positions 181 and 188 of RT are strictly conserved in all HIV-1 isolates, whereas isoleucine/valine or leucine are highly conserved at these positions, respectively, in HIV-2 RT.32 As shown in Figure 5, reciprocal chimeric enzymes were constructed in both HIV-1 and HIV-2 backgrounds with substitutions of corresponding amino acid residues from the other viral RT at or surrounding residues 181 and

188.33

However, these mutant enzymes and resistant variants remained as sensitive to foscarnet and AZTTP as wild type HIV-1 RT (Table 3).34"35 A particularly interesting observation is that sensitivity to nevirapine and TIBO can be conferred onto HIV-2 RT. This sensitivity resulted from substitution of residues 176-190 from HIV-1 RT to HIV-2 RT.33 This result corroborates with the earlier observation that nevirapine and TIBO share the same binding site in HIV-1 RT. Moreover, these studies provide a specific site for identification and characterization of potential clinical isolates resistant to nonnucleoside therapy for the treatment of AIDS.

CONCLUSION

Nevirapine, a potent nonnucleoside inhibitor of HIV-1 RT, exhibits an IC5() 84 nM and 40 nM in an RT enzyme assay and against virus replication in cell culture, respectively. This inhibitory activity is highly specific since RT from HIV-2, SIV, and FeLV are unaffected. Additionally, eukaryotic DNA polymerases alpha, beta, delta, and gamma are also not inhibited by =

nevirapine. The inhibitory activity of nevirapine is not competitive with respect to template primer and nucleoside triphosphates, indicating that this inhibitor does not act directly at the catalytic site of RT. An important consequence of this is that HIV-1 mutants

which are resistant to AZT, a potent nucleoside RT inhibitor, remain fully sensitive to nevirapine.12 These experimental findings indicate that nevirapine, as well as the reported TIBO compounds, interacts with HIV-1 RT in a region containing Tyr,8, and Tyr,88. This region (residues 176-190) is highly conserved in other retroviral RTs and also is present in viral RNA polymerases. A highly conserved sequence motif, YM/GDD, flanked by hydrophobic amino acids is found within this region. One prediction of secondary structure for this motif has been made28 where the DD residues are located in a beta turn bracketed by beta strands; this model could bring tyrosines 181 and 188 into close proximity. This hypothesis is consistent with the observation that both tyrosines 181 and 188 are labeled by a single photoaffinity probe. We conclude that Tyrm and Tyr,88 are important components of the nevirapine binding site on HIV-1 RT. Importantly, particular amino acid substitutions at these positions results in an

pmol Y//////À

DPM

250

30000

200 Y

20000

10000

GlnAsnProAsp

Ile Val Ile

Tyr Gin TyrMetAspAspLeu Tyr Val Gly Ser AspLeu Glu Ile Gly Gin

His

Arg

Residues 174-199 FIG. 4. Amino acid sequence of primary labeled peptide. Approximately 200 pmol of the major labeled peptide was sequenced by Edman degradation analysis. During each cycle of Edman degradation, 40% of the PTH-amino acids released was injected on-line to a PTH-amino acid analyzer for identification and quantification. The remaining 60% was collected and subjected to liquid scintillation counting. The radioactivity measured in the four Edman cycles following Tyr 181 can be attributed to carryover of labeled Tyr 181 into succeeding residues as a result of incomplete Edman degradation at each step. (Reproduced with permission by Journal of Biological Chemistry.) 560

RT1 wt

559

RT2 wt 440

RT1 440 181

RT1-Y181I 181

RT1-Y181V 188

RT1-Y188L 181

188

RT1-Y181I,Y188L RT1 176-190 176-190

RT2 176-190 RT2-I181Y

RT2-L188Y 188

RT2-I181Y.L188Y 181

188

Schematic representation of chimeric constructs between HIV-1 and HIV-2 RTs. The open box indicates the HIV-1 RT sequence. The shaded box indicates the coding sequences of HIV-2Rod RT. The position number indicates amino acid residues at which the substitution was made, eg. "RT1-Y181I" is HIV-1 RT in which tyrosine (Y) was replaced with isoleucine (I). Other abbreviations are valine(V) and leucine(L). RT1 176-190 has amino acids 176to 190, inclusive, from HIV-2 substituted into HIV-1 RT. The amino acid sequence of peptide 176-190 from HIV-1 RT: PDIVIYQYMDDLYVG—HIV-2 RT: KDVIIIQYMDDILIA.

FIG. 5.

coding

NEVIRAPINE

151

Table 3.

Enzyme* RT1 wt RT1 440 RT1-Y181I

RT1-I-Yb RT1-Y181V RT1-Y188L RT1-Y181I Y188L RT1 176-190 RT2 wt RT2-I181Y RT2-L188Y RT2-I181YL188Y RT2 176-190

"See

Figure

IC50s

for

HIV-1/HIV-2 RT Chimeras

BIRG587

R829I3

PFA

AZTTP

(ßM)

(ßM)

(ßM)

(ßM)

0.25 ± 0.01 0.11 ±0.01 73+4 0.28 ± 0.05 56 + 4 >200 >200 >200 >200 >200 >200 121 +21 0.8 ±0.1

1.7 ± 0.1 ND 36 ± 1 ND 60 ± 10 60 ± 10 >200 >200 >200 >200

>200 8.2 ± 0.4 0.52 ±0.10

29 24 65 ± 20:

6 12 30 3 17 ± 10 17 ± 10 60 ± 8 ND 4.8 ± 0.7 ND ND ND ND

ND 0.02 ± 0.01 ND ND ND 0.02 ± 0.01 0.02 ± 0.01 0.11 ±0.03 ND ND ND ND

1 for mutant construction.

bThe symbol (I—Y) indicates the back mutation of mutant 1-181

enzyme which exhibits full polymerase activity but is significantly less sensitive to nevirapine, the TIBO compounds, and L-693,593.25'35 For this reason, it may be expected that HIV-1 mutants may arise upon long-term exposure to these nonnucleoside RT inhibitors. The clinical ramifications of this will be the need for combination drug therapy where the appearance of virus resistant to both antiviral therapeutic agents will be minimized. The nonnucleoside inhibitors have already proved useful tools in understanding RT structure-function relationships. These compounds are currently undergoing clinical evaluation for their potential as the next generation of anti-AIDS

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0.05 ± 0.01

to

wild-type residue.

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