(+)-Calanolide a of Wild-Type and Mutated Forms of HIV-1 Reverse Transcriptase

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Antiviral Therapy 4: 203–209 Sensitivity and resistance to (+)-calanolide A of wild-type and mutated forms of HIV-1 reverse transcriptase

Yudong Quan1, Dimitrios Motakis1, Robert Buckheit Jr2, Ze-Qi Xu3, Michael T Flavin3, Michael A Parniak1 and Mark A Wainberg1*

1McGill University AIDS Centre, Lady Davis Institute, Jewish General Hospital, 3755 Côte Ste-Catherine Road, Montréal, Québec, Canada H3T 1E2 2Southern Research Institute, 431 Aviation Way, Frederick, MD 21701-4756, USA 3Sarawak MediChem Pharmaceuticals Inc, 12305 South New Avenue, Lemont, IL 60439, USA

*Corresponding author: Tel: +1 514 340 8260; Fax: +1 514 340 7537; E-mail: [email protected]

We have tested both wild-type and drug-resistant susceptibility to the latter drug. In contrast, the Y188H mutated, recombinant human immunodeficiency virus substitution in RT resulted in about 30-fold resistance type 1 (HIV-1) reverse transcriptase (RT) molecules for to (+)-calanolide A in these assays but did not result in sensitivity to each of two non-nucleoside RT inhibitors diminished sensitivity to nevirapine. Tissue culture (NNRTI), (+)-calanolide A and nevirapine, in primer results indicated that the combination of (+)-calanolide extension assays. We found that RT containing either A and nevirapine possessed an additive to weakly syner- the V106A or Y181C substitutions, associated with gistic effect in blocking replication of HIV-1 in tissue NNRTI resistance, displayed ≈90-fold resistance to nevi- culture. These results suggest that (+)-calanolide A and rapine but remained fully sensitive to (+)-calanolide A nevirapine might have rationale as a combination and that the Y181C mutation marginally enhanced therapy for HIV disease.

Introduction

Drug development for human immunodeficiency virus tance to other NNRTIs might have affected sensitivity (HIV) disease relies on an understanding of HIV to (+)-calanolide A. We also studied the combination of patterns of susceptibility and resistance to antiviral (+)-calanolide A and nevirapine in both cell-free assays compounds. Viral replication can be inhibited by both as well as in tissue culture studies to assess their effect nucleoside analogue reverse transcriptase (RT) on viral replication. inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI), but resistance has developed against each of Materials and Methods the drugs currently in use after prolonged chemotherapy [1–9]. Primer extension assay Extensive cross-resistance exists among members of A 20-nucleotide DNA primer termed PA (CCCATC- the NNRTI family of drugs because these compounds GATCTAATTCTCCC), complementary to 5′ end of share both a common inhibitory mechanism and the gag region of the HIV-1 genome, was end-labelled binding site within the enzyme’s catalytic domain and an RNA template, spanning the R region through [10–13]. However, (+)-calanolide A may represent a a portion of gag, was prepared as previously reported distinct NNRTI since it has been reported to retain [18–19]. Complexes between template and primer effectiveness against HIV variants that displayed resis- were also prepared as described [18–19]. The RT tance to other members of this class [14–17]. Likewise, product initiated by the PA primer is 376 bases in full- viruses resistant to (+)-calanolide A have been shown length. Mutated and wild-type recombinant RTs were to retain sensitivity to other NNRTIs. To investigate prepared as described [3]. this subject further, we performed cell-free assays with The RT reaction mixture contained 50 mM recombinant wild-type and mutated forms of RT to Tris–HCl pH 7.8, 5 mM MgCl2, 60 mM KCl, 10 mM determine whether mutations in RT that encode resis- DTT, 0.1 mM dNTPs, 50 nM HIV-1 RNA, 100 nM

Table 1. IC50 values of wild-type and mutated RTs for (+)-calanolide A and nevirapine (+)-Calanolide A Nevirapine

IC50 (µM)* Fold resistance IC50 (µM)* Fold resistance Wild-type 0.34±0.06 — 0.044±0.01 — M184V 0.36±0.07 1.2† 0.046±0.01 1.05 E89G 0.36+0.05 1.2 0.22±0.01 5.0 E89G-M184V 0.45±0.06 1.45 0.14±0.01 3.2 K103T 1.05±0.08 3.4 0.56±0.03 12.7 V106A 0.71±0.05 2.3 3.9±0.4 88.6 Y181C 0.09±0.02 0.35 4.1±0.2 93.2 Y188H 9.80±0.86 31.6 0.051±0.01 1.2

*IC50±SD. †Fold resistance was obtained by dividing the mean IC50 obtained with mutated RT by that obtained with wild-type enzyme. primer and 50 nM RT in a total volume of 20 µl. strain of HIV-1 [4]. The cells were maintained in RPMI Varying concentrations of RT inhibitors, (+)-calanolide 1640 medium supplemented with 10% foetal bovine A and nevirapine were used in these assays and the serum. The HIV inhibitory activity of the compounds reaction mixtures were incubated for 60 min at 37°C. was evaluated in a microtitre anti-HIV assay by quanti- Products were extracted with phenol–chloroform, fying the ability of a compound to inhibit HIV-induced boiled for 5 min and electrophoresed in a 5% dena- cell killing or HIV replication. Quantification was turing polyacrylamide gel. The band densities of performed using the tetrazolium dye XTT, which is full-length radioactive products were analysed by metabolized to a coloured, formazan product by viable molecular imaging (Bio-Rad). cells. Statistical evaluations were performed using MacSynergy II [20]. The results of the combination Effect of drug on viral replication assays are presented in three dimensions for each The effects of drug combinations or single drugs on combination and concentration, yielding a surface of HIV replication were assessed in CEM-SS cells, an activity extending above (synergy) or below (antago- established human cell line acutely infected with the IIIB nism) the plane of additive interaction.

Figure 1. Inhibition of wild-type and mutated RT by (+)-calanolide A in a primer extension assay

125

◆ ■ ▼ ✖ ▼ 100 ● ■ ▲ ▼ ❒ ◆ ❍

▲ ■ ▼ 75 ✖ ❒ ◆ ❒ Wild-type ❍

✖ M184V

❍ 50 E89G ▲ ▲

Relative DNA synthesis ❍ 89G–184V ❒ ■ ● K103T ■ ◆ V106A 25 ● Y181C ● ■ ▼ Y188H ◆ ❍ ▼ ✖ ▲ ◆ ❒ ❍✖ ● ▲❒ 0 ● 0 0.01 0.10 1 10 100 (+)-Calanolide A (µM)

The RT reaction mixture contained 50 mM Tris–HCl pH 7.8, 5 mM MgCl2, 60 mM KCl, 10 mM DTT, 0.1 mM dNTPs, 50 nM HIV-1 RNA, 100 nM primer PA and 50 nM RT in a total volume of 20 µl. The RT product of the (–) strong-stop DNA product generated in this reaction is 376 nucleotides long. Varying concentrations of (+)-calanolide A were used in these assays and reaction mixtures were incubated for 60 min at 37°C. Products were electrophoresed in a 5% denaturing polyacrylamide gel and the full-length DNA generated was then analysed by molecular imaging.

Figure 2. Inhibition of wild-type and mutated RT by nevirapine in a primer extension assay

125

● ● 100 ◆ ◆

◆

●

75 ■

■

❍ ◆ ▲ ❒ Wild-type ▼ ● 50 ✖ ✖ M184V ❒ ❍ Relative DNA synthesis ▼ ❍ E89G ▲ ■ ✖ ❒ ▲ 89G–184V

■ ▼ 25 K103T ● ✖ ◆ ◆ V106A ❒ ❍ ● Y181C ▲ ■ ▼ Y188H ▼❒✖ ■ ▲❍✖❒❒▼ ▼✖▲ 0 ❍ 0 0.01 0.1 1.0 10.0 Nevirapine (µM)

The experimental conditions used were the same as described for Figure 1. Products were electrophoresed in a 5% denaturing polyacrylamide gel and the full-length DNA generated was analysed by molecular imaging.

Results can indeed confer resistance to a variety of RT inhibitors including several NRTIs and NNRTIs [21]. Effects of mutations in RT on sensitivity to (+)-calanolide A Effects of mutations in RT on sensitivity to We tested the sensitivity of wild-type recombinant RT nevirapine as well as several mutated forms of RT to (+)-calanolide We also tested the sensitivity of the various RT species A in cell-free primer extension assays. The enzymes to nevirapine. In contrast to results obtained with (+)- tested included several known to be resistant to various calanolide A, both the Y181C and V106A mutations NNRTIs containing the mutations K103T, V106A, conferred high-level resistance to nevirapine, increased Y181C or Y188H. We also tested RT molecules that IC50 values of about 90-fold compared with wild-type contained mutations that confer resistance to various RT. The K103T substitution conferred moderate resis- NRTIs; M184V and M184V/E89G. The results of gels tance to nevirapine (approximately 12-fold increased analysed by molecular imaging (Figure 1) show that the IC50), whereas the Y188H RT was equally sensitive as Y188H substitution conferred fairly high resistance to wild-type enzyme (Figure 2 and Table 1). (+)-calanolide A, with an increase in IC50 of about 30- Consistent with previously reported viral sensitivity fold compared to wild-type enzyme (Table 1). The analysis [21], the E89G-containing RT displayed low- sensitivities of RTs containing either the V106A and level resistance to nevirapine (≈fivefold), whereas RT K103T substitutions were only slightly altered, with containing the M184V mutation was as sensitive to increases in IC50 values limited to two- to threefold. In nevirapine as wild-type RT (Figure 2 and Table 1). contrast, RT containing the Y181C mutation, associ- However, the doubly mutated E89G-M184V enzyme ≈ ated with resistance to several NNRTIs, displayed displayed a threefold increase in IC50 in regard to enhanced sensitivity to (+)-calanolide A, with an IC50 nevirapine, consistent with previously reported studies value that was diminished ≈threefold in comparison on the dominance of E89G over M184V in regard to with wild-type enzyme (Table 1). None of the RTs enzyme processivity [19]. containing NRTI resistance-associated mutations; The above data suggest that (+)-calanolide A and M184V, E89G or both M184V/E89G within the same nevirapine may interact differently with HIV-1 RT and enzyme, showed altered sensitivity to (+)-calanolide A are consistent with previously reported results showing (Figure 1 and Table 1), although the E89G substitution that HIV strains that were resistant to (+)-calanolide A

Antiviral Therapy 4:4 205 Y Quan et al.

Figure 3. Combinatory inhibition of wild-type and mutated RT activity by nevirapine and calanolide A

(a) (b) Wild-type V106A Y181C Y188H 0 0.05 0.2 0 0.8 3.0 10 µ 0 Nev (µM) Nev ( M) 0 0.8 3.0 10 0.005 0.2 0.8 µ 0 0.1 0.4 1.5 0 0.1 0.4 1.5 0 0.1 0.4 0 0.1 0.4 1.5 0 0.1 0.4 0 0.1 0.4 1.5 0 0.1 0.4

1.5 1.5 1.5 Cal ( M) 0 1.5 5.0 20

µ 0 0.1 1.5 0 1.5 5.0 20 0 0.1 0 1.5 5.0 20 0 0.1 0 1.5 5.0 20 0.4 0 0.1

Cal ( M) 1.5 1.5 0.4 0.4 1.5 0.4

Continued opposite

remained sensitive to nevirapine [22,23]. Our finding both drugs are increased. Thus, the additive to syner- that RT containing the Y181C mutation possessed gistic effect against wild-type RT was observed when enhanced sensitivity to (+)-calanolide A is also consis- nevirapine was used at concentrations of 0.05 and 0.2 tent with observations that nevirapine-resistant viruses µM together with (+)-calanolide A at 0.1, 0.4 and 1.5 containing Y181C were cross-resistant to many µM (Figure 3a,c), since the extent of inhibition in NNRTIs, but not to (+)-calanolide A [4]. combination was greater than that of either drug alone The effects of (+)-calanolide A and nevirapine at the same concentrations. With RT that contained the combinations on RT activity were also studied. The V106A substitution, a mutation conferring twofold results of Figure 3 show by gel and Dixon plot analysis resistance to (+)-calanolide A and 10-fold resistance to that (+)-calanolide A and nevirapine in combination nevirapine, respectively, a similar additive to synergistic had an additive to synergistic effect in regard to both effect was observed for the combination only when wild-type and mutated forms of RT. This effect nevirapine was used at higher concentrations, 3.0 and appeared to be concentration-dependent in regard to 10.0 µM (Figure 3c). This is indicated by the fact that RT molecules that possessed the V106A, Y181C and the value of 1/V did not increase significantly when low Y188H mutations. In the Dixon plots, an increase in concentrations of either drug were employed, a finding the value of 1/V designates a decrease in the speed of attributable to the resistance-conferring potential of the enzyme catalysis. This is seen as the concentrations of V106A substitution (Figure 3c).

Figure 3. Continued from opposite page

Wild-type V106A (c) 5 5 Nevirapine (0) Nevirapine (0) 4 Nevirapine (0.05) Nevirapine (0.8) Nevirapine (0.2) Nevirapine (3.0) 3 2.5 Nevirapine (10) 1/v 2 1/v 1 0 0

–0.5 0 0.5 1.0 1.5 2.0 –0.5 0.0 0.5 1.0 1.5 2.0 Calanolide A Calanolide A

Y181C Y188H 4 5 Nevirapine (0) Nevirapine (0) Nevirapine (0.8) 3 Nevirapine (0.05) Nevirapine (3.0) Nevirapine (0.2) Nevirapine (10) Nevirapine (0.8) 1/v 2.5 2 1/v 1

0 0

–0.5 0 0.5 1.0 1.5 2.0 –5 0 5 10 15 20 25 Calanolide A Calanolide A Nev, nevirapine. Cal, (+)-calanolide A. Experimental conditions were the same as described in Figure 1, except that variable concentrations of both nevirapine and (+)-calanolide A were used. (a) Products of reactions with either wild-type enzyme or mutated V106A enzyme were electrophoresed in a 5% denaturing gel and displayed by autoradiography. (b) Same as (a) with mutated Y181C and Y188H enzymes. (c) Data from (a) and (b) above were analysed by molecular imaging and are shown in Dixon plots for wild-type RT and RT molecules containing each of the V106A, Y181C and Y188H substitutions.

With regards to RT molecules that possessed the the other, the additive to synergistic effect was not Y181C and Y188H mutations, which were only resis- observed until the concentrations of the less effective tant to one of the drugs tested but fully susceptible to drug approached its IC50 values (Figure 3c). For

Figure 4. Combinatory inhibition of HIV replication in a microtitre assay performed with CEM-SS cells

50 50 50 40 40 40 30

30 Inhibition (%) 30 20 20 20 10 10 10 0 0 0 –10 0.8 0.5 0.4 0.2 0.1 ) 0.25 0.05 µM Nevirapine0.13 concentration ( 0.025 0.013 0.06 0.006 0.03 0.003 0 0 µM) Calanolide A concentration ( Quantification was achieved by measuring the coloured formazan product of the tetrazolium dye XTT when metabolized by viable cells. The results of the combination assays are presented three dimensionally for each combination concentration, yielding a surface of activity extending above (synergy) or below (antagonism) the plane of additive interaction (see Materials and Methods).

Antiviral Therapy 4:4 207 Y Quan et al.

instance, against Y181C mutant RT, addition of nevi- E89G mutation is not commonly found in the plasma rapine at a concentration of 0.8 µM, fivefold below the of patients receiving antiretroviral therapy, studies on µ IC50 value of 4.1 M (see Table 1), had no effect in RT molecules containing this substitution provide regard to (+)-calanolide A, whereas an additive to valuable insight into the catalytic activity of RT. The synergistic effect was observed at a nevirapine concen- current work corroborates earlier findings [22] that the tration of 3.0 µM (Figure 3c). In regard to RT Y188H mutation, along with Y188C, is likely to be of containing the Y188H substitution, no significant clinical significance in patients who receive (+)-calano- increase in 1/V values was observed at low concentra- lide A for treatment of HIV disease. tions (<5 mM) of (+)-calanolide A for the same reason. Acknowledgements Tissue culture studies A slightly synergistic effect was observed for this This work was supported by grants to Michael A combination in cell culture (Figure 4). At relatively low Parniak and Mark A Wainberg from the Medical concentrations of (+)-calanolide A, 0.003–0.05 µM, Research Council of Canada. and relatively high concentrations of nevirapine, 0.13 –0.25 µM, the combinations yielded results that were References above the surface of activity (Figure 4). This was also µ 1. Boucher CAB, Cammack N, Schipper P, Shuurman R, true for the combination of 0.03 M nevirapine and Rouse P, Wainberg MA & Cameron JA. High-level resis- 0.1 µM (+)-calanolide A. tance to (–) enantiomeric 2′-deoxy-3′-thiacytidine in vitro is due to one amino acid substitution in the catalytic site of human immunodeficiency virus type 1 reverse transcrip- Discussion tase. Antimicrobial Agents Chemotherapy 1993; 37:2231–2234. 2. 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Received 29 July 1999; revised 19 August 1999; accepted 10 September 1999

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