Proc. Natl. Acad. Sc i. USAVol. 93, pp. 969-973, February 1996Biochemistry

The in vitro ejection of zinc from human immunodeficiency virus(HIV) type 1 nucleocapsid protein by disulfide benzamides withcellular anti-HIV activity

[N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide]

PETER J. TUMMINO*t, JEFFREY D. SCHOLTEN*, PATRICIA J. HARVEY*, TOD P. HOLLER*, LISA MALONEY*,Rocco GOGLIOTrIt, JOHN DOMAGALAt, AND DONALD HUPE*Departments of *Biochemistry and tChemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, MI 48105

Communicated by Vincent Massey, University of Michigan Medical School, Ann Arbor, MI, October 25, 1995 (received for reviewSeptember 5, 1995)

ABSTRACT Several disulfide benzamides have beenshown to possess wide-spectrum antiretroviral activity in cellculture at low micromolar to submicromolar concentrations,inhibiting human immunodeficiency virus (HIV) type 1(HIV-1) clinical and drug-resistant strains along with HIV-2and simian immunodeficiency virus [Rice, W. G., Supko, J. G.,Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Gra-ham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gog-liotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder,R. C., II, Arthur, L. 0. & Henderson, L. E. (1995) Science 270,1194-1197]. Rice and coworkers have proposed that thecompounds act by "attacking" the two zinc fingers of HIVnucleocapsid protein. Shown here is evidence that low micro-molar concentrations of the anti-HIV disulfide benzamideseject zinc from HIV nucleocapsid protein (NCp7) in vitro, asmonitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structur-ally similar disulfide benzamides that do not inhibit HIV-1 inculture do not eject zinc, nor do analogs of the antiviralcompounds with the disulfide replaced with a methylenesulfide. The kinetics of NCp7 zinc ejection by disulfide ben-zamides were found to be nonsaturable and biexponential,with the rate of ejection from the C-terminal zinc finger 7-foldfaster than that from the N-terminal. The antiviral com-pounds were found to inhibit the zinc-dependent binding ofNCp7 to HIV W RNA, as studied by gel-shift assays, and thedata correlated well with the zinc ejection data. Anti-HIVdisulfide benzamides specifically eject NCp7 zinc and abolishthe protein's ability to bind I RNA in vitro, providing evidencefor a possible antiretroviral mechanism of action of thesecompounds. Congeners of this class are under advancedpreclinical evaluation as a potential chemotherapy for ac-quired immunodeficiency syndrome.

Development of clinical resistance by human immunodefi-ciency virus (HIV) type 1 (HIV-1) in response to nucleosideinhibitors (1, 2), nonnucleoside reverse transcriptase inhibitors(3), and a protease inhibitor (4) has greatly weakened theprospects for effective monotherapy against acquired immu-nodeficiency syndrome (AIDS). With combination therapy forAIDS more likely (5), there is increased interest in anti-HIVcompounds targeted against novel retroviral targets.

Selected compounds were tested against HIV-1 in cellculture by the National Cancer Institute HIV Screening Pro-gram, and a series of disulfide benzamides (Fig. 1) fromParke-Davis Pharmaceutical Division of Warner-LambertCompany was shown to possess anti-HIV activity at lowmicromolar to submicromolar concentrations (6). These com-

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S-SHN 0 HN 0

I

0=S=O O=S=ONH2 NH2

PD022551

HN. NNH

NS-SH2N 0 0 NH2

PD024886

S-SHN 0 0 NH

HO OH

PD159206

- SHN 0 HN 0

X XO=S=O OJ=S=Oo=s= 0=5=0TNH 0'YNH

PD156202

FIG. 1. Several disulfide benzamides with cellular anti-HIV activ-ity. The compound PD153548 is an analog of PD022551 with theamides in the para position relative to the disulfide, instead of in theortho position. PD153550 is also a PD022551 analog, with the disulfide(S-S) replaced by a methylene sulfide (CH2-S).

pounds were found to inhibit both laboratory strains andclinical isolates of HIV-1, including strains resistant to thenucleoside inhibitor 3'-azido-3'-deoxythymidine (AZT) or tothe nonnucleoside reverse transcriptase inhibitors pyridinoneand nevirapine. The antiviral activity was found to be inde-pendent of cell type, and the compounds were also found to besynergistic with AZT, 2',3'-dideoxycytidine (ddC), or theprotease inhibitor KNI-272. Rice and coworkers have pro-posed that the disulfide benzamides exert an antiviral effect by"attacking" the two zinc fingers of the HIV-1 nucleocapsidprotein (NCp7) and ejecting zinc (6). Nucleocapsid protein ispresent in the core of all retroviruses bound to the dimeric viralRNA genome (7). HIV-1 NCp7 is a small basic protein withtwo CX2CX4HX4C (CCHC) sequences that each tetrahedrallycoordinate a zinc ion (8, 9). Mutagenesis of any of the cysteinesor histidines in the zinc fingers of HIV-1 NCp7 yields virionswith defective RNA encapsidation and noninfectious particles(10, 11). These facts suggest that compounds which specificallydisrupt zinc coordination to NCp7 will have an antiviral effect.There are two previous reports of compounds that either ejector coordinate zinc from HIV proteins. Rice et al. (12) reportedthat low millimolar concentrations of 3-nitrosobenzamide will

Abbreviations: HIV, human immunodeficiency virus; NCp7, nucleo-capsid protein; TSQ, N-(6-methoxy-8-quinolyl)-p-toluenesulfon-amide.tTo whom reprint requests should be addressed.

969

970 Biochemistry: Tummino et al.

eject HIV NCp7 zinc from purified virions, and Otsuka et al.(13) described zinc chelators that inhibit the DNA-bindingactivity of the zinc finger-containing HIV enhancer bindingprotein.The purpose of the studies described here is to determine

whether the anti-HIV disulfide benzamides eject zinc coordi-nated to HIV-1 NCp7 in vitro. For this purpose, a continuousfluorescence-based assay was developed that measures therelease of zinc from the protein. It was found that anti-HIVdisulfide benzamides eject zinc coordinated to NCp7, whilestructurally similar compounds that are not antiviral do not.The kinetics of zinc ejection indicate that the zinc coordinatedin the C-terminal zinc finger is ejected faster than the zinc inthe N-terminal zinc finger. Furthermore, the in vitro zinc-dependent binding of NCp7 to T RNA [a 44-nucleotidesequence that contains the HIV T site (14)], is inhibited by theanti-HIV disulfide benzamides.

MATERIALS AND METHODSCloning, Expression, and Isolation of NCp7. A synthetic

gene encoding a variant of the 55-amino acid NCp7 proteinfrom pNL4-3 (15), which contains a mutation of the firstresidue (His to Met) was designed and constructed by usingpublished methods (16). The gene was inserted into pET-21a(Novagen) to form pET21a-NCp7, which was used to trans-form Escherichia coli strain BL21 (DE3). BL21 (DE3) har-boring pET21a-NCp7 was grown at 37°C in a 2-liter fermentor(VirTis) containing superbroth (Digene Diagnostics, Belts-ville, MD) with ampicillin sodium salt at 100 jig/ml. When theOD600 reached 8, expression was induced by addition ofisopropyl ,B-D-thiogalactoside (IPTG) to a final concentrationof 1 mM. The cells were harvested 2 h after induction bycentrifugation at 5000 x g for 20 min, frozen in liquid nitrogen,and stored at -80°C. The isolation procedure was based upontwo published protocols (17, 18). Cells were resuspended at4°C in lysis buffer [50 mM Tris HCl, pH 7.8/200 mM NaCl/1mM diethylenetriaminepentaacetic acid (DTPA)J and lysedwith a French press at 12,000-14,000 psi (1 psi = 6.89 kPa).Magnesium chloride and bovine pancreatic DNase were addedto 10 mM and 0.4 jig/ml, respectively, and the mixture wasstirred at room temperature for 20 min and then clarified bycentrifugation at 15,000 x g and 4°C for 1 h. Polyethylenei-mine, dialyzed overnight against water, was added to a finalconcentration of 0.5% and the precipitate was removed bycentrifugation at 10,000 x g for 20 min. Solid ammoniumsulfate was added to the supernatant to 40% saturation at 4°Cand the precipitate was removed by centrifugation at 10,000 xg for 20 min. Remaining protein was precipitated by additionof solid ammonium sulfate to 80% saturation and centrifuga-tion at 10,000 x g for 20 min. The NCp7-containing pellet wasresuspended in 100 ml of buffer A [50 mM Tris-HCl, pH7.8/10% (vol/vol) glycerol, 5 mM dithiothreitol (DTT)/1 mMDTPA] and dialyzed for 4 h at 4°C against 4 liters of buffer B[50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (Ches), pH9.8/10% glycerol/5 mM DTT/1 mM DTPA] containing 100mM NaCl. The dialysate was diluted with 225 ml of buffer Bcontaining 100 mM NaCl and stirred with 40 ml (packedvolume) of preequilibrated Q Sepharose (Pharmacia) for 20min. The gel was removed by filtration and washed with 100 mlof buffer B containing 100 mM NaCl. The combined liquidswere stirred with 40 ml of sulfopropyl Sephadex (Pharmacia)for 20 min and the gel was packed into a column. The columnwas washed with 100 ml of buffer B containing 100 mM NaCland eluted with a gradient of 100-700 mM NaCl in buffer B.The protein solution was stored at -80°C. NCp7 aliquots wereprepared for HPLC by the addition of guanidine hydrochlorideto 6 M, DTT to 250 mM, and 2 M Tris base until the pHreached 8.7, followed by warming to 55°C for 20 min. HPLCwas performed with a 1.0 x 25 cm C18 column fitted with a 1.0

x 5 cm guard (Rainen Instruments), 0.1% trifluoroacetic acid(TFA) in water as buffer C, 0.1% TFA in acetonitrile as bufferD, and a flow rate of 2.5 ml/min. Prereduced protein solution(5 ml) was injected into the column and eluted with theprogram 0-15 min 0% buffer D, 15-25 min, 0-18% buffer D,and 25-55 min, 18-23% buffer D. Fractions (3 ml) werecollected and analyzed by SDS/PAGE, and appropriate frac-tions were pooled. The pooled fractions were found to beessentially pure by SDS/PAGE. Zinc chloride was added to thepooled fractions in a 2:1 molar ratio to NCp7, which waslyophilized and stored at -80°C. The concentration of NCp7was determined by using an extinction coefficient of 8280 =6050 M-1cm- 1

HIV NCp7 Zinc Ejection Assay Monitored by N-(6-Methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) and Tryp-tophan Fluorescence. The zinc ejection assay buffer used was10% (vol/vol) glycerol/50mM Tris HCl buffer, pH 7.6 at 24°C.The ejection of zinc from the protein was monitored by thechange in fluorescence of the zinc-selective fluorophore TSQ(Molecular Probes) (19) in the assay buffer. The zinc ejectionassay was initiated by the addition of 10 ,uM final concentrationof a disulfide benzamide or control compound to 2.0 ,tM finalconcentration of NCp7 (with a Zn-to-protein molar ratio of2:1) and 50 ,tM final concentration of TSQ in assay buffer toa total volume of 200 ,ul in a 96-well plate. Immediately afterreaction initiation, the TSQ fluorescence was monitored con-tinuously for 100 min (excitation filter 355 nm, emission filter460 nm) by a Labsystems (Needham Heights, MA) FluoroskanII 96-well plate fluorescence reader. The zinc chloride stan-dard curve was generated from the same 96-well plate underthe same conditions in the absence of NCp7 and disulfidebenzamide. To control for time-dependent fluorescencechanges in the assay not due to the ejection of zinc, the aboveexperiment was run with each compound and apo-NCp7, andno significant fluorescence changes were observed over the100-min time course for any of the compounds. The kinetics ofzinc ejection by PD156202 was measured on a Perkin-ElmerLS 50 luminescence spectrometer, using 355-nm excitation and490-nm emission. The tryptophan fluorescence experimentwas performed on the same instrument, with 280-nm excita-tion and 360-nm emission. Other experimental conditionswere the same as described above.HIV NCp7-T RNA Gel-Shift Assays. The RNA-binding

experiments were carried out by incubating 1.0 ,tM NCp7 with5.0 p,M compound in zinc ejection assay buffer at roomtemperature. After incubation of NCp7 and compound for 0min, 4 h, and 24 h, an aliquot was taken and added to an equalvolume of 32P-labeled T RNA (<2 pmol) in zinc ejection assaybuffer containing yeast tRNA at 25 [kg/ml, 200 mM KCl, and40 mM MgCl2. After mixing, the solutions were incubated atroom temperature for 10 min. The NCp7-RNA complexeswere then analyzed by electrophoresis in a nondenaturing 6%polyacrylamide gel; the running buffer was 23 mM Trisborate/0.5 mM EDTA, pH 8.0 at 24°C. Gels were pre-run at100 V at 4°C and continued for 90 min at 100 V at 4°C afterloading 10 ,ul per well. The data were analyzed by autoradio-gram. The T RNA is a 44-nucleotide sequence (14) which waschemically synthesized by using an Applied Biosystems 394DNA/RNA synthesizer. The RNA was 32P-labeled at the 5'end and purified by PAGE using standard protocols.

RESULTSZinc Ejection from HIV NCp7 by Disulfide Benzamides.

The zinc-selective fluorophore TSQ, previously used only tomeasure the tissue (19, 20) and cellular (21, 22) distribution ofzinc, was found to yield a reproducible relationship betweenlow micromolar zinc and fluorescence. To obtain a standardcurve from 0.5 to 4.0 ,uM, as shown in Fig. 2, an excess of 50,uM TSQ was required. The fluorescence response time after

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 971

u

u0)v:

V)

45 -

35 -

25 -

15 -

0

A100 -

c)

,

N

1 2 3 4 5 6

[Zinc], jM

FIG. 2. Zinc standard curve measuring TSQ fluorescence. Thefluorescence from various concentrations of zinc chloride with 50 ,uMTSQ in zinc ejection assay buffer was measured as described in the text.The data were obtained from the same 96-well plate as the zincejection assay was performed in (Fig. 3), and fluorescence wasmeasured at 100 min after initiation of the reaction. The standardcurve was unchanged over the 100-min time course. The data areshown fit to the equation for a simple binding equilibrium (Kd = 6.8,uM), and the fluorescence value is 6.4 at 0.0 ,uM zinc and 35.6 at 4.0,tM zinc.

addition of zinc was found to be less than 2 sec, and it thereforehad no effect on the measured rates of zinc ejection. Also, thefluorescence values for the zinc standard curve were un-changed over the 100-min time course of the assay (data notshown). This assay was used to measure the time-dependentejection of zinc from NCp7.

Fluorescence values were used to approximate the percent-age of zinc ejected by disulfide benzamides from 2.0 ,UM NCp7(which has 4.0 ,uM zinc bound), and results are shown in Fig.3. With no compound added to NCp7 and TSQ, the fluores-cence increases slightly and then remains constant over thetime course. The small fluorescence increase in the control wasfound to be equivalent or smaller in repeat experiments.Combined with the zinc standard curve in Fig. 2, this obser-vation demonstrates that TSQ is sensitive to free zinc only.Addition of 10 ,uM PDO22551 results in time-dependent zincejection from NCp7, while the analogs PD153548 (with theamide substituents para to the disulfide) and PD153550 (witha methylene sulfide in place of the disulfide) do not eject zincover the same time course (Fig. 3A). Similarly (Fig. 3B),PD156202, PDO24886, and PD159206 all demonstrate NCp7zinc ejection. The rate of zinc ejection by PD156202 is mark-edly greater than that observed for the other three activedisulfide benzamides, and the reaction appears to havereached completion in less than 20 min. The active disulfidebenzamides approach 100% zinc ejected, as clearly seen withPD156202 and PDO24886 (the molar ratio of compound toNCp7 zinc fingers is 2.5:1). The titration of zinc ejection byPD156202 yielded a curve where half the zinc was ejected witha compound to NCp7 zinc finger ratio of 1:2 (data not shown),demonstrating a 1:1 stoichoimetry between compound andzinc coordinated to the protein. The structures of the fouractive disulfide benzamides are shown in Fig. 1.The Kinetics of NCp7 Zinc Ejection by PD156202. Time-

dependent zinc ejection by PD156202 monitored by TSQfluorescence was measured and analyzed at eight compoundconcentrations, and three representative curves are shown inFig. 4. The data fit very well to a biexponential curve at allcompound concentrations, with k1 approximately 8-foldgreater than k2 and the magnitude of the fluorescence increaseassociated with each rate approximately equal. The two ob-served rates of zinc ejection were plotted against compoundconcentration, as shown in Fig. 5. The linear relationshipobserved in these plots can be explained either by nonsaturablezinc ejection or by a Kd for the compound-NCp7 binding muchhigher than the concentrations used. Higher concentrations of

80 -

60

40

20 -

0 20 40 60 80 100

Time, minB

100

04)U

U-

80

60

40

20

0

Time, min

FIG. 3. Ejection of zinc from HIV NCp7 by disulfide benzamidesas measured by TSQ fluorescence. The assay was initiated by theaddition of disulfide benzamide or analog to give a final concentrationof 10,uM; the control is without addition of a compound. Fluorescencewas converted to percent zinc ejected by using the fluorescence valuesfrom the zinc standard curve (Fig. 2), with 4.0 ,uM zinc equivalent to100% zinc ejected. (A) w, PD022551; A, PD153548; 0, PD153550; andA, control. (B) w, PD156202; o, PD024886; A, PD159206; and A,control.

PD156202 could not be used due to solubility limits. Fornonsaturable zinc ejection, the bimolecular rate constants arek 2.8 x 103 M-1-sec-I and k2 = 3.9 x 102 M- Isec- .

The same experiment was performed while monitoringfluorescence from Trp-37 to distinguish the kinetics of zincejection from each zinc finger. This is the single tryptophan

400

c)Q

a)

0

300

200

100

6

Time, min

FIG. 4. Kinetics of zinc ejection from HIV NCp7 by PD156202.PD156202, in final concentrations of 2.0-32 ,uM, was added to 2.0 ,uMNCp7 and 50 ,uM TSQ in zinc ejection assay buffer, and TSQfluorescence was measured immediately. The data were fit to thedouble-exponential equation y = A + B[1 - exp(-klx)] + C[1 -

exp(-k2x)I. Shown are the data at three concentrations of PD156202fit to this equation. At 2.0 ,uM PD156202, k1 = 1.03 min-' and k2 =

0.131 min-'; at 16 ,uM PD156202, k1 = 1.92 min-' and k2 = 0.342min-m;andat32,uMPD156202,k1 = 5.52min-m andk2 = 0.709min-1.

Is

Biochemistry: Tummino et al.

-3C-F-'F+_p-3-3, -,

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972 Biochemistry: Tummino et al.

A6 -

5 -

4 -

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o5 1 15o 5 lo 15

A

hi*.

I 2. 3/S B

I0 mill

20 25 30 35

[156202], jM

B

7-Ie

0.8 i

0.6 -

0.4 -

0.2 -

0o -_

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C I *4. 4C - . f:4ti.*...... a&A_trC !.44 .; _ _ _free - 1 2 4 67probe 12314 56 78

S

S1X~-

0 5 10 15 20 25

[156202], AM

24 h

FIG. 7. Gel-shift assay for inhibition of binding of NCp7 to32P-labeled T RNA (32P-T RNA) by disulfide benzamides. The Cl andC2 bands are NCp7-RNA complexes, consistent with Sakaguchi et al.(14). (A) Lane 1, free probe (32P-T RNA); lane 2, 2.8 KM apo-NCp7and 32P-t RNA; lane 3, 1.4 ,tM zinc-containing NCp7 and 32p-TRNA. (B) Lanes 3-8 contain 32P-T RNA, NCp7, and compound. Lane1, free probe (32P-T RNA, no NCp7, no compound); lane 2, control(32p-T RNA, NCp7, no compound); lane 3, PD153548; lane 4,PD153550; lane 5, PDO22551; lane 6, PD159206; lane 7, PDO24886;and lane 8, PD156202.

30 35

FIG. 5. Kinetic constants for zinc ejection vs. PD156202 concen-tration. The values for ki and k2 (as described in Fig. 4) weredetermined at several PD156202 concentrations and plotted vs.PD156202 concentration. For both kinetic constants, the rates increaselinearly, indicating that the observed zinc ejection is nonsaturable. (A)From the linear fit, k1 = 2.8 X 103 M--sec-1.(B) k2= 3.9 X 102M- I sec- 1.

residue in the protein, present in the C-terminal zinc finger,and thus a probe for changes in the C-terminal zinc finger only(23). The tryptophan data are shown in comparison to thosefrom monitoring TSQ fluorescence in Fig. 6, both uponaddition of 8.0 .LM PD156202. The tryptophan fluorescencedecreases substantially upon addition of compound, and thetime course fits well to a monoexponential. This is consistentwith previous reports of lower NCp7 tryptophan fluorescencewhen no zinc is bound to the protein (9, 23). The kineticconstant associated with the tryptophan fluorescence decrease

v-

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0) .3 6 9 12 1 5Time, in

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FIG. 6. Time-dependent ejection of zinc from HIV NCp7 byPD156202 measured by TSQ and tryptophan fluorescence. The sep-arate experiments measuring either TSQ or tryptophan fluorescencewere performed in the same manner, as described for Fig. 4, withPD156202 final concentration = 8.0 ,tM. The TSQ fluorescence dataare shown fit to a biexponential equation (see Fig. 4 legend) with ki= 1.17 min-1 andk, = 0.221 min- 1. The tryptophan fluorescence dataare shown fit to the monoexponential equation y = A + B[1 -

exp(-klx)], with k, = 1.52 min-.

(1.52 min- 1) is clearly consistent with k, from the TSQexperiment (1.17 min-'). It can be concluded from the com-parison of rates that k1, the faster rate, corresponds to ejectionof the C-terminal zinc and k2 to N-terminal zinc ejection.

Rice and coworkers (6) also measured NCp7 zinc ejection bymonitoring tryptophan fluorescence, but the experiments aresignificantly different. The previous studies were performedwith 50 ,uM compound, which was found to artifactuallydecrease the fluorescence of tryptophan by both light scatter-ing (compound insolubility) and UV absorbance. For example,it was observed that the addition of 50 ,tM compoundPD022551, PD024886, or PD156202 to 10 ,uM tryptophan inthe zinc ejection assay buffer decreased fluorescence (excita-tion 280 nm, emission 360 nm) by 40% or greater (data notshown). We corrected for the problem by using a lowercompound concentration, 8.0 ,uM, and by measuring fluores-cence changes only after addition of compound. Therefore, asignificant degree of the fluorescence decrease upon com-pound addition reported previously may be due not to zincejection but to artifactual fluorescence quenching.

Inhibition of HIV NCp7-I RNA Gel Shift by DisulfideBenzamides. The T RNA gel shift was found to be dependenton the concentration of Zn-containing NCp7 (data notshown), with no shift when twice the concentration of apo-NCp7 was used (Fig. 7A), consistent with the findings ofSakaguchi et al. (14). This gel-shift assay was used as afunctional probe for the NCp7 zinc ejection by disulfidebenzamides. In Fig. 7B, lane 2 relative to lane 1 shows the shiftof free probe to Cl and C2 (protein-RNA complexes) byNCp7, which remains constant for 24 h. The negative controlcompounds PD153548 and PD153550 demonstrate no alter-ation of those shifts at the three time points. PD02255 1,PD159206, and PD024886 demonstrate no significant changein the gel shift until the 24-h time point. At 24 h, the latter threecompounds greatly inhibit the gel shift. PD156202 inhibits thegel shift entirely at all time points. This disulfide benzamidedemonstrates complete inhibition of NCp7 binding even at 10min.

DISCUSSIONA series of disulfide benzamides with cellular anti-HIV activityhave been found to stoichiometrically eject zinc from HIV

Proc. Natl. Acad. Sci. USA 93 (1996)

Proc. Natl. Acad. Sci. USA 93 (1996) 973

NCp7 in vitro, while analogs without anti-HIV activity do not.The anti-HIV compounds were also found to abolish thezinc-dependent binding of NCp7 to T RNA.The zinc ejection assay data demonstrate that NCp7 zinc

ejection is specific, since the structurally similar disulfidePD 153548, with the amide substituentpara instead of ortho tothe disulfide, does not eject zinc. The minimal structurecommon to the active compounds is an o-amidophenyl disul-fide. The disulfide itself is required for zinc ejection, since themethylene sulfide analog of the active PD022551, which isPD153550, does not eject zinc. It was also found that thereduced forms of the four active disulfide benzamides, in theabsence or presence of dithiothreitol, have no zinc ejectionactivity (data not shown). With the necessity for a stoichio-metric amount of disulfide, a simple mechanism for zincejection can be proposed wherein a disulfide benzamide/NCp7thiol-disulfide interchange occurs to form a disulfide betweena cysteine residue previously coordinated to a zinc and half ofthe compound (the other half being released as free thiol).With a disulfide formed, the zinc is no longer tetrahedrallycoordinated by four NCp7 residues and is ejected into solution.Clearly, further thiol-disulfide interchanges would then beprobable, both with other cysteine residues intramolecularlyand with other disulfide benzamide molecules.PD156202 demonstrates a much faster rate of zinc ejection

than the other three active compounds, yet the compounddiffers from PD022551 only by the addition of an acetyl groupon each half of the compound. It is unclear what relevance therate of zinc ejection might have in a cellular antiviral assay,since a stoichiometric amount of disulfide benzamide is re-quired, and in fact PD156202 and PD022551 are similarlypotent against HIV-1 (6).The kinetics of NCp7 zinc ejection by PD156202 is biexpo-

nential. Comparison of data from monitoring TSQ and tryp-tophan fluorescence indicates that the faster rate of zincejection, k1, corresponds to ejection from the NCp7 C-terminalzinc finger and the slower rate, k2, to ejection from theN-terminal zinc finger. Surovoy et al. (24) reported that theN-terminal zinc was coordinated with a higher binding affinitythan the C-terminal zinc. Therefore, the more weakly boundzinc, at the C terminus, is more easily ejected by the disulfidebenzamide. The linear relationship between zinc ejection rateand concentration of PD156202 indicates nonsaturable kinet-ics (unless the PD156202-NCp7 interaction is very weak) andwould mean there is no compound-protein binding step priorto zinc ejection. Despite the evidence of simple collisional zincejection and therefore the potential for nonspecific reactionscellularly, these compounds are relatively nontoxic in cells (6).The characteristics of in vitro zinc ejection were found to

correlate with the loss of an NCp7 zinc-dependent function invitro, which is binding to T RNA (14). PD156202 ejects NCp7zinc at a much faster rate than the other anti-HIV disulfidebenzamides, and it abolishes NCp7 binding to T RNA at thefirst time point of 10 min. PD022551, PD024886, andPD159206 at 10 ,tM all require longer than 100 min forcomplete NCp7 zinc ejection and at 5.0 ,uM all of the com-pounds require longer than 4 h for inhibition of T RNAbinding. The difference in rates observed between zinc ejec-tion and gel-shift inhibition could be due to a number offactors, including the possible necessity to eject zinc from agreat majority of the NCp7 pool before any alteration in thegel shift is observed. Similar to the zinc ejection assay, the twonegative control compounds (PD153548 and PD153550) donot affect the NCp7-T RNA binding.The in vitro zinc ejection and gel-shift data reported here

provide evidence for the proposed retroviral target for thedisulfide benzamides of nucleocapsid protein. However, in

vitro studies cannot provide proof of cellular mechanism forthe antiviral activity. Although studies on the viral life cyclehave been performed with HIV containing NCp7 mutants (10,11, 25), it is not yet well understood what characteristics wouldbe observed for a compound that alters zinc-dependent NCp7function. The previously reported synergism between thedisulfide benzamides and 3'-azido-3'-deoxythymidine, 2',3'-dideoxycytidine, or the protease inhibitor KNI-272 not onlyincreases their potential therapeutic value but also providesfurther evidence that the compounds are acting at a noveltarget. Congeners of the disulfide benzamide series are underadvanced preclinical evaluation as a potential therapy forHIV-1 infection.

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