Oct 25, 1995 - ham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gog- liotti, R., Bader ...... Gorelick, R. J., Nigida, S. M., Bess, J. W., Arthur, L. O., Henderson,.
Proc. Natl. Ac ad. Sc i. USA Vol. 93, pp. 969-973, February 1996
Biochemistry
The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular 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 review September 5, 1995)
ABSTRACT Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, 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 the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV W RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind I RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.
S-S
S-S HN
0
0=S=O
HN
HN I HO
0
O=S=O NH2
NH2
0
0
NH OH
PD159206
PD022551
S
-
0
HN
o=s= O=S=O
NS-S H2N
X X
NNH
HN.
0
0
PD024886
NH2
HN
0T
0
OJ=S=O 0=5= 'YNH
0
NH
PD156202
FIG. 1. Several disulfide benzamides with cellular anti-HIV activity. The compound PD153548 is an analog of PD022551 with the amides in the para position relative to the disulfide, instead of in the ortho 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 and clinical isolates of HIV-1, including strains resistant to the nucleoside inhibitor 3'-azido-3'-deoxythymidine (AZT) or to the nonnucleoside reverse transcriptase inhibitors pyridinone and nevirapine. The antiviral activity was found to be independent of cell type, and the compounds were also found to be synergistic with AZT, 2',3'-dideoxycytidine (ddC), or the protease inhibitor KNI-272. Rice and coworkers have proposed that the disulfide benzamides exert an antiviral effect by "attacking" the two zinc fingers of the HIV-1 nucleocapsid protein (NCp7) and ejecting zinc (6). Nucleocapsid protein is present in the core of all retroviruses bound to the dimeric viral RNA genome (7). HIV-1 NCp7 is a small basic protein with two CX2CX4HX4C (CCHC) sequences that each tetrahedrally coordinate a zinc ion (8, 9). Mutagenesis of any of the cysteines or histidines in the zinc fingers of HIV-1 NCp7 yields virions with defective RNA encapsidation and noninfectious particles (10, 11). These facts suggest that compounds which specifically disrupt zinc coordination to NCp7 will have an antiviral effect. There are two previous reports of compounds that either eject or coordinate zinc from HIV proteins. Rice et al. (12) reported that low millimolar concentrations of 3-nitrosobenzamide will
Development of clinical resistance by human immunodeficiency virus (HIV) type 1 (HIV-1) in response to nucleoside inhibitors (1, 2), nonnucleoside reverse transcriptase inhibitors (3), and a protease inhibitor (4) has greatly weakened the prospects for effective monotherapy against acquired immunodeficiency syndrome (AIDS). With combination therapy for AIDS more likely (5), there is increased interest in anti-HIV compounds targeted against novel retroviral targets. Selected compounds were tested against HIV-1 in cell culture by the National Cancer Institute HIV Screening Program, and a series of disulfide benzamides (Fig. 1) from Parke-Davis Pharmaceutical Division of Warner-Lambert Company was shown to possess anti-HIV activity at low micromolar to submicromolar concentrations (6). These com-
Abbreviations: HIV, human immunodeficiency virus; NCp7, nucleo-
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
capsid protein; TSQ, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide.
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-binding activity of the zinc finger-containing HIV enhancer binding protein. The purpose of the studies described here is to determine whether the anti-HIV disulfide benzamides eject zinc coordinated to HIV-1 NCp7 in vitro. For this purpose, a continuous fluorescence-based assay was developed that measures the release of zinc from the protein. It was found that anti-HIV disulfide benzamides eject zinc coordinated to NCp7, while structurally similar compounds that are not antiviral do not. The kinetics of zinc ejection indicate that the zinc coordinated in the C-terminal zinc finger is ejected faster than the zinc in the N-terminal zinc finger. Furthermore, the in vitro zincdependent binding of NCp7 to T RNA [a 44-nucleotide sequence that contains the HIV T site (14)], is inhibited by the anti-HIV disulfide benzamides.
MATERIALS AND METHODS Cloning, Expression, and Isolation of NCp7. A synthetic gene encoding a variant of the 55-amino acid NCp7 protein from pNL4-3 (15), which contains a mutation of the first residue (His to Met) was designed and constructed by using published methods (16). The gene was inserted into pET-21a (Novagen) to form pET21a-NCp7, which was used to transform Escherichia coli strain BL21 (DE3). BL21 (DE3) harboring pET21a-NCp7 was grown at 37°C in a 2-liter fermentor (VirTis) containing superbroth (Digene Diagnostics, Beltsville, MD) with ampicillin sodium salt at 100 jig/ml. When the OD600 reached 8, expression was induced by addition of isopropyl ,B-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The cells were harvested 2 h after induction by centrifugation at 5000 x g for 20 min, frozen in liquid nitrogen, and stored at -80°C. The isolation procedure was based upon two published protocols (17, 18). Cells were resuspended at 4°C in lysis buffer [50 mM Tris HCl, pH 7.8/200 mM NaCl/1 mM diethylenetriaminepentaacetic acid (DTPA)J and lysed with a French press at 12,000-14,000 psi (1 psi = 6.89 kPa). Magnesium chloride and bovine pancreatic DNase were added to 10 mM and 0.4 jig/ml, respectively, and the mixture was stirred at room temperature for 20 min and then clarified by centrifugation at 15,000 x g and 4°C for 1 h. Polyethyleneimine, dialyzed overnight against water, was added to a final concentration of 0.5% and the precipitate was removed by centrifugation at 10,000 x g for 20 min. Solid ammonium sulfate was added to the supernatant to 40% saturation at 4°C and the precipitate was removed by centrifugation at 10,000 x g for 20 min. Remaining protein was precipitated by addition of solid ammonium sulfate to 80% saturation and centrifugation at 10,000 x g for 20 min. The NCp7-containing pellet was resuspended in 100 ml of buffer A [50 mM Tris-HCl, pH 7.8/10% (vol/vol) glycerol, 5 mM dithiothreitol (DTT)/1 mM DTPA] and dialyzed for 4 h at 4°C against 4 liters of buffer B [50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (Ches), pH 9.8/10% glycerol/5 mM DTT/1 mM DTPA] containing 100 mM NaCl. The dialysate was diluted with 225 ml of buffer B containing 100 mM NaCl and stirred with 40 ml (packed volume) of preequilibrated Q Sepharose (Pharmacia) for 20 min. The gel was removed by filtration and washed with 100 ml of buffer B containing 100 mM NaCl. The combined liquids were stirred with 40 ml of sulfopropyl Sephadex (Pharmacia) for 20 min and the gel was packed into a column. The column was washed with 100 ml of buffer B containing 100 mM NaCl and eluted with a gradient of 100-700 mM NaCl in buffer B. The protein solution was stored at -80°C. NCp7 aliquots were prepared for HPLC by the addition of guanidine hydrochloride to 6 M, DTT to 250 mM, and 2 M Tris base until the pH reached 8.7, followed by warming to 55°C for 20 min. HPLC was performed with a 1.0 x 25 cm C18 column fitted with a 1.0
Proc. Natl. Acad. Sci. USA 93
(1996)
x 5 cm guard (Rainen Instruments), 0.1% trifluoroacetic acid (TFA) in water as buffer C, 0.1% TFA in acetonitrile as buffer D, and a flow rate of 2.5 ml/min. Prereduced protein solution (5 ml) was injected into the column and eluted with the program 0-15 min 0% buffer D, 15-25 min, 0-18% buffer D, and 25-55 min, 18-23% buffer D. Fractions (3 ml) were collected and analyzed by SDS/PAGE, and appropriate fractions were pooled. The pooled fractions were found to be essentially pure by SDS/PAGE. Zinc chloride was added to the pooled fractions in a 2:1 molar ratio to NCp7, which was lyophilized and stored at -80°C. The concentration of NCp7 was 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 Tryptophan Fluorescence. The zinc ejection assay buffer used was 10% (vol/vol) glycerol/50 mM Tris HCl buffer, pH 7.6 at 24°C. The ejection of zinc from the protein was monitored by the change in fluorescence of the zinc-selective fluorophore TSQ (Molecular Probes) (19) in the assay buffer. The zinc ejection assay was initiated by the addition of 10 ,uM final concentration of a disulfide benzamide or control compound to 2.0 ,tM final concentration of NCp7 (with a Zn-to-protein molar ratio of 2:1) and 50 ,tM final concentration of TSQ in assay buffer to a total volume of 200 ,ul in a 96-well plate. Immediately after reaction initiation, the TSQ fluorescence was monitored continuously for 100 min (excitation filter 355 nm, emission filter 460 nm) by a Labsystems (Needham Heights, MA) Fluoroskan II 96-well plate fluorescence reader. The zinc chloride standard curve was generated from the same 96-well plate under the same conditions in the absence of NCp7 and disulfide benzamide. To control for time-dependent f luorescence changes in the assay not due to the ejection of zinc, the above experiment was run with each compound and apo-NCp7, and no significant fluorescence changes were observed over the 100-min time course for any of the compounds. The kinetics of zinc ejection by PD156202 was measured on a Perkin-Elmer LS 50 luminescence spectrometer, using 355-nm excitation and 490-nm emission. The tryptophan fluorescence experiment was performed on the same instrument, with 280-nm excitation and 360-nm emission. Other experimental conditions were 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 with 5.0 p,M compound in zinc ejection assay buffer at room temperature. After incubation of NCp7 and compound for 0 min, 4 h, and 24 h, an aliquot was taken and added to an equal volume of 32P-labeled T RNA (
