WO1992017182A1

WO1992017182A1 - Methods and compounds for inhibiting rnase h activity of reverse transcriptase

Info

Publication number: WO1992017182A1
Application number: PCT/US1992/002770
Authority: WO
Inventors: Jay F. Ii Davies; Zuzana Hostomska; Zdenek Hostomsky; Steven R. Jordan; David A. Matthews; Richard C. Ogden; Michael D. Varney; Stephen E. Webber
Original assignee: Agouron Pharmaceuticals LLC
Current assignee: Agouron Pharmaceuticals LLC
Priority date: 1991-04-03
Filing date: 1992-04-03
Publication date: 1992-10-15
Anticipated expiration: 1993-10-03
Also published as: AU1904492A

Abstract

A metal material is provided in an amount sufficient to inhibit RNase H activity of reverse transcriptase of viruses that are reverse transcriptase dependent. Methods are also provided for inhibiting RNase H activity of reverse transcriptase of viruses that are reverse transcriptase dependent by exposing such viruses to a metal material in an amount sufficient to inhibit RNase H activity.

Description

METHODS AND COMPOUNDS FOR INHIBITING RNASE H ACTIVITY

OF REVERSE TRANSCRIPTASE

The present invention relates to metal materials that can be used to inhibit RNase H activity of reverse transcriptase.

Onset of the acquired immunodeficiency syndrome (AIDS) epidemic and its association with human immunodeficiency virus

(HIV) has generated considerable interest in trying to understand enzymatic and structural properties of key retroviral proteins. By determining and understanding such properties, it is hoped that various therapies can be designed against AIDS.

One suggested target is the HIV replication pathway, and in fact, reverse transcription has been suggested as one target in that pathway (Mitsuya et al., Science. 249;1533-1544 (1990)).

Conversion of genomic single-stranded RNA into double-stranded proviral DNA is an essential step in replication of all

retroviruses and hepadnaviruses. Reverse transcription of viral genomic RNA is an essential step in replication of human

immunodeficiency virus (HIV) and other retroviruses or

hepadnaviruses (nonlimiting examples include HIV-1, HIV-2, HTLV-1, HBV, FeLV, or SIV). This process is catalyzed by reverse

transcriptase (RT) (Goff, S., J. AIDS. 2:817-831 (1990)), a multifunctional enzyme with both DNA polymerase and RNase H activities. Three activities are associated with this enzyme: (1) RNA-dependent DNA polymerase; (2) DNA-dependent DNA polymerase; and (3) ribonuclease H (RNase H). Inhibition of any of these activities results in disruption of virus replication thus establishing RT as an important target for therapy of retroviral infections (Mitsuya et al., supra).

To accomplish the conversion of the single-stranded RNA genome into the double-stranded DNA of the provirus, coordination of RNA- and DNA-dependent DNA polymerase and RNase H activities is required (Gilboa et al., Cell, 18:93-100 (1979)). Owing to its ability to selectively cleave phosphodiester bonds in the RNA moiety of the RNA/DNA heteroduplex intermediate (Crouch et al., Eds. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982), p. 211; R. J . Crouch, New Biologist, 2 : 771 (1990)), RNase activity is indispensable at several stages of this complex process. For example, RNase H (1) degrades the RNA template during synthesis of minus-strand DNA from the tRNA primer, (2) generates primer for the synthesis of plus-strand DNA, and (3) specifically removes both primers via an endonucleolytic mechanism (Omer et al., Cell, 30:797 (1982); Rattray et al., J. Virol.,

61: 2843 (1987); Panganiban et al., Science, 241:1064 (1988).). Because of its cmicial role in the life cycle of retroviruses and hepadnaviruses, RT and the RNase H domain of RT are prime targets for antiretroviral and antihepdnaviral therapy, especially in connection with HIV infections and AIDS (Mitsuya et al., supra).

Certain articles refer to targeting the RNase H domain of the RT of viruses for mutations, and those articles note that such mutations resulted in some reduction of RNase H activity. Schatz et al., FEBS Lett., 257:311-314 (1989); Mizrahi et al., Nucl.

Acids Res., 18:5359-5363 (1990); Tisdale et al., J. Cell.

Biochem., Supplement 14D, p. 179 (1990); Kanaya et al., J. Biol. Chem., 265:4615-4621 (1990); and Repaske et al.. Journal of

Virology, 63: 1460-1464 (1989)). At least some of those articles refer to mutations at specific amino acid sites that are predicted to be involved in the active sites of the RNase H domains mutated .

An object of the present invention is to provide materials that inhibit RNase H activity of reverse transcriptase.

Another object according to certain preferred embodiments is to provide materials that bind to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.

Another object is to provide methods of inhibiting RNase H activity of reverse transcriptase by exposing the RNase H domain of reverse transcriptase to materials that inhibit RNase H

activity.

Yet another object is to provide methods of inhibiting RNase

H activity by exposing the RNase H domain of reverse transcriptase to materials that bind to at least a portion of the RNase H domain active site, such that RNase H activity is inhibited.

Still another object is to inhibit viral replication by exposing the RNase H domain of reverse transcriptase to materials that inhibit RNase H activity. According to certain preferred embodiments, those materials bind to at least a portion of the RNase H domain of reverse transcriptase, such that RNase H

activity is inhibited.

These and other objects are obtained by providing metal materials that are capable of inhibiting RNase H activity of reverse transcriptase of viruses that are reverse transcriptase dependent. The metal materials are provided in an amount that is sufficient to inhibit the RNase H activity of reverse

transcriptase.

According to certain preferred embodiments, those metal materials bind to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

Figure 1 is a schematic representation of the subunits in the heterodimer of HIV-1 RT. Relative positions of DNA polymerase and RNase H domains are indicated. The shaded area defines the C- terminal portion of the p66 subunit.

Figure 2 is a stereo drawing of seven invariant residues in retroviral and bacterial RNases H. These conserved residues (Asp443, Glu478, Asp498, Ser499, His539, Asn545, and Asp549) are clustered at one edge of the molecule. The loop containing His539 is disordered in the structure of the HIV-1 RNase H domain. The histidine (yellow) is positioned by analogy with its location in the E. coli RNase H structure. Positions for the other side chains are from refined coordinates of the native structure.

Location of the UO₂F₅ ^3- anion is indicated by a cross.

Figure 3 depicts the substrate which can be used for

detection of RNase activity.

Figure 4 is a picture of the gel after electrophoresis that is further described in Example I below. Lane 1 was the substrate as depicted in Fig. 3 without reverse transcriptase. The

remaining lanes 2 through 8 included the substrate as depicted in Fig. 3 with reverse transcriptase and the following concentrations of K₃UO₂F₅: lane 2, 0 μM; lane 3, 10 μM; lane 4, 40 μM; lane 5, 60 μM; lane 6, 100 μM; lane 7, 125 μM; lane 8, 150 μM. The arrow shows the 28 nucleotides long reaction product.

Figure 5 is a response curve generated from the data obtained in the experiment described in Example I below for the analysis of RNase H activity of HIV-1 reverse transcriptase in the presence of different concentrations of K₃UO₂F₅.

Figure 6 is a response curve generated from the data obtained in the experiment described in Example II below for the analysis of RNase H activity of HIV-1 reverse transcriptase in the presence of different concentrations of copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt.

DESCRIPTION OF PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently

preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the

invention. All references cited herein are hereby incorporated by reference. To the extent any of the cited references are not capable of being incorporated by reference because they have not been published yet, applicants have attached as an appendix copies of the manuscripts submitted to certain publications.

All araino acid abbreviations used herein are the standard three letter code designations known in the art and set forth, e.g., in Lehninger, Biochemistry, 2d Edition, Worth Publishers (1976). As discussed above, reverse transcriptase (RT) is well-known to those of skill in this field, and the RT contemplated by the present invention includes the RT of any virus that is RT

dependent, and according to certain preferred embodiments, encompasses RT of any strain of HIV, including but not limited to HIV-1 and HIV-2. Also, according to certain preferred

embodiments, the present inventors contemplate the RT of other retroviruses and hepadnaviruses, and certain nonlimiting examples include HTLV-1, HBV, Feline Leukemia virus (FeLV) or SIV.

In general, an enzyme includes a binding site where the enzyme binds to the substrate, and includes a catalytic site responsible for the activity of the enzyme. Those sites can be separate or overlapping, but are both required for the enzyme to display its activity on the substrate. However, as defined in the present invention, the generic term "active site" encompasses the binding site and/or the catalytic site, because the binding of the metal materials according to the present invention to either site will inhibit RNase H activity.

The metal materials according to the present invention inhibit RNase activity of RT, and according to certain preferred embodiments bind to at least a portion of the active site of the RNase H domain of RT. The metal can be part of an inorganic or organometallic compound.

Metal materials that should inhibit RNase H activity and may do so by binding to at least a portion of the active site of the RNase H domain of RT include the following materials and

appropriate salts thereof, if any: mercury chloranilate, silver chloranilate, cobalt phthalocyanine, strontium chloranilate, thorium chloranilate trihydrate, manganese phthalocyanine, magnesium phthalocyanine, zirconium phthalocyanine, zinc

phthalocyanine, tin phthalocyanine, silver phthalocyanine, nickel phthalocyanine, lead phthalocyanine, iron phthalocyanine,

palladium phthalocyanine, copper phthalocyanine, phthalocyanine gold, gallium phthalocyanine, chloroindium phthalocyanine, platinum phthalocyanine, calcium phthalocyanine, molybdenum phthalocyanine, dichlorotin phthalocyanine, dilithium

phthalocyanine, dichlorogermanium phthalocyanine, flourochromium phthalocyanine, chloroaluminum phthalocyanine, phthalocyanine green, alcian blue, solvent blue, copper phthalocyanine

tetrasulfonic acid, vanadyl phthalocyanine, chloroaluminium phthalacyanine, meralluride (MERCUROCHROME), aluminum

actylacetonate, aluminum acetate, aluminum phenoxide, aluminum cyclohexanebutyrate, aluminum citrate, aluminum benzoate,

colbaltaluminate, aluminum-2-ethylhexanoate, aluminum galicylate, hexafluoro acetylacetone aluminum, mercuricsodiumparaphenyl sulfonate, zinc acetate, zinc nitrate, silver trifluoroacetate, silver acetate, copper acetate, mercurytrifluoroacetate, aluminum hydroxide, aluminum sulfate, heme, UO₂F₅, V₂O₃, V₂O₄, V₂°5' VCl₂O, VCI₃O, VF₃O, V₂MgO₃, V₂LiO₆, VO₂ (OAc)₂, Mn₂(CO)₁₀, Fe₂(CO)₉, OV - (porphorins), and compounds having the following formulas and appropriate salts thereof, if any:

With the guidance of the present specification, those skilled in this field would be able to routinely test metal materials to determine if they meet the requirements of the present invention.

First, for the embodiments in which activity of RNase H is inhibited by metal material binding to at least a portion of the active site of the RNase H domain of RT, the present inventors have for the first time known to them determined the crystal structure of the RNase H domain of RT of HIV-1 (Hostomska et al., "Proteolytic Release and Crystallization of the RNase H Domain HIV-1 Reverse Transcriptase", J. Biol. Chem.. 266:14697-14702 (1991); and Davies et al., "Crystal Structure of the RNase H Domain of HIV-1 Reverse Transcriptase", Science, 252:88-95

(1991)).

It should first be noted that the mature HIV-1 RT forms a heterodimer composed of two subunits, p66 and p51 (Figure 1) (Di Marzo Veronese et al., Science, 231:1289-1291 (1986); Lightfoote et al., J. Virol., 60:771-775 (1986)), which have identical N-termini. The presence of several domains in HIV-1 RT has been deduced from sequence homology studies (Johnson et al., Proc.

Natl. Acad. Sci. USA. 83: 7648-7652 (1986)) and from experiments involving limited proteolysis (Lowe et al., Biochemistry, 27:8884-8889 (1988)). The N-terminal portion of the p66 subunit

corresponds to the DNA polymerase domain while the C-terminal portion shows homology with RNase H of E. coli as well as with the RNase H domain of RT from Moloney murine leukemia virus (MoMuLV) and other retrovirusea. The p51 subunit of HIV-1 RT apparently results from proteolytic processing of p66, during which the C-terminal 120 amino acid residues are removed (Le Grice et al.,

J. Biol. Chem., 264:14902-14908 (1989); Mizrahi et al., Arch.

Biochem. Biophys., 273:347-358 (1989)).

The C-terminal portion of p66 as an isolated domain (p15) is necessary but not sufficient for RNase H activity. The C-terminal domain of the p66 subunit of HIV-1 RT was expressed separately but, in contrast with MoMuLV, it is not sufficient for RNase H activity. However, the RNase H activity of HIV-1 RT can be reconstituted in vitro by combining isolated p15 with the purified p51 domain (Hostomsky et al., "Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 Reverse Transcriptase", Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991)).

These observations, together with results from deletion and insertion mutagenesis studies (Prasad et al., Proc. Natl. Acad.

Sci. USA. 86:3104-3108 (1989); Hizi et al., Virology, 175:575-580 (1990)), indicate that structural domains of HIV-1 RT are

functionally interdependent. Hence, in the current context, the present inventors use the term RNase H domain in a structural rather than a functional sense, to describe the C-terminal domain of HIV RT that shows sequence homology with other known RNase H domains.

In an effort to better understand the structural features of this unusual interdependence, as well as to establish a structural basis for the design of specific inhibitors directed against RNase H activity, the present inventors determined the 2.4 Å crystal structure of the C-terminal domain of HIV-1 RT. Comparison of this structure with the recently determined three-dimensional structure of E. coli RNase H (Katayanagi et al., Nature (London),

347:306-309 (1990); Yang et al., Science. 249:1398-1405 (1990)) confirms that the C-terminal portion of HIV-1 RT indeed represents an RNase H domain, although it requires interaction with the N-terminal portion of HIV-1 RT for RNase H activity.

From the crystal structure that the present inventors

determined, the present inventors were able to analyze the RNase domain active site. The present inventors analyzed the active site using the UO₂F₅ ^3- anion.

Analysis of the binding site for the UO₂F₅ ^3- anion indicates that four different sidechains, Asn545 and Asp443, Asp498, and

Asp549, closely approach the heavy atom complex. Residues at this site have been implicated in catalysis by mutagenesis studies (Schatz et al., FEBS Lett., 257:311-314 (1989); Mizrahi et al., Nucl. Acids Res., 18:5359-5363 (1990); Tisdale et al., J. Cell. Biochem., Supplement 14D, p. 179 (1990); and Kanaya et al., J. Biol. Chem., 265:4615-4621 (1990)). Difference electron density maps calculated at 2.8Å resolution between the native and the heavy atom derivative data indicate that at least two of the sidechains move upon binding. The carboxylates of Asp498 and Asp443 may displace two of the fluorine atoms bound to uranium, but it is not possible at this resolution to unambiguously define the uranium coordination.

Based on these observations and the data set forth in Example

I below, the present inventors discovered that the UO₂F₅ ^3- anion binds to the active site of the RNase domain of RT and is an RNase H inhibitor. Residues that form the heavy atom binding site are among seven amino acids conserved in all analyzed bacterial and retroviral RNase H sequences (Doolittle et al., The Quarterly

Review of Biology, 64:1-30 (1989)). These residues, Asp443, Glu478, Asp498, Ser499, His539, Asn545, and Asp549, all cluster one face of the molecule, as shown in Figure 2. The position of the histidine in Figure 2, which is disordered in the present model, is inferred from its location in the corresponding

constellation of residues in E. coli RNase H (Katayanagi et al., Nature (London), 347:306-309 (1990); Yang et al., Science,

249:1398-1405 (1990)).

Katayanagi et al., supra, found that magnesium ion bound to residues in E. coli RNase H corresponding to Asp443, Glu478, Asp498, and Asp549 of HIV-1 RNase H. Although the three aspartate residues that participate in ion binding are analogous in the retroviral and bacterial RNase H structures, the bound ions are positioned differently. In the HIV-1 RNase H domain, the UO₂F₅ ^3- interacts with Asn545 and the ion is bound deeper in the catalytic site than is the magnesium ion in E. coli RNase H, where the fourth ligand is a glutamate side chain. In the refined native structure the carboxylate side chains are in several instances within hydrogen bonding distance of one another, suggesting that one or more of these acidic residues must be protonated.

Through the following routine techniques, and in view of the material related to the active site provided in the present specification, those of skill in this field could determine if other metal materials inhibit the RNase H activity of RT, and according to certain preferred embodiments, determine if they bind to the active site of the RNase H domain of RT.

To determine if there is binding to the active site, crystals are grown at 4ºC in hanging drops equilibrated against a reservoir solution containing 0.15 M sodium potassium tartrate, 20 percent

PEG8000 and 0.1 M sodium citrate pH 5.2. The starting drops are composed of equal volumes of stock protein solution, i.e., the

RNase H domain of RT, (protein at 10 mg/ml, 25 mM potassium phosphate pH 7.0) and reservoir solution. Inhibitor (metal material) binding to RNase H is studied using x-ray diffraction data from a crystal soaked in reservoir solutions containing the inhibitor of interest. A difference map calculated with

diffraction data from this crystal and data from the native protein crystal reveals the geometry of inhibitor binding.

After determining that the UO₂F₅ ^3- anion bound to the active site of the RNase H domain of RT, the present inventors tested the effect of that anion on the activity of the RNase H domain of RT.

That experiment is set forth in detail below in Example I.

In general, for testing the effect of other metal materials on the RNase H activity of RT, one could use the following

protocol, although those of skill in this art may know other procedures for making the same determination.

An assay which can be used includes incubating RT with the metal material being tested and with a test RNA/DNA hybrid

substrate. After incubation, the extent of RNase H activity of RT on the substrate is determined by electrophoresis. A fully active

RNase H domain will produce a characteristic pattern of degradation products. Inhibition of such activity can thus be detected by variations from that characteristic pattern.

Although the present inventors should not be limited to a specific concentration range of metal material, it is contemplated that according to certain preferred embodiments, the concentration range of metal material could be from about 10 ^-3 M to about 10^-12

M. That range encompasses all points in between the endpoints listed. According to certain preferred embodiments, the

concentration could be less than 10 ^-12 M. Moreover the term

"about" provides leeway such that minor variations outside the recited range that work according to certain preferred embodiments are included in that recited range.

A general protocol, which is considered by those of skill in this art to be predictive of the effect of test materials in vivo, could be as follows. Three nanograms of the radioactively labeled RNA/DNA substrate set forth in Figure 3 are incubated in 10 μl containing 50 mM Tris-HCl pH 8/ 50 mM KCl/7 mM MgC12/5 mM

dithiothreitol with 0.2 μg of HIV-1 reverse transcriptase for 5 minutes at 37°C at different concentrations of the metal material being tested (for example, 0-400 μM). The reaction is

terminated by adding 2 μl of formamide with bromophenol blue. The material, e.g., 5 μl, is then analyzed by electrophoresis in 10% polyacrylamide gel containing 8 M urea. After electrophoresis, the gel is autoradiographed. The formation of a predominant reaction product 28 nucleotides long is evaluated by scanning of the autoradiograph using laser densitometer (LKB-Pharmacia). The formation of the product in the absence of the compound being tested represents 100% RNase H activity. The amount of inhibition can then be determined.

An even more detailed description of a protocol that can be used follows.

Enzyme Assay For Analysis of RNase H Activity of RT

Analysis of RNase H activity was based on the experiments described by Mizrahi and coworkers (Mizrahi et al.. Biochemistry, 28: 9088-9094 (1989); Dudding et al., Biochem. Biophys. Res.

Commun., 167:244-250 (1990)). Descriptions of the RNA-DNA hybrid substrate preparation are provided below and in Figure 3.

Description of the RNase H assay is provided below.

Substrate for detection of RNase H activity (See Figure 3) A portion of the gag region of HIV-1 [nucleotides 629-694 in the nucleotide sequence of BH-10 (Ratner et al., Nature. 313:277-284 (1985)), shown to contain a cluster of HIV-1 RNase H cleavage sites numbered I-VI (Mizrahi, Biochemistry, 28:9088-9094 (1989)), is cloned in the plasmid pTZ18R (Pharmacia). Uniformly labeled runoff transcripts (boldface) of this region are prepared from th resulting plasmid with an RNA synthesis kit (Stratagene) and UTP[α³⁵S]. The 3' ends of these transcripts are generated by Pvu

II or BamHI digestions of the template DNA in the positions indicated. The transcripts are gel purified and hybridized with complementary synthetic oligodeoxyribonucleotide, the sequence of which is shown in italics. Analysis of RNase H activity

Three nanograms of hybrid substrate (150,000 cpm), prepared as shown in Fig. 3 with the 3' end from either Pvu II or BamHI digested template, are incubated in 10 μl of buffer containing 50 mM Tris-CHI, pH 8.0/50 mM KCl/7 mM MgCl₂/5 mM dithiothreitol, 0.2 μg of RT, and with various concentrations of the test metal material (e.g., 0-400 μM). After incubation for 5 minutes at

37°C reactions are terminated by adding 2 μl of formamide with bromophenol blue and then boiling for 3 minutes. Five microliters of the sample is analyzed by electrophoresis in 10% polyacrylamide gel containing 8 M urea. After electrophoresis, the gel is soaked in Amplify (Amersham), dried and autoradiographed at -70°C.

Determinations on inhibition are then made based on the data generated.

As discussed in the Background and Summary of the Invention section of this specification, RNase H activity is essential for viral replication of viruses that are RT dependent. (Mitsuya, supra; Tisdale et al., supra; and Repaske et al., supra). By exposing such viruses to the metal materials according to the present invention such that RNase H activity of RT is inhibited, those viruses will be unable to replicate. Therefore, the present invention is also directed to inhibition of viral replication that is dependent on RT for replication.

The following is a protocol known to those skilled in this art for detecting inhibition of viral replication that is

dependent on RT. This procedure is described in Ashorn et al.,

Proc. Natl. Acad. Sci. USA. 87:7472-7476 (1990), and is considered by those of skill in this art to be predictive of the effect on viral replication in a patient.

Infection of Peripheral Blood Mononuclear

Cells (PBMCs) with HIV-1

Ficoll/Hypaque-isolated PBMCs are stimulated for 3 days in RPMI/FCS containing phytohemagglutinin (5 μg/ml). The cells are washed and suspended at 10 cells per ml in RPMI/FCS, and HIV-1_LAV is added at the multiplicity of 0.005 TCID₅₀ per cell. After a 2 hour adsorption period, the volume is raised 20-fold with RPMI/FCS supplemented with 10% (vol/vol) interleukin 2-containing

conditioned medium (Boehringer Mannheim). The cells are seeded in 24-well tissue culture plates plus the "test" metal material additions (2.5 × 10⁵ cells/1.25 × 10³ TCID₅₀ of HIV_LAV in a total volume of 1 ml per well). The metal material could be tested in concentrations from 10 -3 M to 10-12 M, although that concentration range should in no way be considered limiting, and according to certain preferred embodiments the concentration could be less than 10 -12 M. After 3 days, the cells are diluted 1:2 in fresh metal material-containing medium. At 6 days, the supernatants are harvested and analyzed for HIV-1p24 and RT activity.

Viability Assays

The relative numbers of viable cells are determined by the

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] oxidation procedure, which has been shown to correlate well with the trypan blue exclusion assay (Berger et al., AIDS Res. Hum.

Retrovir., 6: 795-804 (1990)). Quadruplicate reactions are

initiated by addition of 10 μl of MTT solution [thiazole blue

(Sigma); 5 mg/ml in isotonic phosphate-buffered saline] to 100 μl of cell suspensions in 96-well flat-bottom tissue culture plates.

After 4 hr at 37°C, 100 μl of 0.01 M HCl containing 10% (vol/ vol). SDS is added to each well. Oxidized MTT is allowed to dissolve in medium for 16 hr at 37°C and the absorbance is measured at 590 nm with an ELISA plate reader (V-max; Molecular

Devices, Menlo Park, CA). Relative cell numbers are expressed as percent of the MTT value of control wells that receive an equal number of cells but no virus or metal material.

Because the metal materials according to the present

invention inhibit viral replication, such materials can be used in pharmaceutical compositions to treat patients infected with a retrovirus or a hepadnavirus. In view of the present

specification, those skilled in this art would be able to

formulate such compositions in effective doses with known carriers or excipients. According to certain nonlimiting embodiments, the metal materials could be administered in a concentration range of about 10 ^-3 M to about 10^-12 M, and according to certain preferred embodiments the concentration could be less than 10 ^-12 M.

The following specific examples will illustrate certain embodiments of the invention. However, as described above, it will be appreciated that these teachings apply to all metal materials that inhibit RNase H activity. Various alternatives will be apparent to or could be determined, in view of the present specification, by those of ordinary skill in the art from the teachings herein, and the invention is not limited to the specific illustrative examples. EXAMPLE I

The RNase H activity of HIV-1 reverse transcriptase was assayed with RNA/DNA hybrid substrate in the presence of K₃UO₂F₅. The RNA/DNA substrate is described in detail in Fig. 3 (Hostomsky et al., Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991).

Incubation of this substrate with the RNase H activity leads to the appearance of a characteristic pattern of degradation

products. As discussed above, the assay used and described in this Example (and below in Example II) is considered by those of skill in this art to be predictive of the effect of the metal material in vivo.

Description of the assay

Three nanograms of the radioactively labeled RNA/DNA

substrate were incubated in 10 μl containing 50 mM Tris-HCl pH 8/ 50 mM KCl/7 mM MgCl2/5 mM dithiothreitol with 0.2 μg of HIV-1 reverse transcriptase for 5 minutes at 37°C at different

concentrations of K₃UO₂F₅ (0-400 μM). The reaction was

terminated by adding 2 μl of formamide with bromophenol blue.

Five μl were analyzed by electrophoresis in 10% polyacrylamide gel containing 8 M urea. After electrophoresis, the gel was autoradiographed. The formation of a predominant reaction product that is 28 nucleotides long was evaluated by scanning of the autoradiograph using laser densitometer (LKB-Pharmacia).

The formation of the product in the absence of the compound

K₃UO₂F₅ represents 100% RNase H activity. (See lane 1 of Figure 4). Based on the generated data, 50% inhibition of RNase H activity (IC₅₀) was observed at 50 μM. (See Figure 5). The protein forms visible precipitation at concentrations above 400 μM K₃UO₂F₅.

EXAMPLE II

The RNase H activity of HIV-1 reverse transcriptase was assayed with RNA/DNA hybrid substrate in the presence of copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt.

The RNA/DNA substrate is described in detail in Fig. 3 (Hostomsky et al., Proc. Natl. Acad. Sci. USA. 88:1148:1152 (1991).

products.

Description of the assay

Three nanograms of the radioactively labeled RNA/DNA

concentrations of copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt (0-100 μM). The reaction was terminated by adding 2 μl of formamide with bromophenol blue. Five μl were analyzed by electrophoresis in 10% polyacrylamide gel containing 8

M urea. After electrophoresis, the gel was autoradiographed. The formation of a predominant reaction product that is 28 nucleotides long was evaluated by scanning of the autoradiograph using laser densitometer (LKB-Pharmacia).

The formation of the product in the absence of the compound copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt represents 100% RNase H activity. Based on the generated data, 50% inhibition of RNase H activity (IC₅₀) was observed at μM. (See Figure 6).

It will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. (Appendices A and B follow the claims and Abstract of the Disclosure).

Claims

WHAT IS CLAIMED IS:

1. A metal material that inhibits RNase H activity of reverse transcriptase of a virus that is reverse transcriptase dependent, in an amount sufficient to inhibit RNase H activity.

2. A metal material as in claim 1, wherein the metal material is at a concentration of about 10^-3 M to about 10-12 M.

3. A metal material as in claim 1, wherein the metal material is at a concentration of about 10^-3 M.

4. A metal material as in claim 1, wherein the metal material is at a concentration of about 10^-6 M.

5. A metal material as in claim 1, wherein the metal material is at a concentration of about 10 M.

6. A metal material as in claim 1, wherein the metal material is at a concentration of less than or equal to about 10^-12 M.

7. A metal material as in claim 1, wherein the metal material binds to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.

8. A metal material as in claim 1, wherein the metal material is selected from the group consisting of a metal phthalocyanine containing compound and UO₂F₅ ^3- salt.

9. A metal material as in claim 8, wherein the metal of the metal phthalocyanine containing compound is selected from the group consisting of mercury, silver, cobalt, strontium, thorium, manganese, magnesium, zirconium, zinc, tin, nickel, lead, iron, palladium, copper, gold, gallium, chloroindium, platinum, calcium, molybdenum, dichlorotin, dilithium, dichlorogermanium, vanadyl, flourochromium, and chloroaluminum.

10. A metal material as in claim 1, wherein the metal material is selected from the group consisting of K₃UO₂F₅ and copper phthalocyanine-3,4',4",4"' tetrasulfonic acid, tetrasodium salt.

11. A method of inhibiting RNase H activity of reverse transcriptase comprising exposing reverse transcriptase of a virus that is reverse transcriptase dependent to a metal material in an amount sufficient to inhibit RNase H activity.

12. A method as in claim 11, wherein said metal material binds to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.

13. A method as in claim 11, wherein said metal material is selected from the group consisting of a metal phthalocyanine containing compound and UO₂F₅ ^3- salt.

14. A method as in claim 13, wherein the metal of the metal phthalocyanine containing compound is selected from the group consisting of mercury, silver, cobalt, strontium, thorium,

manganese, magnesium, zirconium, zinc, tin, nickel, lead, iron, palladium, copper, gold, gallium, chloroindium, platinum, calcium, molybdenum, dichlorotin, dilithium, dichlorogermanium, vanadyl, flourochromium, and chloroaluminum.

15. A method as in claim 11, wherein said exposing includes using the metal material in a concentration of about 10^-3 M to about 10^-12 M.

16. A method as in claim 11, wherein said exposing includes using the metal material in a concentration of about 10^-3 M.

17. A method as in claim 11, wherein said exposing includes using the metal material in a concentration of about 10^-6 M.

18. A method as in claim 11, wherein said exposing includes using the metal material in a concentration of about 10^-9 M.

19. A method as in claim 11, wherein said exposing includes using the metal material in a concentration of less than or equal to about 10^-12 M.

20. A method of inhibiting replication of a reverse

transcriptase dependent virus comprising exposing the virus to the metal material of claim 1.

21. The method of claim 20, wherein the virus is a

retrovirus or a hepadnavirus.

22. The method of claim 21, wherein the virus is selected from the group consisting of HIV-1, HIV-2, HTLV-1, HBV, FeLV, or SIV.

23. The method of claim 20, wherein the viral replication inhibited in vitro.

24. The method of claim 20, wherein the viral replication inhibited in vivo.

25. A pharmaceutical composition for the treatment of patients infected with a reverse transcriptase dependent virus comprising a metal material according to claim 1 in an amount sufficient to inhibit viral replication in vivo, and a

pharmaceutically-acceptable carrier.

26. A method of treating patients infected with a reverse transcriptase dependent virus comprising administering an effective amount of the pharmaceutical composition of claim 25.

27. A complex comprising a metal material bound to at least a portion of the active site of the RNase H domain of reverse transcriptase, such that RNase H activity is inhibited.