WO2003012131A2 - Assays for protease inhibitors, methods and means - Google Patents

Abstract

A fusion protein is provided in cells in culture that comprises (i) an autoproteolytic precursor of a cytotoxic protease and (ii) a reporter protein, which reporter protein provides a quantitative signal indicative of amount of reporter protein present within the cells, allowing for assays to identify inhibitors of the protease and potential therapeutics useful in targeting pathogens, especially viruses such as HIV.

Description

ASSAYS FOR PROTEASE INHIBITORS, METHODS AND MEANS

The present invention relates to assays for protease inhibitors, especially inhibitors of proteases of pathogens, such as viruses. Preferred embodiments of the present invention involve the HIV protease and are concerned with assays for inhibitors of that protease, which have therapeutic potential .

The present invention also allows for determining susceptibility of a protease to a known protease inhibitor, e.g. in monitoring of mutation of HIV-1 protease in different isolates. Information on inhibitor-susceptibility is useful in planning of therapeutic strategies for individuals and populations.

The human immunodeficiency virus (HIV) -1 protease is essential for production of infectious virus and is therefore a major target for drug development against AIDS. Cellular proteins are also cleaved by the protease, which explains its cytotoxic activity and the consequent failure to establish convenient cell-based protease assays. The present invention exploits this toxicity in providing new protease assays involving expression of an artificial protease precursor harboring a reporter protein, such as the green fluorescent protein (GFP- PR) . The precursor is activated in vivo by autocatalytic cleavage resulting in rapidly elimination of protease expressing cells. Treatment with protease inhibitors results in a dose-dependent accumulation of the reporter protein that can be easily detected and quantified, for example by flow cytometric and fluorimetric assays. The precursor provides a convenient and non-infectious model for high throughput screenings of substances that can interfere with the activity of the protease in living cells. The protease encoded by the human immunodeficiency virus (HIV) -1 plays an essential role in the retroviral life cycle by processing the viral p55Gag and pl60Gag-Pol polyprotein precursors into structural proteins and enzymes . The activity of the protease is required for conformational rearrangement of the immature virion and production of infectious virus particles, thus providing an attractive target for development of antiviral agents against acquired immune deficiency syndrome (AIDS) and related disorders (30) .

Several potent HIV-1 protease inhibitors (PI) are widely used in the clinics (7, 10) . However, the constant emergence of resistant strains due to the additive effect of multiple amino acid substitutions within and outside the catalytic site motivates the urge for continuous development of new protease inhibitors (26) . The availability of reliable and convenient assays for protease activity is, in this context, of great importance .

The majority of assays available today are based on trans- or autocatalytic cleavage of reporter proteins in bacteria, yeast or in vi tro (1, 8, 17, 24) or on the in vi tro hydrolysis of synthetic peptides encompassing the scissile bonds in p55Gag and pl60Gag-Pol (3, 14, 23) .

However, none of these assays allows probing of all the native HIV-1 protease specificity sites under physiologic conditions, a situation for which a human cell environment would be required. An important reason for the lack of convenient mammalian cell-based assays is the cytotoxicity observed upon expression of the protease in cells. Thus, while this retroviral aspartic protease possesses unique structural and functional properties that distinguish it from its cellular counterparts (6) , several cellular proteins are efficient substrates of the protease.

Among those are cytoskeletal proteins such as vimentin, actin, troponin and tropomyosin (20, 22), microtubule-associated proteins (28) , precursors of NF- B (19) and bcl-2 (25) , providing a likely explanation for the capacity of the protease to induce apoptosis.

Five inhibitors of HIV protease (PR) have received regulatory approval for clinical use. They are all peptidomimetic, competitive inhibitors acting to prevent cleavage of HIV polyproteins . Emergence of drug-resistant variants has been analysed both in patients and in tissue culture replication studies, demonstrating that high-level resistance results from the combined effects of multiple substitutions. The high rate of virus production coupled with a high mutation rate yields a population of closely related variants ^■ referred to as quasispecies . It is assumed that an HIV population in the patient has acquired optimal fitness and antiviral therapy results in the selection of mutant viruses with decreased sensitivity against the inhibitor, but also impaired enzyme function. Such variants exhibit a lower fitness compared to the wild-type population and need to acquire compensatory mutations to restore fitness. Accordingly, , resistance development in vivo occurs by stepwise accumulation of mutations both in the PR gene itself and its targets: primary mutations affect the substrate-binding pocket of PR and lead to a decreased binding affinity for the inhibitor, while secondary mutations map outside the active site and compensate for a loss in enzymatic function, and further mutations affect the PR cleavage sites. Many substitutions exhibit considerable overlap among different PI and the mutational patterns selected by various drugs often become indistinguishable as resistance evolves.

The emerging picture of PI resistance is more complex than in the case of inhibitors of the reverse transcriptase, where one or a few mutations in the coding region are sufficient for drug resistance.

In order to optimise treatment and to ensure a long term antiviral response, it appears crucial to monitor antiviral resistance and to adjust the therapeutic regimen accordingly. Retrospective studies have shown a good correlation between resistance detected before treatment change, and subsequent response to that change .

However, it is still unclear which assays are best suited for therapeutic adjustment. The usefulness of genotypic assays, detecting the presence of resistance-associated mutations, is limited by incomplete understanding of the mechanisms leading to resistance and cross-resistance. PI resistance is the consequence of the stepwise accumulation of mutations within PR, leading to a gradual increase of the level of resistance. Some of the mutations impair enzymatic activity of PR, thus reducing the replicative capacity (fitness) of the virus, and compensatory mutations in PR itself or its targets are required to restore fitness. Several algorithms for prediction of resistance against anti-HIV drugs have been developed and are currently tested in clinical settings. However, the complexity of the mutational pattern renders interpretation difficult and the combined and subsequent use of different PI makes it less reliable. Furthermore, the result of genotyping reflects only the predominant virus species in the sample and the emergence of new mutations and combinations of mutations able to confer resistance to PI is continuing. Phenotypic assays, which measure actual susceptibility of viral strains to anti-retroviral agents, are currently the only way to directly determine resistance and give a rationale for the therapeutic adjustment. Known assays are based on inserting PCR-amplified PR sequences from patient plasma virus into a proviral clone defective in the PR gene, followed by transfection and analysis of the resulting virus population for susceptibility to various drugs. A number of adaptations of this recombinant virus assay (RVA) have been described. The introduction of the RVA has considerably improved resistance testing, but this test can only be performed in specialised laboratories, requires several weeks for readout, and is quite expensive.

The present inventors have developed a new reporter system that allows monitoring of HIV-1 protease activity and the effect of protease inhibitors in living cells. Embodiments of the invention may involve transient expression of a non-toxic protease precursor harboring a reporter molecule such as the green fluorescent protein (GFP) . Autocatalytic cleavage of the protease releases toxicity and the consequent disappearance of fluorescence provides a simple means to quantify protease activity and to search for inhibitors of this important enzyme.

Brief Description of the Figures

Figure 1 shows a schematic representation of the GFP-PR chimera. The plasmid was constructed by linking the HIV-1 protease coding and flanking sequences to the 3' end of the GFP open reading frame. The 5' and 3' flanking sequences of the protease are marked as p6 and RT, respectively. The amino acid sequences of the protease borders are indicated with the cleavage sites underlined. The scissile bonds are marked by an asterisk.

Figure 2 shows results of densitometric quantification of the GFP-PR and GFP bands in a Western Blot on which lysates of HeLa cells transiently transfected with GFP-PR were analysed using an anti-GFP antibody. The transfected cells were incubated with increasing concentrations of ritonavir for 24h. The GFP-PR precursor and the GFP released upon autocatalytic cleavage are indicated. One representative experiment out of three .

Figure 3 shows results of experiments in which HeLa cells transiently transfected with pcDNA3/GFP-PR were treated with increasing concentrations of indinavir, nelfinavir, ritonavir and saquinavir for 24 h and analysed by flow cytometry. Nelfinavir was toxic at concentrations higher then 10 μM. Fluorescence is expressed as fold induction over transfected cells that were cultured in the absence of inhibitor. Mean ± standard deviation of three experiments .

Figure 4 shows results of fluorimetric analysis of HeLa cells transiently transfected with pcDNA3/GFP-PR. Cells were treated for 24 h with the HIV-1 protease inhibitors indinavir (10 μM) , nelfinavir (10 μM) , ritonavir (10 μM) , saquinavir (10 μM) , the aminopeptidase inhibitor bestatin (1 mM) , the cysteine protease inhibitor leupeptin (1 mM) and the proteasome inhibitor Z-L3-VS. (8 μM) . Mean ± standard deviation of three experiments. Excitation and emission wavelengths are 480 nm and 510 nm, respectively.

Figure 5 shows results of experiments in which HeLa cells transiently transfected with GFP-PRwt were treated with increasing concentrations of iopinavir and analysed by flow cytometry. FACS data are shown as points and fitted data are shown as a curve. From these data, Y_max ^{^} was observed to be

3048+ 129 and EC_Ξ0 was observed to be 22+4 nM.

According to one aspect of the present invention there is provided an assay method for screening for an inhibitor of a cytotoxic protease, the method comprising: providing within cells in culture a fusion protein that comprises (i) an autoproteolytic precursor of the protease and (ii) a reporter protein, which reporter protein provides a quantitative signal indicative of amount of reporter protein present within the cells, contacting with a test substance cells containing the fusion protein, determining levels of signal in cells in the presence or absence of test substance, and/or different dosages of test substance, wherein level of the signal is indicative of amount of inhibition of the protease.

An inhibitor of the protease inhibits release of the protease from the precursor, and inhibits cell-killing, allowing for detection of an increased level of signal provided by the reporter protein.

In the absence of inhibitor, the autoproteolytic protease acts autocatalytically on the precursor, freeing the protease and allowing the protease to act cytotoxically to kill the cells. Then, there is little or no detectable signal from the reporter protein.

Compared with this, an increased signal is detected when a test substance or agent is an inhibitor of the protease, since the autocatalytic activity of the protease is inhibited, release of the protease is inhibited and cell-killing is reduced, and may be wholly or partially abolished. Cells that are not killed continue to contain and preferably express reporter protein, the signal from which is therefore detectable.

In preferred embodiments of the present invention, the protease is an HIV-1 protease polypeptide. This may be full- length or a sufficient portion or fragment to contain the requisite autoproteolytic activity and cytotoxicity.

Other potential proteases that could be used in the invention include those of other pathogens, especially retroviruses such as HIV-2, HTLV or hepatitis C virus, and other cytotoxic proteases of medicinal importance such as caspases, cathepsins, proteases of the thrombolytice cascade, and rennin may also be subject to the present invention, all of these being toxic under appropriate conditions for cells in which they are overexpressed as recombinant proteins .

Reporter proteins that may be employed in fusion proteins and assays of the present invention include any that provides a signal, for example an enzymatic, fluorescent or bioluminensent signal . Examples include green fluorescent protein (GFP) , coral red fluorescent protein, luciferase, β- galactosidase, β-lactamase and chloramphenicol acetyltransferase. A detectable signal is required, preferably a signal that is proportional in level to the amount of reporter protein present, and can thus be assayed quantitatively under comparable conditions and using appropriate controls as is standard for experimental protocols in the art . The perfect stoichiometry between toxic protease and the reporter accomplished by a precursor fusion protein is advantageous in assays of the invention, and may be used to give better results than co-transfection of independent plasmids harboring the reporter protein and protease open reading frames. The invention can be applied to any pathogenic proteases provided that overexpression of these proteases has a cytotoxic effect and that insertion of the reporter moiety does not disturb autocatalytic release of the protease from the precursor.

In a further aspect, the present invention provides a method of determining susceptibility of a protease of a pathogen, which protease exists in more than one variant or mutant isoform, to an agent that inhibits at least one isoform of the protease, the method comprising providing within cells in culture a fusion protein that comprises (i) a precursor of a test protease isoform and (ii) a reporter protein, which reporter protein provides a quantitative signal indicative of amount of reporter protein present within the cells, contacting with the agent cells containing the fusion protein, determining levels of signal in cells in the presence or absence of test substance, and/or different dosages of test substance, wherein level of the signal is indicative of amount of inhibition of the protease isoform.

Sensitivity of different isoforms of the protease can be compared, and new mutants or variants can be identified.

Acquisition of mutations within a protease, such as that of HIV-1, in an infected individual can be monitored, as a means for monitoring for pathogen evasion of treatment regimens and in determining appropriate adjustment of therapy. For instance, embodiments of the present invention may be used to assess susceptibility or the development of resistance to antiviral drugs such as Saquinavir, Ritonavir, Indinavir and Lopinavir .

In assays and methods of the invention, preferred cells are human cells, especially where the protease is of a pathogen of humans, e.g. HIV. Appropriate cells include human lymphocytes and neurons, HeLa cells and COS cells. Any cell may be used, e.g. human or non-human or mammalian, as long as the protease of concern is toxic to that cell. Mammalian cell lines available in the art for expression of a fusion protein and use in an assay include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. HeLa and COS cells are preferred in assaying for inhibitors of a human pathogen protease .

A fusion protein for use in an assay and method of the present invention itself represents an aspect of the present invention, in which there is provided a fusion protein comprising (i) an autoproteolytic precursor of a cytotoxic protease and (ii) a reporter protein, which reporter protein provides a detectable signal . The protease and the reporter protein may be any protease or any reporter protein disclosed herein, or any other that can be employed in an assay of the invention.

Further provided as an aspect of the present invention is nucleic acid encoding a fusion protein of the invention. Such nucleic acid can be used in production of the fusion protein by recombinant expression, e.g. to provide purified or isolated fusion protein. Moreover, in an assay of the invention, the fusion protein may be provided within cells in culture by introduction of encoding nucleic acid into the cells and production of the fusion protein by recombinant expression from the nucleic acid.

Polynucleotides with nucleic acid sequences encoding a fusion protein comprising protease and reporter can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, 1992) . These techniques include the use of the polymerase chain reaction (PCR) to amplify nucleic acid from appropriate sources, chemical synthesis, and preparation of cDNA sequences. Modifications to sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified polypeptides or to take account of codon preference in the host cells used to express the nucleic acid. In general, short sequences for use as primers can be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides can be produced using recombinant means, for example using a PCR (polymerase chain reaction) cloning technique. This may involve making a pair of primers (e.g. of about 15-50 nucleotides) based on available sequence information to a region of the RNA or DNA which it is desired to clone, bringing the primers into contact with RNA or DNA obtained from the relevant source, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that- the amplified DNA can be cloned into a suitable cloning vector.

Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, phagemid or baculoviral, cosmids, YACs, BACs, or PACs as appropriate, depending on the host cell.

For expression in human cells, e.g. for assaying for inhibitors of HIV-1 or other human pathogen, preferred vectors allow for high levels of expression in the particular cell line. Expression may be transient following transfection or may be inducible. Inducible cell lines are particular useful for high throughput screening as all cells will produce the signal (e.g. be fluorescent) when the protease is inhibited, improving improve the signal/background ratio. Examples of inducible systems are the Tet on/off systems where expression of the gene of interest is regulated by addition/withdrawal of tetracycline, but any inducible systems are also available or will be produced in the future and will be useful to those skilled in the art in performing embodiments of the present invention.

Vectors may be provided with an origin of replication, optionally a promoter for the expression of .the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, such as a neomycin resistance gene for a mammalian vector. Vectors may be used to transfect or transform a host cell.

Vectors may be transformed into a suitable host cell as described above to provide for expression of a fusion protein of the invention. Thus, in a further aspect the invention provides a process for producing a fusion protein according to the invention which includes cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the fusion protein. This may be followed by recovering the expressed fusion protein, or may be followed by performance of an assay of the invention by contacting with a test agent the host cell containing the fusion protein.

Further aspects of the present invention provide the use of a fusion protein of the invention as disclosed, and/or encoding nucleic acid therefor, in screening or searching for and/or obtaining/identifying a substance, e.g. peptide or chemical compound, which inhibits the activity of a pathogenic protease. For instance, a method according to one aspect of the invention includes providing a fusion protein of the invention and bringing it into contact .with a substance, which contact may result in inhibition of the activity of the protease within the fusion protein.

The precise format of an assay of the invention may be varied by those of skill in the art using routine skill and knowledge, in particular employing suitable control experiments and comparable conditions in order to elucidate information of activity of a test substance. A method of screening for a substance which modulates activity of a protease may include contacting one or more test substances with a fusion protein as disclosed, within suitable host cells, determining the presence or absence, and/or amount of detectable signal provided by the reporter protein, and comparing that signal with signal for comparable host cells untreated with the test substance or substances . A difference in reporter activity between the treated and untreated cells is indicative of a modulating effect of the relevant test substance or substances.

Combinatorial library technology (Schultz, JS (1996)

Biotechnol . Prog. 12:729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate protease activity.

The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.1 nM to 10 μM concentrations of a test compound (e.g. putative inhibitor) may be used. Greater concentrations may be used when a peptide is the test substance.

Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants which contain several characterised or uncharacterised components may also be used.

Other candidate inhibitor compounds may be based on modelling the 3-dimensional structure of a protease and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.

Following identification of a substance which modulates or affects protease activity, the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals .

Thus, the present invention extends in various aspects not only to a substance identified as a modulator, especially inhibitor, of protease activity, in accordance with what is disclosed herein, and a substance obtained by a method of the invention, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for treatment (which may include preventative treatment) of a disease or condition associated with or caused by the protease activity, use of such a substance in manufacture of a composition for administration, e.g. for treatment of a disease or condition, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

A substance identified using as a protease inhibitor may be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses . Accordingly, a mimetic or mimic of the substance

(particularly if a peptide) may be designed for pharmaceutical use. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. some peptides are not well suited as active agents for oral compositions as they may be quickly degraded by proteases in the alimentary canal.

Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore" .

Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. •

Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of substances identified as having ability to modulate polypeptide activity using a screening method as disclosed herein are included within the scope of the present invention. A polypeptide, peptide or substance able to inhibit protease activity according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use.

Further aspects and embodiments of the present invention will be apparent to those skilled in the art . The following experiments provide support for and exemplification by way of illustration of aspects and embodiments of the invention. All documents mentioned in this specification are incorporated by reference.

EXPERIMENTAL

MATERIALS AND METHODS

Plasmids

The HIV-1 protease coding and flanking regions were amplified from the pK-HIV plasmid (12) using the sense primer 5 ' -AGCTGTACATTTGGGGAAGAGACAACAACT CCCT-3' (SspBI site underlined) and antisense primer

5'-CGAGATATCTTTTGGGCCATCCATTCCTGGCTTTA-3' (EcoRV site underlined) . The PCR product was cioned in frame with the GFP open reading frame from EGFP-N1 (Clontech, Palo Alto,

California) into the pcDNA3 vector (Invitrogen) yielding the pcDNA3/GFP-PR construct. For the pcDNA3/PR construct the protease was amplified using the primers 5'-CCCAAGCTTATGGAATTCCCTCAGATCACTCTTTGGCAGCG-3' (Hindlll site underlined and start codon in bold) and

5'-CCCGCGGCCGCTTAAAAATTTAAAGTGCAGCCAATCTG-3' (Not I site underlined and stop codon in bold) and cloned in pcDNA3. The identities of the plasmids were confirmed by DNA sequencing using dye terminator cycle sequencing (Applied Biosystems) . The plasmid pT7-HIVlGag was kindly provided by S. Schwartz (Uppsala University, Sweden) .

The HIV protease amino acid and nucleotide sequences are available from GenBank.

Trans feet ion and vaccinia infections

HeLa (human cervical carcinoma cell line) and COS-1 cells (green monkey kidney cell line) were grown in Iscove's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (Life Technologies, Grand Island, New York) .

The cells were transfected with a mixture of plasmid DNA and Lipofectamine (Life Technologies) as recommended by the supplier. Stable sub-lines were generated by selection in 500 μg ml -1 G418 (Sigma, St. Louis, MO) and screened by flow cytometry. The vaccinia-T7 RNA polymerase-based expression system was utilised for transient high level expression of the pT7-HIVlGag as described (9) . HeLa cells were infected with the recombinant virus VTF7-3 (provided by S. Schwartz) for 2 h before transfection. Where indicated, the transfected cells were treated with protease inhibitors for 24 h before harvesting. For protease inhibitor treatment of cultured cells, the inhibitors were initially dissolved in DMSO and diluted to appropriate concentrations in Iscove's modified Eagle's medium supplemented with 10% fetal calf serum. Where indicated, the transfected cells were treated with protease inhibitors immediately after transfection until cells were harvested 24 h later.

Western blot analysis

Lysates of 10⁵ HeLa cells were fractionated by SDS-PAGE and blotted onto Protan BA 85 nitrocellulose filters (Schleicher & Schuell Keene, New Hampshire) . The filters were probed with a rabbit polyclonal anti-GFP serum (Molecular Probes Europe, Leiden, The Netherlands) or anti-capsid (p24) serum (15) . The filters were developed by enhanced chemiluminiscence (ECL, Amersham, Aylesbury, United Kingdom) . Quantification of Western blot bands was performed by densitometry (Molecular Dynamics) .

Flow cytometry, fluorescence microscopy and fluorimetric analysis

Expression of GFP was detected 24 h after transfection using a

FACSort flow cytometer (Beckton & Dickinson, Mountain View, California) and Cellquest software. For fluorescence microscopy, the cells were grown on coverslips, fixed with 4% paraformaldehyde in PBS and counterstained with Hoechst33258 (SIGMA-Aldrich) . A LEITZ-BMRB fluorescence microscope (Leica, Heidelberg, Germany) was used with appropriate filter setting for GFP or Hoechst staining.

Photographs were taken with a Hamamatsu 800 cooled CCD camera (Hamamatsu, Osaka, Japan) and processed with the Adobe Photoshop software. Fluorimetric analyses were performed with an LS-50B luminescence spectrometer (Perkin Elmer, Beaconsfield, United Kingdom) , with excitation wavelengths at 480 nm and emission at 510 nm.

RESULTS

Construction of the GFP-PR reporter

The toxicity of the HIV-1 protease has prevented the development of convenient assays for protease inhibitors in living cells. In the present invention, the inventors have employed expression of a precursor in which the protease is fused to a reporter protein circumventing this problem since the chimera is detected only when the activity of the protease is inhibited.

The autofluorescent green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a good reporter protein from among those available, because its expression can be easily monitored and quantified in vivo (27) . The GFP-PR chimera was generated by fusing a PCR product containing the HIV-1 protease open reading frame and the flanking sequences coding for 23 amino acids upstream and 20 amino acids downstream from the protease to the 3 ' end of the GFP open reading frame (Figure 1) . The flanking regions contain the endogenous p6/PR and PR/RT cleavage sites that are used for generation of an enzymatic active protease in virus infected cells.

The HIV-1 protease is activated in vivo by autocatalytic cleavage of the GFP-PR chimera

HeLa cells were co-transfected with the pcDNA3/GFP-PR plasmid and the plasmid pT7-Gag that expresses the HIV-1 polyprotein p55Gag, a natural substrate of the protease (30) . Processing of p55Gag yields the p41 (matrix and capsid protein) and p24 (capsid protein) products that can be detected in Western blots with a polyclonal antibody specific for p24 (15) .

High levels of unprocessed p55Gag were detected in HeLa cells transiently transfected with pT7-Gag and infected with a recombinant vaccinia virus expressing the T7 polymerase.

Lysates of HeLa cells cotransfected with GFP-PR and the HIV-1 p55Gag were analysed by Western blot . Increasing concentrations of the protease inhibitor saquinavir were added to the culture medium immediately after transfection. The p55Gag precursor and the specific cleavage products p41Gag and p24Gag were seen on the blot. In addition, some low molecular weight species were also detected by the anti p24 antibody, probably due to processing of the over-expressed polyprotein by cellular proteases.

A characteristic band corresponding to the p24 capsid protein was readily detected in cells coexpressing the GFP-PR reporter (Figure IB) . Similar results were obtained upon co- transfection with the pcDNA3/PR plasmid that expresses an enzymatic active HIV-1 protease devoid of flanking sequences.

Furthermore, the generation of p24 from the p55Gag precursor was inhibited in GFP-PR expressing cells by the HIV-1 protease inhibitors saquinavir, ritonavir, nelfinavir or indinavir in a dose-dependent manner (Figure IB and data not shown) , further confirming that the GFP-PR reporter harbours authentic protease activity.

The detection of HIV-1 protease activity in transfected cells indicates that processing of the chimera and activation of the protease occurs in vivo . This was investigated by probing Western blots of transiently transfected HeLa cells with polyclonal antibodies to GFP, which bind the intact chimera and its processed product GFP. The intact GFP-PR chimera was not detected while a weak band corresponding to the GFP moiety of the reporter was identified. The absence of detection of intact GFP-PR provides indication that the reporter may be rapidly processed in the transfected cells. To test this, increasing concentrations of the specific protease inhibitors saquinavir or ritonavir were added to the culture medium immediately after transfection. A dose-dependent increase in the intensity of a band corresponding to the intact GFP-PR was observed, indicating that processing of the chimera is blocked by the inhibitor. Densitometric quantification of the GFP and GFP-PR specific bands confirmed that accumulation of the precursor was accompanied by disappearance of the GFP cleavage product. (HeLa cells transiently transfected with GFP-PR were treated for 24 h with increasing concentrations of indinavir and the accumulation of GFP was monitored by flow cytometry. The percentage and mean fluorescence intensity of cells located in the upper right quadrant were determined.)

Taken together these results indicate that the GFP-PR reporter behaves as a bona fide protease precursor, which is activated in vivo by autocatalytic cleavage of the protease. The HIV-1 protease is toxic in GFP-PR expressing cells

The GFP reporter allows accurate quantification in living cells. Thus, accumulation of GFP fluorescence provides a convenient method for monitoring for presence of the HIV-1 protease in cells expressing GFP-PR. However, only a small number of weakly fluorescent cells were detected by FACS analysis and fluorescence microscopy of HeLa cells transfected with the pcDNA3/GFP-PR plasmid. The poor fluorescence was not due to inefficient transfection since dose dependent increase in the number of fluorescent cells and fluorescence intensity of the positive cells was observed upon treatment with the HIV-1 protease inhibitor indinavir and similar results were obtained on transfection of COS-1 cells. A dramatic increase of fluorescence intensity was also detected by fluorescence microscopic analysis of cells treated with 1 μM saquinavir.

Failure to accumulate GFP in the absence of protease inhibitors could be due to inactivation of GFP or early elimination of the GFP expressing cells by the cytotoxic effect of the protease. To discriminate between these possibilities, HeLa cells were transfected with plasmids encoding the GFP-PR chimera or GFP alone and the yield of fluorescent clones was' compared after selection in G418. In anticipation of the cytotoxic effect of GFP-PR, a parallel selection was performed in continuous presence of the HIV-1 protease inhibitor saquinavir. Transfection with the GFP expressing plasmid yielded a high proportion of clones that stably expressed high levels of GFP (Table I) . In contrast, only two poorly fluorescent clones out of 34 selected were obtained from GFP-PR transfected cells. Inclusion of saquinavir throughout the selection procedure improved the yield of fluorescent clones and the percentage of fluorescent cells in each clone but these remained in all cases far less than observed in cells transfected with GFP alone. (Table I) . Furthermore, the fluorescence was gradually lost and attempts to select stable populations of fluorescent cells by FACS sorting or cloning by limiting dilution failed, suggesting that the fluorescent cells may be progressively eliminated due to toxicity of the protease. This was confirmed by analysis of one fluorescent GFP-PR clone, which was originally selected with 1 μM saquinavir and later maintained in the presence of

10 μM ritonavir. At the time of first testing the clone contained approximately 40% fluorescent cells but the number decreased progressively and the fluorescent population was completely lost within a few weeks. Omission of ritonavir at the time when only 12% of the cells remained fluorescent resulted in disappearance of the fluorescent cells within 24h. The loss was irreversible since subsequent administration of 10 μM ritonavir did not lead to reappearance of the fluorescent population.

Ti tration of HIV-1 protease inhibi tors in GFP-PR expressing cells The effect of indinavir nelfinavir, ritonavir and saquinavir was monitored in pcDNA3/GFP-PR transfected HeLa cells. Increasing concentrations of the inhibitors were added immediately after transfection and the cells were cultured for 24 h before analysis by flow cytometry. The inhibitors induced a dose dependent increase in fluorescence intensity of the positive cells (Figure 3) . Significant accumulation of the reporter was detected in cells treated with concentrations of the inhibitor as low as 0.01 μM, which is lower than the effective plasma concentration achieved in protease inhibitor treated patients (29) . A maximum of approximately nine-fold increase in fluorescence was reached at 10-50 μM of inhibitor (Figure 3) . The possibility of using the GFP-PR reporter in fluorimetric assays would facilitate its employment in high throughput screens. The inventors compared the fluorescence intensity of GFP-PR expressing cells in response to the HIV-1 protease inhibitors, indinavir, nelfinavir, saquinavir and ritonavir and three irrelevant inhibitors of aminopeptidases (bestatin) , cysteine proteases (leupeptin) and a proteasome inhibitor (carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone, Z-L3-VS (5)) .

Approximately three-fold increase of total fluorescence was demonstrated by fluorimetric analysis of GFP-PR transfected cells following treatment with the four HIV-1 protease inhibitors, whereas bestatin, leupeptin and Z-L3-VS had no effect (Figure 4) . The level of fluorescence accumulation detected by fluorimetry was well in line with the approximate nine fold increase of mean fluorescence intensity recorded in parallel experiments where the effect of HIV-1 protease inhibitors on the accumulation of GFP was monitored by FACS analysis . Although a stronger effect was measured by flow cytometry, the clear and highly reproducible increase detected by fluorimetric analysis in transiently transfected cells indicates that the GFP-PR reporter can be used in high throughput screens of protease inhibitors using a multiwell plate fluorimeter.

DISCUSSION

The present invention provides among various embodiments a new and convenient method for monitoring HIV-1 protease activity in human cells, which is based on expression of a precursor protein harboring the viral protease fused to a reporter protein such as GFP. As noted, the invention may be used with any of a variety of different proteases and any of a variety of different reporter proteins.

Expression of reporter ensures a 1:1 stoichiometry between the viral protease and the reporter protein. Thus, where the reporter is GFP, quantification of the amount of GFP by its emitted fluorescence^' is directly correlated to the amount of protease in reporter-expressing cells. The chimeric reporter is enzymatic active in vivo due to autocatalytic activation of the protease. As high intracellular levels of the protease are only tolerated when its enzymatic activity is inhibited, expression of GFP is a reliable parameter of intracellular HIV-1 protease activity. A clear inverse correlation was observed between the emitted fluorescence and the activity of the HIV-1 protease in protease inhibitor-treated cells.

Transient transfection experiments showed that titration of HIV-1 protease inhibitors leads to an approximate nine-fold increase in the mean fluorescence intensity as measured by flow cytometry. Furthermore, measurement of the total fluorescence by fluorimetry revealed a reproducible approximate three-fold increase indicating that the reporter system can be adapted to high throughput screenings of protease inhibitors using microtiter plate readers. The system was shown to be very sensitive, responding to concentration of HIV-1 protease inhibitors in the therapeutic range of the different inhibitors. Importantly, no effect was observed when cells were treated with different classes of unrelated inhibitors showing that this reporter is specific for HIV-1 protease inhibitors .

The reporter system offers several distinct advantages over the broad array of existing screening assays for HIV-1 protease activity. The currently available detection methods are mainly performed in yeast and bacteria cells or in vi tro . The most simple in vivo assays exploit the inherent toxicity of the protease for bacterial cells (2) and more sophisticated strategies have involved the introduction of HIV-1 protease cleavage sites into selectable markers such as β-galactosidase (1) , tetracycline resistance protein (4) , thymidylate synthase

(13) , galactokinase (24) , transcription factors of

Saccharomyces cerevisiae (17) and cl repressor of bacteriophage λ (21) . These assays usually monitor only one or few of the native HIV-1 protease specificity sites. Furthermore, the activity of the protease is tested under non- physiologic conditions that may modify the catalytic properties of the enzyme.

Assays according to embodiments of the present invention monitor the activity of the protease where therapeutic interference is most desired, in human cells. The exact sequence of the proteolytic events in virus infected cells is not known but the protease is believed to act mainly during the budding process when it is located at the intracellular face of the cell membrane. Moreover, the toxicity caused by the viral protease in infected cells is suspected to contribute to the complex pathogenesis of AIDS (11) . Since the GFP readout is inversely correlated to the cytotoxic effect of the protease, the use of this system in various HIV-1 susceptible cells can provide detailed information as to what extent different candidate inhibitors are able to suppress these cell-associated effects. Parameters that are expected to vary between different cell types, such as permeability to the inhibitor or metabolic stability, are conveniently evaluated by measuring GFP fluorescence.

The present invention accordingly provides a major tool in the identification of candidate HIV-1 protease inhibitors. The invention also allows for the identification and analysis of HIV-1 protease variants. Treatment with protease inhibitors often results in accumulation of multiple mutations in the protease due to ongoing replication of incompletely suppressed virus (16) . This consecutive accumulation of mutations that are often located in regions distant from the active site confers broad resistance to various protease inhibitors. In order to optimise treatment and to ensure a long-term antiviral response, it appears crucial to monitor antiviral resistance and to adjust the therapeutic regimen accordingly.

Phenotypic assays are presently the only way to directly determine resistance and give a rationale for therapeutic adjustments since they measure susceptibility of the actual viral strains to anti-retrovirals (18) . These assays are based on inserting PCR-amplified protease sequences from patient blood into a proviral clone defective in the protease gene, followed by transfection and analysis of the resulting virus population for susceptibility to various drugs. Although effective, these tests can only be performed in specialised laboratories, require several weeks for readout, and are quite expensive. Thus, there is still an urgent need for faster and cheaper assays, which accurately monitor the development of resistance in vivo . Assays according to embodiments of the present invention allow rapid evaluation of the sensitivity of different protease mutants by replacing the original protease open reading frame with the mutated variants, representing a contribution for the development of a reliable, non-infectious system for analysis of viral resistance in AIDS patients treated by HIV-1 protease inhibitors.

Construction of GFP-PR reporters containing drug -resistant PR species

GFP-PR reporters were generated by fusion of GFP and the HIV-1 protease open reading frame and the flanking sequence coding for 12 amino acids upstream p6/PR cleavage site that is used for generation of an enzymatic active protease in virus infected cells.

1. HIV-1 PR variants from clinical samples

Clinical PR variants were obtained by PCR amplification of HIV-PR genes from the blood samples of HIV positive individuals using specific primers derived from the conservative sequences upstream and downstream from the PR coding region:

HIV-lb 5'-T Aga att cat atg AGA GAC AAC AAC TCC CCC T-3' HIV-2 5'-GGg gat ccT TAC TAT GGT ACA GTC TCA ATA GG-3'

The PCR products contained useful EcoRI, BamHI and Ndel restriction sites for easy subcloning of individual PR mutants into the reporter plasmid. The PCR products cleaved by EcoRI and BamHI were first cloned into pUC19 and sequenced to verify the presence of DNA mutations conferring the drug resistance. Subsequently, the drug-resistant PR species were subcloned into the reporter plasmid pEGFP-C3 (Clontech Labs) , cleaved by EcoRI and BamHI .

The following resistant PR variants were used for the validation of the assay:

PRA E35D, N37S, L63P, I72V PRB M46L, L63P, A71V, L90M, I93L PRC M46L, I54V, K55R, D60E, Q61E, I62V, L63P, A71V, I72V,V82A,L90M, I93L

PRA is the PR coding region from an HIV positive patient that has not been treated by PR inhibitor ("naive patient") . PRB and PRC represent PR variants obtained from patients treated for prolonged period of time with Saquinavir, Ritonavir and Indinavir, and exhibiting clear clinical signs of resistance towards these antiviral drugs (increased viraemia, decreased number of CD4+ cells) .

2 . HIV-PR variants representing characteristic resistant mutations

For validation purpose, in order to develop the assay using well-characterized mutants, laboratory-derived resistant mutants of HIV PR known to represent the most common mutations conferring the resistant genotype of HIV (Gulnik et al . , 1995) were cloned into the reported plasmid (see above) . The drug resistant mutants were prepared by site-directed mutagenesis of the laboratory strain of HIV known as pNL4 -3. The cloning into the reporter plasmid was performed as described above .

The following mutants were used for the validation analysis: GFP-PR2 G48V, L90M (saquinavir resistant) GFP-PR5 I82A (ritonavir resistant)

Validation of the reporters Transfection HeLa (human cervical carcinoma cell line) were grown in

Iscove's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics (Life Technologies, Grand Island, New York) . The cells were transfected with a mixture of plasmid DNA and Lipofectamine (Life Technologies) as recommended by the supplier. For protease inhibitor treatment of cultured cells, the inhibitors were initially dissolved in DMSO and diluted to appropriate concentrations in Iscove's modified Eagle's medium supplemented with 10% fetal calf serum. Where indicated, the transfected cells were treated with protease inhibitors immediately after transfection until cells were harvested 24 h later.

Flow cytometry Expression of GFP was detected 24 h after transfection using a FACSort flow cytometer (Beckton & Dickinson, Mountain View, California) and Cellquest software.

Resul ts and data analysis 1. All the protease mutants capable of liberating themselves from the precursor were cytotoxic, showing fluorescence increase after an inhibitor treatment.

2. The increase of mean fluorescence value (Y_M) as determined by FACS analysis in presence of an HIV PR inhibitor was dose- dependent and showed saturation kinetics. An example of an inhibition curve obtained in experiments performed by the inventors is given in Figure 5. The dependence of the fluorescence intensity change Y_M on the inhibitor concentration I in the assay can be fitted to the Hill- Langmuir equation.

Y*T = Ymax^{^} . l/ (I + EC₅₀ . ) (1)

Y_M" = Y_mean ( in the presence of appropriate inhibitor concentration) - Y_mean (in the absence of the inhibitor)

Y_max is the fluorescence when the enzyme is saturated by the inhibitor, I is the inhibitor concentration and EC₅₀ - the 50% of the maximal effective inhibitor concentration, quantifying thus the enzyme - inhibitor interaction.

EC50: • EC₅₀ is independent on experimental conditions. Its value is the quantitative parameter of the reporter - inhibitor interaction.

• The lower EC₅₀, the higher affinity of the inhibitor to the enzyme .

From the FACS analysis, the dependence of the reporter fluorescence Ymean on the inhibitor concentration was obtained.

The "baseline" was subtracted (the baseline is the fluorescence of cells untreated with an inhibitor) and the resulting data were fitted to the equation (1) .

The FACS data are shown in Figure 5 as points and fitted data as a curve. In the results shown in Figure 5, Y_max"=3048+ 129 and EC₅₀=22+4 nM.

Inhibi tion analysis of resistant PR species using FACS analysis of the reporter system

To validate the usefulness of the reporter for the analysis of resistant PR species and their inhibition by various PR inhibitors, three protease variants were analysed as described above. The wild-type PR, the PR variant isolated from a patient (PRC) as well as a mutant harboring characteristic mutations known to confer resistance towards Saquinavir were cloned into the reporter plasmid, transfected into mammalian cells and grown in the presence of various concentrations of Saquinavir as described above. The transfected cells were quantified by FACS and the fluorescence values analysed by Hill-Langumir kinetic plot as described above. The results are summarised in Table 2. The EC₅o values show two order of magnitude increase for the saquinavir resistant mutant PR2 (G48V, L90M) . Therefore, the read-out from the reporter system corresponded well with the experimentally observed resistant phenotype. The patient- derived PR showed only limited resistance towards Saquinavir in this assay (5x increase in EC50 value) .

In a further series of experiments, the same PR clones were analysed in the reporter in the presence of other PR inhibitor, lopinavir, that has been reported to inhibit most of the resistant PR clones known to date. The results are summarised in Table 3.

As seen from the relatively low EC₅₀ , Lopinavir is an example of inhibitor active also against the drug resistant mutants and could be thus recommended for the clinical treatment of HIV positive patient C.

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TABLE 1

Comparison of yield of fluorescent clones

^a Clone showed irreversible loss of fluorescence once the inhibitor was omitted ^b Clone died after a few generations

TABLE 2 Hill -Langmuir parametres for three different mutants treated wi th PR inhibi tor saquinavir.

TABLE 3 Hill -Langumir parameters for inhibi tion curves of three different HIV mutants and PR inhibi tor lopinavir .

Claims

CLAIMS :

1. An assay method for screening for an inhibitor of a cytotoxic protease, the method comprising: providing within cells in culture a fusion protein that comprises (i) an autoproteolytic precursor of the protease and (ii) a reporter protein, which reporter protein provides a quantitative signal indicative of amount of reporter protein present within the cells, contacting with a test substance cells containing the fusion protein, determining levels of signal in cells in the presence or absence of test substance, and/or different dosages of test substance, wherein level of the signal is indicative of amount of inhibition of the protease.

2. A method according to claim 1 comprising identifying an inhibitor of the cytotoxic protease.

3. A method of determining susceptibility of a protease of a pathogen, which protease exists in more than one variant or mutant isoform, to an agent that inhibits at least one isoform of the protease, the method comprising providing within cells in culture a fusion protein that comprises (i) a precursor of a test protease isoform and (ii) a reporter protein, which reporter protein provides a quantitative signal indicative of amount of reporter protein present within the cells,- contacting with the agent cells containing the fusion protein, determining levels of signal in cells in the presence or absence of test substance, and/or different dosages of test substance, wherein level of the signal is indicative of amount of inhibition of the protease isoform.

4. A method according to claim 3 comprising comparing sensitivity of different isoforms of the protease to the agent .

5. A method according to claim 3 comprising comparing sensitivity of an isoform of the protease to a plurality of different agents.

6. A method according to any one of claims 1 to 5 wherein the cells are human cells.

7. A fusion protein comprising (i) an autoproteolytic precursor of a cytotoxic protease and (ii) a reporter protein, which reporter protein provides a detectable signal .

8. A nucleic acid encoding a fusion protein according to claim 7.

9. An expression vector comprising nucleic acid according to claim 8.

10. A process for producing a fusion protein according to claim 7 which comprises cultivating a host cell transformed or transfected with an expression vector comprising nucleic acid encoding the fusion protein under conditions to provide for production of the fusion protein.

11. A method according to claim 10 wherein the host cell is human.

12. A method according to claim 10 further comprising recovering the fusion protein.

13. A method according to claim 10 further comprising

5 contacting with an agent the host cell containing the fusion protein, and determining level of the signal provided by the reporter protein.

14. A method according to claim 13 comprising identifying an 10 inhibitor of the protease.

15. A method according to claim 2 or claim 14 further comprising formulating the inhibitor into a composition comprising at least one additional component.

15.

16. Use of a fusion protein according to claim 7 and/or encoding nucleic acid for the fusion protein, in screening for and/or obtaining a substance which inhibits the activity of a pathogenic protease. 0