Patent Application
20170166950
This invention relates to molecular and cellular biology, biochemistry, molecular genetics, and drug design and discovery. In one aspect, the invention is directed to compositions to screen for compounds such as small molecule drugs that inhibit, act as competitor, or modulate an enzyme such as a protease, e.g., a protease, proteinase or peptidase or the like, e.g., viral proteases, e.g., an HIV-1 protease. In alternative embodiment, the compositions and methods of the invention are adapted for high through-put or multiplexed screening of compounds, e.g., drug molecules that can modulate or inhibit enzymes.
The human immunodeficiency virus (HIV), identified as the causative agent of AIDS in 1981, has resulted in over 33 million deaths since then. Three decades of HIV research have resulted in antivirals targeting the viral proteins necessary for HIV infection, mainly Protease, Reverse Transcriptase, and recently, Integrase and Envelope (Env). In total 32 inhibitors have been approved by the FDA since Saquinavir, the first Protease inhibitor, in 1995. Inhibitors supplied as a cocktail of three or more inhibitors in the form of Highly Active Anti-retroviral Therapy (HAART) have resulted in a drastic reduction in the number of AIDS-related deaths. Despite the significant progress achieved with the development of HAART, AIDS still remains a devastating disease. Emergence of resistant strains together with the terrible side-effects of existing drugs, and lack of success with vaccine development, highlights the need for novel antivirals as well as innovative methods to facilitate their discovery.
The invention provides compositions, assays and cell-based methods for monitoring the activity of an enzyme, e.g., a protease, proteinase or peptidase or the like, e.g., a viral protease or an HIV-1 protease (PR). The invention provides compositions, assays and cell-based methods for detecting or assaying for modulators (e.g., inhibitors or competitors, or activators) of the activity of an enzyme, e.g., a protease, proteinase or peptidase or the like, e.g., a viral protease or an HIV-1 protease (PR). Thus, in one embodiment, invention provides compositions, assays and cell-based methods for drug discovery.
In alternative embodiments, the invention provides chimeric or recombinant scaffold proteins comprising:
wherein: when the scaffold protein is expressed on the cell surface the amino acid motif or subsequence susceptible to cleavage by the enzyme or protease (“the cleavable target motif”) is positioned within the scaffold protein such that at least one of the detectable moieties is distal to the cleavable target motif (with respect to the cell), and at least one of the detectable moieties is between the cleavable target motif and the cell surface when the scaffold protein is expressed extracellularly,
thus, if the enzyme or protease is active in the cell, the detectable moiety or moieties distal to the cleavable target motif will not be present (or part of) the scaffold protein expressed on the cell surface, but when an effective amount of an inhibitor or a competitor to the enzyme or protease is present in the cell, the cleavable target motif is not cleaved and the detectable moiety or moieties distal to the cleavable target motif will be present (or part of) the scaffold protein expressed on the cell surface;
(b) the chimeric or recombinant scaffold protein of (1), wherein the scaffold protein comprises all of, substantially all of, or part of: a murine CD8a homolog or a Lyt2 transmembrane domain glycoprotein receptor polypeptide as the scaffold;
(c) the chimeric or recombinant scaffold protein of (a) or (b), wherein the enzyme or protease is a viral enzyme or protease, or an HIV enzyme or protease, or a furin, or a proteinase or peptidase; or
(d) the chimeric or recombinant scaffold protein of any of (a) to (c), wherein the amino acid motif or subsequence susceptible to cleavage (“cleavable target motif”) is from or is derived from a virus of the family Flaviviridae, or a Dengue virus, a Hepatitis C Virus, a West Nile virus, a Yellow fever virus, a Japanese encephalitis virus, a Tick-borne encephalitis virus, a Kyasanur Forest disease virus, a Murray Valley encephalitis virus, a St. Louis encephalitis virus, a bovine viral diarrhoea virus, a Rio Bravo virus, a Culex flavivirus or pegivirus, an influenza virus, a papilloma virus, a Sindbis virus and/or an Ebola virus; or, an amino acid motif or subsequence susceptible to cleavage as illustrated in
In alternative embodiments, the invention provides isolated, recombinant or synthetic nucleic acids encoding a scaffold protein of the invention,
wherein optionally the nucleic acid is operatively linked to a transcriptional regulatory unit, and optionally the transcriptional regulatory unit comprises a promoter, and optionally the promoter is a constitutive or an inducible promoter,
and optionally the nucleic acid further comprises a sequence encoding the enzyme or protease, and optionally the enzyme or protease-coding sequence is operatively linked to the same or a similar transcriptional regulatory unit as the nucleic acid encoding the scaffold protein of the invention.
In alternative embodiments, the invention provides a vector, expression cassette, cosmid or plasmid comprising the isolated, recombinant or synthetic nucleic acid of the invention.
In alternative embodiments, the invention provides a cell comprising:
(a) the chimeric or recombinant scaffold protein of the invention; the isolated, recombinant or synthetic nucleic acid of the invention; and/or, the vector, expression cassette, cosmid or plasmid of the invention;
(b) the cell of (a), wherein the enzyme or protease is heterologous to the cell and the cell further comprises a heterologous nucleic acid encoding the heterologous enzyme or protease,
and optionally the enzyme or protease-coding sequence is operatively linked to the same or a similar transcriptional regulatory unit as the nucleic acid encoding the scaffold protein of the invention,
and optionally the enzyme or protease-coding sequence is contained in or is part of the same or a different vector, expression cassette, cosmid or plasmid of the invention;
(c) the cell of (a) or (b), wherein the cell constitutively or inducibly expresses the chimeric or recombinant scaffold protein of the invention; or
(d) the cell any of (a) to (c), wherein the cell is a mammalian cell, a monkey cell, or a human cell, or a lymphocyte or a T-cell.
In alternative embodiments, the invention provides a cell line, or a stable cell line, derived from the cell of the invention.
In alternative embodiments, the invention provides a non-human transgenic animal comprising: the chimeric or recombinant scaffold protein of the invention; the isolated, recombinant or synthetic nucleic acid of the invention; a cell line or a stable cell line of the invention; and/or, the vector, expression cassette, cosmid or plasmid of the invention.
In alternative embodiments, the invention provides a cell-based method for monitoring the activity of an enzyme, a protease, a viral protease, or an HIV-1 protease (PR), comprising:
(a) (i) providing a cell of the invention, or a cell line of the invention, or non-human transgenic animal of the invention, wherein the cell or cell line expresses the chimeric or recombinant scaffold protein of the invention,
wherein optionally the enzyme or protease is endogenous to the cell or is heterologous to the cell; and
(b) determining whether the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein is expressed on the scaffold protein on the extracellular surface of the cell, or whether or not the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein are or are not be present (or part of) the scaffold protein expressed on the cell surface,
wherein lack of detection of the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein indicates that the enzyme or protease is active in the cell, and detection of the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein indicates the presence of an inhibitor or a competitor of the enzyme or protease,
wherein extracellular detection of the detectable moiety or moieties positioned on the scaffold protein between the cleavable target motif and the cell surface when the scaffold protein is expressed on the cell surface indicates or confirms that the scaffold protein is expressed on the cell surface.
In alternative embodiments, methods of the invention further comprise screening for a putative inhibitor or competitor of an enzyme, a protease, a viral protease, an HIV protease, or an HIV-1 protease, by:
(a) providing a compound to be screened as an inhibitor or a competitor of an enzyme, a protease, a viral protease, an HIV protease, or an HIV-1 protease; or a nucleic acid encoding a protein to be screened as an inhibitor or a competitor of an enzyme, a protease, a viral protease, an HIV protease, or an HIV-1 protease;
(b) contacting a plurality of the cells with the compound or nucleic acid of (a),
wherein optionally the contacting is either before, during and/or after expression of the scaffold protein-expressing nucleic acid in the cell,
and optionally the nucleic acid encoding a protein to be screened as an inhibitor or a competitor of an enzyme, a protease, a viral protease, an HIV protease, or an HIV-1 protease is expressed before, during and/or after expression of the scaffold protein-expressing nucleic acid in the cell; and
(c) determining whether the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein is expressed on the scaffold protein on the extracellular surface of the cell, or whether or not the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein are or are not be present (or part of) the scaffold protein expressed on the cell surface,
wherein lack of detection of the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein indicates that the enzyme or protease is active in the cell, and detection of the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein indicates that the compound is acting as an inhibitor or a competitor of the enzyme or protease (assuming that the same cells cultured under or exposed to the same conditions but not exposed to or contacted with or expressing the putative inhibitor or competitor do not express the detectable moiety or moieties distal to the cleavable target motif on the scaffold protein),
wherein extracellular detection of the detectable moiety or moieties positioned on the scaffold protein between the cleavable target motif and the cell surface when the scaffold protein is expressed on the cell surface indicates or confirms that the scaffold protein is expressed on the cell surface.
In alternative embodiments, methods of the invention further comprise running a negative control comprising dividing the plurality of the cells and not adding the compound to be screened as an inhibitor or competitor to one of the divided cell samples, or not expressing the putative inhibitor or competitor in one of the divided cell samples.
In alternative embodiments, methods of the invention further comprise running a positive control comprising dividing the plurality of the cells and adding a compound known to be an inhibitor or competitor of the enzyme or protease to one of the divided cell samples, or expressing a known inhibitor or competitor of the enzyme or protease in one of the divided cell samples.
In alternative embodiments, detectable moiety is detected or measured on the extracellular surface of the cell by a high throughput screen (HTS), a flow cytometry or a microscope visualization.
In alternative embodiments, the compound to be screened as an inhibitor of the enzyme, protease, viral protease or HIV-1 protease comprises a small molecule, a nucleic acid, a polypeptide or peptide, a peptidomimetic, a polysaccharide or a lipid.
In alternative embodiments, the compound to be screened as an inhibitor of the enzyme, protease, viral protease or HIV-1 protease is a member of a library of compounds to be screened, or a member of a random peptide library or a chemical compound.
In alternative embodiments, the compound to be screened comprises a plurality of compound comprising or from a combination of: retroviral random peptide libraries; combinatorial compound libraries; and/or, endogenously expressed random peptide libraries specifically targeted to the compartment where Env processing occurs.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.
As example,
Like reference symbols in the various drawings indicate like elements.
The invention provides methods and compositions, including chimeric recombinant proteins, nucleic acids that encode them, and cells and kits comprising them, to screen for compositions, e.g., small molecule drugs, that can modulate, e.g., inhibit, compete or modulate, e.g., enhance, enzymes, e.g., proteases, proteinases or peptidases or the like, e.g., viral proteases, including retroviral (e.g., HIV) proteases. In alternative embodiments, the invention provides cells and cell-based assays for monitoring the activity of enzymes, e.g., proteases, proteinases or peptidases or the like, e.g., viral proteases, e.g., HIV-1 protease (PR), which is an aspartyl protease. In one embodiment, these cells and cell-based assays are used to screen for and identify novel viral protease, e.g., PR, inhibitors or modulators, such as competitors. In one embodiment, assays of the invention effectively couple the surface (extracellular) expression of a protein used as a scaffold (a scaffold protein), and expression of detectable markers, or “tags” on the scaffold, with the activity of the enzymes, e.g., proteases, proteinases or peptidases or the like, e.g., viral protease, e.g., PR. In alternative embodiments, the compositions (e.g., scaffolds), the methods (e.g., assays for drug discovery) and cells of the invention comprise use of at least two markers on the extracellularly expressed scaffold, i.e., the scaffold is “double-tagged”, wherein an cleavable target, e.g., a protease target, is placed on the scaffold between at least two of the tags, and when an effective amount of an inhibitor or a competitor of the enzyme (e.g., a protease, such as a furin) is present in the cell, the enzyme will not cleave the target, and the tag positioned distal (from the cell) on the scaffold (e.g., a FLAG tag) is not released, and only at least a second tag (positioned between the cleavable target and the cell surface) remains on the cell surface. Alternatively, if the enzyme (e.g., protease) is not inhibited, the target is cleaved and the distal tag will not remain on the scaffold or be associated with the cell's surface. Thus, in these embodiments, drug discovery assays screen for tag-positive FLAG positive cells, e.g., by FACS or equivalents, to screen for putative drugs, i.e., the inhibitors or competitors of the enzymes or proteases.
In alternative embodiments, compositions and assays of the invention, scaffolds of the invention comprise use of at least recognizable or detectable two tags expressed as a part of the scaffold that utilizes the classical secretory pathway for transport into the cell surface. In alternative embodiments, compositions and assays of the invention can use any recognizable or detectable marker, or “tag”. One exemplary composition and assay of the invention, as described in the Examples, is based on two independent TAGs; HA and FLAG. However, compositions and assays of the invention are adaptable to any recognizable or detectable marker, e.g., tag or fluorescent protein, of choice. The tags, separated by a putative cleavable site within the ER/Golgi/TGN, can be any tag of choice, provided they are recognizable or detectable. In alternative embodiments, they can be any antigen or TAGs, e.g., when antibodies that recognize them are available, or they can be a fluorescent compound or proteins.
In alternative embodiments, compositions and assays of the invention can detect a modulator, e.g., inhibitor or activator, of any enzyme, e.g., proteases, proteinases or peptidases or the like. One exemplary composition and assay of the invention, as described in the Examples, is based on a scaffold with two tags, separated by a putative cleavable site within the ER/Golgi/TGN compartment. While the data described in the Example shows a cleavable site specifically cleaved by Furin and other protein convertases (PCs), alternative embodiments of compositions and/or assays of the invention can be designed as a platform for any putative or known cleavable site in the ER/Golgi/TGN compartment. In alternative embodiments, the assay can monitor cleavage by any enzyme, e.g., a protease, proteinase or peptidase or the like, provided the enzyme is expressed in (and can be recombinantly expressed or naturally expressed, or functions and/or resides in the ER/Golgi/TGN compartment.
In alternative embodiments, compositions and assays of the invention can be used to assay for or detect any modulator for any enzyme cleavable target or substrate. The exemplary assay as described in the Examples relies on the cleavage of the HIV envelope gp120/gp41 boundary, chosen as proof of principle. This exemplary target is a well-recognized target of Furin/PCs cleaved in the ER/Golgi/TGN compartment. However, in alternative embodiments, compositions and assays of the invention are adaptable to any target of choice; whether viral or of cellular organisms, provided it can be cleaved in the ER/Golgi/TGN compartment. In alternative embodiments, compositions and assays of the invention provide a platform for the monitoring of cleavage of proteins processed, cleaved and/or matured during the travelling or residency within the ER/Golgi/TGN compartment.
In alternative embodiments, compositions and assays of the invention can be used to study of the cleavage and or transport process. In alternative embodiments, compositions and assays of the invention can be used to monitor the cleavage/maturation process as well as the process of transport to the cell surface. In alternative embodiments, compositions and assays of the invention can be used to study the proteins and/or factors that influence or are required for the processes of protein cleavage, processing and/or maturation in the ER/Golgi/TGN compartment. As the compositions and assays of the invention comprise use of a scaffold protein that travels to the cell surface, they can also be used to learn about the factors and/or proteins required for transport to the cell surface.
In alternative embodiments, compositions and assays of the invention can be used to for drug discovery. In alternative embodiments, compositions and assays of the invention can be used to screen for inhibitors and/or competitors against the process of cleavage of the substrate inserted between the two detectable markers, e.g., flags, and/or against the activity of the enzymes responsible for their cleavage, provided the enzyme is expressed in (and the enzyme can be recombinantly expressed or homologous to the cell of the assay), or reside and/or function in the ER/Golgi/TGN compartment.
In one embodiment, the scaffold is engineered for its conditional expression on the surface of a cell, e.g., a eukaryotic, a yeast or a mammalian cell. For that purpose, in one embodiment, the scaffold is fused to a signal sequence to enable efficient and/or directed transport, and a transmembrane domain (e.g., an Lyt2, the murine CD8 molecule, and the like) is used to enable subsequent insertion in the cell membrane. In one embodiment, at least two detectable markers, e.g., “tag”, including a FLAG tag, is added to the scaffold downstream of the signal sequence for detection, e.g., for antibody detection, e.g., through flow cytometry or equivalent visualization.
In one embodiment, the assay co-expresses both the scaffold protein and the enzyme or protease, e.g., viral protease, e.g., the HIV-1 PR, which if active will bind to and cleave the scaffold at the protease recognition sequence.
In one embodiment, both scaffold and protease are co-expressed in T cells, e.g., SupT1 T-cells, in an inducible off/on-based vector system (e.g., activated upon addition of tetracycline or doxycycline). Inducible expression of protease, e.g., PR, helps avoid its possible cytopathic effects.
In one embodiment, the logic behind the engineering of the scaffold as a membrane-expressed protein is as follows: in the presence of the active viral protease, the proteolytic enzyme will cleave the scaffold, resulting in the loss of transmembrane domain, thus preventing tag cell surface expression. In the absence of protease, or when protease is blocked or inhibited, the scaffold will be intact and incorporated into the membrane. As a result, the surface expression of the scaffold can be determined by flow cytometry allowing the discrimination between active and inactive or blocked protease.
The assay is cell-based, and can be easily implemented for a high throughput screen, e.g., FACS. As such, the assay is invaluable for drug discovery, and can be utilized in biological screens aimed at finding novel enzyme, e.g., protease, inhibitors or modulators through random peptide libraries or chemical compounds libraries.
In one embodiment, the invention provides assays that can be adapted for a high throughput manner using e.g. flow cytometry such as FACS, and can discriminate between active and non-active or blocked protease. In one embodiment, the invention provides assays that can be easily adapted for high throughput screening. In one embodiment, the invention provides assays of this invention can be used to screen for novel enzyme or protease inhibitors or modulators.
In one embodiment, the invention provides assays of this invention adapted for the screen of random peptide libraries or chemical compounds for drug discovery.
In one embodiment, the methods of the invention use a random peptide library or any peptide of choice, which can be introduced ‘in cis’, replacing the p2/p7 recognition/cleavage site, enabling the discovery of higher affinity sites for enzyme or protease, e.g., PR, which can be the basis for the development of competitor peptidomimetic drugs. In one embodiment, the random peptide library is expressed ‘in trans’, enabling the discovery of competitors/inhibitors or modulators for enzyme or protease, e.g., PR, which can be the basis for peptidomimetic drugs.
In one embodiment, the non-biased approach of the invention permits the rescue of peptides or chemicals targeted not necessarily to the catalytic site of enzyme or protease, e.g., PR. Thus, the assays of the invention provide for extensive characterization of enzyme or protease or PR, facilitating the elucidation of interactions of enzyme or protease or PR with cellular targets, its mode of action and modulation, in the context of the host cell. Assays of this invention will permit the replacement of PR with PR from different viral strains or clades, or truncated versions of PR, enabling further dissection of PR activity, and study its modulation through co-expression of cellular factors or addition of drugs.
The assays of this invention can be further adapted to proteases of different viruses such as Hepatitis C by just exchanging the recognition/cleavage site segment of the scaffold. The assays of this invention can thus be exploited for the search for protease inhibitors or modulators against any of the known viral pathogens that utilize their own protease/s as part of their lifecycles.
The assays of this invention can be adapted for the search of HIV envelope processing inhibitors or modulators. One of the HIV proteins, envelope, is processed by furin and other cellular convertases. By just exchanging the recognition/cleavage segment of the scaffold with the envelope recognition site, the assay can be further utilized for the finding of envelope processing inhibitors or modulators. This same scaffold is useful for the search of transport inhibitors or modulators, as envelope is transported through the ER, trans-Golgi network in order to be inserted within the cell membrane.
In alternative embodiments, the assays of this invention comprise expression of a scaffold designed for expression in the cytoplasm that is able to be exported into the cell membrane.
In alternative embodiments, assays of this invention comprise expression of both enzyme, e.g., viral enzyme, such as a PR, and a scaffold of the invention, in an off/on system for inducible expression, or alternatively, for constitutive expression.
In alternative embodiments, assays of this invention comprise expression of a protein that is expressed on the surface of the mammalian cell only when not cleaved by a protease, e.g., an HIV protease.
In alternative embodiments, assays of this invention can be adapted for the screen of random peptide libraries or chemical compounds.
In alternative embodiments, assays of this invention can be implemented in mammalian cells and other cells, e.g., yeast or bacterial cells.
In alternative embodiments, methods provide for the construction of the scaffold and its expression on the cell surface. In alternative embodiments, the p2/p7 scaffold has been engineered as described and effectively expressed on the cell surface. In alternative embodiments, the scaffold has been introduced in a retroviral vector.
We utilized a novel cell-based assay that exploits the classical secretory pathway for the elucidation of the processes involved in HIV-1 envelope (Env) maturation. HIV-1 relies on proteases for the processing of its proteome. While the viral protease cleaves most of the recognition sites within the viral proteome, the site within Env is cleaved by the host enzymes Furin and similar protein convertases (PCs) within the Trans-Golgi Network (TGN). Processing at the gp120/gp41 boundary within the gp160 Env poly-protein is necessary for the production of infectious viral particles. In one embodiment, the recognition of gp160 by Furin is a target for drug development against HIV-1.
We describe an assay based on the engineering of a scaffold protein that will place the gp120/gp41 boundary within the lumen of the Trans-Golgi Network (TGN) where it can be recognized and then processed. The well-established topology of the murine CD8a homolog, Lyt2, transmembrane domain glycoprotein receptor, prompted us to choose Lyt2 as the basic scaffold for the assay. In one embodiment, a Lyt2 FLAG-tagged molecule is fused to the prolactin signal sequence to ensure both antibody-based recognition and proper insertion into the Endoplasmic Reticulum (ER) for transport to the cell surface. Additionally, the gp120/gp41 boundary is introduced between the FLAG tag and an HA tag fused to the Lyt2 transmembrane domain, ensuring that the Env segment faces the lumen of the ER/TGN. In this manner, if Furin/PCs recognize and cleave the gp120/gp41 boundary, the FLAG tag will be released. However, if blocked or inhibited, the FLAG tag will remain attached to the HA-tagged scaffold. The double-tagged engineered scaffold will thus allow for the discrimination between cleaved and non-cleaved events based on the cell surface expression of one or two tags, respectively. Results show a drastic reduction of FLAG surface expression with a scaffold containing the wild-type gp120/41 boundary in comparison to its mutant counterpart, proving the utility of FLAG cell-surface expression as a biosensor, e.g., for the activity of Furin.
This exemplary assay, developed in T-cells to provide the natural milieu of HIV-1 infection, can elucidate the still unclear mechanisms of gp160 maturation, which is a target for the inhibition of HIV infection. This will be the first assay of its kind to be developed in a relevant cellular context, facilitating the discovery of drugs specifically inhibiting the recognition/cleavage of Env rather than the activity of cellular enzymes and will thus be aimed at discovering competitors rather than inhibitors of Furin.
In alternative embodiments, exemplary assays of the invention are adapted to host substrates that utilize the classical secretory pathway. These include targets of the three domains of life, Bacteria, Archaea and Eukarya. These include targets but are not restricted to, enzymes that cleave in the way to the cell surface, at the cell surface, or at the extracellular matrix. These include, but are not restricted to some of the possible substrates, known to be cleaved by Furin and similar enzymes, shown in
In alternative embodiments, exemplary assays of the invention are used to monitor other enzymes involved in the classical secretory pathway. In alternative embodiments, exemplary assays of the invention are easily adaptable to monitor enzymes and/or their required factors that reside within the secretory pathway, within the membranes of the secretory pathway, cell surface, or the extracellular matrix. These enzymes/factors include, but are not restricted to protein convertases, peptide peptidases, peptide peptide-peptidases, alpha secretases, beta secretases and gamma secretases. For instance, the amyloid precursor protein (APP) involved in Alzheimer's is cleaved by alpha, beta and gamma secretases.
In alternative embodiments, exemplary assays of the invention are adapted to any detection technique. The assay can be analyzed with any detection technique available, whether it is flow cytometry, microscopy, imaging-based coupled flow cytometry, or any other.
In alternative embodiments, exemplary assays of the invention are used as a platform for drug screening. The utility of the assay, as cell-based, can be adaptable to any screen, including chemical compound libraries, combinatorial libraries, peptide libraries or retrovirally-expressed peptide libraries.
In alternative embodiments, exemplary assays of the invention are used as a platform for target discovery. Targets may include, but not be restricted to: a) enzymes or factors involved in the recognition of the substrate under study (inserted between the two tags), b) enzymes or factors involved in the cleavage of the substrate under study (inserted between the two tags), c) factors involved in the Endoplasmic Reticulum/Golgi/TransGolgi Network, d) factors involved in insertion or targeting to the Endoplasmic Reticulum, e) factors involved in the transport to the cell surface, f) cofactors required for any of the processes mentioned above or any of their combinations.
In alternative embodiments, exemplary assays of the invention are used as a platform for target discovery. Targets may include, but not be restricted to: a) enzymes or factors involved in the recognition of the substrate under study (inserted between the two tags), b) enzymes or factors involved in the cleavage of the substrate under study (inserted between the two tags), c) factors involved in the Endoplasmic Reticulum/Golgi/TransGolgi Network, d) factors involved in insertion or targeting to the Endoplasmic Reticulum, e) factors involved in the transport to the cell surface, f) cofactors required for any of the processes mentioned above or any of their combinations.
In alternative embodiments, exemplary assays of the invention are used as a platform for target discovery utilizing any molecular biology tool for their discovery. The assay can be easily coupled for target discovery with complementary DNA (cDNA) libraries, knockdown based technologies such as siRNA, shRNA, knock-in, knock out, overexpression of genes/proteins of interest, and others.
In alternative embodiments, exemplary assays of the invention are coupled to the event of cleavage itself. This exemplary assay, in contrast to others, pinpoints at the specific cells where the event of cleavage occurs. This is possible as the cleaved scaffold is not lost and it is detectable by one tag (HA as example). The assay is retrovirally engineered so one can back track and rescue any cell of interest within a population of cells.
The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.
The invention provides compositions and assays for screening for inhibitors or modulators of enzymes or proteases, e.g., viral proteases such as HIV-1 protease (PR) (an aspartyl protease). PR is required for the efficient processing of the Gag and Gag-Pol precursor polyproteins; a critical step in the viral life cycle. In alternative embodiments, the invention provides compositions and assays for: (1) Discerning the effects of protease, e.g., PR, on signaling cascades of the host cell, and (2) Developing novel cell-based assays to enable screening of peptide libraries for the search of novel protease, e.g., PR, inhibitors or modulators. In alternative embodiments, a protease, e.g., PR, is expressed as a fusion protein in the presence of limiting levels of inhibitors or modulators, in different cellular compartments and in an inducible manner.
This invention provides a novel cell-based assay to provide insight into the mechanisms of HIV-1 envelope maturation. Envelope maturation, which occurs during classical transport through the Endoplasmic Reticulum-TransGolgi Network, is absolutely necessary for the production of infectious viral particles. Assays and compounds of this invention will facilitate the search for inhibitors/competitors of Envelope maturation, and as such will be the first of its kind, with huge impact in the fight against HIV-1.
In alternative embodiments, the invention provides cell-based assays that facilitate high throughput screening (HTS), in order to identify novel inhibitory compounds targeted against the production of infectious HIV-1 particles.
In alternative embodiments, the invention provides assays in an appropriate host cell context to screen for compounds that inhibit the recognition and/or cleavage of the gp120/gp41 boundary within the viral envelope. In alternative embodiments, fluorescence is utilized as the assay read-out, demonstrating its suitability for flow cytometry, and thus search for inhibitory compounds in a high throughput manner.
In alternative embodiments, the invention provides a rapid screening method for novel inhibitors of HIV-1 envelope processing, which is a crucial step in the production of infectious viral particles. In alternative embodiments, the invention provides an assay developed in the natural context, i.e. T-cells, and adaptable to flow cytometry, to enhance the utility and optimization of the assay for HTS, in turn drastically enhancing the chances of discovery of efficient compounds targeting HIV-1 envelope processing.
In alternative embodiments, the invention provides assays adapted to HTS platforms and in appropriate cellular contexts to facilitate identification of active and specific inhibitors of HIV-1 envelope processing. In alternative embodiments, the invention provides an assay that monitors HIV-1 envelope processing—recognition and/or cleavage of the gp120/gp41 boundary.
In alternative embodiments, the invention provides assays that specifically monitor the recognition and/or cleavage of the gp120/gp41 boundary within HIV-1 envelope. As such, these exemplary assays will greatly facilitate the discovery of a novel set of antivirals targeting envelope processing, with huge impact in the fight against HIV/AIDS.
We constructed a scaffold that can be used as the vector backbone for exemplary assays of the invention. In alternative embodiments, plasmids needed for the assays include an Lyt2/Env scaffold and relevant controls (with/out gp120/gp41 boundary and/or mutated boundary site). Their expected behavior can be corroborated in transient experiments at first and analyzed by flow cytometry.
In alternative embodiments, clonal cell lines are adapted for use in assays of the invention for HTS. Clones expressing the Lyt2/Env scaffold and relevant controls are selected and amplified. The utility of these clones can be ensured in e.g., 384- and 1536-well plate formats, and all parameters needed for HTS can be calibrated.
In alternative embodiments, assays of the invention screening with one or more libraries of chemical compounds, e.g., small molecules, e.g., using the NIH Molecular Libraries and Imaging Roadmap Initiative, or a chemical-compound library provided by the Molecular Libraries Production Centers Network to screen the selected clones, in order to e.g., identify potential novel inhibitory compounds targeted against the recognition and/or cleavage of the gp120/gp41 boundary.
In alternative embodiments, the invention provides cell-based assays in relevant host cell contexts. In alternative embodiments, assays of the invention monitor the activity of the viral protease in T-cells; the T-cell context provides the natural milieu necessary for HIV infection. In alternative embodiments, exemplary assays are engineered in T-cells. As gp160 processing occurs in infected cells, cell-based assays of this invention performed in an appropriate cell context will greatly enhance the study of HIV Env processing and maturation, a complex process still remaining to be fully elucidated. Moreover, as Env processing is critical for the production of infectious viral particles, Env processing and particularly Env recognition by Furin/PCs is an attractive target for antivirals. Exemplary assays can greatly enhance the discovery of a new kind of drugs specifically blocking the recognition and/or cleavage of the gp120/gp41 boundary, and assays of this invention can be a tool for their discovery. In alternative embodiments, a combination of combinatorial compound libraries and endogenously expressed random peptide libraries specifically targeted to the compartment where Env processing occurs are used, drastically increasing the chances of discovering novel drug candidates.
In alternative embodiments, cell-based assays are designed to specifically target Env processing, and can be suitable for HTS. Exemplary cell-based assays can monitor the cleavage of the gp120/gp41 boundary, greatly facilitating the search for HIV-1 Env processing inhibitors, which represents a completely novel kind of antivirals. Importantly, exemplary cell-based assays can facilitate the discovery of antivirals that inhibit the recognition/cleavage of Env rather than the activity of cellular enzymes, which would be probably detrimental to the cell. In alternative embodiments, cell-based assays are aimed at competitors for Env recognition rather than Furin inhibitors. In alternative embodiments, cell-based assays rely on the expression of a surface tag, and can be readily adaptable to a flow cytometry, enhancing its high throughput capabilities for drug discovery. In alternative embodiments, retroviral random peptide libraries are used for the screen of Env competitors for Furin/PCs recognition, which can be engineered to specifically localize to the ER/TGN luminal compartment.
In alternative embodiments, cell-based assays of the invention comprise use of a double-tagged engineered scaffold (of the invention) that allows discrimination between cleaved and non-cleaved events based on the cell surface expression of one or two tags, respectively. In alternative embodiments the scaffold protein travels to the cell surface and retains the FLAG tag only when processing of the gp120/gp41 boundary within the gp160 Env precursor is inhibited, e.g., by a putative drug. In alternative embodiments, flow cytometry-based detection of the cell surface-expressed FLAG-tag serves as a biosensor for the gp160 boundary recognition and cleavage by Furin and related enzymes, i.e., cell surface FLAG expression directly correlates with blocked/inhibited gp120/gp41 boundary processing. A representation of an exemplary assay is depicted in
Env processing is critical for the production of infectious viral particles, rendering this process an attractive target for antivirals. In alternative embodiments, different gp120/gp41 Env boundaries with increasing sizes including different domains of gp41 are used and can be easily analyzed in the context of an assay of the invention. The robustness of the assay, together with its simplicity and the fact it is performed in the natural cellular milieu, all make the assays of the invention a perfect tool for elucidating the requirements for Env processing and trafficking, shedding light into one of the most complex processes in the HIV-1 life cycle. In alternative embodiments assays are engineered as an inducible retroviral system, ensuring stability of clones for long-term usage. These embodiments can be used for screening of a novel kind of drugs targeting the specific action of enzymes or proteases, e.g., Furin and similar PCs on Env processing.
In alternative embodiments, a combination of combinatorial compound libraries and random peptide libraries is expressed inside the cell and targeted to the ER-TGN compartment to drastically facilitate the discovery of drugs. In alternative embodiments the retrovirally delivered peptide libraries are expressed endogenously and are specifically engineered to be localized/targeted to the ER lumen. As each random peptide adopts a specific structure in space, it is expected that some peptides within the libraries (with expected complexities of several millions) to bear the right conformation needed to block the interaction between Env and Furin/PCs and/or cleavage. In alternative embodiments if/when a drug is found, peptidomimetics and biochemical studies are performed to further convert a putative peptide into a deliverable drug. In alternative embodiments assays of the invention are used as a platform for the study of processing of any viral envelope or cellular protein provided recognition and cleavage occurs in the ER-TGN.
Design of Lentiviral Constructs for the Expression of the Minimal Gp120/Gp41 Boundary Scaffold in T-Cells:
The murine CD8a homolog Lyt2 glycoprotein receptor was chosen as a scaffold for two reasons: First, its well-established topology and transport from the ER to the Golgi and trans-Golgi network (TGN) [53] for subsequent insertion into the outer cell membrane, and second, our extensive expertise with Lyt2-based engineering. We have exploited a construct we have previously developed where the green fluorescent protein citrine was fused to the Lyt2 TM at its C′ terminus and to the prolactin signal sequence fused to the FLAG tag at its N′ terminus, as illustrated in
The prolactin signal sequence ensures proper insertion into the Endoplasmic Reticulum (ER) for transport to the cell surface while the FLAG tag ensures antibody-based recognition. For this exemplary assay, the citrine sequence of the basic scaffold construct (
Transient Experiments Corroborate the Expected Results of the Engineered Assay:
While the assay is intended for stable expression in mammalian cells, we first analyzed the behavior of the engineered constructs in transient expression experiments. 293T cells transfected with pBMN-gp160 min-wt and pBMN-gp160 min-mut, were analyzed by flow cytometry following staining with APC-coupled HA antibodies, FITC-coupled FLAG antibodies or both. (In the basic citrine scaffold construct APC-coupled FLAG antibodies were used instead). The experiment demonstrates beyond any doubt that the mutated gp120/gp41 boundary is recognized by both HA and FLAG antibodies (
Selected Clones Corroborate Robustness of the Engineered Assay:
The transient expression experiments clearly demonstrated that the assay behaved as expected and reassured us to proceed with the stable expression experiments aimed at obtaining stable clones. Clonal populations should drastically increase robustness as 100% of the cells are expected to express the assay scaffold. For that purpose we utilized our extensive expertise with retroviral technology [54-56] in order to transfer the assay element into SupT1 T-cells for its stable expression. Selected clones sorted into a 96-well plate, were amplified for a period of one month and analyzed by flow cytometry and fluorescence microscopy. Flow cytometry analysis shows a high level of scaffold expression in both wild type (wt) and mutant gp120/gp41 boundary-expressing clones, as seen by approximately 100% staining in the HA-APC axis (
Inhibited Wt Construct Further Corroborates the Robustness and Utility of the Assay:
In an attempt to further demonstrate the robustness of the assay and its utility for future drug discovery it was important to prove whether an inhibitor can reverse the observed trend. For that purpose, clones expressing the wt gp120/41 boundary were analyzed by flow cytometry following incubation with increasing concentration of the Furin inhibitor DCK. FLAG surface expression is progressively recovered at 10 mM and 50 mM DCK, increasing form 0.9% to 39% and then 90% (
Tet-on System for Inducible Expression:
In alternative embodiments, constructs are expressed in an inducible manner to be able to turn on the expression of the scaffold proteins only when desired. As preliminary studies we have tested the tetracycline inducible system (Tet-On), adapted from Clontech. The system relies on the reverse tetracycline transactivator (rtTA), allowing induction of expression only upon addition of Tet or doxycycline (Dox). rtTA binds to the Tetracycline Responsive Element (TRE), which we have introduced in an HIV-based self-inactivating vector, with most of the 3′ HIV Long Terminal Repeat (LTR) U3 sequence deleted for safety reasons (
Assay Overview:
In order to establish a reliable and reproducible cell-based assay to monitor the recognition/cleavage of the gp120/gp41 Env boundary, facilitate the study of Env maturation and maximize throughput capabilities, the following points are addressed: Stable expression of the assay elements using retroviral vectors and selection of clones; Adaptation to inducible expression to avoid the possible toxic side effects of an ER-TGN-targeted protein; Adaptation to other strains of HIV; Establishment of the assay in T-cells, a cell-type that mimics the natural environment of HIV infection; Adaptation of the assay to 96 and 384-well formats facilitating HTS. Studies demonstrate beyond any doubt the robustness of the assay for the assessment of cleavage by Furin/PCs, at least of the minimal gp120/gp41 HIV-1 Env boundary.
Adaptation to an Inducible System:
As continuous expression of the scaffold may overwhelm the ER transport machinery and have cytotoxic effects, they can be expressed in an inducible manner via the Tet-On system, e.g., as adapted from Clontech. This will allow to turn expression on only when needed, upon addition of Dox. The fusion protein of the original vector [51] can be replaced with a Lyt2/Env scaffold and transferred into an rtTA-expressing SupT1 cell line. Since these are retroviral vectors, they enable the stable expression of the desired proteins, which optimizes clone selection.
Corroboration of Reproducibility and Stability of the Assay Over Time:
In order to ensure the reproducibility of the assay over time and the stability and utility of the clonal cell populations for long term usage, chosen clones can be analyzed over a period of time, throughout which they will be treated with Dox, stained and analyzed by flow cytometry at two-week intervals. As the constructs used are retroviral/lentiviral in nature, routinely used for the expression of ectopic information for months to years, this assures that the clones will be stable and functional for long periods of time.
Implementation of the Assay in 96 and 384-Well Format:
In alternative embodiments, the assay is intended for drug discovery, so it can be important to calibrate the system in large-number-of-well plate format. In alternative embodiment, the assay uses cell populations in 96-well to a 384-well or 1536-well plate format. The sample volume acquired by flow in an HTS set-up represents 10-20% of the total working volume of the well; 4-to-8 μl for a 384-well plate. Thus around 4,000-to-8,000 cells can be plated per well in the latter, enabling a concentration of about 100-to-200 cells/μl, sufficient for staining and flow cytometry analysis. Exact conditions for proper staining and washing in 96 and 384-well plates can be defined. In order to ensure reproducibility across the plate, experiments can be done in triplicates; three 384-well plates can be loaded with 5,000-10,000 cells/well and treated with 1 μg/mL Dox, with/out peptidyl inhibitor (when needed). Plates can then be stained with fluorophore-coupled anti-FLAG antibody and analyzed by flow cytometry to corroborate that fluorescence is similar across the entire plate and across different plates.
Analysis of Env Elements Required for Furin Cleavage:
In alternative embodiments, the largest portion of gp160 that still allows recognition and cleavage by Furin/PCs in the context of the assay is used, with the goal of mimicking wild-type conformation. The HXB2 T-tropic wild-type HIV-1 sequence can be used as consensus to engineer our constructs. In alternative embodiments, a minimal gp120/gp41 boundary is used, or an increasing number of motifs known to be important for Furin recognition and tertiary structure can be used. In alternative embodiments the goal is to mimic, as much as possible, the natural requirements for cleavage. In alternative embodiments, for each construct, a version with the mutated cleavage site is included. Their expected behavior can be corroborated in transient experiments; and also can be transferred to SupT1 through viral transduction for the selection of clones.
Construction of Env-Based Proteins:
In alternative embodiments, several versions of the gp120/gp41 boundary are used in order to calibrate and ensure the utility and robustness of the assay. All the outlined constructs can be amplified by PCR from the same template and mutated versions will be constructed by site-directed mutagenesis. For each of the constructs both wild type (wt) and mutated sites (indicated as “m”) can be included (
Also illustrated in
Analysis of the Expression Pattern:
Once the recombinant lentiviral vectors have been generated, transient transfection experiments with HEK293T adherent cells can corroborate their expression (Western blot), specifically, their expression on the cell surface (flow cytometry). To verify intracellular expression and localization confocal microscopy analysis with fluorescence-coupled anti-FLAG antibodies can be performed. As performed with the minimal region, wild-type with mutated versions of the scaffolds can be compared, as well as wild-type with and without the commercially available DCK inhibitor (Calbiochem, shown to bind to the catalytic site of Furin and block its activity [45]).
The minimal construct can be used as control as has been proven to travel to the surface and lose the FLAG tag unless inhibited or mutated, as illustrated in
Establishment of Clonal Cell Lines Expressing Individual Scaffold Proteins:
The lentiviral constructs shown to behave as expected in transient experiments can be transduced through lentiviral particles into the rtTA-expressing SupT1 cell line. Three days post-transduction, individual cells are sorted into individual wells of a 96-well-plate, and amplified. Dox can be added to induce scaffold expression and cell surface expression analyzed by flow cytometry following anti-FLAG antibody staining. Clones can be chosen based on highest cell-surface expression relative to their cell-surface expression when treated with DCK and/or compared to their mutated counterparts will be chosen, expanded and frozen in aliquots for future use.
Screening for the Discovery of Novel Drugs Against Gp160 Processing:
In alternative embodiments, assays of the invention can screen a variety of libraries, e.g., a combinatorial chemical library (e.g., as the Torrey Pines institute for Molecular Studies (TPIMS) combinatorial chemical library) as well as the retroviral peptide libraries. This screen can be performed with the minimal gp120/gp41 boundary (Aim 1) and/or with any of the larger fragments.
Libraries for Screening:
In alternative embodiments different libraries are screened, increasing the chances of finding putative new drugs. In alternative embodiments, each screen is independent of each other and can be done independently. In alternative embodiments, libraries include:
1. The Combinatorial Library of TPIMS:
TPIMS libraries include more than five million small molecule compounds assembled as systematically arranged mixtures of approximately 2,000 compounds each in average. Arranged in 96-well plates, they will be de-convoluted at different stages during the screening process based on positive FLAG staining. This approach allows the assay to produce hits without first biasing the screening collection.
2. Retroviral ER-TGN-Retained Random Peptide Library:
For the expression of a random peptide library localized to the ER/TGN luminal compartment, a retroviral vector carrying a protein scaffold composed of a signal sequence, the site for library insertion, a long flexible link and a KDEL ER retention signal (construct a in
In alternative embodiments, a long flexible link, composed of four repetitions of the Glycine/Glycine/Glycine/Serine motif, between the random peptide library and the KDEL motif, is used to ensure relative freedom of movement and flexibility within the ER/TGN lumen, most probably required to interfere with Furin/PC recognition and/or cleavage of HIV Env. The rationale behind the construction of a library localized to the ER/TGN luminal compartment is obviously to increase the chances of physical interaction between the players involved in Env processing. Moreover, the retroviral nature of the library ensures the continuous expression of the peptide fusion required for optimal competition. As the retroviral plasmid carries an IRES-mCherry cassette, cells expressing a peptide will fluoresce red.
3. Retroviral Secreted Random Peptide Library:
In alternative embodiments, for the expression of a secreted random peptide library, a random sequence is introduced into a vector carrying a protein scaffold very similar to the one described for the ER-TGN-retained library. In alternative embodiments, the scaffold will not contain the flexible link or the KDEL retention signal and will be allowed to travel to the cell surface. The engineered retroviral vector can carry a protein scaffold composed of a signal sequence, the site for library insertion, and a stop codon (construct b in
Transfer of the Peptide Libraries into Clones Carrying the Assay:
The peptide libraries carried by the retroviral vectors can be transferred into the selected SupT1 clonal populations expressing the assay. For the production of retroviral particles packaging cell lines can be transfected with the library-carrying retroviral vectors. The resulting viral particles can be collected and used to transduce target cells. As the peptide libraries are coupled to the IRES-mCherry cassette, cells expressing a peptide can become red fluorescence. Transduction can be performed in a very low multiplicity of infection so cells will be infected by a single viral particle to facilitate the propagation of clonal populations when putative hits are encountered. For that purpose at least 300 million cells can be infected at an expected infection rate of 10%. Following a period of two-three weeks, red fluorescent cells can be sorted out and amplified for further screening.
The Screening Process:
For the screening of the TPIMS libraries, cells harboring the assay alone can be screened. Cells can be transferred into pre-set library 96-well plates, which contain around 200 compounds per well. Following induction with Dox, and staining with FITC-coupled anti-FLAG antibody based on the conditions discussed above, the plate can be analyzed by flow cytometry. Wells showing any FLAG staining above background can be further de-convoluted. The process can be repeated until reaching plates with one compound per well, provided FLAG positive stain is observed along the way. For the screening of the peptide libraries, the cells carrying both the assay and the libraries can be used. An outline of the screening process is shown in
Corroboration of Hits:
As schematically illustrated in
In alternative embodiments, a HXB2 T-tropic wild-type HIV-1 sequence is used, but the assay can use other strains or clades of HIV. While the Furin recognition site is very conserved among the different strains, amino acid substitutions are found within gp120 and gp41 motifs known to influence the three-D structure of Env, and thus the recognition by Furin/PCs. The assays of the invention thus can be adapted to HIV-1 and HIV-2 as well as other clades of HIV-1 making it into a platform for the study of Env processing and drug discovery against all HIV viruses spread around the world. Assays of the invention can be adapted to other viruses that rely on processing in the ER-TGN compartment, such as members of the Flaviviridae. If putative hits obtained from the random peptide-library-based screen is not intended to find the final drug product and peptidomimetics and biochemical studies can be performed to further convert a putative peptide into a deliverable drug.
Production of Retroviral Particles:
For the production of MLV-based viral particles, Phoenix-GP packaging cell line (kindly provided by Garry Nolan, Stanford University, CA) was transfected with retroviral vectors. For the production of HIV-based virus particles, 293T cells were transfected with pH-GFP transfer vector, pCI-VSVg, and pCMV.Δ8.2 (Didier Trono, EPFL, Switzerland). In each case, viral supernatant was collected at 48 hours post-transfection. Viral supernatant was used to transduce SupT1 cells by centrifugation at 1500×g, at 32° C. for 80 minutes. Cells harboring rtTA were further transduced with viral particles carrying the wt gp120/gp41 boundary (pBMN-gp160 min-wt), or the mutant (pBMN-gp160 min-mut).
Flow cytometry and sorting: Flow cytometry is performed on a BD FACSAria™ and/or FACSCanto (SDSU FACS core facility) with 488 nm and 633 nm lasers. Data is collected using FACSDiva 6.1.1™ software (BD Biosciences, San Jose, Calif.) and analyzed by FLOWJO™ (Tree Star, Inc., Ashland, Oreg.).
In alternative embodiments, assays of the invention can be adapted to any high-number well format for high content, high throughput adaptations.
In alternative embodiments, multiplexing can be used to enhance high-throughput capabilities. The assay, in a multiplexed format, can be used as a platform that allows the analysis or investigation of multiple targets/substrates supposedly cleaved or recognized within the secretory pathway, in one sample. The assay can be coupled with barcoding, genetic or other, to increase multiplexing capabilities. Genetic fluorescent barcoding through retroviral technology, for example, allows multiplexing of the assay without further manipulations.
As example,
In order to further prove the adaptability of exemplary assays of the invention to a multiplexed format, the same three populations where analyzed by flow cytometry independently or mixed in the same sample.
In alternative embodiments, exemplary assays of the invention are used for drug discovery. As a proof of principle, the same experiment with the three bar-coded populations was repeated in the presence of DCK, an inhibitor of Furin and similar protein convertases that are active in the Golgi/TransGolgi network. DCK treatment reconstitutes the second tag (FLAG) cell surface expression only with wildtype HIV1-envelope boundary, proving that Furin or similar enzymes are responsible for the cleavage of the HIV-1 envelope boundary. Interestingly, while DenV pr-M was expected to be cleaved by Furin, it was not inhibited by DCK, proving that this exemplary assay can pinpoint specificity of substrate recognition or specificity of enzyme involved in cleavage.
In alternative embodiments, exemplary assays of the invention are used for pinpointing substrate and/or enzyme specificity with the use of drugs or other compounds. Interestingly, while DenV pr-M was expected to be cleaved by Furin, it was not inhibited by DCK, proving that this exemplary assay can pinpoint specificity of substrate recognition or specificity of enzyme involved in cleavage.
In alternative embodiments, exemplary assays of the invention are used for pinpointing substrate, enzyme specificity, and/or requirement of factors with the use of drugs and/or RNA-knockdown-based techniques or similar. The assay can be couple to RNAi technologies for example to pinpoint specificity of protease cleavage of substrates and elucidate unknown targets, proteases, and/or cleavage events.
Knockdown of Furin reconstitutes FLAG expression with wildtype HIV-1 envelope boundary for a period of at least five days, as graphically illustrated in
This exemplary assay's versatility can be further proved as it can be easily adapted to any substrate of interest, viral or host. To prove versatility of the assay, other viral substrates were analyzed. The substrates that were included in the flow cytometry analysis are:
The WNV pr-M wt substrate is: CTKTRHSRRSRRSLTVQTHG (SEQ ID NO:8)
The DenV pr-M wt substrate is: CTTTGEHRREKRSVALVPHV (SEQ ID NO:9)
The DenV pr-M mut substrate is: CTTTGEHRREKSVALVPHV (SEQ ID NO:10)
The HIV Env wt substrate is: KRRVVQREKRAVGIGAL (SEQ ID NO:11)
The HIV Env mut substrate is: KRRVVQREKSAVGIGAL (SEQ ID NO:12)
This includes not only HIV wild type and mutant boundaries, and wild type DenV pr-M boundary, but also a mutant DenV pr-M boundary as additional control and the pr-M boundary of West Nile virus. As shown in the flow cytometry data schematically illustrated in
In alternative embodiments, exemplary assays of the invention are adapted to other viral substrates that utilize the classical secretory pathway. HIV-1 utilizes the pathway for the transport of its envelope protein to the cell surface and in its way it is cleaved by Furin and similar protein convertases. All viruses of the Flaviviridae family are tightly associated with the Endoplasmic Reticulum and/or the Golgi TransGolgi network.
While we have already adapted the assay to HIV-1 envelope and to Dengue virus and West Nile virus pr-M boundaries, the assay can be adapted to other viral segments that serve as putative substrates of enzymes that are active in the classical secretory pathway. These include viruses or all viral families that exploit the secretory pathway, such as Flaviviridae, which include, but are not restricted to: Dengue virus, Hepatitis C Virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus, Tick-borne encephalitis virus, Kyasanur Forest disease virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, bovine viral diarrhoea virus, Rio Bravo virus, Culex flavivirus or pegivirus. Other examples include viral proteins from other families, some of which are listed in the table of
Dengue Virus pr-M
Influenza A Virus HA
Ebola Virus (Zaire) GP
Shigella dysentariae Shiga Toxin subunit A
Bacillus anthracis Anthrax Toxin Protective
Corynebacterium diphtheria Diptheria Toxin
Pseudomona aeroginosa exotoxin A
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/012148 | 1/18/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61754573 | Jan 2013 | US |
