Methods according to the present invention are generally useful for studying kinase activity in situ and for screening molecules that modulate kinase activities in situ.
Optimal drug design largely depends upon drug specificity in the complex context of a living cell. Anti-tumor chemotherapeutic drugs, for example, ideally destroy malignant cells while having a minimal damaging effect on healthy cells. However, most chemotherapeutic drugs have limited specificity and are toxic to both normal and malignant cells. Examples of such side-effects on healthy cells include direct myocardial damage, heart rhythm disturbances, pericarditis, pulmonary fibrosis, hemorrhage, nausea, vomiting, dyspnea, alopecia, peripheral and central neuropathies, pain, nephropathies, stomatitis, diarrhea, fever, immunosuppression, and changes in the state of consciousness. Therefore, cytotoxic side-effects of these chemotherapeutics greatly limit their efficacy.
Many cytostatic drugs, including those used in chemotherapy, function by inducing programmed cell death (apoptosis). However, since many tumor cells arise because of failure to respond to natural cues for apoptosis, they tend to be resistant to chemotherapeutic drugs that aim at triggering apoptotic cues. Therefore, a key strategy of the pharmaceutical industry for treating tumor cell growth is to pre-sensitize cells to apoptotic cues. A means for doing this is to block the protein kinases that inhibit apoptosis, thereby either directly inducing cell death or sensitizing cells to other anti-tumor drugs. Such kinases include the survival kinases AKT, IKK, ERK, Raf-1, PI 3-kinase, PDK-1 and others. Up-regulation of these kinases blocks apoptosis, and is often associated with tumors in humans and other mammals, further suggesting that identification and inhibition of these kinases will be of therapeutic benefit, (e.g., by enhancing the apoptosis-inducing effects of current anti-tumor therapeutics). There is also much interest in finding molecules that inhibit kinases that control other cell functions such as inflammation signaling, cell growth, and cell metabolism. Such inhibitors need to be highly selective in targeting specific kinases in situ.
Presently, most kinase activity measurements are carried out on recombinant proteins, produced and purified from insect cells or from mammalian cells in culture. In vitro assays such as radiometric assays or in-plate binding assays with read-outs are then used to measure the activity of these purified kinases. These in vitro assays are performed under conditions that only marginally reproduce the context of a live cell and are likely to have only marginal biological relevance. Therefore, even when a drug molecule is identified based on its in vitro specificity for a particular kinase, the in situ or in vivo specificity of the molecule remains extremely difficult to assess. Drugs developed using in vitro assays often turn out to have little or no effect in vivo or to have highly toxic side effects such as those mentioned above.
Realizing the importance of examining biological activities inside cells, the pharmaceutical industry is moving towards cell-based screens. However, developing a whole cell screening assay that monitors kinase activity, e.g., in response to an inhibitory molecule, is particularly difficult because of the large number of different kinases within the cell and because of the structural similarities of the catalytic regions of many of these kinases. One approach has been to look at fixed cell imaging of activated kinases. However, this approach only measures whether a kinase has been phosphorylated by an upstream activator kinase. Other approaches rely on a reporting system that is hard to duplicate for multiple kinases, such as the use of fluorescence resonance energy transfer (FRET) technology, which examines an isolated protein-protein interaction that is regulated by a kinase. Because these assays evaluate only a single kinase at a time, they have limited utility for purpose of drug discovery. Further, reporter systems such as FRET are not easily amenable to high-throughput or multiplexing approaches often needed in today""s drug discovery programs.
There is, therefore, a need for an in situ kinase assay that determines kinase specificity within a living cell. In particular, an assay is needed that provides information on multiple protein kinases simultaneously, and that provides real-time determination of kinase specificity.
The present invention provides kinase assays that are cell-based, and that allow for the discovery of compounds capable of modulating kinase activity in situ. It is an object of the invention to provide methods that can be adapted to assay the activities of different kinases in a cell with relative ease. It is a further object of the invention to provide methods that can screen a candidate molecule, e.g., a small molecule, peptide or drug candidate, regarding its ability to modulate multiple kinases simultaneously. The invention also provides compounds and molecules identified through these methods.
In a preferred embodiment, these and other objects of the invention are accomplished by providing assays based on a cellular signaling event between a signaling enzyme and its substrate. One example of such a signaling event is the binding between the signaling enzyme ubiquitin E3 (E3) ligase, and its substrate. After the binding, the E3 substrate is subject to transubiquitination and targeted by the degradation pathway. Another example of a signaling event on which the invention may be based is part of a peptide translocation pathway. Specifically, the signaling event can be the binding of a transporting protein to a traffic signaling domain of its substrate. After binding takes place, the substrate is eventually transported from a first subcellular area to a second area.
According to the invention, either the signaling enzyme or its substrate is altered so that their interaction is regulated by a kinase of interest. A label is associated with the signaling substrate so that the kinase activity of interest is monitored through the expression of the label as the signaling pathway now targets both the substrate and the label, for example, by degrading or transporting the substrate and the label. Because the signaling pathway takes place in a living cell, monitoring of the kinase activity through the label expression is carried out in situ. When a cell is exposed to a candidate molecule, changes in the expression of the label are indicative of whether the candidate molecule modulates the kinase activity of interest. Because the assay is conducted in live cells, results from the assay provide reliable and relevant information on biological functions and drug specificity.
According to one aspect of the invention, a signaling substrate is altered. In one embodiment, the kinase recognition domain of signaling substrate is modified. For example, an adapter module, e.g., a consensus recognition motif for a kinase of interest, is incorporated into a wild type kinase recognition domain. Alternatively, random mutagenesis can be performed on the wild type kinase recognition domain to produce specificity for the kinase of interest, which can be verified through subsequent screening. Through one or both of the above methods of modification, binding between the altered signaling substrate and the signaling enzyme becomes regulated by the kinase of interest. Using recombinant DNA technologies, an adapter module can be easily incorporated into a peptide. Because the consensus recognition motifs for many kinases are known, methods of the invention generally provide assay systems that can be routinely modified to test large numbers of kinases. These kinases include, but are not limited to, survival kinases implicated in apoptosis, thereby allowing discovery of drugs such as those that can be used in anti-tumor therapies. In an embodiment, a signaling substrate is altered such that its enzyme binding region is flanked by two sequestering motifs that interact with each other. The interaction between the sequestering motifs prevents the signaling substrate from being recognized or bound by the signaling enzyme. The interaction between the sequestering motifs is regulated by a kinase of interest. As a result, binding between the altered signaling substrate and the signaling enzyme is also regulated by the kinase of interest.
In an exemplary method, a candidate molecule is exposed to a cell that expresses a phosphorylation substrate having a kinase recognition domain. The kinase recognition domain is altered to be recognized by a kinase of interest that does not recognize the substrate in its unaltered state. A detectable label is associated with the phosphorylation substrate. This method of the invention further includes determining whether the candidate molecule causes a change in the expression of the label in order to identify a molecule that is capable of modulating the activity of the kinase of interest in situ.
In one embodiment of the invention, the phosphorylation substrate is also the substrate for an E3 ligase. In its unaltered state, binding of the phosphorylation substrate to the E3 ligase is regulated by a wild type kinase, and after the binding takes place, the substrate is eventually degraded by the proteosome. Methods of the invention provide various ways of altering the phosphorylation substrate so that binding between E3 and the substrate, and the ensuing ubiquitin-mediated degradation of the substrate are preserved and regulated by at least one kinase of interest that normally does not regulate the E3 binding event. A label is associated with the substrate, allowing monitoring of E3 binding and providing a readout as a consequence of the cell""s exposure to a candidate molecule.
In another embodiment of the invention, the phosphorylation substrate is also the substrate for a transporting protein that causes the substrate to be translocated from a first subcellular area to a second are. In its unaltered state, the phosphorylation substrate""s binding with the transporting protein is regulated by a wild type kinase, e.g., through an allosteric modification that affects the structural conformation of the phosphorylation substrate. The modification may change the accessibility by a transporting protein to different traffic signaling regions on the substrate. Binding of the transporting protein to a different signaling region results in translocation of the substrate to a different subcellular area, such as mitochondria, endoplasmic reticulum (ER) or the extracellular space. Methods of the invention provide various ways of altering the phosphorylation substrate such that binding between the transporting protein and the substrate, and the ensuing translocation of the substrate, are preserved and regulated by at least one kinase of interest that normally does not regulate the binding event. A label is similarly associated with the substrate for allowing monitoring the signaling event and any modulation of the signaling event due to cellular exposure to a candidate molecule.
In another embodiment according to the first aspect of the invention, a candidate molecule is exposed to a cell that expresses a signaling substrate whose enzyme binding region is flanked on both sides by two sequestering motifs. When the pair of sequestering motif interact with each other, they prevent the enzyme binding region from binding with the signaling enzyme, for example, because of conformational changes to the substrate. The interaction between the sequestering motifs is regulated by a phosphorylation event that a kinase of interest is responsible for. A detectable label is associated with the signaling substrate and by determining whether the candidate molecule changes the expression of the label in the cell, a molecule capable of modulating the activity of the kinase of interest in situ can be identified. Examples of the signaling substrate include those for an E3 ligase involved in a ubiquitin-mediated degradation pathway, and those for a transporting protein involved in a peptide translocation pathway.
According to another aspect of the invention, the signaling enzyme is altered. In one embodiment, an adapter module, whose ability to recognize and bind to its ligand is regulated by a kinase of interest, is incorporated into the signaling enzyme. Through the adapter module, the altered signaling enzyme becomes capable of recognizing and binding the ligand of the adapter module, subject to regulation by the kinase of interest. In another embodiment, random mutagenesis is performed on a portion of the signaling enzyme, preferably the substrate-binding portion, so that the mutant enzyme recognizes and binds a phosphorylation substrate having a particular phosphorylation state.
In one embodiment, a candidate molecule is exposed to a cell that expresses a signaling enzyme that is altered to bind a phosphorylation substrate for a kinase of interest that, in its unaltered state, the signaling enzyme does not bind. A detectable label is associated with the phosphorylation substrate. Further, binding between the altered signaling enzyme and the substrate is regulated by a kinase. This method of the invention further includes determining whether the candidate molecule causes a change in the expression of the label in order to identify a molecule that is capable of modulating the kinase activity in situ. Examples of the signaling enzyme include an E3 ligase involved in a ubiquitin-mediated degradation pathway, and a transporting protein involved in a peptide translocation pathway.
According to another aspect of the invention, assays according to any of the above-described embodiments of the invention are multiplexed to study multiple kinases by using differentiable labels that are each associated with a different kinase substrate. For example, multiple phosphorylation substrates may each be mutated to contain a kinase recognition domain for a different kinase, each substrate associated with a differentiable label. Examples of such labels include GFP and its variants, which fluoresce at differentiable wavelengths. Expressing these multiple kinase substrates in one of the cell-based assay systems described above allows monitoring of kinase regulation of a signaling event and any modulation thereof by a candidate molecule to which the cell is exposed. An application of the multiplexed embodiment is the screening for a molecule for specificity for multiple kinases in the same signaling pathway.
The invention also provides molecules identified through one of the methods described wherein the molecule is capable of modulating a kinase activity in situ. The invention also provides fusion proteins useful for the methods described, isolated genetic molecules encoding the fusion proteins, vectors capable of expressing the genetic molecules, and cells transfected with at least one of such vectors.