Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2000-12-27
2004-07-13
Kishore, Goilamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C264S004100, C264S004300
Reexamination Certificate
active
06761902
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to proteoliposomes, their construction and use. Preferably the proteoliposome contains an integral membrane protein, having at least one transmembrane domain.
BACKGROUND OF THE INVENTION
Advances in genomics have resulted in the discovery and identification of numerous proteins. These advances have made it possible to obtain transcripts and DNA encoding a range of proteins, including putative integral membrane proteins having multiple transmembrane domains, as well as the proteins themselves. The availability of such proteins makes it possible to identify ligands that interact with these proteins, permitting one to better understand the biology of these proteins and/or screen for compounds that modulate the function of such proteins. However, there are increasing problems in knowing what a specific protein actually does and/or finding simple and accurate methods that actually identify the ligands that interact with a particular protein. For example, a protein such as a receptor protein for which a ligand has not yet been identified is referred to as an “orphan protein”. Such orphan proteins are becoming more numerous as more DNA sequences, including DNA sequences encoding putative receptors, become available. In these cases, the DNA and proteins are classified based upon homologies to known proteins. For example, one can recognize conserved sequences that resemble a known domain, such as a transmembrane domain, thus indicating that the identified protein resides in a membrane (i.e., is an integral membrane protein).
Transmembrane proteins or integral membrane proteins are amphipathic, having hydrophobic domains that pass through the membrane and interact with the hydrophobic lipid molecules in the interior of the bilayer, and hydrophilic domains which are exposed to the aqueous environment on both sides of the membrane (for example, the aqueous environments inside and outside of the cell). The biological activities of integral membrane proteins (e.g., ligand binding) are dependent upon the hydrophilic domains; in some cases, the membrane—spanning regions contribute to function.
Despite our ability to predict extra-membrane protein regions with some confidence, the prediction of the actual structure of these regions and the ligands bound thereto is much more tenuous. For the most part, the identification of natural and unnatural ligands of integral membrane proteins is an empirical process.
The identification of ligands and the study of their binding properties is more complicated for integral membrane proteins than for water-soluble proteins. Water-soluble proteins can be readily purified in aqueous buffers and maintained in a native conformation under such circumstances. Integral membrane proteins cannot be solubilized in aqueous buffers but must be maintained in an environment that allows the membrane-spanning region to maintain hydrophobic contacts. This is most often accomplished by including detergents in the solubilization buffer. When mixed with integral membrane proteins, the hydrophobic regions of the detergent bind the transmembrane region of the protein, displacing the lipid molecules of the membrane.
Although solubilizing transmembrane proteins in detergents in theory allows their purification, in practice, it is typically difficult to effectively isolate that protein from other membrane proteins while retaining native conformation for extended periods of time. For example, the calcium pump from the sarcoplasmic reticulum can only be isolated with its native structure intact when maintained within the context of the sarcoplasmic reticular membrane (Zhang et al. (1998), Nature 392: 835-39). Similarly, a three-dimensional map of the plasma membrane H+-ATPase was only possible when two-dimensional crystals were grown directly on electron microscope grids (Auer et al. (1998), Nature 392: 840-3). For many other transmembrane proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR), it has not yet been possible to purify the protein for extended periods of time while maintaining the wild-type conformation.
Additionally, identifying the actual ligands that interact with such a transmembrane protein, while extremely important, has many difficulties. For example, the transmembrane protein needs to be in the proper conformation in order to interact with ligands. Yet part of the way that transmembrane proteins maintain their conformation is by being part of a cellular membrane. The current solutions to this problem are less than optimal. For some integral membrane proteins that span the membrane only once, the extracellular and/or intracellular domains can be synthesized as independent entities and, in some cases, will fold properly. However, this is not always true. Furthermore, the post-translational modifications made to soluble versions of the extracellular or intracellular domains often differ from those of the full-length membrane-bound protein. These differences can exert profound effects on ligand binding or other functional properties. For the vast majority of integral membrane proteins, which span the membrane more than once, even this less-than-ideal solution is not feasible. Typically, cell-based screens are utilized to identify ligands of interest with these proteins. Cell lines that express the integral membrane protein of interest are established and compared to a parental cell line not expressing the protein. However, in such cases, it is difficult to effectively isolate the protein of interest from other proteins that are also present in the cell membrane. In many cases, the protein of interest is expressed in lower amounts than other integral membrane proteins. Thus, there can be interference caused by a compound or ligand interacting with an entirely different protein. For phenotypic screens, it may be that one protein is involved in one stage of a large pathway involving multiple proteins. In such cases, the readout in the screening assay may be affected even when the protein of interest is not directly affected. Accordingly, it would be desirable to have a method to look at a specific integral membrane protein in its native confirmation where it can be isolated from other competing proteins.
Seven-transmembrane segment, G protein-coupled receptors (GPCRs) represent approximately 1-2 percent of the total proteins encoded by the human genome and are important targets for pharmaceutical intervention. GPCRs have seven transmembrane domains, and also include chemokine receptors such as CCR5 and CXCR4, which have been identified as cofactors in permitting the human immunodeficiency virus (HIV) to enter cells. Generally low levels of expression and the dependence of the native conformation of GPCRs on the hydrophobic, intramembrane environment have complicated the study of these proteins. Analysis of ligand interactions with GPCRs and screening for inhibitors of such interactions are commonly conducted using live cells or intact cell membranes. Typically, the binding of radiolabeled ligand with the cells or the induction of intracellular calcium levels by the ligand are used as readouts in such screens. A significant drawback of such assays are the extremely large number of cells required for high-throughput screening. Furthermore, such studies can be complicated by the presence of numerous cell surface proteins, many of which are expressed at much higher levels than the GPCR of interest. Thus, certain approaches, such as using the GPCR-expressing cells to identify either natural or synthetic ligands in a complex mixture, are precluded. In addition, the generation of monospecific antibodies directed towards a particular GPCR in the complex cell membrane environment is inefficient. Furthermore, for some GPCRs, like the chemokine receptors, multiple ligands bind a single receptor, and conversely, a single ligand can bind multiple receptors. Therefore, if the cell expresses more than one receptor for the ligand being studied, interpretation of the results can be complicated.
Tr
Mirzabekov Tajib
Sodroski Joseph G.
Dana-Farber Cancer Institute Inc.
Kishore Goilamudi S.
Nixon & Peabody LLP
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