Source: http://www.google.com/patents/US7968350?dq=6,757,682
Timestamp: 2014-10-25 21:20:08
Document Index: 183734194

Matched Legal Cases: ['Application No. 2', 'Application No. 02795636', 'Application No. 02795636', 'Application No. 02795636', 'Application No. 04776693', 'Application No. 04776693', 'Application No. 2', 'Application No. 2', 'Application No. 04776693']

Patent US7968350 - Aqueous partitioning; drug discovery - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsThe invention involves obtaining signatures of species (including chemical, biological, or biochemical molecules) and/or signatures of interactions between species and using them to characterize species, characterize interactions, and/or identify species that could be useful in a variety of settings....http://www.google.com/patents/US7968350?utm_source=gb-gplus-sharePatent US7968350 - Aqueous partitioning; drug discoveryAdvanced Patent SearchPublication numberUS7968350 B2Publication typeGrantApplication numberUS 10/293,959Publication dateJun 28, 2011Filing dateNov 12, 2002Priority dateNov 12, 2001Also published asCA2466663A1, DE60235642D1, EP1444515A2, EP1444515B1, US8211714, US20030162224, US20110218738, US20120245856, WO2003042694A2, WO2003042694A3, WO2003042694B1Publication number10293959, 293959, US 7968350 B2, US 7968350B2, US-B2-7968350, US7968350 B2, US7968350B2InventorsArnon Chait, Boris ZaslavskyOriginal AssigneeAnaliza, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (30), Non-Patent Citations (65), Referenced by (3), Classifications (18), Legal Events (1) External Links: USPTO, USPTO Assignment, EspacenetAqueous partitioning; drug discoveryUS 7968350 B2Abstract The invention involves obtaining signatures of species (including chemical, biological, or biochemical molecules) and/or signatures of interactions between species and using them to characterize species, characterize interactions, and/or identify species that could be useful in a variety of settings. Signatures can be obtained using aqueous multi-phase partitioning and can be used to predict molecular interactions for applications such as drug discovery. A plurality of aqueous multi-phase partitioning arrangements can define an overall system providing an information-intensive signature, maximizing precision and sensitivity.
1. A method of determining a property of a species, comprising:
determining a relative measure of interaction between, at least, a species and first and second phases of a first aqueous multi-phase partitioning system, in which each of the first and second phases is able to interact with the species;
repeating the determining, with the species, in a second aqueous multi-phase partitioning system including at least one phase different from the first and second phases;
deriving an interaction signature which is a numerical construct that is assembled from the totality of the individual relative measures of interaction of the individual phase in the first and second aqueous multi-phase partitioning systems; and
determining a property of the species from the interaction signature, wherein the property is the molecular identity of the species.
2. A method as in claim 1, wherein the species is provided in combination with at least one auxiliary molecule that affects interaction between the species and the first and second phases.
3. A method of determining a property of a species, comprising:
determining a property of the species from the interaction signature, wherein the property is a conformational state of the species.
4. A method of determining a property of a species, comprising:
determining a property of the species from the interaction signature, wherein the property is one of interaction between the species and at least one additional species.
5. A method as in claim 1, comprising:
(a) providing the species;
(b) contacting the species with a first phase and at least one second phase, wherein the species can interact with each phase and wherein the species and phases form an aqueous multi-phase partitioning system;
(c) determining a relative measure of interaction between the species and each phase;
(d) calculating a coefficient which defines a ratio of the relative measure of interaction between the species and the first phase and the relative measure of interaction between the species and the second phase;
(e) repeating, at least one additional time, (b)-(d), wherein at least one different phase is used, in step (b), in each successive repeating step; and
(f) constructing an interaction signature using numerical, mathematical and/or visual representations of the coefficient from (d).
6. A method as in claim 5, comprising repeating (b)-(d) at least three times.
7. A method as in claim 5, wherein (b) comprises:
i) forming an aqueous partitioning system capable of separation into two or more immiscible phases;
wherein the coefficient of (d) is a partition coefficient.
8. A method as in claim 5, wherein (b) comprises:
wherein the coefficient of (d) is an apparent partition coefficient.
9. A method as in claim 8, wherein (c) comprises monitoring retention of the species.
10. A method as in claim 9, wherein retention of the species is measured as a function of time or volume of the mobile first phase.
11. A method as in claim 8, wherein the method is a chromatographic method.
12. A method as in claim 5 or 6, wherein the phases of (b) differ in their ability to interact with the species on the basis of the topography and type of groups on the species that are solvent-accessible.
13. A method as in claim 6, comprising:
repeating (a)-(f), wherein the species in (a) is varied; and
14. A method as in claim 13, wherein the group of species are a mixture of biomolecules that are structurally closely related.
15. A method as in claim 13, wherein the group of species are a microheterogeneous mixture of biomolecules.
16. A method as in claim 13, wherein the group of species are biomolecules bound to at least one low molecular weight ligand.
17. A method as in claim 13, wherein the group of species are biomolecules bound to other biomolecules.
18. A method as in claim 13, wherein the group of species comprises proteins bound to other proteins.
19. A method as in claim 13, wherein a biological activity level of at least one of the species is known.
20. A method as in claim 19, wherein the species are selected from the group consisting of biomolecules, mixtures of closely related biomolecules, microheterogeneous mixtures of biomolecules, biomolecules bound to at least one low molecular weight ligands and biomolecules bound to other biomolecules.
21. A method as in claim 1, wherein the identity of the species is unknown.
providing a species having an unknown conformational state and/or structure;
determining a relative measure of interaction including, at least, the species and a first phase and the species and a second phase, in a first aqueous multi-phase partitioning system in which each of the first and second phases is able to interact with the species;
repeating the determining phase, with the species, in a second aqueous multi-phase partitioning system including at least one different phase;
deriving, from the relative measures of interaction of the species with the phases in the different systems, an interaction signature indicative of a property of the species which is a numerical construct that is assembled from the totality of the individual relative measures of interaction of the individual phases in the first and second aqueous multi-phase partitioning systems; and
determining a conformational state and/or a structure of the species based on the interaction signature.
23. The method of claim 1, further comprising calculating an Euclidian distance for each signature with respect to a reference signature.
24. The method of claim 1, wherein the signature is a mathematical matrix.
25. A method of determining a property of a species, comprising:
deriving an interaction signature which is a numerical construct that is assembled from the totality of the individual relative measures of interaction of the individual phase in the first and second aqueous multi-phase partitioning systems, wherein the signature is a single numerical value; and
determining a property of the species from the interaction signature.
26. The method of claim 1, wherein the signature is expressed visually.
27. A method as in claim 3, wherein the species is provided in combination with at least one auxiliary molecule that affects interaction between the species and the first and second phases.
28. A method as in claim 3, comprising:
29. A method as in claim 28, comprising repeating (b)-(d) at least three times.
30. A method as in claim 28, wherein (b) comprises:
31. A method as in claim 28, wherein (b) comprises:
32. A method as in claim 28, wherein the phases of (b) differ in their ability to interact with the species on the basis of the topography and type of groups on the species that are solvent-accessible.
33. A method as in claim 29, comprising:
34. A method as in claim 33, wherein the group of species are a mixture of biomolecules that are structurally closely related.
35. A method as in claim 33, wherein the group of species are a microheterogeneous mixture of biomolecules.
36. A method as in claim 33, wherein the group of species are biomolecules bound to at least one low molecular weight ligand.
37. A method as in claim 33, wherein the group of species are biomolecules bound to other biomolecules.
38. A method as in claim 33, wherein the group of species comprises proteins bound to other proteins.
39. A method as in claim 3, wherein the identity of the species is unknown.
40. The method of claim 3, further comprising calculating an Euclidian distance for each signature with respect to a reference signature.
41. The method of claim 3, wherein the signature is a mathematical matrix.
42. The method of claim 3, wherein the signature is expressed visually.
43. A method as in claim 4, wherein the species is provided in combination with at least one auxiliary molecule that affects interaction between the species and the first and second phases.
44. A method as in claim 4, comprising:
45. A method as in claim 44, comprising repeating (b)-(d) at least three times.
46. A method as in claim 44, wherein (b) comprises:
47. A method as in claim 44, wherein (b) comprises:
48. A method as in claim 44, wherein the phases of (b) differ in their ability to interact with the species on the basis of the topography and type of groups on the species that are solvent-accessible.
49. A method as in claim 45, comprising:
50. A method as in claim 49, wherein the group of species are a mixture of biomolecules that are structurally closely related.
51. A method as in claim 49, wherein the group of species are a microheterogeneous mixture of biomolecules.
52. A method as in claim 49, wherein the group of species are biomolecules bound to at least one low molecular weight ligand.
53. A method as in claim 49, wherein the group of species are biomolecules bound to other biomolecules.
54. A method as in claim 49, wherein the group of species comprises proteins bound to other proteins.
55. A method as in claim 4, wherein the identity of the species is unknown.
56. The method of claim 4, further comprising calculating an Euclidian distance for each signature with respect to a reference signature.
57. The method of claim 4, wherein the signature is a mathematical matrix.
58. The method of claim 4, wherein the signature is expressed visually.
59. A method as in claim 25, wherein the species is provided in combination with at least one auxiliary molecule that affects interaction between the species and the first and second phases.
60. A method as in claim 25, comprising:
61. A method as in claim 60, comprising repeating (b)-(d) at least three times.
62. A method as in claim 60, wherein (b) comprises:
63. A method as in claim 60, wherein (b) comprises:
64. A method as in claim 60, wherein the phases of (b) differ in their ability to interact with the species on the basis of the topography and type of groups on the species that are solvent-accessible.
65. A method as in claim 61, comprising:
66. A method as in claim 65, wherein the group of species are a mixture of biomolecules that are structurally closely related.
67. A method as in claim 65, wherein the group of species are a microheterogeneous mixture of biomolecules.
68. A method as in claim 65, wherein the group of species are biomolecules bound to at least one low molecular weight ligand.
69. A method as in claim 65, wherein the group of species are biomolecules bound to other biomolecules.
70. A method as in claim 65, wherein the group of species comprises proteins bound to other proteins.
71. A method as in claim 25, wherein the identity of the species is unknown.
72. The method of claim 25, further comprising calculating an Euclidian distance for each signature with respect to a reference signature.
73. The method of claim 25, wherein the signature is a mathematical matrix.
74. The method of claim 25, wherein the signature is expressed visually.
RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. �119(e) to the following U.S. provisional patent application Ser. No. 60/336,895, filed Nov. 12, 2001, Ser. No. 60/352,473, filed Jan. 28, 2002, Ser. No. 60/361,661, filed Mar. 4, 2002, and Ser. No. 60/412,754, filed Sep. 23, 2002 each of which is hereby incorporated herein by reference in its entirety.
FIELD OF THE INVENTION The invention is generally related to characterization of physical and structural properties of molecules and interactions between molecules. More particularly, the invention is related to developing and using signatures obtained, for example, via aqueous multi-phase partitioning, which reflect structural and functional characteristics of biomolecules and/or molecules which interact with biomolecules.
BACKGROUND OF THE INVENTION This invention relates generally to the analysis and characterization of biomolecules, complexes comprising biomolecules or analogous species thereof. The results of the analysis, represented as signatures, can be used to establish relationships between properties of large numbers of species which allows selection of species for specific uses based upon the correlation of the species' properties which have been analyzed and characterized using the methods of the invention.
SUMMARY OF THE INVENTION The present invention provides a variety of techniques involving characterization of species, conformational aspects of species, interaction between species, and the like. A wide variety of the species including chemical species (such as small molecules), biological species such as proteins, and the like can be used in accordance with the invention, i.e., can realize benefits associated with the invention. In one aspect, the invention relates to a method for characterizing a species. In one embodiment, the method includes determining a relative measure of interaction between, at least, a species and a first interacting component and the species and a further interacting component, in a first system in which each of the first and second interacting components is able to interact with the species. The determining phase is repeated, with the species, in a second system including at least one different interacting component. The method also involves constructing, from relative measures of interaction of the species with interacting components in different systems, an interaction signature indicative of a property of the species. The property can be the molecular identity of the species, a conformational property of the species, a property of interaction between the species and at least one additional species, and/or other properties. The interacting components may contain, in addition to their constitutive components, also other molecules which may further interact with the species, e.g., formulation excipients, chaperon proteins, etc.
FIG. 1 is a visual representation of the signatures, using normalized bar graphs, whose numerical values are listed in Table 1. The values 1-4 on the abscissa refer to the PEG-phosphate, Dex-Ficoll, Dex-Ficoll-NaSCN, and Dex-PEG systems, respectively.
FIG. 2 is a visual representation of the signatures, using normalized radar plots, whose numerical values are listed in Table 2. The values 1-4 in each of the radar plots refer to the Dex-PEG, PEG-3350-phosphate, Dex-Ficoll, and Dex-Ficoll-Na2SO4 systems, respectively.
FIG. 3 is a visual representation of the relative contribution of each system to the signatures whose numerical values are listed in Table 2. The values 1-4 on the abscissa refer to the Dex-PEG, PEG-3350-phosphate, Dex-Ficoll, and Dex-Ficoll-Na2SO4 systems, respectively.
DETAILED DESCRIPTION OF THE INVENTION The present invention involves techniques for determining information about molecules and/or interactions between molecules. More particularly, the invention is related to developing and using signatures obtained, for example, via aqueous multi-phase partitioning, which reflect structural and functional characteristics of biomolecules or molecules which interact with biomolecules. These signatures can be used for the purposes of establishing relationships between structure and function or for the purposes of establishing functional and structural relationships to activity or to conditions.
As used in the specification and the appended claims, the singular forms �a,� �an� and �the� include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to �a biomolecule� can include mixtures of a biomolecule, and the like.
�Analyte,� �analyte molecule,� or �analyte species� refers to a molecule, typically a macromolecule, such as a polynucleotide or polypeptide, whose presence, amount, and/or identity are to be determined.
�Antibody,� as used herein, means a polyclonal or monoclonal antibody. Further, the term �antibody� means intact immunoglobulin molecules, chimeric immunoglobulin molecules, or Fab or F(ab′)2 fragments. Such antibodies and antibody fragments can be produced by techniques well known in the art which include those described in Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)) and Kohler et al. (Nature 256: 495-97 (1975)) and U.S. Pat. Nos. 5,545,806, 5,569,825 and 5,625,126, incorporated herein by reference. Correspondingly, antibodies, as defined herein, also include single chain antibodies (ScFv), comprising linked VH and VL domains and which retain the conformation and specific binding activity of the native idiotype of the antibody. Such single chain antibodies are well known in the art and can be produced by standard methods. (see, e.g., Alvarez et al., Hum. Gene Ther. 8: 229-242 (1997)). The antibodies of the present invention can be of any isotype IgG, IgA, IgD, IgE and IgM.
�Aqueous,� as used herein, refers to the characteristic properties of a solvent/solute system wherein the solvating substance has a predominantly hydrophilic character. Examples of aqueous solvent/solute systems include those where water, or compositions containing water, are the predominant solvent.
�Aqueous multi-phase system,� as used herein, refers to an aqueous system which consists of greater than one aqueous phase in which an analyte species can reside, and which can be used to characterize the structural state of the analyte species according to the methods described herein. For example, this includes aqueous system which can separate at equilibrium into two, three, or more immiscible phases. Aqueous multi-phase systems are known in the art and this phrase, as used herein, is not meant to be inconsistent with accepted meaning in the art.
An �interacting component� means a component, such as a phase of an aqueous multi-phase system, that can interact with a species and provide information about that species (for example, an affinity for the species). Multiple interacting components, exposed to a species, can define a system that can provide a �relative measure of interaction� between each component and the species. An interacting component can be aqueous or non-aqueous, can be polymeric, organic (e.g. a protein, small molecule, etc.), inorganic (e.g. a salt), or the like, or any combination. A set of interacting components can form a system useful in and in part defining any experimental method which is used to characterize the structural state of a species such as an analyte species according to the methods described herein. Typically, a system of interacting components can measure the relative interaction between the species and at least two interacting components. An aqueous multi-phase system is a species of a system of interacting components, and it is to be understood that where �Aqueous system� or �Aqueous multi-phase system� is used herein, this is by way of example only, and any suitable system of interacting components can be used.
Both aqueous two-phase and aqueous multi-phase systems, as used herein, also refer to systems analogous to those comprising only aqueous solutions or suspensions. For example, an aqueous two-phase system can include non-aqueous components in one or more phases that are not liquid in character. In this aspect, aqueous phase systems also refers to related techniques that rely on differential affinity of the biomolecule to one media versus another, wherein the transport of the biomolecule between one medium and, optionally, another medium occurs in an aqueous environment. Examples of such �heterogeneous phase systems� include, but are not limited to, HPLC columns or systems for liquid-liquid partition chromatography as are known to those of skill in the art.
�Partition coefficient,� as used herein, refers to the coefficient which is defined by the ratio between the concentrations of the solute in the two immiscible phases at equilibrium. For example, the partition coefficient (K) of an analyte in a two-phase system is defined as the ratio of the concentration of analyte in the first phase to that in the second phase. For multi-phase systems, there are multiple partition coefficients wherein each partition coefficient defines the ratio of analyte in first selected phase and a second selected phase. It will be recognized that the total number of partition coefficients in any multi-phase system will be equal to the total number of phases minus one.
For heterogeneous phase systems, an �apparent partition coefficient,� as used herein, refers to a coefficient which describes information obtained from alternative techniques which is correlated to the relative partitioning between phases. For example, if the heterogeneous two-phase system used is an HPLC column, this �apparent partition coefficient� can be the relative retention time for the analyte. It will be recognized by those of skill in the art that retention time of an analyte reflects the average partitioning of the analyte between a first, mobile phase and a second, immobile phase. Also, it will be recognized that other similarly determinable properties of analytes can also be used to quantify differences in physical properties of the analytes and are, therefore, suitable for use as apparent partition coefficients.
�Bind,� as used herein, means the well understood receptor/ligand binding as well as other nonrandom association between an a biomolecule and its binding partner. �Specifically bind,� as used herein describes a binding partner or other ligand that does not cross react substantially with any biomolecule other than the biomolecule or biomolecules specified.
Generally, molecules which preferentially bind to each other are referred to as a �specific binding pair.� Such pairs include, but are not limited to, an antibody and its antigen, a lectin and a carbohydrate which it binds, an enzyme and its substrate, and a hormone and its cellular receptor. As generally used, the terms �receptor� and �ligand� are used to identify a pair of binding molecules. Usually, the term �receptor� is assigned to a member of a specific binding pair, which is of a class of molecules known for its binding activity, e.g., antibodies. The term �receptor� is also preferentially conferred on the member of a pair that is larger in size, e.g., on lectin in the case of the lectin-carbohydrate pair. However, it will be recognized by those of skill in the art that the identification of receptor and ligand is somewhat arbitrary, and the term �ligand� may be used to refer to a molecule which others would call a �receptor.� The term �anti-ligand� is sometimes used in place of �receptor.�
�Biomolecule,� as used herein, means; peptides, polypeptides, proteins, protein complexes, nucleotides, oligonucleotides, polynucleotides, nucleic acid complexes, saccharides, oligosaccharides, carbohydrates, lipids and combinations, derivatives and mimetics thereof.
�Detectable,� as used herein, refers the ability of a species and/or a property of the species to be discerned. One method of rendering a species detectable is to provide further species, that bind or interact with the first species, that comprise a detectable label. Examples of detectable labels include, but are not limited to, nucleic acid labels, chemically reactive labels, fluorescence labels, enzymic labels and radioactive labels.
�Mimetic,� as used herein, includes a chemical compound, or an organic molecule, or any other mimetic, the structure of which is based on or derived from a binding region of an antibody or antigen. For example, one can model predicted chemical structures to mimic the structure of a binding region, such as a binding loop of a peptide. Such modeling can be performed using standard methods (see for example, Zhao et al., Nat. Struct. Biol. 2: 1131-1137 (1995)). The mimetics identified by methods such as this can be further characterized as having the same binding function as the originally identified molecule of interest according to the binding assays described herein.
�Solid support,� as used herein, means the well-understood solid material to which various components of the invention are physically attached, thereby immobilizing the components of the present invention. The term �solid support,� as used herein, means a non-liquid substance. A solid support can be, but is not limited to, a membrane, sheet, gel, glass, plastic or metal. Immobilized components of the invention may be associated with a solid support by covalent bonds and/or via non-covalent attractive forces such as hydrogen bond interactions, hydrophobic attractive forces and ionic forces, for example.
�Heterogeneous aqueous systems,� as used herein, refers to systems wherein in addition to an aqueous or largely aqueous component, there is also a largely non-aqueous component. The non-aqueous component can be capable of immobilizing or interacting with analytes. Examples of the non-aqueous component may include a solid support.
�Structure�, �structural state�, or �conformation,� as used herein, all refer to the commonly understood meanings of the terms. Most specifically, the meaning of the terms as they apply to biomolecules such as proteins and nucleic acids, but also to pharmacologically active small molecules. In different contexts, the meaning of these terms will vary as is appreciated by those of skill in the art. For instance, the use of the terms; primary, secondary, tertiary or quaternary in reference to protein structure have accepted meanings within the art, which differ in some respects from their meaning when used in reference to nucleic acid structure (see Cantor & Schimmel, Biophysical Chemistry, Parts I-III). Unless otherwise specified, the meanings of these terms will be those generally accepted by those of skill in the art.
As used herein, �signature� refers to a particular representation of desired information, which can be defined as a set of relative measures of interaction described above obtained from experiments with different interacting components. Typically, a signature is used in place of more detailed information when the latter is difficult to obtain, or when it is not necessary to completely describe such information in order to make use of it. For example, fingerprinting individual people is a well-recognized technique to uniquely identify an individual (to a reasonable certainty), providing a conveniently obtained and conveniently dense information set instead of describing the individual using other representations, e.g., genetic makeup, or by using exhaustively physical description and other information.
An �interaction signature,� a used herein, means a signature characteristic of interaction of a species with at least one other species, optionally also characteristic of interaction of either or both species with another species or an environment (medium) in which the species exist and/or with which the species interact. For example, an interaction signature may characterize interaction of a species with one phase of a multi-phase system, and with another phase of the multi-phase system, with an overall interaction signature characteristic of the relative interaction of the species with the two (or more) phases. As further examples, an interaction signature can be characteristic of interaction of a species with any number of phases of a multi-phase system, and interaction signatures can exist for interaction between and among a variety of species and a variety of phases of a multi-phase system.
Partitioning in the aqueous polymer two-phase systems is a highly efficient, versatile, and cost-effective method for detecting changes in a receptor induced by its binding to a binding partner. Aqueous two-phase systems arise in aqueous mixtures of different water-soluble polymers or a single polymer and specific salts. For example, dextran and polyethylene glycol (�PEG�) are mixed in water above certain concentrations, the mixture separates into two immiscible aqueous phases separated by a clear interfacial boundary. These two separated phases are said to have resolved. In one phase, the solution is rich in one polymer and, on the other side of this boundary in a second phase, the solution is rich in the other polymer. The aqueous solvent in both phases provides media suitable for biological products such as proteins or for other biomolecules.
Evaluation of data from partitioning of a biomolecule can involve use of the partition coefficient (�K�), which is defined as the ratio between the concentrations of the biomolecule in the two immiscible phases at equilibrium. For example, the partition coefficient, K, of a protein is defined as the ratio of the protein in first phase to that in the second phase in a biphasic system. When multiple phase systems are formed, there can be multiple independent partition coefficients that could be defined between any two phases. From mass balance considerations, the number of independent partition coefficients will be one less than the number of phases in the system.
It will be recognized that the partition coefficient K for a given biomolecule of a given conformation will be a constant if the conditions and the composition of the two-phase system to which it is subjected remain constant. Thus, if there are changes in the observed partition coefficient K for the protein upon addition of a potential binding partner, these changes can be presumed to result from changes in the protein structure caused by formation of a protein-binding partner complex. �K�, as used herein, is used as specifically mathematically defined below, and in all instances also includes, by definition, any coefficient representing the relative measure of interaction between a species and at least two interacting components.
In order to determine the partition coefficient K of a protein or a mixture of a protein with another compound to be analyzed, concentrated stock solutions of all the components (polymer 1, e.g., dextran; polymer 2, e.g., PEG, polyvinylpyrrolidone, salts, etc.) in water can be prepared separately. The stock solutions of phase polymers, salts, and the protein mixture can be mixed in the amounts and conditions (e.g., pH from about 3.0 to about 9.0, temperature from about 4� C. to 60� C., salt concentration from 0.001 to 5 mole/kg) appropriate to bring the system to the desired composition and vigorously shaken. The system can then be allowed to equilibrate (resolve the phases). Equilibration can be accomplished by allowing the solution to remain undisturbed, or it can be accelerated by centrifugation, e.g., for 2-30 minutes at about 1000 to 4000 g or higher. Aliquots of each settled (resolved) phase can be withdrawn from both the upper and lower phases. The concentration of biomolecule can be determined for both the upper and lower phases.
Once the set of partition coefficient values is obtained, the signature of the structure of the biomolecule, or of the biomolecule-ligand complex, or of the mixture of biomolecules is available as a set of numerical values. Multiple ways to further express this information are then available�the particular choice of which is primarily dependent upon the complexity of the set (e.g., how many K values are available?) and on the manner in which the signature is used (e.g., would simple visual representation suffice?). Different arrangements of this information and selection of those most suitable are within the skill of one of the art. One simple way to express the signature is to write down the K values for each case in a row. Different cases, corresponding to different structural states of the biomolecule in response to, e.g., different ligands, are written in sequential rows, with the columns representing the various aqueous systems that are used to construct the signature. In this case just described, the representation is a matrix of numbers that allows easy comparison of case against all other signatures. Alternatively, this information could be expressed visually, e.g., by using bar graphs for each state, where the location of each bar denotes a particular system and its height the actual value of K. If the range of K values obtained from a set of aqueous systems is large, it can be difficult to visually represent all of the values using linear abscissa scale. In such cases, it is easy to first transform the data using simple shift, logarithmic, or normalization operations.
These mapping techniques could be represented, without a loss of generality, via the following equations:
∑ i = 1 n ⁢ ⁢ { K i ⁢ ⁢ 1 K i ⁢ ⁢ 2 ⋮ K il } , { BI i ⁢ ⁢ 1 BI i ⁢ ⁢ 2 ⋮ BI im } ⇒ { C 1 C 2 ⋮ C m } Where the symbol BI represents the biological index, e.g. activity level in a specific tissue, the symbol C is the coefficient that results from the mapping, the index n refers to the number of samples, the index m refers to the number of different biological activities related to the particular conformational state (e.g., in different tissues), and the index 1 refers to the number of K values (extent of the signature). Once the set of coefficients C (which could be as small as one, when only one biological activity level is relevant) is calculated, it could be used in reverse to predict the set of biological activity levels, BI, from the measured signature for an unknown compound, represented by its K values according to the reverse mapping:
{ K 1 K 2 ⋮ K l } , { C 1 C 2 ⋮ C m } ⇒ { BI 1 BI 2 ⋮ BI m } Other techniques to condense the information space provided by the signatures are available. For example, matrix rotation and projection techniques are well known to those skilled in the art. Using principal component analysis and/or singular value decomposition, the information embedded in the signature can be optimally rotated and presented in, e.g., a set of eigenvalues of decreasing values corresponding to the principal components (eigenvectors). These eigenvalues could be expressed numerically or visually for each signature.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in � C. or is at ambient temperature, and pressure is at or near atmospheric pressure.
EXPERIMENTAL EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in � C. or is at ambient temperature, and pressure is at or near atmospheric.
Example 1 In this example, the signature of a structural state of a biomolecule was created. The signature created can be shown numerically and visually. Binding of different ligands induced different conformational changes, which were then monitored to obtain signatures.
PEG/Phosphate
Dex-Ficoll
Dex-Ficoll-NaSCN
1.796 � 0.005
0.836 � 0.003
0.451 � 0.002
0.180 � 0.008
1.858 � 0.016
0.812 � 0.006
0.468 � 0.002
0.196 � 0.001
1.714 � 0.012
0.823 � 0.018
0.420 � 0.001
0.103 � 0.001
Bromoethanol
1.904 � 0.013
0.848 � 0.009
0.486 � 0.005
0.183 � 0.001
This example illustrates that the use of a series of different aqueous two-phase systems significantly improves our ability to describe the conformational changes induced in a protein by binding of different ligands. Different systems to be used for the purpose of the present invention, as shown in this example, may include those formed by a single polymer and inorganic salt(s), by different pairs of polymers, and by the same pair of polymers and different salt additives. The analytical assay technique used for the determination of the receptor concentration may also be different�in this case using fluorescamine-based assay provided results identical to those obtained with direct absorbance measurements at �280 nm.
Example 2 In this example, it was demonstrated how to use a structural signature to discern between specific versus non-specific binding of ligands to a biomolecule by observing the structural signatures of the conformational state that is induced by such binding.
The aqueous two-phase system contained 12.33 wt. % Dextran-64 (molecular weight of about 64,000), 6.05 wt. % PEG-6000 (molecular weight of about 6,000), 0.15M NaCl, and 0.01 M sodium phosphate buffer (pH 7.4). Each system was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed by liquid handling workstation Hamilton H-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 800 μL. A varied amount (50, 100, 150, and 200 μl) of the lysozyme solution or that of a mixture of lysozyme with a ligand and the corresponding amount (150, 100, 50, and 0 μl) of water were added to a system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 30 min. at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. Tubes were taken out of the centrifuge, and aliquots of 100 μl for direct spectrophotometric analysis from the top and the bottom phases were withdrawn in duplicate and each diluted and mixed with appropriate reagents as indicated below and used for further analysis performed as described below.
The other aqueous two-phase system contained 8.88 wt. % Dextran-64 (molecular weight of about 64,000), 12.86 wt. % Ficoll-400 (molecular weight of about 400,000) and 0.01 M sodium phosphate buffer (pH 8.6). Each system was prepared by mixing the appropriate amounts of stock polymer and buffer solutions dispensed by liquid handling workstation Hamilton H-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 820 μL. A varied amount (40, 80, 120, and 160 μl) of the lysozyme solution or that of a mixture of lysozyme with a ligand (140, 100, 60, and 20 μl) of water were added to each system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 30 min. at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. The tubes were taken out of the centrifuge, aliquots of a given volume (100 μl for direct spectrophotometric analysis and 150 μl for fluorescence analysis) were removed from the top and the bottom phases in duplicate and each aliquot was diluted and mixed with appropriate reagents and used for further analysis performed as described below.
PEG-3350-
Dex-Ficoll-
2.728 � 0.010
2.278 � 0.006
0.890 � 0.007
1.239 � 0.008
2.645 � 0.025
2.119 � 0.035
0.829 � 0.005
1.206 � 0.024
2.848 � 0.028
2.311 � 0.022
0.811 � 0.005
1.236 � 0.012
2.860 � 0.035
2.402 � 0.002
0.818 � 0.003
1.214 � 0.010
2.563 � 0.032
1.311 � 0.013
0.826 � 0.001
1.177 � 0.009
N,N′,N″-
2.033 � 0.052
0.461 � 0.006
0.803 � 0.004
1.106 � 0.007
In this particular representation of the signature, non-specific binding events were recognized as those which do not result in significant visual (or numerical) deviations from the signature of the free receptor alone, while the three ligands that are known to be of an increased degree of specificity (from N to N,N′ to N,N′,N″, respectively) produced signature that increasingly deviate from that of the free receptor. However, by using the graphical representations of the signature it was also easy to determine with component of the signature did not provide information useful to the providing a useful signature. For a signature to be useful, it must provide one-to-one correspondence with the underlying information, even if the basis by which the underlying information gives rise to the signature is not known. Thus, if we plot, e.g., the variation of the components of all the signatures for each system separately (FIG. 3), it becomes evident that the last two systems (Dex-Ficoll-NaSCN and Dex-PEG) are less useful for discerning structural differences amongst the different ligands.
Example 3 In this example, it was shown how sensitive conformational information can be obtained using a signature and how different states of the structure of biomolecules can be compared against a reference state. It was also demonstrated how to condense the difference between the signatures of a different conformation into a simple numerical value. Further, several representative applications for such condensed information sets which reflect differences between signatures are disclosed.
The partition coefficients for the examined proteins are given in Table 3. The data presented in Table 3 indicate clearly that there is a difference in the partitioning of apo-transferrin and transferrins saturated with different metals. The differences observed are due to different conformations of transferrins induced by binding of different metals. The data given in Table 3 also show that the changes in the partition coefficient value of a protein induced by binding of a binding partner depend on the particular two-phase system employed, although the changes are observed in any of the systems employed.
Table 3. Partition coefficients of human apo-transferrin and different metal-transferrins in different aqueous two-phase systems
Fe3+ Al3+ Cu2+ Bi3+ Ca2+ I
1.068 � 0.021
0.655 � 0.004
0.692 � 0.001
0.717 � 0.019
0.749 � 0.007
0.759 � 0.005
1.305 � 0.027
1.134 � 0.014
1.038 � 0.003
1.104 � 0.012
1.121 � 0.001
1.045 � 0.002
1.232 � 0.008
1.152 � 0.004
0.966 � 0.010
1.075 � 0.003
1.056 � 0.002
1.012 � 0.007
0.467 � 0.013
0.514 � 0.024
0.528 � 0.001
0.537 � 0.002
0.514 � 0.002
0.570 � 0.003
0.296 � 0.002
0.319 � 0.003
0.419 � 0.009
0.345 � 0.007
0.294 � 0.003
0.321 � 0.002
0.343 � 0.004
0.403 � 0.019
0.350 � 0.007
0.401 � 0.004
1.138 � 0.020
0.961 � 0.015
1.274 � 0.018
1.068 � 0.018
0.446 � 0.015
0.243 � 0.015
0.488 � 0.005
0.459 � 0.009
I - Dex-Ficoll-buffer, pH 8.6;
II - Dex-Ficoll-Na2SO4-buffer, pH 8.6;
III - Dex-Ficoll-Li2SO4-buffer, pH 8.6;
IV - Dex-Ficoll-CsCl-buffer, pH 8.6;
V - Dex-Ficoll-NaClO4-buffer, pH 8.6;
VI - Dex-Ficoll-NaSCN-buffer, pH 8.6;
VII - Dex-PEG-buffer, pH 7.01;
VIII - Dex-PVP-buffer, pH 7.4
The data presented in Table 3 represent a structural signature corresponding to each ligand. This data could be visually displayed in several ways, some of which were outlined in the previous examples. Sometimes it is convenient to further reduce the complexity of the signature itself. One way to accomplish signature condensation is to calculate a (normalized) Euclidian distance for each signature versus a reference case. In the following we chose to describe the distance between each conformational state corresponding to each ligand, against that of the apo-Tf signature. One formula for calculating such distance is:
d i , j = ∑ k = 1 n ⁢ ( c j , k max ⁡ ( c , k ) - c i , k max ⁡ ( c , k ) ) 2 Where the distance is calculated between any signature j and the reference signature i, for n aqueous systems. Applying such distance measurement to the first 3 ligands in Table 4 produces the following data:
Fe3+ to apo-Tf: 0.62 Al3+ to apo-Tf: 0.54 Cu2+ to apo-Tf: 0.64 The above data could be interpreted individually for each ligand as a measure of the overall similarity between the conformation induced by that ligand to that of the free receptor. Other data transformation and condensation methods could be readily devised, depending on the ultimate use of the signature. Thus, for example, if one wishes to rapidly compare the similarity of one ligand-induced conformation to another, or versus a reference state which might be that for a ligand whose biological activity is known, then the distance measure is one convenient way to express the similarity in a compact manner. Another application could be the use of the similarity distance to assess how close the signature of a particular isoform or a modified form or a biomolecule (e.g., with a single point mutation) to that of the intact biomolecule. Yet another possibility to use the distance measure of a signature similarity is to conveniently compare lots of microheterogeneous proteins (e.g., glycoproteins) that were produced using recombinant DNA techniques in non-mammalian host cells. In this case, the signature of each lot, representing the average conformation state of a mixture, could be readily compared against that obtained from a well-characterized lot of known biological activity level.
Example 4 In this example, it is shown how small differences in the primary structure of biomolecules, if they lead to significant changes in the conformation, can be detected using a signature.
β-Lactoglobulin A and β-lactoglobulin B from bovine milk were purchased from Sigma Chemical Company (St. Louis, Mo., USA) and used without further purification. The proteins differ by two amino acid residues�β-lactoglobulin A has Asp-residue in position 64 and Val-residue in position 118, while β-lactoglobulin B has Gly-residue in position 64 and Ala-residue in position 118. Stock solutions of β-lactoglobulin A and β-lactoglobulin B in water were prepared at the protein concentration of 5.0 mg/ml. Stock solution of β-lactoglobulin A and stock solution of β-lactoglobulin B were added to an aqueous two-phase system as described below.
The aqueous two-phase system contained 12.33 wt. % Dextran-64 (molecular weight of about 64,000), 6.05 wt. % PEG-6000 (molecular weight of about 6,000), 0.15M NaCl, and 0.01 M sodium phosphate buffer (pH 7.4). Each system was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed by liquid handling workstation Hamilton ML-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 800 μL. A varied amount (50, 100, 150, and 200 μl) of the each protein solution and the corresponding amount (150, 100, 50, and 0 μl) of water were added to a system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 30 min at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. Tubes were taken out of the centrifuge, and aliquots of 100 μl for direct spectrophotometric analysis from the top and the bottom phases were withdrawn in duplicate and each diluted and mixed with appropriate reagents as indicated below and used for further analysis performed as described below.
The other aqueous two-phase system contained 12.33 wt. % Dextran-64 (molecular weight of about 64,000), 6.05 wt. % PEG-6000 (molecular weight of about 6,000), and 0.11 M sodium phosphate buffer (pH 7.4). Each system was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed by liquid handling workstation Hamilton ML-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 800 μL. A varied amount (50, 100, 150, and 200 μl) of the each protein solution and the corresponding amount (150, 100, 50, and 0 μl) of water were added to a system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 30 min. at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. Tubes were taken out of the centrifuge, and aliquots of 100 μl for direct spectrophotometric analysis from the top and the bottom phases were withdrawn in duplicate and each diluted and mixed with appropriate reagents as indicated below and used for further analysis performed as described below.
The other aqueous two-phase system contained 12.33 wt. % Dextran-64 (molecular weight of about 64,000), 6.05 wt. % PEG-6000 (molecular weight of about 6,000), 0.1M Li2SO4, and 0.01 M sodium phosphate buffer (pH 7.4). Each system was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed by liquid handling workstation Hamilton ML-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 800 μL. A varied amount (50, 100, 150, and 200 μl) of the each protein solution and the corresponding amount (150, 100, 50, and 0 μl) of water were added to a system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 30 min. at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. Tubes were taken out of the centrifuge, and aliquots of 100 μl for direct spectrophotometric analysis from the top and the bottom phases were withdrawn in duplicate and each diluted and mixed with appropriate reagents as indicated below and used for further analysis performed as described below.
β-lactoglobulin B
PEG-600-NaPB
3.596 � 0.010
1.840 � 0.006
Dex-PEG-NaCl-NaPB
0.048 � 0.005
0.067 � 0.003
Dex-PEG-NaPB
0.097 � 0.011
0.123 � 0.007
Dex-PEG-Li2SO4-NaPB
0.748 � 0.015
0.404 � 0.011
Dex-PEG-NaSCN-NaPB
0.096 � 0.002
0.167 � 0.013
Example 5 In this example, it is shown how small differences in the primary structure of biomolecules, if they lead to significant changes in the conformation, can be detected using a signature.
Human recombinant insulin was purchased from ICN Biomedicals Inc. (Aurora, Ohio, USA), bovine and porcine insulins were purchased from Calbiochem Corporation (San Diego, Calif., USA) and used without further purification. The proteins differ by two or three amino acid residues�human insulin has Thr-residue in position 8 and Ile-residue in position 10 in the A-chain, and Thr-residue in position 30 in the B-chain, while porcine insulin differs from human insulin by Ala-residue in position 30 in the B-chain, and bovine insulin has Ala-residue in position 8 and Val-residue in position 10 in the A-chain, and Ala-residue in position 30 in the B-chain. Stock solutions of all insulins in 0.009M HCl were prepared and titrated with 0.01M NaOH to pH �6.0 with the final protein concentration of ca. 1.0 mg/ml. Stock solution of human insulin, stock solution of porcine insulin, and stock solution of bovine insulin were added to an aqueous two-phase system as described below.
The other aqueous two-phase system contained 13.67 wt. % Dextran-69 (molecular weight of about 69,000), 18.34 wt. % Ficoll-70 (molecular weight of about 70,000), 0.33M NaSCN, and 0.01 M sodium phosphate buffer (pH 7.4). Each system was prepared by mixing the appropriate amounts of stock polymer, salt, and buffer solutions dispensed by liquid handling workstation Hamilton ML-2200 into a microtube of a total volume of 1.2 mL up to a total volume of a system of 880 μl. A varied amount (0, 24, 48, 72, 96, and 120 μl) of each insulin solution and the corresponding amount (120, 96, 72, 48, 24, and 0 μl) of water were added to a system. The ratio between the volumes of the two phases of each system of a final volume of 1.00 mL was as 1:1. The system was vigorously shaken and centrifuged for 40 min. at about 4000 rpm in a centrifuge with a bucket rotor to speed the phase settling. Tubes were taken out of the centrifuge, and aliquots of 50 μl from the top and the bottom phases were withdrawn in duplicate and each diluted and mixed with appropriate reagents as indicated below and used for further analysis performed as described below.
Human Insulin Bovine Insulin Porcine Insulin System K-value K-value K-value Dex-Ficoll-NaCl- 1.262 � 0.010 1.512 � 0.012 1.350 � 0.015 NaPB Dex-Ficoll-NaSCN- 0.849 � 0.014 0.968 � 0.005 0.911 � 0.006 NaPB Dex-PEG-NaClO4- 0.776 � 0.019 0.702 � 0.024 0.656 � 0.020 NaPB Dex-PEG-NaSCN- 1.898 � 0.022 1.848 � 0.021 1.514 � 0.019 NaPB This example illustrates that small differences in the protein 3-D structure corresponding to human, bovine, and porcine insulins, are displayed via different partitioning behavior pattern when a set of different two-phase systems is used. In a manner analogous to the previous examples, the signature could also be visualized, if so desired. Since structural variations might exist within each insulin sample, the use of a signature comprised of information obtained from multiple systems provides a much more robust means to reliably distinguish or classify unknown samples.
Example 6 This example shows how the conformational signature can be used to assess the biological activity of an unknown drug, using signatures of known drugs as reference cases.
Sometimes, in the course of discovery of new drugs for a known target (e.g., a receptor), there are one or more previous drugs already available, with well-characterized biological efficacy profiles. Assuming that the biological activity of a receptor is reflected in its conformational state, then the problem at hand is how to use conformational information that is already available for previous drugs (ligands) for the same receptor to rapidly evaluate the anticipated biological activity of a new drug candidate. More generally, the question is how to predict a profile of conformationally-related bio-activities of a new drug candidate, if such profiles already exist for other drugs. Examples of conformationally-related bio-activities are biological activity level and toxicity, the latter expressed as a consequence of undesired binding to other receptors that produce conformational states that activate undesired biological activities. This particular embodiment is of significant value for analysis of many classes of receptors that undergo multiple conformational changes in response to bindings to different drugs. For example, many transcription factors, e.g., estrogen receptor, are known to exhibit multiple conformational states in response to binding to different estrogens. The discovery of new estrogens that modulate the estrogen receptor is of great current interest since it is widely recognized that the intermediate conformations are of practical interest for discovering new estrogens that exhibit favorable biological activity profile in various tissues in the body. Thus, instead of merely �turning on� the receptor upon binding, many drugs could be tailored to induce specific conformational states that could result in improved bio efficacy.
System Drug A B C D Activity Level X 1 0.3 0.6 1 10 Y 0.5 0.6 1 0.7 0 Z (unknown) 0.7 1 0.8 0.9 ? Using, e.g., the formula in Example 4, the distance of the signature of Z to the two known drugs, X and Y is:
Z-X:
Z-Y:
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