Methods for high throughput chemical screening using magnetic resonance imaging

• US 6,307,372 B1
• Filed: 11/02/1999
• Issued: 10/23/2001
• Est. Priority Date: 11/02/1999
• Status: Expired due to Fees

• Current Assignee: Smithkline Beecham Corporation (Smithkline Beecham PLC)
• Original Assignee: Glaxo Wellcome Incorporated (GlaxoSmithKline PLC)
• Inventors: Sugarman, Jeffrey H., Star-Lack, Joshua M.
• Primary Examiner(s): ARANA, LOUIS M
• Application Number:
  US09/432,492
• Time in Patent Office: 721 Days
• Field of Search: 324/300, 324/321, 324/322, 324/318, 324/312, 324/314, 324/306, 324/307, 324/309, 422/67, 422/102, 436/173
• US Class Current: 324/321
• CPC Class Codes: G01N 24/085 Analysis of materials for the purpose of controlling industrial production systems
• CPC Class Codes: G01R 33/44 using nuclear magnetic resonance [NMR] G01R33/24, G01R33/62 take precedence

Abstract

A screening method is provided that comprises the steps of performing an assay in which one or more chemicals and/or entities are present in a mixture to produce an outcome. A static magnetic field B₀ is applied to the mixture, and one or more RF magnetic pulses are also applied to the mixture. Multiple assays may be conducted simultaneously through the use of magnetic resonance imaging techniques.

Claims

1. A screening method comprising:

performing an assay in which one or more chemicals and/or entities are present in a mixture to produce an outcome;

applying a static magnetic field (B₀) to the mixture;

applying one or more RF magnetic pulses to the mixture; and

measuring and evaluating resulting FID or echo signals to evaluate the outcome of the assay.

2. A method as in claim 1, further comprising performing the assay in a plate having a plurality of wells, wherein the mixture in each well includes a different chemical and/or entity, and further comprising applying spatially dependent B₀ gradient fields to the wells and measuring and evaluating FID or echo signals from the wells.

3. A method as in claim 1, wherein the mixture includes a test compound that causes a detectable nuclear magnetic resonance signal to be produced that is dependent on the degree of interaction of the test compound with the mixture.

4. A method as in claim 3, wherein the test compound comprises a test product, and wherein the entities includes one or more cells that produce the test product.

5. A method as in claim 2, wherein the different entities comprise cells which produce a test product with a chemical shift different from that of water protons.

6. A method as in claim 5, wherein the test product is selected from a group of products consisting of choline, lactate, N-acetyl-aspartate, N-acetyl-glutamate, ATP, ADP, alanine, creatine, phosphocreatine, phosphocholine, citrate, myo-inositol, methanol, and ethanol.

7. A method of screening comprising:

combining a test compound with another chemical and/or entity to form a mixture, wherein the test compound causes a detectable nuclear magnetic resonance signal to be produced that is dependent on the amount of interaction of the test compound with the mixture;

applying a static magnetic field (B₀) to the mixture;

applying one or more RF magnetic pulses to the mixture; and

measuring and evaluating resulting FID or echo signals to obtain information as to whether the test compound is biologically active in the mixture.

8. A method as in claim 7, wherein different test compounds are provided in multiple wells, and further comprising applying spatially dependent B₀ gradient fields to the wells, and measuring and evaluating the FID or echo signals from each of the wells at substantially the same time.

9. A method as in claim 8, wherein the wells are arranged in a three dimensional array.

10. A method as in claim 7, wherein the combining step further comprises combining the test compound with at least one cell in an aqueous medium, wherein the test compound is biologically active when binding to a receptor on the cell surface or within the cell to produce an enzyme that indirectly affects the relaxation properties of water protons to indicate that the test compound is biologically active.

11. A method as in claim 10, wherein the enzyme modifies the relaxation properties of water protons by cleaving a probe molecule in the mixture, thereby increasing the exposure of a paramagnetic atom in the probe molecule to water protons and decreasing the T1 relaxation rate of the water protons.

12. A method as in claim 11, wherein the enzyme comprises beta-galactosidase and the probe molecule comprises a chelated gadolinium atom complexed with a galactose molecule, whereby the galactosidase causes the galactose molecule to be cleaved from the chelated gadolinium.

13. A method as in claim 7, wherein the combining step further comprises mixing the test compound in an aqueous medium with a chemical or entity that when reacting with the test compound changes the pH of the medium, and wherein the change is measurable using nuclear magnetic resonance to indicate that the test compound is biologically active.

14. A method as in claim 13, wherein the change in pH of the aqueous medium causes a change to the ionization of polymer molecules also in the aqueous medium, thereby changing the viscosity of the aqueous medium and affecting nuclear magnetic resonance relaxation properties, magnetization transfer properties, spin density and/or the diffusion coefficient of the water protons constituting the aqueous medium.

15. A method as in claim 13, wherein the change in pH of the aqueous medium causes a change to the volume or configurations of gel particles also in the medium, thereby affecting nuclear magnetic resonance relaxation properties, magnetization transfer properties, spin density and/or the diffusion coefficient of the water protons constituting the aqueous medium.

16. A method as in claim 13, wherein the aqueous medium contains a pH-sensitive contrast agent that causes the nuclear magnetic resonance relaxation properties of water protons or other nuclei constituting the aqueous medium to be changed in a manner that is reflective of pH.

17. A method as in claim 16, wherein the contrast agent contains a lanthanide metal selected from a group consisting of Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb or another paramagnetic element selected from a group consisting of Fe, Mn or ¹⁷O.

18. A method as in claim 17, wherein the contrast agent comprises Fe(III)meso-tetra(4-sulfonatophenyl)porphine (Fe-TPPS4).

19. A method as in claim 17, wherein the contrast agent comprises H₂¹⁷O.

20. A method as in claim 13, wherein the aqueous medium comprises water protons or other nuclei, and wherein the aqueous medium contains a shift reagent affecting the chemical shift properties of the water protons or other nuclei in a manner that is reflective of pH.

21. A method as in claim 20, wherein the shift reagent contains a lanthanide metal selected from a group consisting of Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, and Yb or another paramagnetic element.

22. A method as in claim 21, wherein the shift reagent comprises Tm(DOTP)⁵⁻

, the thulium (III) complex of 1,4,7,10-tetraazacylododecane-N,N′

,N″

,N′

″

-tetra(methylenephospate).

23. A method as in claim 21, wherein the shift reagent comprises Dy(PPP)₂⁷-dysprosium tripolyphosphate.

24. A method as in claim 7, wherein the combining step further comprises placing the test compound into an aqueous medium, and wherein the chemical and/or entity is selected such that when reacting with the test compound changes the temperature of the medium, and wherein the temperature is measurable using nuclear magnetic resonance to indicate that the test compound is biologically active.

25. A method as in claim 24, wherein the aqueous medium comprises water protons, and wherein the change in temperature of the aqueous medium is reflected by a change in the nuclear magnetic resonance relaxation rates, diffusion constant or spin density of the water protons.

26. A method as in claim 24, wherein the aqueous medium comprises water protons or other nuclei, and wherein the change in temperature of the aqueous medium is reflected by a change in the chemical shift of the water protons or other nuclei.

27. A method as in claim 24, wherein the aqueous medium comprises water protons or other nuclei, and wherein the aqueous medium contains a contrast agent affecting the chemical shift properties of the water protons or other nuclei in a manner that is reflective of temperature.

28. A method as in claim 27, wherein the contrast agent contains a lanthanide metal selected from a group consisting of Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb.

29. A method as in claim 28, wherein the contrast agent comprises Pr[MOE-DO3A].

30. A method as in claim 7, wherein the entity comprises a cell in an aqueous medium, and wherein the cell is configured to produce a test product that has a chemical shift different from that of water protons to indicate that the compound is biologically active.

31. A method as in claim 30, wherein the test product comprises a molecule that is selected from a group consisting of choline, lactate, N-acetyl-aspartate, N-acetyl-glutamate, ATP, ADP, alanine, creatine, phosphocreatine, phosphocholine, citrate, myo-inositol, methanol, and ethanol.

32. A method as in claim 7, wherein the mixture includes an aqueous medium, and wherein the combining step comprises combining the test compound with a cell that when reacting with the test compound causes the concentration of one or more intracellular ions to change in a manner that is detectable by nuclear magnetic resonance.

33. A method as in claim 32, wherein the intracellular ions are selected from a group consisting of ²³Na, ⁷Li, ²⁵Mg, ³⁹K, ⁴³Ca, ⁸⁷Rb or ¹¹³Cd.

34. A method as in claim 7, wherein the mixture includes one or more cells in an aqueous medium, and wherein the aqueous medium contains a shift reagent allowing for separation of extracellular and intracellular ions by their different chemical shifts.

35. A method as in claim 34, wherein the shift reagent contains a lanthanide metal selected from a group consisting of Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb.

36. A method as in claim 35, wherein the shift reagent is selected from a group consisting of Dy(PPP)₂⁷⁻

-dysprosium tripolyphosphate, Tm(DOTP)⁵⁻

, the thulium (III) complex of 1,4,7,10-tetraazacylododecane-N,N′

,N″

,N′

″

-tetra(methylenephospate), Dy(DPA)₃³⁻

-dysprosium dipicolinate pyridine-2,6-dicarboxylate, Dy(NTA)₂³⁻

, dysprosium nitrilotriacetate, Dy(TTHA)³⁻

-dysprosium triethylenetetraminehexaacetate, Tm(PPP)₂⁷⁻

, and Tm(TTHA)³⁻

.

37. A method as in claim 34, wherein the extracellular and intracellular ions are differentiated by their T2 relaxation properties.

38. A method as in claim 34, wherein the extracellular and intracellular ions are differentiated by the use of a multiple quantum filter.

39. A method as in claim 34, further comprising washing the extracellular ions out of the aqueous medium before nuclear magnetic resonance measurements are made.

40. A method as in claim 7, wherein the mixture includes an aqueous medium comprising water protons, and further comprising introducing a contrast agent to the aqueous medium that changes conformation upon binding to an ion in the mixture, thereby changing the relaxation properties of the water protons.

41. A method as in claim 40, wherein the contrast agent contains a lanthanide metal selected from a group consisting of Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb or other paramagnetic element selected from a group consisting of Fe and Mn.

42. A method as in claim 41, wherein the contrast agent comprises DOPTA-Gd.

43. A method of screening comprising:

providing a plurality of plates that each include multiple wells;

introducing a chemical and/or entity into at least some of the wells of each plate;

introducing a test compound into wells containing the chemical and/or entity;

stacking the plates on top of each other; and

inserting the plates into a nuclear magnetic resonance device and screening for any biologically active test compounds.