US 6,569,631 B1
`(10) Patent No.:
`a2 United States Patent
`Pantoliano etal.
`(45) Date of Patent:
`*May 27, 2003
`
`
`US006569631B1
`
`(54) MICROPLATE THERMAL SHIFT ASSAY
`FOR LIGAND DEVELOPMENTUSING5-(4"-
`DIMETHYLAMINOPHENYL)-2-(4'-PHENYL)
`OXAZOLE DERIVATIVE FLUORESCENT
`DYES
`
`(75)
`
`Inventors: Michael W. Pantoliano, Avondale, PA
`(US); Eugenio C. Petrella, Wayne, PA
`(US); F. Raymond Salemme, Yardley,
`PA (US); Barry A.Springer,
`Wilmington, DE (US)
`
`(73) Assignee: 3-Dimensional Pharmaceuticals, Inc.,
`Yardley, PA (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`USC. 154(b) by 0 days.
`This patent is subject to a terminal dis-
`claimer.
`
`9
`.
`(21) Appl. No.: 09/438,357
`(22)
`Filed:
`Noy. 12, 1999
`
`(60)
`
`Related U.S. Application Data
`Provisional application No. 60/108,085, filed on Nov. 12,
`1998.
`(51) Unt. C1 cece GOIN 33/53; GOIN 21/76
`(52) US. Che accccccccccsscssesssscsecee 435/7.1; 435/7.4; 435/4;
`435/DIG. 1: 435/DIG. 14; 435/DIG.41:
`” 436/172: 436/501: 436/518
`(58) Field of Search uo... 435/4, 7.1, 7.4,
`435/DIG. 1, DIG. 12, DIG. 41, DIG. 14;
`436/501, 518, 172
`
`(56)
`
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`Primary Examiner—Padmashri Ponnaluri
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`& Fox P.L.L.C.
`
`U.S. PATENT DOCUMENTS
`
`(57)
`
`ABSTRACT
`
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`.
`
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`The present invention provides a method for ranking the
`affinity of each of a multiplicity of different molecules for a
`target molecule which is capable of unfolding due to a
`th
`1
`ch
`Th
`thod
`.
`tactine
`th
`ermal change.
`The method
`comprises (a) contacting
`the
`target molecule with one molecule of a multiplicity of
`different molecules,
`in the presence of a 5-(4"-
`dimethylaminopheny-2-(4'-phenyl)oxazole derivative dye,
`in each of a multiplicity of containers; (b) simultaneously
`heating the multiplicity of containers; (c) measuring the
`fluorescence in each of the containers; (d) generating ther-
`mal unfolding information for the target molecule as a
`function of temperature for each of the containers;
`(¢)
`:
`:
`.
`:
`RG
`comparing the thermal unfolding information obtained for
`each of the containers tothe thermal unfolding information
`for the target molecule in the absence of any of the mol-
`ecules in the multiplicity of different molecules, and (f)
`ranking the affinities of each of the molecules according to
`the difference in the thermal unfolding information between
`the target molecule in each of the containers and the target
`molecule in the absence of any of the molecules in the
`ulliplcity ofdifferent molecules
`12 Claims, 43 Drawing Sheets
`
`Agilent Exhibit 1253
`Page 1 of 82
`
`Agilent Exhibit 1253
`Page 1 of 82
`
`

`

`US 6,569,631 B1
`Page 2
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