(12) Umted States Patent
`(10) Patent N0.:
`US 6,569,631 B1
`
`Pantoliano et al.
`(45) Date of Patent:
`*May 27, 2003
`
`USOO6569631B1
`
`(54) MICROPIAATE THERMAL SHIFT ASSAY
`FOR LIGAND DEVELOPMENT USING 5.(4".
`DIMETHYLAMINOPHENYL)-2-(4'—PHENYL)
`OXAZOLE DERIVATIVE FLUORESCENT
`DYES
`
`.......... 364/496
`10/1995 Agrafiotis et al.
`5,463,564 A
`3/1996 Schacher
`..................... 422/64
`5,496,519 A
`.
`(L'
`d
`1“ commue 0“ “6’“ page‘)
`FOREIGN PATENT DOCUMENTS
`
`(75)
`
`Inventors: Michael W. Pantoliano, Avondale, PA
`(US), Eugenio C. Petrella, Wayne, PA
`(US), F. Raymond Salemme, Yardley,
`PA. (95); 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
`U.S.C. 154(1)) by 0 days.
`
`This patent is subject to a terminal dis—
`claimer.
`
`3
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`0 640 828 A1
`0 770 876 A1
`W0 92/03542 A1
`W0 93/147231
`W0 94/05394
`wo 95/18969
`WO 96/23879
`W0 97/09342
`W0 97/14038
`W0 97/20952
`W0 97/42500
`W0 98/13/969 A2
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`1:;133
`3/1995
`5/1997
`3/1992
`8/1993
`3/1994
`7/1995
`8/1996
`3/1997
`4/1997
`6/1997
`11/1997
`4/1998
`
`ggggg
`$833123: A3
`OTHER PUBLICATIONS
`
`(21) Appl. NO" 09/438’357
`(22)
`Filed:
`Nov. 12, 1999
`
`(60)
`
`Related U.S. Application Data
`Provisional application No. 60/108,085, filed on Nov. 12,
`1998~
`Int. Cl.7 ......................... G01N 33/53; G01N 21/76
`(51)
`(52) US. Cl.
`............................. 435/7.1; 2135/74; 435/4;
`435/DIG. 1; 435/DIG. 14; 435/DIG. 41;
`436/172; 436/501; 436/518
`(58) Field of Search .............................. 435/4, 7.1, 7.4,
`2135/1310. 1, DIG. 12, DIG. 41, DIG. 14;
`436/501, 518, 172
`
`(56)
`
`References Cited
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`‘
`'
`Primary Examiner—Padmashri Ponnaluri
`(74) Attorney, Agent, or Firm—Sterne, Kessler, Goldstein
`& Fox P.L.L.C.
`
`(57)
`
`ABSTRACT
`
`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
`thermal 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;
`(e)
`comparing the thermal unfolding information obtained for
`each of the containers to the 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 aflinities 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
`multiplicity of different molecules.
`
`12 Claims, 43 Drawing Sheets
`
`Agilent Exhibit 1253
`
`Page 1 of 82
`
`Agilent Exhibit 1253
`Page 1 of 82
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`

`

`us 6,569,631 B1
`Page 2
`
`U.S. PATENT DOCUMENTS
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