(12) United States Patent
`Pant0lian0 et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,569,631 B1
`*May 27, 2003
`
`US006569631B1
`
`(54) MICROPLATE THERMAL SHIFT ASSAY
`FOR LIGANI) DEVELOPMENT USING 5_(4"_
`DIMETHYLAMINOPHENYL)-2-(4'-PHENYL)
`OXAZOLE DERIVATIVE FLUORESCENT
`DYES
`
`5,463,564 A 10/1995 Agra?otis et al. ........ .. 364/496
`5,496,519 A
`3/1996 Schacher ................... .. 422/64
`(List Continued on next page)
`'
`FOREIGN PATENT DOCUMENTS
`
`(75) Inventors: Michael W. Pant0lian0, Avondale, PA
`(US); Eugenio C. Petrella, Wayne, PA
`_
`(US), F. Raymond Salemme, Yardley,
`PA_ (Us); Barry A- SPnnger>
`Wilmington, DE (Us)
`
`(73) Assignee: 3-Dimensi0nal 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(k)) by 0 days'
`
`EP
`EP
`EP
`EP
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`W0
`
`0 512 334 B1
`0 613 007 A2
`0 640 828 A1
`0 770 876 A1
`WO 92/03542 A1
`W0 93/14781
`WO 94/05394
`W0 95/153969
`WO 96/23879
`WO 97/09342
`W0 97/ 14038
`W0 97/20952
`WO 97/42500
`WO 98/15969 A2
`
`11/1992
`8/1994
`3/1995
`5/1997
`3/1992
`8/1993
`3/1994
`7/1995
`8/1996
`3/1997
`4/1997
`6/1997
`11/1997
`4/1998
`
`This patent is subject to a terminal dis
`claimer.
`
`(21) App1'NO"09/438’357
`(22) Filed;
`Nov, 12, 1999
`
`Related US. Application Data
`(60) Provisional application No. 60/108,085, ?led on Nov. 12,
`1998-
`(51) Int. c1.7 ....................... .. G01N 33/53; G01N 21/76
`(52) US. Cl. ........................... .. 435/7.1; 435/7.4; 435/4;
`435/DIG_ 1; 435/DIG_ 14; 435/DIG_ 41;
`
`A3
`OTHER PUBLICATIONS
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`evolutionary relationships among three modular proteins
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`Escherichia COli K—12,” Science 277:1453—1462, American
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`
`(58) Field of Search ............................ .. 435/4, 7.1, 7.4,
`435/DIG. 1, DIG. 12, DIG. 41, DIG. 14;
`436/501, 518, 172
`
`(56)
`
`References Cited
`
`(List Continued on next page)
`
`Primary Examiner—Padmashri Ponnaluri
`(74) Attorney, Agent, or Firm—Sterne, Kessler, Goldstein
`& Fox P.L.L.C.
`
`U.S. PATENT DOCUMENTS
`
`(57)
`
`ABSTRACT
`
`4/1986 Patel ......................... .. 356/39
`4,580,895 A
`4,626,684 A 12/1986 Landa """" "
`250/328
`2 15/
`glarlgetnket atl'
`4356/47‘
`4’778’763 A lojlgss Mzkiiigiciieetaa'lt"x
`43447
`4j859:609 A
`8/1989 Dull et at ______ __
`436/501
`4,963,263 A 10/1990 Kauvar .... ..
`210/635
`5,096,807 A
`3/1992 Leaback
`435/6
`5,133,866 A
`7/1992 Kauvar .... ..
`210/635
`5,200,504 A
`4/1993 Ghadiri
`530/304
`52177869 A
`6/1993 Kauvar ~~~~ ~~
`- 435/79
`5’255’976 A 10/1993 connelly ' ' ' ' ' ' ' ' ' '
`' ' ' " 374/31
`gantt?h?lnotetlal' "
`435/79
`4/1994 KZLVZI
`436/43
`5/1994 WeyrauCh et at
`6/1994 Kawase et al. .......... .. 435/794
`
`5’300’425 A
`5:314:825 A
`5,324,635 A
`
`The present invention provides a method for ranking the
`af?nity 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 meleeilles, 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
`?uorescence 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 H101
`
`
`ecules in the multiplicity of different molecules; and 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
`hiitipiieity ei eieeeht heieeiiee
`
`
`
`6/1994 Fratantoni et a1, 8/1994 Kauvar et al.
`
`
`
`5,325,295 A 5,338,659 A
`
`
`364/413_08 ...... .. 435/7.1
`436/172
`8/ 1994 Eden -------------- -
`5,340,747 A
`~~ 356/317
`53557215 A 10/1994 Schroeder 6i a1~
`5,356,784 A 10/1994 Kauvar ..................... .. 435/7.9
`21333123? 2
`111332 2235551211: """" ":11: 38/232
`
`5,415,839 A
`5,436,718 A
`
`5/1995 Zaun et al. . . . . . . .
`. . . .. 422/64
`7/1995 Fernandes et a1. .......... .. 356/73
`
`12 Claims, 43 Drawing Sheets
`
`THERMO FISHER EX. 1027
`
`

`
`US 6,569,631 B1
`Page 2
`
`US. PATENT DOCUMENTS
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`4/1996 Potter et a1. ................. .. 435/4
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`6/1996 Danssaert et a1.
`422/99
`5,525,300 A
`9/1996 Wechsler et a1.
`356/318
`5,557,398 A
`5,567,317 A 10/1996 Kauvar ........... ..
`210/635
`5,585,277 A 12/1996 Bowie et a1. ..
`436/518
`5,587,293 A 12/1996 Kauvar et a1. ........... .. 435/7.21
`5,589,351 A 12/1996 Harootunian .............. .. 435/29
`5,599,504 A
`2/1997 Hosoi et al.
`422/82.08
`5,620,901 A
`4/1997 Kauvar ....... ..
`436/518
`5,631,734 A
`5/1997 Stern et al.
`356/317
`5,679,582 A 10/1997 Bowie et al. ............. .. 436/518
`6,020,141 A
`2/2000 Pantoliano et a1. ........ .. 435/7.1
`6,036,920 A
`3/2000 Pantoliano et a1. ..
`422/67
`6,214,293 B1
`4/2001 Pantoliano et a1. ..
`422/67
`6,232,085 B1
`5/2001 Pantoliano et a1. ..
`435/7.1
`6,268,158 B1
`7/2001 Pantoliano et a1. ..
`.. 435/7.1
`6,268,218 B1
`7/2001 Pantoliano et a1. ..
`436/86
`6,291,192 B1
`9/2001 Pantoliano et a1. ..
`435/7.1
`6,303,322 B1
`10/2001 Pantoliano et a1. ........ .. 435/7.1
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