(12) United States Patent
`Pantoliano et al.
`
`US006303322B1
`US 6,303,322 B1
`Oct. 16, 2001
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`(54) METHOD FOR IDENTIFYING LEAD
`COMPOUNDS
`
`(75) Inventors: Michael W. Pantoliano, Avondale, PA
`(US); Alexander W. Rhind,
`Libertyville, IL (US); Francis R.
`Salemme, Yardley, PA (US)
`
`(73) Assignee: 3-Dimensi0nal Pharmaceuticals, Inc.,
`EXton, PA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/478,216
`(22) Filed:
`Jan. 5, 2000
`
`Related US. Application Data
`
`(62) Division of application No. 08/853,464, ?led on May 9,
`1997, now Pat. NO. 6,020,141.
`(60) Provisional application No. 60/017,860, ?led on May 9,
`1996.
`
`(51) Int. Cl.7 ............................ .. G01N 33/53; C12Q 1/68
`(52)
`. 435/7.1; 435/6
`(58) Field of Search ......................................... .. 435/7.1, 6
`
`(56)
`
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`
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`(List continued on neXt page.)
`
`Primary Examiner—Eggerton A. Campbell
`(74) Attorney, Agent, or Firm—Sterne, Kessler, Goldstein
`& Fox, PLLC
`(57)
`
`ABSTRACT
`
`The present invention is a method for ranking the affinity of
`each of a multiplicity of different molecules for a target
`molecule Which is capable of denaturing due to a thermal
`change. The method comprises contacting the target mol
`ecule With one molecule of the multiplicity of different
`molecules in each of a multiplicity of containers, simulta
`neously heating the multiplicity of containers, measuring in
`each of the containers a physical change associated With the
`thermal denaturation of the target molecule resulting from
`the heating in each of the containers, generating a thermal
`denaturation curve for the target molecule as a function of
`temperature for each of the containers and determining a
`midpoint temperature (Tm) therefrom, comparing the Tm of
`each of the thermal denaturation curves With the Tm of a
`thermal denaturation curve obtained for the target molecule
`in the absence of any of the molecules in the multiplicity of
`different molecules, and ranking the affinities of the multi
`plicity of different molecules according the change in Tm of
`each of the thermal denaturation curves.
`
`16 Claims, 40 Drawing Sheets
`
`THERMO FISHER EX. 1005
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`

`
`US 6,303,322 B1
`Page 2
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`

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`Page 3
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`
`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 1 of 40
`
`US 6,303,322 B1
`
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`THERMO FISHER EX. 1005
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 2 of 40
`
`US 6,303,322 B1
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`THERMO FISHER EX. 1005
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`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 3 of 40
`
`US 6,303,322 B1
`
`TEMPERATURE (°c)
`
`TH + 309 4026 0 57,111
`Tm ~ 65.8 mm = 3.4)
`Kd ~ 6.5 nM 0 310°K
`
`15 104
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`TEMPERATURE (°1<)
`
`THERMO FISHER EX. 1005
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`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 4 0f 40
`
`US 6,303,322 B1
`
`TEMPERATURE ("0)
`6O
`55
`5O
`35
`4O
`45
`30
`25
`||l|||TlTT||l||||l||l|l||||||||||||||||
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`—-— HS_5000
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`+ 134 [LM ANS
`Tm ~ 59.7:0.5°c \
`
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`‘ 3
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`Kd ~ 4.9 11M 0 298°K
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`
`6 (TI 0 |E|l|||l|||1||||||||1
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`yo.) : yu+
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`(Y/ " Yu)
`
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`
`lIIIIIIIIIIIIIIIIIIIIIIIIllllllllllIlll
`295
`500
`505
`310
`515
`520
`525
`550
`555
`TEMPERATURE (°K)
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 5 0f 40
`
`US 6,303,322 B1
`
`TEMPERATURE ('c)
`
`_
`
`-
`
`j
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`[FACTOR 0] = 4 0M IN 10 uL DROPS(LID DIMPLES);
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`325
`330
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`I
`
`|
`
`335
`
`TEMPERATURE ('K)
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`061. 16, 2001
`
`Sheet 6 6f 40
`
`US 6,303,322 B1
`
`TEMPERATURE ('c)
`
`I6I5
`
`I
`
`I
`
`I I7I5 I
`
`I
`
`I I8I0
`
`+FXO
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`68 pg/mL
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`Trn =337.9 1 0.3 ‘K
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`
`_________~___________ 0 AU 0 AU 0
`
`0
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`320
`325
`330
`335
`340
`TEMPERATURE ('K)
`
`skémaédm
`355
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 7 of 40
`
`US 6,303,322 B1
`
`TEMPERATURE ('0)
`
`40
`
`45
`
`50
`
`55
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`65
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`1,8 ANS]=100 MM
`3DP-4660]=50 MM
`BUFFER=50 mM Hepes, pH 7.5
`0.1 M NoC|
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`Tm =3”-9 * 0-4 K
`
`Tm =332.2 : o.5°1<
`
`Kd @ Tm = 15 nM
`
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`
`300
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`310
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`325
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`330
`
`335
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`340
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`TEMPERATURE ('1<)
`
`F|G.7
`
`THERMO FISHER EX. 1005
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 8 of 40
`
`US 6,303,322 B1
`
`TEMPERATURE ('0)
`
`25
`
`30
`
`35
`
`40
`
`45
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`50
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`1400
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`[LlCAND]=300uM
`BUFFER=50 mM Hepes, pH 7.5,
`01 M NoC|
`
`Tm =311.6 t 0.1
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`
`295
`
`300
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`THERMO FISHER EX. 1005
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 9 of 40
`
`US 6,303,322 B1
`
`20
`
`30
`
`40
`
`TE_MPERATURE, (°c)
`50
`so
`70
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`
`90
`
`100
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`—-o— pH 7/0.1 1111131
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`(IN 95 WELL
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`[Urokinase/CMK]=74 pM
`[L8 ANS]=200 p.M
`
`BUFFER=50 mM MOPS pH 7.0,
`100 mM NaCl
`
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`
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`
`310
`
`320
`
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`340
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`350
`
`350
`
`370
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`TEMPERATURE.
`
`(°K)
`
`9
`
`20
`
`30
`
`TEMPERATURE.
`40
`
`(°c)
`50
`
`60
`
`70
`
`MEAN = 322.75 ‘'11; s.d. +/— 0.90
`
`——o— 2uL
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`3
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` (49.s1°c) RELATIVEFLUORESCENCE,0460nm
`
`.300
`
`310
`
`320
`
`530
`
`340
`
`TEMPERATURE, CK)
`
`1 0
`
`THERMO FISHER EX. 1005
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 10 0f 40
`
`US 6,303,322 B1
`
`TEMPERATURE, ("0)
`7o
`60
`40
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`
`320
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`TEMPERATURE, (°K)
`
`330
`
`340
`
`FIG.11
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 11 0f 40
`
`US 6,303,322 B1
`
`2.8.82 0.8 @ mozmommg:
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`FIG. 1 2
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 12 0f 40
`
`US 6,303,322 B1
`
`45
`
`TEMPERATURE ('C)
`50
`55
`
`60
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`65
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`
`335
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`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 13 0f 40
`
`US 6,303,322 B1
`
`TEMPERATURE ('c)
`5O
`55
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`
`335
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`340
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`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`0a. 16, 2001
`
`Sheet 14 0f 40
`
`US 6,303,322 B1
`
`
`
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`TEMPERATURE, "K
`
`__.____________________
`
`THERMO FISHER EX. 1005
`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 15 of 40
`
`1
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 16 of 40
`
`US 6,303,322 B1
`
`a
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`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 17 of 40
`
`US 6,303,322 B1
`
`10
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 18 of 40
`
`US 6,303,322 B1
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 19 of 40
`
`US 6,303,322 B1
`
`F|G.2O
`
`TEMPERATURE,"K
`
`325
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`320
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 20 of 40
`
`US 6,303,322 B1
`
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 21 of 40
`
`US 6,303,322 B1
`
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`
`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 22 of 40
`
`US 6,303,322 B1
`
`THERMO FISHER EX. 1005
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`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 23 of 40
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`US 6,303,322 B1
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`U.S. Patent
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`Oct. 16, 2001
`
`Sheet 24 of 40
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`US 6,303,322 B1
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`U.S. Patent
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`Oct. 16, 2001
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`Sheet 25 of 40
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`US 6,303,322 B1
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`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 26 of 40
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`US 6,303,322 B1
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`H-H-H—H-H-H-
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`US 6,303,322 B1
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`U.S. Patent
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`Oct. 16, 2001
`
`Sheet 28 of 40
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`US 6,303,322 B1
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 29 of 40
`
`US 6,303,322 B1
`
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 30 of 40
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`US 6,303,322 B1
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`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 31 of 40
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`US 6,303,322 B1
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 32 of 40
`
`US 6,303,322 B1
`
`ZQQQM
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 33 of 40
`
`US 6,303,322 B1
`
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`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 34 of 40
`
`US 6,303,322 B1
`
`HEAT CONDUCIING
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`
`THERMO FISHER EX. 1005
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`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 35 of 40
`
`US 6,303,322 B1
`
`3370?.
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`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 36 of 40
`
`US 6,303,322 B1
`
`START
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`3302
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`START TEMP
`
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`3806
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`OVER SAMPLE OR
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`ROW OF SAMPLES
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`TRANSMIT
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`EXCITATORY
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`LIGHT
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`THERMO FISHER EX. 1005
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`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 37 of 40
`
`US 6,303,322 B1
`
`r)90O
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`3902
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`PROFILE
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`
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`
`TEMP
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`
`3916
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`THERMO FISHER EX. 1005
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`THERMO FISHER EX. 1005
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`

`
`U.S. Patent
`
`Oct. 16, 2001
`
`Sheet 38 of 40
`
`US 6,303,322 B1
`
`
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