US 20160282360A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0282360 A1 Dart et al. (43) Pub. Date: Sep. 29, 2016 (54) LUCIFERASE-BASED THERMAL SHIFT Publication Classification ASSAYS (51) Int. Cl. (71) Applicant: Promega Corporation, Madison, WI GOIN 33/68 (2006.01) (US) GOIN 2L/64 (2006.01) GOIN 33/58 (2006.01) (72) Inventors: Melanie Dart, Madison, WI (US); (52) U.S. Cl. Lance P. Encell, Fitchburg, WI (US); CPC ........ G0IN 33/6845 (2013.01); G0IN 33/581 Thomas Kirkland, Atascadero, CA (2013.01); G0IN 33/582 (2013.01); C12Y (US); Thomas Machleidt, Madison, WI 113/12007 (2013.01); G0IN 21/6428 (US); Matthew Robers, Madison, WI (2013.01); G0IN 202 1/6432 (2013.01); G0IN (US); Brock F. Binkowski, Sauk City, 2333/90241 (2013.01); G0IN 2500/04 WI (US); Keith Wood, Mt. Horeb, WI (2013.01); G0IN 202 1/6439 (2013.01); G0IN (US); Ce Shi, San Luis Obispo, CA 2500/10 (2013.01) (US) (22) Filed: Feb. 5, 2016 O O Provided herein are systems and methods for characterizing Related U.S. Application Data target/ligand engagement. In particular, luciferase-labeled (60) Provisional application No. 62/112,518, filed on Feb. polypeptide targets are used to detect or quantify target/ 5, 2015. ligand engagement (e.g., within a cell or cell lysate). Patent Application Publication Sep. 29, 2016 Sheet 1 of 64 US 2016/0282360 A1 FG. A he a A is So lite Cei Count 44: 86 4.8 8 52 5 4 56 S 8 SO 62 64, 66 68 e in pe atti e C x Ces w8- Centrifuge d Ce Peiet Patent Application Publication Sep. 29, 2016 Sheet 2 of 64 US 2016/0282360 A1 FIG. B He la Celi Wiability i 25 7 5 5 25 4 S 8 SO S S A SS S 8 6 62 62. 33 8 Temperature C w8x Ces *8- Centrifuged Ceil Peilet Patent Application Publication US 2016/0282360 A1 Patent Application Publication Sep. 29, 2016 Sheet 4 of 64 US 2016/0282360 A1 FIG. 2A Therm a Stability of Warious Nuc Fusions live He la Ces 5. slot fused Nic Prix: C. K. 2...Nc *x* R-N3.c 25 was K. R. -8- SRCN: c are EZ-2-3 (t s * 8. C. CAC 53 25 42 44 46 48 so S2 S. S6 S8 s s2 s SS temperature C Patent Application Publication Sep. 29, 2016 Sheet 5 of 64 US 2016/0282360 A1 FIG. 2B Therm at Stability of Warious Nius-Fusions Digiton in freated He la Cei is 153 s NC-C K2 N-H fr SRC-N tic E23-NC c - AC N - BR 4 7 5 s 3 42 a 4. As 88 50 52 54. 56. 58 6 52 a 6s Temperature C Patent Application Publication Sep. 29, 2016 Sheet 6 of 64 US 2016/0282360 A1 g : g s ce y d c o s c c sa Ecs m wa were w 4W { %) & 3 A. e. a Patent Application Publication Sep. 29, 2016 Sheet 7 of 64 US 2016/0282360 A1 : ---------issy.---------, ----------&--------- s s s s ne { %) & & As 3 e 8 & Patent Application Publication US 2016/0282360 A1 Patent Application Publication Sep. 29, 2016 Sheet 10 of 64 US 2016/0282360 A1 s as {}{}} na. { %) & 8 A3e 8 Patent Application Publication Sep. 29, 2016 Sheet 11 of 64 US 2016/0282360 A1 VS"OIH co s ce n §2 age , f & A. ei & Patent Application Publication US 2016/0282360 A1 Patent Application Publication Sep. 29, 2016 Sheet 13 of 64 US 2016/0282360 A1 tes (%) is a A. ei is Patent Application Publication Sep. 29, 2016 Sheet 14 of 64 US 2016/0282360 A1 8?i 2# s c NA Patent Application Publication Sep. 29, 2016 Sheet 15 of 64 US 2016/0282360 A1 t Ecs a. s , f is a A3 is Patent Application Publication Sep. 29, 2016 Sheet 16 of 64 US 2016/0282360 A1 g s: s d :a o s & s { % 3 A et é & Patent Application Publication Sep. 29, 2016 Sheet 17 of 64 US 2016/0282360 A1 F.G. 6A CDK 2-N luc (1:00) in Hella Known Positive ligands 25 xx RS C C or ro e O -- 00m M. Staro sporine *& N or 3 if 75 * 1 OM A2D 538 e SO 25 O as 42 44, 4S, AB S. 52 5A S6. S8 6. S2 8 S6. S8 Temperature C FIG. 6B cDK 2-Niuc (1:100 in HeLa Know in Negative Compounds 25 w8r C. N. S. C. C or tro e ox OO 8s a ti is se asa Nior is - S at 80 y A V. G. 548 s ww SB2O3S 80 50 25 40 2 & 4 48 A8 S0 S2 S4 S8 58 60 62 64 66 88 Temperature C Patent Application Publication Sep. 29, 2016 Sheet 18 of 64 US 2016/0282360 A1 FIG. 6C Niu c-A Bi-1 ( : 100) in Hella Positive ligands s w8w S. Cotg e 25 - 100 m i? Staro sporifie 0.8 ** 1 OOA as at wax Of it? it 3. s *8- 10 A PC as e 50 c 2 5 4 42 4 4.6 as 50 S2 is a SS 58 6. S2 64 SS. 6.8 e imperature C FG, 6D Niu c-A B L 1 (1:100) in Hella Negative ligands 15 ac-8-ca. S C. C. to 125 -- M. A. W. G 548 10 xx - i? SB 2.358 x O C M A Z 54.38 FS s e 5 2 s a 42 A4 46 8 50 S2 5A S6 58 58. 82 64 86 68 Temperature C Patent Application Publication Sep. 29, 2016 Sheet 19 of 64 US 2016/0282360 A1 eTHu?(0\,:?.)oniN-?VG 08 09 0Z 0}, { %) ? e A3 ele Patent Application Publication Sep. 29, 2016 Sheet 20 of 64 US 2016/0282360 A1 da s s :-----8XX---- ------- s d 5. & s s N p. 838. o 3 p 3 no. 6); a et S %) 3 A. ea & Patent Application Publication Sep. 29, 2016 Sheet 21 of 64 US 2016/0282360 A1 d d ed s c ad is s s sa N N w w stry g pea & E E ( 3 g is no if x 3 eq S { %) n & a A et a Patent Application Publication Sep. 29, 2016 Sheet 22 of 64 US 2016/0282360 A1 FIG. 9 C DK 2-N Lu c 156 + pep 86 Detection Reagent Digiton in Treated HeLa C e i is 1 50 xx y SO Contro 25 --- rism OO M A Z 5433 t O O 57 O5 25 4 42 AA 4 S. 48 S. 52 54. 53 S 8 SO 82 SA S-S 68 emperature C Patent Application Publication Sep. 29, 2016 Sheet 23 of 64 US 2016/0282360 A1 FIG. OA C DK 2-pep 86 + pept 1 S. Detection Digiton in Treated HeLa Ces 400 BOC 3. 3200 0 003 8 -2 0 - to 2 1 3 AZD 5438, a M Patent Application Publication Sep. 29, 2016 Sheet 24 of 64 US 2016/0282360 A1 FIG. OB C K2 -pep 88 - pep S Detection Digito n in Treated. He a C e is 23888 238.338 S. 888 BB 88 S8388 2 18 3 3. A Z 5438, u A Patent Application Publication Sep. 29, 2016 Sheet 25 of 64 US 2016/0282360 A1 F.G. 10C CD K2 peg 86 + pep S. Detection Reagent ig to n in Treated he la C e is 25 & *8* 8, 14 M A Z 5438 w:x : C G 8 A2 Sa: 33 s s 25 3 4 as S S S S4 SS 53 6s 82. S. 86 SS is in p: { a } Patent Application Publication Sep. 29, 2016 Sheet 26 of 64 US 2016/0282360 A1 FG. A CDK 2-pep 1 S + pep 86 Detection Reagent Digiton in Treated he La Ce is 3 O B. 2000 6 2. 6 5 0.33 -2 10 : 30 0 102 3 AZE) 5438, uM Patent Application Publication Sep. 29, 2016 Sheet 27 of 64 US 2016/0282360 A1 FIG 11 B C C K 2 -pep S + pep 8s 3 etection Reas ent Dig if on in feated he la C e is 8 3. 83.38 8 88 28 03 88 - : 8 - R 3 AZE 5438, is M Patent Application Publication Sep. 29, 2016 Sheet 28 of 64 US 2016/0282360 A1 FIG C CD K2-pep i t S + pep 8 6 Detection Reagent Digiton in treated Heia Cei is 25 x 58 A2: S:38 w:x 338, 3.38 g 5 s 8 s 42 & 3 SR 33 3.4 S S 53 S 8 is 3 SS S3 e i g { a } Patent Application Publication US 2016/0282360 A1 (0g vi O 9) in a tu Patent Application Publication Sep. 29, 2016 Sheet 31 of 64 US 2016/0282360 A1 FG. 3 - R F at He a igitan in : 3 ( - A + 8.5x life Tech Rye 8 is 3.38 88: wer Stat: rasgo rise s w : 33::::::: -38 x 8.33 is 3: Y. 8: 8: - 13 2. 38 g 338; 3:3: 38 -- R F ea - Big it an in : NEA + 5x ilife Tech ye 88: w8x A: i:38 -Sir Sia 33 g spa is 88: wx 333:8: s: ww. iiii is 8: 28. 8: - ; 3 -2 38 * $38g & 888 is $8 Patent Application Publication Sep.