(12) Patent Application Publication (10) Pub. No.: US 2016/0282360 A1 Dart Et Al

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

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

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

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 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

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

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

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

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

:-----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

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

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

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

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

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: - 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. 29, 2016 Sheet 32 of 64 US 2016/0282360 A1

FG. 4A

C K2-83 it c : : {3} in his 33 k is own Positi e iiga is

88 xx x . S & 3rs: -Br 3838: Sta:3sg&rine 383 *&^ 3:3 assi: & 83.38 23:38 s 388

s : 388

8 2 & 3 SE 8. 2 ... is 8 8 3:2 . E8 38 : 283 & 3:33 e. g.

CB K2-8 tu c : : 88 in. He a K.F. & W 8: egative $g 3 r is

38 anxi : S : w8x : 3 & S3: a 38 are 3 3:3: 3 x 333 - 3 3: A 3:8 288 xx 3: S S 3.8

e : 3:

. . . 8 SE. S. S. S8, S8: 83 SE 8. S. SS eige 3: re. Patent Application Publication Sep. 29, 2016 Sheet 33 of 64 US 2016/0282360 A1

F.G. 14B

*; it c - A R x 3 : : { } is Hea. Pasitive liga is is

is axx 3:3: $.333i: 888 ^^ 38 33 is was 8888; 8 gi: s *8* 3: Fis 3:::: s -- $8,388 3 33 -xx 8:8; 33.283

8 3 & B. S. S. s. 38 3 & 8 & 38 age F3 : £e &

8 it c-s: A P K 4 ; ; ; ) { } irs. He a i e g a tiwe ligan cis

3. x8x 3: S: , ; } 88 stor : 33:8: S38; 8sg3; :38 s w : 3888 & 3.38 3 se 38

38 & 3. S & 3.8 s is: 88, S8 8: 8 is 88 S3 e333; as a Eurs : Patent Application Publication Sep. 29, 2016 Sheet 34 of 64 US 2016/0282360 A1

$•¿• $No.?sagae &{}\\?shs-e- §?.No.?Å3** ºff{ºg§

Patent Application Publication US 2016/0282360 A1

: s £3.83. th y §§ is $f 3.3 8 kit Patent Application Publication Sep. 29, 2016 Sheet 36 of 64 US 2016/0282360 A1

c a N c m ------it Patent Application Publication Sep. 29, 2016 Sheet 37 of 64 US 2016/0282360 A1

§9?$|€£ §33333833?å?k???.

c g : 3} f : 8 Patent Application Publication US 2016/0282360 A1

8$$Z9§?$.{}{$

C;338;8&2{

c {}{}{ s s

Patent Application Publication Sep. 29, 2016 Sheet 44 of 64 US 2016/0282360 A1

F.G. 19

hi DAC 1 - Niu c 1:10 0} in He a

* 25. O 8S O C or to: ic ceir sat 03 O ASC contro heat or y OO w w a ceting stat he at only 5

s 58 s 2s

a 42 & 4 as 48 5 52 5A. S. 5 is 82 a 68 3 emperature C

H DAC i-Niuc (1:10 0} in Heia

AO as R. S. O. Contro w8- 13, oceticssia 3. -k- O MS O control heat only *- : G. R. Mocetinostat heat ciny

4 4: 44 48 48 2: Sa SS 58 SO 2 8, 88 The rature C Patent Application Publication Sep. 29, 2016 Sheet 45 of 64 US 2016/0282360 A1

------yyya

& { % f : 8 A3 ea Patent Application Publication Sep. 29, 2016 Sheet 46 of 64 US 2016/0282360 A1

S. s se

8 s 3 g 3- s o) : & o a we a .

2 m : S is to O 8.

CN8 if a. a st CD a a . w

------&- - 8 w s

a N yya 4-ye t %} f : 8 A38 & X Patent Application Publication Sep. 29, 2016 Sheet 47 of 64 US 2016/0282360 A1

Patent Application Publication US 2016/0282360 A1 Patent Application Publication Sep. 29, 2016 Sheet 49 of 64 US 2016/0282360 A1

0000SD

As Patent Application Publication Sep. 29, 2016 Sheet 50 of 64 US 2016/0282360 A1

8OOOSO ##9 Patent Application Publication Sep. 29, 2016 Sheet 51 of 64 US 2016/0282360 A1

#88*** Caes«»

33s 83.38: Patent Application Publication Sep. 29, 2016 Sheet 52 of 64 US 2016/0282360 A1

†7ZOOSO 344%

SS 8383 Patent Application Publication Sep. 29, 2016 Sheet 53 of 64 US 2016/0282360 A1

i8 x :- a, ; : {

3 & 83. Patent Application Publication Sep. 29, 2016 Sheet 54 of 64 US 2016/0282360 A1

9800SO Patent Application Publication Sep. 29, 2016 Sheet 55 of 64 US 2016/0282360 A1

OOTOSO

Patent Application Publication Sep. 29, 2016 Sheet 56 of 64 US 2016/0282360 A1

cits

33.8 Patent Application Publication Sep. 29, 2016 Sheet 57 of 64 US 2016/0282360 A1

8700SO

Patent Application Publication Sep. 29, 2016 Sheet 58 of 64

£/OOSO Patent Application Publication Sep. 29, 2016 Sheet 59 of 64 US 2016/0282360 A1

9600SO fS Patent Application Publication Sep. 29, 2016 Sheet 60 of 64 US 2016/0282360 A1

ZITOSO

the Patent Application Publication Sep. 29, 2016 Sheet 61 of 64 US 2016/0282360 A1

a. fast:3t eases or Asts agressist Patent Application Publication Sep. 29, 2016 Sheet 62 of 64 US 2016/0282360 A1

F.G. 35

COK2-Nuc CS0036 42 °C 125000 O ...8s. 6.666667 10000 :- 4.444444 2.962963 75000 1975309 3.16872 50000 X & :- 0.877915 & O 25000

wavelength, mm

CDK2-Nuc CS003.656C

1O 6666667 4.444444 2.962963 1975309 1316872 O877915 O

650 wavelength, nm Patent Application Publication Sep. 29, 2016 Sheet 63 of 64 US 2016/0282360 A1

F.G. 36

CDK2-Nuc CSOO96 42 °C Dye (uM)

500 525 550 575 600 625 650 675 700 725 Wavelength (nm)

CDK2-Nuc CS0096 56C Dye (M)

500 525 550 575 600 625 650 675 700 725 Wavelength (nm) Patent Application Publication Sep. 29, 2016 Sheet 64 of 64 US 2016/0282360 A1

FIG. 37

Nic WAPK4 CS0048 42°C 2000 10 6.668667 e 4.444444 150000 2.98.2983 1975309 136872 : 600 0.87795 O

e 50000 i.

50 550 600 650 700 wavelength, nm.

Nic WAPK14 CS004.852 °C 2000 10 ... 6,686667 4.444444 150000 2.962963 1975309 136872 10000 O.877915 3. O se d 50000 i.

500 550 6 650 700 wavelength, nm

US 2016/0282360 A1 Sep. 29, 2016

LUCFERASE-BASED THERMAL SHIFT unfolded target protein significantly more than to the folded ASSAYS target protein (e.g., 2-fold increase, 3-fold increase, 4-fold increase, 5-fold increase, 6-fold increase, 7-fold increase, CROSS-REFERENCE TO RELATED 8-fold increase, 9-fold increase, 10-fold increase, 20-fold APPLICATIONS increase, 50-fold increase, 100-fold increase, 1000-fold 0001. The present application claims the priority benefit increase, or more, or ranges therein). In some embodiments, of U.S. Provisional Patent Application 62/112,518, filed Feb. systems further comprise a ligand or test ligand for the target 5, 2015, which is incorporated by reference in its entirety. protein. In some embodiments, systems further comprise a substrate for the bioluminescent reporter. FIELD 0006. In some embodiments, provided herein are systems 0002 Provided herein are systems and methods for char comprising: (a) a fusion of a target protein and a biolumi acterizing target/ligand engagement. In particular, nescent reporter, wherein the bioluminescent reporter pro luciferase-labeled polypeptide targets are used to detect or tein has an emission spectra that encompasses a wavelength quantify target/ligand engagement (e.g., within a cell or cell X; and (b) a fluorescent dye that: (1) binds nonspecifically lysate). to aggregated proteins and/or hydrophobic peptide seg ments, and (2) has an excitation spectra that encompasses the wavelength X. In some embodiments, the emission BACKGROUND spectra of the bioluminescent reporter protein and the exci 0003 Multiple challenges face drug development today tation spectra of the fluorescent dye overlap such that the including high costs and long development cycles for new bioluminescent reporter excites the fluorescent dye by therapeutics. Methods that promote accelerated drug devel BRET. In some embodiments, the bioluminescent reporter opment are urgently needed. The efficacy of therapeutics is and the fluorescent dye comprise a BRET pair. In some dependent on a drug binding to its target. Due to its embodiments, the bioluminescent reporter is a BRET donor, simplicity, a protein thermal shift assay (TSA) is a com and the fluorescent dye is a BRET acceptor. In some monly used method for screening libraries and validating embodiments, the system is a cell, e.g., a live, intact cell, cell hits in drug discovery programs. The most common method lysate (e.g., cells lysed by chemical methods (e.g., lytic of TSA in use today can only be performed using purified NANOGLO reagent, cells lysed by sonication, etc.), or protein, which has several disadvantages. Recently, the reaction mixture. In some embodiments, the bioluminescent cellular thermal shift assay (CETSA) was developed to reporter is a luciferase. In some embodiments, the luciferase detect endogenous target protein within cells or cell lysates, is a variant Oplophorus gracihrostris luciferase (Og|Luc) thereby alleviating the need to create purified proteins and (e.g., >60%, 70%, 80%, 90%, or 95% identity with SEQID allowing for target engagement analysis in complex envi NO: 1, SEQ ID NO: 2, etc.). In some embodiments, the ronments that are more biologically relevant. However, the bioluminescent reporter is a peptide or polypeptide tag that CETSA is a multi-step assay in which the analysis of target forms a bioluminescent complex upon interaction with a engagement relies upon western blot or AlphaScreen (Perkin complement polypeptide or peptide (See, e.g., U.S. Pub. No. Elmer) technologies, both of which have several disadvan 2014/0363375; herein incorporated by reference in its tages, e.g., cost, insensitive, multi-stepped protocols, entirety). In some embodiments, the system further com requires cell lysis and spin steps, low throughput (e.g., prises complement polypeptide or peptide that forms a Western blot), dependent upon antibody recognition of bioluminescent reporter with the target portion of the fusion. native protein state, cell line optimization, requires a large In some embodiments, the fluorescent dye is an environ amount of cells, etc. What is needed is a simple, homoge mentally-sensitive dye. In some embodiments, the fluores neous, rapid, and inexpensive TSA, whereby target engage cent dye is hydrophobic. In some embodiments, the fluo ment can be characterized in complex environments, such as rescence of the fluorescent dye is quenched by water (e.g., cells and cell lysate. fluorescence is Suppressed when the dye is free in aqueous Solution and enhanced when the dye is in a hydrophobic SUMMARY environment). In some embodiments, the fluorescent dye is 0004 Provided herein are systems and methods for char fluorogenic. In some embodiments, binding between the acterizing target/ligand engagement. In particular, fluorescent dye and the reporter protein is altered by changes luciferase-labeled polypeptide targets are used to detect or in target protein structure (e.g., unfolding). In some embodi quantify target/ligand engagement (e.g., within a cell, e.g., in ments, the fluorescence of the fluorescent dye is quenched or an intact live cell, or cell lysate). Suppressed when free in aqueous Solution and enhanced 0005. In some embodiments, provided herein are systems when the dye bound to a hydrophobic composition. In some comprising: (a) a fusion of a target protein and a biolumi embodiments, the fluorescent dye is SYPRO Orange, nescent reporter; and (b) a fluorescent dye; wherein the SYPRO Red, Nile Red, ANS, Bis-ANS, SYPRO Ruby, bioluminescent reporter and the fluorescent dye comprise a SYPRO Tangerine, and/or Dapoxyl Sulfonic Acid Sodium bioluminescence resonance energy transfer BRET pair (e.g., Salt, PROTEIN THERMAL SHIFT Dye (Life Technolo the bioluminescent reporter is the BRET donor and the gies), PROTEOSTAT Dye (Enzo), or combinations thereof. fluorescent dye is the BRET acceptor); and wherein the In some embodiments, the system further comprises a ligand fluorescent dye interacts with exposed hydrophobic regions of the target or a test ligand. of the target protein upon unfolding, denaturation, and/or 0007. In some embodiments, methods are provided for aggregation of the target protein. In some embodiments, the detecting the interaction of a ligand and target protein based fluorescent dye does not interact with the natively folded (or upon the stabilization of the target protein upon formation of otherwise stably folded) target protein. In some embodi a target/ligand complex comprising: (a) incubating a fusion ments, the fluorescent dye binds to the unfolded or partially of the target protein and a bioluminescent reporter with a US 2016/0282360 A1 Sep. 29, 2016

substrate for the bioluminescent reporter and a fluorescent approximate melting temperature of the target protein. In dye that binds to hydrophobic peptide sequences when they Some embodiments, a plurality of test samples is produced are exposed by protein unfolding; and (b) detecting BRET using a plurality of test ligands. from the bioluminescent reporter to the fluorescent dye in the presence and absence of the ligand or test ligand. In some 0009. In some embodiments, provided herein are meth embodiments, signal is detected over a range of denaturing ods to detect a target/ligand interaction, comprising the steps conditions (e.g., two or more conditions (e.g., ranging from of: (a) incubating a fusion of a target protein and a biolu non-denaturing to highly-denaturing)). In some embodi minescent reporter: (i) in the presence of a ligand to produce ments, due to the proximity limitation of BRET (e.g., donor a test sample, and (ii) in the absence of a ligand, to produce to acceptor distance of about 1-10 nm), emission from the a control sample; (b) contacting the test and control samples fluorescent dye as a consequence of BRET from the biolu with an environmentally-sensitive hydrophobic dye, minescent reporter is only detected when the fluorescent dye wherein the emission spectra of the bioluminescent reporter is bound to the target protein. In some embodiments, binding and the excitation spectra of the environmentally-sensitive of the ligand or test ligand to the target protein stabilizes the hydrophobic dye overlap; (c) treating said test and control protein and increases the degree of the denaturing conditions samples under conditions that cause the target protein to (e.g., temperature, concentration of denaturant, increased unfold to an appropriate extent; (d) measuring signal from pressure, etc.) required to unfold the target protein, allow the environmentally-sensitive hydrophobic dye in said test binding of the fluorescent or fluorogenic dye to the exposed and control samples (e.g., emission resulting from energy hydrophobic portions of the target protein, producing a transfer from the active bioluminescent reporter after addi tion of Substrate); and (e) comparing the measurement made detectable signal resulting from BRET from the biolumi in step (d) between the test and control samples, wherein nescent reporter to the target-bound fluorescent dye. In some alteration of the signal from said environmentally-sensitive embodiments, the fusion, fluorescent dye, Substrate, and hydrophobic dye in the test sample compared to the control ligand (when present) are combined in any suitable order of sample indicates the presence of a target/ligand interaction. addition. In some embodiments, steps (a), (b), and (c) are performed 0008. In some embodiments, provided herein are meth in any suitable order (e.g., a-b-c, a-c-b,b-a-c, b-c-a,c-a-b, or ods to detect a target/ligand interaction, comprising the steps c-b-a). In some embodiments, the fusion is within a cell, e.g., of: (a) incubating a fusion of a target protein and a reporter a live intact cell, cell lysate, or reaction mixture. In some polypeptide: (i) in the presence of a ligand to produce a test embodiments, the fusion is expressed within the cell, e.g., a sample and (ii) in the absence of a ligand to produce a live intact cell, cell lysate, or reaction mixture. In some control sample; (b) treating said test and control samples embodiments, the ligand is added exogenously to the cell, under conditions that cause the target protein to unfold (e.g., e.g., a live intact cell, cell lysate, or reaction mixture. In to an appropriate extent); (c) measuring signal from the Some embodiments, the environmentally-sensitive hydro reporter polypeptide in said test and control samples; and (d) phobic dye is added exogenously to the cell, e.g., a live comparing the measurement made in step (c) between the intact cell, cell lysate, or reaction mixture. In some embodi test and control samples, wherein alteration of the signal ments, the bioluminescent reporter is a luciferase. In some from said reporter polypeptide in the test sample compared embodiments, the luciferase is a variant Oplophorus gracil to the control sample indicates the presence of a target/ irostris luciferase (Og|Luc) (e.g., >60%, 70%, 80%, 90%, or ligand interaction. In some embodiments, the fusion is 95% identity with SEQ ID NO: 1, SEQID NO: 2, etc.). In within a cell, e.g., a live, intact cell, cell lysate, or reaction Some embodiments, the bioluminescent reporter is a peptide mixture. In some embodiments, the fusion is expressed or polypeptide tag that forms a bioluminescent complex within the cell, e.g., a live intact cell, cell lysate, or reaction upon interaction with a complement polypeptide or peptide. mixture. In some embodiments, the ligand is added exog In some embodiments, the complement polypeptide or pep enously to the cell, e.g., a live intact cell, cell lysate, or tide is added exogenously to the test and control samples reaction mixture. In some embodiments, the reporter poly prior to step (d). In some embodiments, the complement peptide is a luciferase. In some embodiments, the luciferase polypeptide or peptide is expressed within the cell, e.g., a is a variant Oplophorus gracihrostris luciferase (Og|Luc) live intact cell, cell lysate, or reaction mixture. In some (e.g., >60%, 70%, 80%, 90%, or 95% identity with SEQID embodiments, the conditions that cause the target protein to NO: 1, SEQ ID NO: 2, etc.). In some embodiments, the unfold to an appropriate extent comprise elevated tempera bioluminescent reporter is a peptide or polypeptide tag that ture, increased pressure, and/or a denaturant. In some forms a bioluminescent complex upon interaction with a embodiments, elevated temperature comprises one or more complement polypeptide or peptide (See, e.g., U.S. Pub. No. temperatures above physiologic temperature. In some 2014/0348747; herein incorporated by reference in its embodiments, elevated temperature comprises one or more entirety). In some embodiments, the complement polypep temperatures near the approximate melting temperature of tide or peptide is added exogenously to the test and control the target protein. In some embodiments, the environmen samples prior to step (c). In some embodiments, the comple tally-sensitive hydrophobic dye binds nonspecifically to ment polypeptide or peptide is expressed within the cell, cell hydrophobic Surfaces. In some embodiments, the environ lysate, or reaction mixture. In some embodiments, the con mentally-sensitive hydrophobic dye binds preferentially to ditions that cause the target protein to unfold to an appro the folded, unfolded, or molten globule states of the protein. priate extent comprise elevated temperature, increased pres In some embodiments, the fluorescence of the environmen Sure, and/or a denaturant. In some embodiments, elevated tally-sensitive hydrophobic dye is quenched by water. In temperature comprises one or more temperatures above Some embodiments, the environmentally-sensitive hydro physiologic temperature for the protein in question. In some phobic dye is Sypro Orange, SYPRO Red, Nile Red, ANS, embodiments, elevated temperature comprises one or more Bis-ANS, SYPRO Ruby, SYPRO Tangerine, and/or temperatures near (e.g., +/-1, 2, 3, 4, 5, 10, 15, 20°C.) the Dapoxyl Sulfonic Acid Sodium Salt, Protein Thermal US 2016/0282360 A1 Sep. 29, 2016

ShiftTM Dye (Life Technologies), PROTEOSTAT Dye test sample; and (d) detecting target/ligand interaction based (Enzo), or combinations thereof. Other suitable dyes for use on the signal from said reporter polypeptide in the test in embodiments described herein include, but are not limited sample. to: Styryl dyes, asymmetric cyanines, oxazole dyes (DapoX 0013. In some embodiments, provided herein are meth yls), azo dyes, and other dyes Such as Thioflavin T and ods to detect a target/ligand interaction, comprising the steps (Dicyanovinyl)ijulolidine (DCVJ). Suitable dyes for use in of: (a) incubating a fusion of a target protein and a biolu embodiments described herein are also described in, for minescent reporter in the presence of a ligand to produce a example: Kovalska et al. Dyes and Pigments 67 (2005) test sample; (b) contacting the test sample with an environ 47-54.; Volkova et al. Bioorganic & Medicinal Chemistry 16 mentally-sensitive hydrophobic dye, wherein the emission (2008) 1452-1459.: Volkova et al. J. Biochem. Biophys. spectra of the bioluminescent reporter and the excitation Methods 70 (2007) 727-733.: Hawe et al. Pharmaceutical spectra of the environmentally-sensitive hydrophobic dye Research, Vol. 25, No. 7, July 2008. U.S. Pub. No. 2011/ overlap; (c) treating the test sample under conditions that O130305; PCT Pub. WO 2006/079334; Diwu et al. Photo cause the target protein to unfold to an appropriate extent; chemistry and Photobiology, 1997, 66(4): 424-431.; herein (d) exposing the test sample to the substrate of the biolu incorporated by reference in their entireties. minescent reporter; (e) measuring signal from the environ 0010. In some embodiments, methods of screening a mentally-sensitive hydrophobic dye in said test sample; and group of test ligands for interaction with a target protein are (f) detecting target/ligand interaction based on the signal provided. In some embodiments, a fusion of the target from said environmentally-sensitive hydrophobic dye in the protein and a bioluminescent reporter is combined with a test sample. fluorescent dye that binds to exposed hydrophobic portions 0014 Based on the successful use of a direct luciferase of proteins (e.g., upon exposure of the hydrophobic portions fusion or BRET to detect ligand-mediated thermal stabili due to protein denaturation) and a substrate for the biolu Zation, other proximity-based reporter chemistries are minescent reporter, in the presence and absence of one or understood to be useful. For example, in certain embodi more of the test ligands. In some embodiments, multiple ments, an epitope tag is attached to the protein of interest. assays, each comprising a different test ligand of set of test Following a thermal denaturation step, addition of a detec ligands are performed in parallel (e.g., in a high throughput tion antibody labeled with a donor fluorophore (e.g., ter method). Fluorescence emission from the fluorescent dye as bium, europium, etc.) is used in a FRET assay with a a result of BRET from the bioluminescent reporter is denaturation/aggregation-sensitive dye as a FRET acceptor. detected. Decrease of BRET-induced fluorescence in the Ligand-mediated thermal stabilization results in a loss of presence of the one or more test ligands, and/or an increase FRET/TR-FRET signal. In other embodiments, a combina in the temperature or amount of denaturant required to tion of labeled antibodies (e.g. donor-labeled anti-FLAG and generate a BRET-induced fluorescent signal, indicates inter acceptor labeled anti-V5) and a tandem epitope (e.g. FLAG action of one or more of the test ligands with the target V5) tethered to the target protein are used as a detection protein. system. When stabilized, the target is stabilized by binding of a ligand, the epitope is presented and both antibodies 0011. In some embodiments, provided herein are meth bind, generating a proximity-based signal (e.g. FRET. TR ods of Screening a group of test ligands for binding to a FRET). Upon thermal denaturation, the epitopes are unavail target protein comprising: (a) creating a plurality of test able to the antibody pair, resulting in a loss of proximity samples each comprising at least one test ligand and a fusion based signal. In some embodiments, various detection of a target protein and a bioluminescent reporter; (b) creating chemistries are applied (e.g. TR-FRET proximity ligation, at least one control sample comprising a fusion of a target singlet oxygen transfer/alphaScreen, etc.). protein and a bioluminescent reporter in the absence of a test 0015. In some embodiments, methods are provided for ligand; (c) contacting the test and control samples with an the detection of target/ligand interactions within a cell. In environmentally-sensitive hydrophobic dye, wherein the Some embodiments, methods allow detection of target/li emission spectra of the bioluminescent reporter and the gand interactions within live, intact cells. In some embodi excitation spectra of the environmentally-sensitive hydro ments, all steps (e.g., target/reporter expression, reagent phobic dye overlap; (d) treating the test and control samples (e.g., ligand, Substrate, dye, etc.) addition, target denatur under conditions that cause the target protein to unfold to an ation, signal detection, etc.) are performed to or within the appropriate extent; (e) exposing the test and control samples live, intact cells. In some embodiments, the target/ligand to the substrate of the bioluminescent reporter, (f) measuring interaction is initiated within the live, intact cells, but one or signal from the environmentally-sensitive hydrophobic dye more steps of the assay (e.g., Substrate addition, target in said test and control samples; and (g) comparing the denaturation, signal detection, etc.) are performed in a lysate measurement made in step (f) between the test and control of the cells (e.g., following lysis of the live, intact cells to samples, wherein alteration of the signal from said environ produce a cell lysate). In some embodiments, all steps (e.g., mentally-sensitive hydrophobic dye in one or more test target/reporter expression, reagent (e.g., ligand, Substrate, samples compared to the control sample indicates the pres dye, etc.) addition, target denaturation, signal detection, etc.) ence of a target/ligand interaction. are performed to or within the cell lysate. The following 0012. In some embodiments, provided herein are meth paragraphs provide exemplary methods for performing Such ods to detect a target/ligand interaction, comprising the steps assays. The following embodiments are not limiting, and of: (a) incubating a fusion of a target protein and a reporter may be combined and/or modified with other embodiments polypeptide in the presence of a ligand to produce a test described herein. sample; (b) treating said test sample under conditions that 0016. In some embodiments, provided herein are meth cause the target protein to unfold to an appropriate extent; ods to detect a target/ligand interaction within a live, intact (c) measuring signal from the reporter polypeptide in said cell, comprising the steps of: (a) providing a live, intact cell US 2016/0282360 A1 Sep. 29, 2016

expressing a fusion of a target protein and a bioluminescent bioluminescent reporter in the cell lysate. In some embodi reporter; (b) allowing time for the target protein to interact ments, methods further comprise a step of adding the ligand with the ligand within the live, intact cell; (c) contacting the to the live, intact cell. In some embodiments, the ligand is live, intact cell with an environmentally-sensitive hydropho endogenous to the live, intact cell. In some embodiments, bic dye, wherein the emission spectra of the bioluminescent the signal from said environmentally-sensitive hydrophobic reporter and the excitation spectra of the environmentally dye in the cell lysate is compared to a control sample without sensitive hydrophobic dye overlap; (d) treating the live, the ligand. intact cell under conditions that cause the target protein to unfold to an appropriate extent; (e) exposing the live, intact 0019. In some embodiments, provided herein are meth cell to the substrate of the bioluminescent reporter, (f) ods to detect a target/ligand interaction within a live, intact measuring signal from the environmentally-sensitive hydro cell, comprising the steps of: (a) providing a live, intact cell phobic dye and bioluminescent reporter in the live, intact expressing a fusion of a target protein and a bioluminescent cell; and (g) detecting target/ligand interaction based on the reporter; (b) allowing time for the target protein to interact ratio from the signals from said environmentally-sensitive with the ligand within the live, intact cell; (c) contacting the hydrophobic dye and bioluminescent reporter in the live, live, intact cell with an environmentally-sensitive hydropho intact cell. In some embodiments, methods further comprise bic dye, wherein the emission spectra of the bioluminescent a step of adding the ligand to the live, intact cell. In some reporter and the excitation spectra of the environmentally embodiments, the ligand is endogenous to the live, intact sensitive hydrophobic dye overlap; (d) exposing the live, cell. In some embodiments, the signal from said environ intact cell to the substrate of the bioluminescent reporter; (e) mentally-sensitive hydrophobic dye in the live, intact cell is measuring signal from the environmentally-sensitive hydro compared to a control sample without the ligand. phobic dye and bioluminescent reporter in the live, intact cell under pre-denaturing conditions; (f) treating the live, 0017. In some embodiments, provided herein are meth intact cell under conditions that cause the target protein to ods to detect a target/ligand interaction within a cell, com unfold to an appropriate extent, (g) measuring signal from prising the steps of: (a) providing a live, intact cell express the environmentally-sensitive hydrophobic dye and biolu ing a fusion of a target protein and a bioluminescent minescent reporter in the live, intact cell under post-dena reporter; (b) allowing time for the target protein to interact turing conditions; and (h) detecting target/ligand interaction with the ligand within the live, intact cell; (c) lysing the cell based on the difference or ratio in signals from said envi to produce a cell lysate; (d) contacting the cell lysate with an ronmentally-sensitive hydrophobic dye and bioluminescent environmentally-sensitive hydrophobic dye, wherein the reporter under the pre-denaturing and post-denaturing con emission spectra of the bioluminescent reporter and the ditions in the live, intact cell. In some embodiments, meth excitation spectra of the environmentally-sensitive hydro ods further comprise a step of adding the ligand to the live, phobic dye overlap; (e) treating the cell lysate under con intact cell prior to step (e). In some embodiments, the ligand ditions that cause the target protein to unfold to an appro is endogenous to the live, intact cell. In some embodiments, priate extent; (f) exposing the cell lysate to the substrate of the difference or ratio of the signal is compared to a the bioluminescent reporter, (g) measuring signal from the difference or ratio of signal in a control sample without the environmentally-sensitive hydrophobic dye and biolumines ligand. cent reporter in the cell lysate; and (h) detecting target/ligand interaction based on the ratio from the signals from said 0020. In some embodiments, provided herein are meth environmentally-sensitive hydrophobic dye and biolumines ods to detect target/ligand interactions within live, intact cent reporter in the cell lysate. In some embodiments, cells using luminescence-based readout. For example, in methods further comprise a step of adding the ligand to the Some embodiments, provided herein are methods compris live, intact cell. In some embodiments, the ligand is endog ing the steps of: (a) providing a live, intact cell expressing enous to the live, intact cell. In some embodiments, the a fusion of a target protein and a bioluminescent reporter; (b) signal from said environmentally-sensitive hydrophobic dye allowing time for the target protein to interact with the in the cell lysate is compared to a control sample without the ligand within the live, intact cell; (c) treating the live, intact ligand. cell under conditions that cause the target protein to unfold 0018. In some embodiments, provided herein are meth to an appropriate extent; (d) exposing the live, intact cell to ods to detect a target/ligand interaction within a cell, com the Substrate of the bioluminescent reporter; (e) measuring prising the steps of: (a) providing a live, intact cell express signal from the bioluminescent reporter in the live, intact ing a fusion of a target protein and a bioluminescent cell; and (f) detecting target/ligand interaction based on the reporter; (b) allowing time for the target protein to interact signal from said bioluminescent reporter in the live, intact with the ligand within the live, intact cell; (c) contacting the cell. live, intact cell with an environmentally-sensitive hydropho 0021. In some embodiments, provided herein are meth bic dye, wherein the emission spectra of the bioluminescent ods to detect target/ligand interactions in live cells using reporter and the excitation spectra of the environmentally lytic endpoint luminescence-based readout. For example, in sensitive hydrophobic dye overlap; (d) lysing the cell to Some embodiments, methods comprise the steps of: (a) produce a cell lysate; (e) treating the cell lysate under providing a live, intact cell expressing a fusion of a target conditions that cause the target protein to unfold to an protein and a bioluminescent reporter; (b) allowing time for appropriate extent, (f) exposing the cell lysate to the Sub the target protein to interact with the ligand within the live, strate of the bioluminescent reporter, (g) measuring signal intact cell; (c) treating the live, intact cell under conditions from the environmentally-sensitive hydrophobic dye and that cause the target protein to unfold to an appropriate bioluminescent reporter in the cell lysate; and (h) detecting extent; (d) lysing the cell to produce a cell lysate; (e) target/ligand interaction based on the ratio from the signals exposing the cell lysate to the substrate of the biolumines from said environmentally-sensitive hydrophobic dye and cent reporter, (f) measuring signal from the bioluminescent US 2016/0282360 A1 Sep. 29, 2016 reporter in the lysate; and (g) detecting target/ligand inter sensitive hydrophobic dye, wherein the emission spectra of action based on the signal from said bioluminescent reporter the bioluminescent reporter and the excitation spectra of the in the lysate. environmentally-sensitive hydrophobic dye overlap; (e) 0022. In some embodiments, provided herein are meth exposing the live, intact cell to the substrate of the biolu ods to detect target/ligand interactions within a cell lysate minescent reporter, (f) measuring signal from the environ using luminescence-based readout. For example, in some mentally-sensitive hydrophobic dye and bioluminescent embodiments, methods comprise the steps of: (a) providing reporter in the live, intact cell; and (g) detecting target/ligand a live, intact cell expressing a fusion of a target protein and interaction based on the rationetric signal from said envi a bioluminescent reporter; (b) lysing the cell to produce a ronmentally-sensitive hydrophobic dye and bioluminescent cell lysate; (c) allowing time for the target protein to interact reporter in the live, intact cell. with the ligand within the cell lysate; (d) treating the cell 0026. In some embodiments, provided herein are meth lysate under conditions that cause the target protein to unfold ods to detect target/ligand interactions in live cells using to an appropriate extent; (e) exposing the cell lysate to the lytic endpoint BRET readout with dye addition. For Substrate of the bioluminescent reporter, (f) measuring sig example, in Some embodiments, methods comprise the steps nal from the bioluminescent reporter in the lysate; and (g) of: (a) providing a live, intact cell expressing a fusion of a detecting target/ligand interaction based on the signal from target protein and a bioluminescent reporter; (b) allowing said bioluminescent reporter in the lysate. time for the target protein to interact with the ligand within 0023. In some embodiments, provided herein are meth the live, intact cell; (c) treating the live, intact cell under ods to detect target/ligand interactions in live, intact cells conditions that cause the target protein to unfold to an using luminescence-based readout with rationetric analysis. appropriate extent; (d) lysing the cell to produce a cell For example, in some embodiments, methods comprise the lysate; (e) contacting the cell lysate with an environmen steps of: (a) providing a live, intact cell expressing a fusion tally-sensitive hydrophobic dye, wherein the emission spec of a target protein and a bioluminescent reporter; (b) allow tra of the bioluminescent reporter and the excitation spectra ing time for the target protein to interact with the ligand of the environmentally-sensitive hydrophobic dye overlap: within the live, intact cell; (c) exposing the live, intact cell (f) exposing the cell lysate to the substrate of the biolumi to the substrate of the bioluminescent reporter; (d) measur nescent reporter, (g) measuring signal from the environmen ing signal from the bioluminescent reporter in the live, intact tally-sensitive hydrophobic dye and bioluminescent reporter cell under pre-denaturing conditions; (e) treating the live, in the cell lysate; and (h) detecting target/ligand interaction intact cell under conditions that cause the target protein to based on the rationetric signal from said environmentally unfold to an appropriate extent, (f) measuring signal from sensitive hydrophobic dye and bioluminescent reporter in the bioluminescent reporter in the live, intact cell under the cell lysate. post-denaturing conditions; and (g) detecting target/ligand 0027. In some embodiments, provided herein are meth interaction based on the difference or ratio in signal from the ods to detect target/ligand interactions in cell lysate using bioluminescent reporter under the pre-denaturing and post BRET readout with dye addition. For example, in some denaturing conditions in the live, intact cell. embodiments, methods comprise the steps of: (a) providing 0024. In some embodiments, provided herein are meth a live, intact cell expressing a fusion of a target protein and ods to detect target/ligand interactions in a cell lysate using a bioluminescent reporter; (b) lysing the cell to produce a luminescence-based readout with rationnetric analysis. For cell lysate; (c) contacting the cell lysate with an environ example, in some embodiments, methods comprise the steps mentally-sensitive hydrophobic dye, wherein the emission of: (a) providing a live, intact cell expressing a fusion of a spectra of the bioluminescent reporter and the excitation target protein and a bioluminescent reporter; (b) lysing the spectra of the environmentally-sensitive hydrophobic dye cell to produce a cell lysate; (c) allowing time for the target overlap; (d) allowing time for the target protein to interact protein to interact with the ligand within the cell lysate; (d) with the ligand within the cell lysate; (e) treating the cell exposing the cell lysate to the substrate of the biolumines lysate under conditions that cause the target protein to unfold cent reporter; (e) measuring signal from the bioluminescent to an appropriate extent, (f) exposing the cell lysate to the reporter in the cell lysate under pre-denaturing conditions; Substrate of the bioluminescent reporter, (g) measuring (f) treating the cell lysate under conditions that cause the signal from the environmentally-sensitive hydrophobic dye target protein to unfold to an appropriate extent; (g) mea and bioluminescent reporter in the cell lysate; and (h) Suring signal from the bioluminescent reporter in the cell detecting target/ligand interaction based on the rationetric lysate under post-denaturing conditions; and (h) detecting signal from said environmentally-sensitive hydrophobic dye target/ligand interaction based on the difference or ratio in and bioluminescent reporter in the cell lysate. signal from the bioluminescent reporter under the pre denaturing and post-denaturing conditions in the cell lysate. BRIEF DESCRIPTION OF THE DRAWINGS 0025. In some embodiments, provided herein are meth ods to detect target/ligand interactions in live, intact cells 0028 FIGS. 1A-C show the effects of heat treatment on using BRET readout with dye addition following a partial HeLa cell viability (FIGS. 1A and 1B) and NANOLUC denaturation step (e.g., post-heat). For example, in some activity (RLU) within HeLa cells after exposure to a tem embodiments, methods comprise the steps of: (a) providing perature gradient (FIG. 1C). a live, intact cell expressing a fusion of a target protein and 0029 FIGS. 2A-B show analysis of melting temperatures a bioluminescent reporter; (b) allowing time for the target of several example target-NANOLUC (Nluc) fusions across protein to interact with the ligand within the live, intact cell; a range of target classes as determined by NANOLUC (c) treating the live, intact cell under conditions that cause activity (RLU) in live, intact cells (FIG. 2A) and digitonin the target protein to unfold to an appropriate extent; (d) treated (FIG. 2B) mammalian cells when exposed to a contacting the live, intact cell with an environmentally temperature gradient. US 2016/0282360 A1 Sep. 29, 2016

0030 FIGS. 3A-D show a detection of an increase in spontaneous binary complementation after incubation in the melting temperature for CDK2-Nluc and Nluc-CDK2 as presence of stabilizing ligand in digitonin-treated mamma determined by NANOLUC activity (RLU) after binding to lian cells Subsequently exposed to a temperature gradient. a stabilizing ligand in live, intact cells (FIGS. 3A-B) and 0040 FIG. 13 shows detection of ligand binding through digitonin-treated mammalian cells (FIGS. 3BC-D) and a change in melting temperature for CDK2-Nluc and LCK Subsequently exposed to a temperature gradient. Nluc as determined by bioluminescence resonance energy 0031 FIG. 4 shows a detection of an increase in melting transfer (BRET) after incubation in the presence of stabi temperature for CDK2-Nluc as determined by NANOLUC lizing ligand in digitonin-treated mammalian cells Subse activity (RLU) after binding to a stabilizing ligand in live quently exposed to a temperature gradient. mammalian cells subsequently lysed with lytic NANOGLO 0041 FIGS. 14A-B show analysis of isothermal dose Detection Reagent (Promega) and Subsequently exposed to response curves for a panel of kinase inhibitors with CDK2 a temperature gradient. Nluc and LCK-Nluc as determined by BRET after incuba 0032 FIGS. 5A-F show detection of an increase in melt tion in the presence of different concentrations of the indi ing temperature for a panel of kinases as determined by vidual compounds while time of heating and temperature NANOLUC activity (RLU) after binding to stabilizing were kept constant. This highlights the concentration depen ligands in digitonin treated mammalian cells and Subse dence of the thermal stabilization and allows for compound quently exposed to a temperature gradient. affinity signatures to be obtained. 0033 FIGS. 6A-D show detection of an increase in 0042 FIG. 15 shows detection of an increase in melting melting temperatures or no change in melting temperatures temperatures (Stabilizing ligand) or no change in melting for CDK2-Nluc and Nluc-ABL1 as determined by NANO temperatures (non-binding) for cytoplasmic target fusions LUC activity (RLU) after incubation with a panel of com CDK2-Nluc and Nluc-MAPK14 as determined by BRET pounds thus displaying compound selectivity for the cyto after incubation with a panel of compounds thus displaying plasmic targets in mammalian cells Subsequently exposed to compound selectivity in mammalian cells Subsequently digitonin and a temperature gradient. exposed to digitonin and a temperature gradient. 0034 FIG. 7 shows detection of an increase in melting 0043 FIGS. 16A-F show detection of an increase in temperatures or no change in melting temperatures for melting temperatures (stabilizing ligand) or no change in HDAC1-Nlucas determined by NANOLUC activity (RLU) melting temperatures (non-binding) for the nuclear target after incubation with a panel of compounds thus displaying fusion HDAC1-Nluc as determined by BRET after incuba compound selectivity for the nuclear target in mammalian tion with a panel of compounds in mammalian cells Subse cells and Subsequently exposed to digitonin and a tempera quently exposed to digitonin and a temperature gradient. ture gradient. 0044 FIGS. 17A-B show detection of an increase in 0035 FIGS. 8A-B show detection of an increase in melting temperature for CDK2-Nluc as determined by melting temperature for the targets KDR-Nluc and DHFR BRET after incubation with stabilizing ligand (stauro Nluc as determined by NANOLUC activity (RLU) after sporine) in mammalian cells Subsequently exposed to digi incubation with stabilizing ligand in mammalian cells Sub tonin and a temperature gradient using three different envi sequently harvested with the compound being washed out ronmentally-sensitive acceptor dyes reporting on protein and then subjected to a temperature. With this compound folding status as BRET acceptor dyes. Dye examples washout step, the experiment is analyzed not under com included: Protein Thermal ShiftTM Dye (Life Technologies), pound equilibrium conditions and has the ability to analyze SYPRO Orange protein gel stain, and SYPRO Red protein binding kinetics. KDR is a membrane protein highlighting gel stain. B is acceptor dye dose dependent, but that there another Subcellular location that the assay is able to monitor. is no change in the apparent melting temperature (T). This assay was performed with live, intact cells throughout 0045 FIG. 18 shows detection of an increase in melting the whole assay conditions. temperature for CDK2-Nluc as determined by BRET after 0036 FIG. 9 shows detection of an increase in melting incubation with stabilizing ligand (AZD5438) in digitonin temperature for CDK2-pep86 and pep86-CDK2 as deter treated mammalian cells Subsequently exposed to a tem mined by luciferase activity through spontaneous binary perature gradient and using two different environmentally complementation after incubation in the presence of a sta sensitive dyes to report on the folded protein status and serve bilizing ligand in digitonin-treated mammalian cells Subse as BRET acceptor dyes which was either added before or quently exposed to a temperature gradient. after the heating step. Dye examples included: the dye 0037 FIGS. 10A-C show detection of an increase in included in the Life Technologies Thermal Shift Assay Dye melting temperature for CDK2-NLuc156 as determined by Kit and the dye included in the Enzo PROTEOSTAT Ther luciferase activity through spontaneous binary complemen mal Shift Assay Kit. tation after incubation in the presence of a stabilizing ligand 0046 FIG. 19 shows detection of an increase in melting in digitonin-treated mammalian cells Subsequently exposed temperature for CDK2-Nluc as determined by NANOLUC to a temperature gradient. activity (RLU) and BRET (mBU) after incubation with 0038 FIGS. 11A-C show temperature and stabilizing stabilizing ligand in mammalian cells Subsequently exposed ligand concentration effects on melting temperature of to a temperature gradient and either analyzed immediately or CDK2-pep86 as determined by luciferase activity through incubated at room temperature for 3 minutes prior to analy spontaneous binary complementation after incubation in the S1S. presence of stabilizing ligand in digitonin-treated mamma 0047 FIGS. 20A-B show detection of an increase in lian cells Subsequently exposed to a temperature gradient. melting temperature for the nuclear target fusion HDAC1 0039 FIGS. 12A-B show temperature and stabilizing Nluc as determined by NANOLUC activity (RLU) and ligand concentration effects on melting temperature of BRET (mBU) after incubation with stabilizing ligand in CDK2-11S as determined by luciferase activity through mammalian cells Subsequently exposed to a temperature US 2016/0282360 A1 Sep. 29, 2016 gradient and either analyzed immediately or incubated at chemical synthesis, etc.). A “wild-type polypeptide' or room temperature for 3 minutes prior to analysis. “wild-type luciferase' is the most common variant occurring 0048 FIG. 21 displays prophetic examples of alternative in nature; whereas a “variant polypeptide' or “mutant poly energy transfer strategies combined with environmentally peptide' refers to a polypeptide having an amino acid sensitive dyes to use in a thermal shift assay. sequence that is distinct from the wild-type sequence. A 0049 FIG. 22 is Table 1, providing the order of addition variant or mutant polypeptide may be natural or synthetic. of the components for the experiments depicted in FIGS. The aforementioned terms (e.g., “natural”, “synthetic', 1-20. “wild-type”, “variant, and “mutant”) have the same mean 0050 FIGS. 23-34 show graphs depiction characteriza ings when used in reference to nucleic acids, genes, peptide, tion of dyes synthesized in Example 18: A left) BRET be proteins, etc. used to detect ligand binding through a change in relative 0057. As used herein, the term “sequence identity” refers BRET compared to DMSO controls (Unt) as exampled with to the degree to which two polymer sequences (e.g., peptide, CDK2-Nluc target fusions +/-Staurosporine (SSP) in live polypeptide, nucleic acid, etc.) have the same sequential or lytic conditions +/-digitonin (Dig). As expected, the composition of monomer Subunits. The term "sequence shape of the BRET curves are bell-shaped due to the loss of similarity refers to the degree with which two polymer Nluc signal with protein unfolding and increasing tempera sequences (e.g., peptide, polypeptide, nucleic acid, etc.) tures or dye dissociation upon protein aggregation or both; have similar polymer sequences. For example, similar amino Bmiddle) the fold change in BRET signal at 56°C. over acids are those that share the same biophysical characteris the background BRET signal at 42°C. for cells transfected tics and can be grouped into the families, e.g., acidic (e.g., with CDK2-Nluc target that were treated with DMSO and aspartate, glutamate), basic (e.g., lysine, arginine, histidine), varying dye concentrations. The larger the signal/back non-polar (e.g., alanine, Valine, leucine, isoleucine, proline, ground (S/B), the more signal window to allow for deter phenylalanine, methionine, tryptophan) and uncharged polar mining compound binding stability effects. This also allows (e.g., glycine, asparagine, glutamine, cysteine, serine, threo optimal dye concentrations to be determined; Cright) the nine, tyrosine). The “percent sequence identity” (or “percent fold change in BRET signal at 52° C. over the background sequence similarity') is calculated by: (1) comparing two BRET signal at 42° C. for cells transfected with Nluc optimally aligned sequences over a window of comparison MAPK14 target that were treated with DMSO and varying (e.g., the length of the longer sequence, the length of the dye concentrations. The larger the signal/background (S/B), shorter sequence, a specified window, etc.), (2) determining the more signal window to allow for determine compound the number of positions containing identical (or similar) binding stability effects, allowing determination of optimal monomers (e.g., same amino acids occurs in both sequences, dye concentrations. similar amino acid occurs in both sequences) to yield the 0051 FIG. 35 shows fluorescent mode analysis of number of matched positions, (3) dividing the number of CSOO36. matched positions by the total number of positions in the 0052 FIG. 36 shows fluorescent mode analysis of comparison window (e.g., the length of the longer sequence, CSOO96. the length of the shorter sequence, a specified window), and 0053 FIG. 37 shows fluorescent mode analysis of (4) multiplying the result by 100 to yield the percent CSOO48. sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in DEFINITIONS length and have identical amino acids at all but 1 position, 0054 As used herein, the terms “fusion,” “fusion pro then peptide A and peptide B have 95% sequence identity. If tein, and “fusion polypeptide' synonymously refer to a the amino acids at the non-identical position shared the same chimera of heterologous first and second protein or poly biophysical characteristics (e.g., both were acidic), then peptide segments, for example, a chimera of a protein of peptide A and peptide B would have 100% sequence simi interest (e.g., target protein) joined to a reporter protein (e.g., larity. As another example, if peptide C is 20 amino acids in luciferase). The second protein is typically fused to the length and peptide D is 15 amino acids in length, and 14 out N-terminus or C-terminus of the first protein, but may also of 15 amino acids in peptide D are identical to those of a be inserted internally within the sequence of the first protein. portion of peptide C, then peptides C and D have 70% 0055 As used herein, the term “luciferase” refers to any sequence identity, but peptide D has 93.3% sequence iden of a variety of monooxygenase enzymes that catalyze the tity to an optimal comparison window of peptide C. For the conversion (e.g., oxidation) of a Substrate (e.g., firefly purpose of calculating percent sequence identity” (or “per luciferin, latia luciferin, bacterial luciferin, coelenterazine, cent sequence similarity') herein, any gaps in aligned dinoflagellate luciferin, vargulin, and derivatives thereof) sequences are treated as mismatches at that position. into an excited-energy-state product that emits energy in the 0.058 As used herein, the term “sample is used broadly form of light upon decaying to its ground state. to refer to any of biological samples (e.g., fluids, tissues, 0056. As used herein, the term “natural polypeptide’ as etc.) and environmental samples as well as reaction mixtures used herein refers to a polypeptide that exists in nature. For or other multicomponent Solutions and mixtures. example, a “natural luciferase' is a luciferase polypeptide 0059. As used herein, the term “complex sample” refers with a sequence and/or any other relevant features (e.g., to a sample comprising a large number and variety of post-translational modifications) that exists in nature. The different compounds, polymers, macromolecules, com term “synthetic polypeptide' refers to a polypeptide having plexes, etc. A complex sample may comprise buffers, salts, an amino acid sequence that is distinct from those found in peptides, polypeptides, proteins (including also enzymes), nature (e.g., not natural). When used in this context, the term carbohydrates (complex and simple carbohydrates), lipids, “synthetic” does not relate to the method by which a fatty acids, fat, nucleic acids, organelles and other cellular polypeptide is produced (e.g., recombinant technology, components, etc. Examples of complex samples include US 2016/0282360 A1 Sep. 29, 2016 cells, e.g., live intact cells, cell lysates, body fluids (e.g., which is herein incorporated by reference in its entirety) is blood (or blood products), Saliva, urine, etc.), tissues (e.g., monitored over temperature range sufficient to result in biopsy tissue), cells grown in vitro and Subsequently unfolding and aggregation of the unbound target protein, injected into animal in vivo and recollected for ex vivo both in the presence and absence of a ligand or test ligand analysis, cells in 3D culture, cells in tissues, reaction mix for the target protein. An altered or shifted luminescent tures, etc. In particular embodiments, a complex samples signal over the range of temperatures indicates interaction contain a target protein as well as additional non-target between the target protein and ligand. In some embodi peptides, polypeptides, and/or proteins. ments, the luminescent signal (e.g., relative light units 0060. As used herein, the term “quenched” refers to a (RLU)) from the luciferase reporter portion of the fusion is decrease in fluorescence emission from a fluorescent entity directly monitored. In some embodiments, bioluminescence (e.g., dye) upon interaction of a particular substance (e.g., resonance energy transfer (BRET) from the luciferase water) or condition, relative to the fluorescence emission reporter portion of the fusion to an environmentally sensitive from the fluorescent entity when not interacting with the dye (e.g., a dye that interacts with the hydrophobic portions particular substance or condition. The term “quenched of the target protein as they become exposed at higher itself does not place any limitation on the extent of the temperatures, a protein-aggregation detection dye, etc.) is decrease in fluorescence. The degree of quenching may be measured. expressed at a percentage of fluorescence in the quench state 0065. In some embodiments, the target/luciferase fusion compared to the unquenched State (e.g., 10 RLU in the is expressed in cells (e.g., a live intact cell or a relevant cell quenched state compared to 100 RLU in the unquenched line), and the cells and/or a cell lysate thereof, is exposed to state is 90% quenching). a ligand of interest (e.g., ligand for the target protein, test 0061. As used herein, the term “BRET is used to ligand (e.g., potential drug), etc.) and Subsequently exposed describe the occurrence of bioluminescence resonance to conditions (e.g., increased temperature) capable of caus energy transfer between a bioluminescent donor (e.g., a ing the unfolding and/or aggregation of the unbound target luciferase protein) and an acceptor fluorophore. It is a protein. In Such embodiments, a target protein that has been distance-dependent interaction in which energy is trans stabilized by interaction with ligand in the cell or cell lysate ferred from the donor bioluminescent protein and substrate will exhibit an altered or shifted melting temperature as to an acceptor fluorophore without emission of a photon. detected by alteration of the luminescent or BRET signal in The efficiency of BRET is dependent on the inverse sixth the presence and absence of ligand. power of the intermolecular separation, making it useful 0066. In some embodiments in which luciferase signal is over distances comparable with the dimensions of biological directly detected as a measure of thermal shift, luciferase macromolecules (e.g., within 30-80 A, depending on the Substrate (and purified complementary peptide/polypeptide degree of spectral overlap). in the case complementation assays (e.g., using NLPep and 0062. As used herein, the term “ligand for a target NLPoly (Promega Corp., Madison, Wis.); see, e.g., U.S. protein’ refers to a molecular entity that binds to a target Pub. No. 2014/0348747, which is herein incorporated by protein. The ligand may be a small molecule, peptide, reference in its entirety)) is added to the samples (e.g., cells, antibodies, macromolecules (e.g., nucleic acid, viral pro cell lysate, etc.) for analysis. In other embodiments in which teins, bacterial proteins, polysaccharides, synthetic poly BRET signal from the luciferase of the fusion to an envi mers), or other molecular entity that bind the target protein. ronmentally sensitive dye (e.g., a dye that interacts with the The term “test ligand refers to a molecular entity that is hydrophobic portions of the target protein as they become being assayed for the capacity to bind the target protein. exposed at higher temperatures, a protein-aggregation detec tion dye, etc.) is detected as a measure of thermal shift, DETAILED DESCRIPTION luciferase Substrate (and purified complementary peptide? 0063 Provided herein are systems and methods for char polypeptide in the case complementation assays (e.g., using acterizing target/ligand engagement. In particular, NLPep and NLPoly)) and the environmentally sensitive dye luciferase-labeled polypeptide targets are used to detect or are added to the samples (e.g., cells, cell lysate, etc.) for quantify target/ligand engagement (e.g., within a cell or cell analysis. In some embodiments, detergents (e.g. Digitonin), lysate). lysis buffer (e.g. NANOGLO Lytic reagent), and/or other 0064. In some embodiments, compositions (e.g., fusions reagents are added to the sample. In some embodiments, of target polypeptides and luciferase reporter), Systems (e.g., luminescence and/or BRET is analyzed using, for example, kits or reaction mixtures comprising, for example, test by a luminometer at one or more specific temperatures (e.g., ligands, luciferase Substrates, assay reagents, cells, e.g., live encompassing a temperature in which the target protein intact cells, cell lysates, etc.) and methods are provided for unfolds). Shift of the signal (e.g., luciferase signal or BRET monitoring (e.g., detecting, quantitating, etc.) target/ligand signal) in the presence Vs. absence of ligand indicates engagement in complex (cellular) environments (e.g., based interaction (e.g., binding) of the target and ligand. on the biophysical principle of ligand-induced thermal sta 0067. It has long been recognized that the binding of low bilization of target proteins). In some embodiments, the molecular weight ligands increases the thermal stability of a luminescent signal generated by a fusion of a target protein protein (Koshland (1958). Proc Natl Acad Sci USA. 44 (2): and luciferase (e.g., NANOLUC (Promega Corp., Madison, 98-104.: Linderstrøm-Lang & Schellman (1959). The Wis.); see, e.g., U.S. Pat. No. 8,557,970 and U.S. Pat. No. Enzymes. 1(2) 443-510.; herein incorporated by reference in 8,669,103, both of which are herein incorporated by refer its entirety). A TSA detects this stabilizing effect by mea ence in their entireties) or a luciferase peptide/polypeptide Suring the thermal stability of a target protein in the presence interacts with a complement polypeptide?peptide to produce and absence of a ligand, thereby detecting or quantifying the an active enzyme (e.g., NLPep and NLPoly (Promega Corp., target/ligand interaction. A traditional TSA is a fluorescence Madison, Wis.); see, e.g., U.S. Pub. No. 2014/0348747, based method for monitoring target/ligand interactions (WO US 2016/0282360 A1 Sep. 29, 2016

1997/020952; herein incorporated by reference in its 0069. Although the reporter of a fusion construct may be entirety); the primary weakness of Such an assay is that it one that exhibits any suitably detectable activity, in some detects the unfolding of any proteins present in the sample embodiments, the reporter is a luciferase enzyme. Suitable being assayed (e.g., target and non-target protein), and luciferase enzymes include those selected from the group therefore can only be used to analyze purified protein. consisting of: Photinus pyralis or North American firefly Because target/ligand interactions may be affected by a luciferase; Luciola cruciata or Japanese firefly or Genji multitude of factors in vivo, a traditional TSA of purified botaru luciferase; Luciola italic or Italian firefly luciferase: protein may be of limited informational value. Other TSA Luciola lateralis or Japanese firefly or Heike luciferase: type assays have been developed (See, e.g., Moreau MJ, et Luciola ming relica or East European firefly luciferase; Pho al. Quantitative determination of protein stability and ligand turis pennsylvanica or Pennsylvania firefly luciferase: Pyro binding using a green fluorescent reporter system. Mol. phorus plagiophthalamus or Click beetle luciferase: Phrixo thrix hiritus or Railroad worm luciferase: Renilla reniformis Biosyst. (6): 1285-1292. 2010; and CETSA, U.S. Pub No. or wild-type Renilla luciferase; Renilla reniformis Rluc8 2014/0057368; herein incorporated by reference in their mutant Renilla luciferase: Renilla reniformis Green Renilla entireties); however, these assays rely on post-denaturation luciferase; Gaussia princeps wild-type Gaussia luciferase; quantification of soluble vs. denatured protein as a measure Gaussia princeps Gaussia-Dura luciferase; Cypridina noc of target protein stability and require multiple purification tiluca or Cypridina luciferase; Cypridina hilgendorfii or and/or detection steps. Provided herein are assays that can be Cypridina or Vargula luciferase; Metridia longa or Metridia used to identify ligands that bind to a target protein and/or luciferase; and Oplophorus luciferase (e.g., Oplophorus to quantify the affinity of Such interactions. The assays gracilirostris (Og|Luc luciferase), Oplophorus grimaldii, described herein are distinct from other methods (e.g., Oplophorus spinicauda, Oplophorus foliaceus, Oplophorus traditional thermal shift, GFP reporter systems, CESTA, noraeZeelandiae, Oplophorus typus, Oplophorus noraeze etc.), for example, because: (1) the assays allow character landiae or Oplophorus spinous). For any of the above ization of target/ligand interactions with non-purified pro luciferases, fusions may contain a wild-type or naturally tein; (2) the assays can be conducted in a cell, cell lysate, or occurring variant of the luciferase or may comprise a syn other complex liquid containing many different biomol thetic version (e.g., optimized for one or more characteristics ecules; (3) the assay technology does not rely upon anti (e.g., emission, stability, etc.). In some embodiments, assays bodies recognizing target protein epitopes; (4) the assays are are carried out both in the presence and absence of a ligand homogeneous; (5) the assays use common and simple for the target protein (or a test ligand) and in the presence of reagents, materials and instruments (e.g., a luminometer); the appropriate Substrate for the luciferase. A negative and/or (6) assays are not limited to a single read. control assay may be performed in the absence of Substrate. A positive control may be performed with the un-fused 0068. In some embodiments, the systems and methods luciferase and appropriate Substrate in the presence and described herein utilize fusion constructs (e.g., fusion poly peptides and nucleic acids and vectors encoding them) absence of ligand and/or in the presence an absence of comprising a target protein (e.g., the binding characteristics un-fused target protein. of which are being examined) and a reporter peptide or 0070. In some embodiments, a substrate for the reporter polypeptide. In some embodiments, any protein of interest (e.g., bioluminescent reporter (e.g., luciferase, etc.), etc.) is may find use as the target protein. Assays described herein provided. In some embodiments, a bioluminescent reporter provide for analysis of the binding characteristics (e.g., converts the Substrate into a reaction product and releases potential ligands, binding affinity, etc.) of Such proteins. A light energy, e.g., luminescence, as a byproduct. In some reporter of the fusion construct may be any peptide or embodiments, the substrate is a substrate for a luciferase polypeptide, the activity of which can be detected within a enzyme. Appropriate Substrates for known reporters are cell, e.g., a live intact cell, or cell lysate, in real-time. While understood in the field. the scope of embodiments herein is not limited by the (0071. In some embodiments, a fusion construct com identity of the reporter, many embodiments utilize a prises a target protein fused to a Oplophorus grachlorostris luciferase reporter. In Such embodiments, a suitable luciferase (Og|Luc). The fusion may comprise a natural (e.g., luciferase is fused (e.g., directly or by a linker (e.g., peptide wild-type or variant) Ogluc sequence or may comprise a or other linker moiety)) to the N-terminus or C-terminus of synthetic Og|Luc (e.g., optimized for one or more charac a target protein (or inserted internally within the target teristics (e.g., luminescence, signal stability, protein stabil protein) to generate a fusion polypeptide for use in the ity, etc.), etc.). The natural wild-type Og|Luc sequence is systems and methods herein. In some embodiments, nucleic given in SEQ ID NO: 1. Some suitable Oplophorus acid constructs are provided that encode fusion polypeptides luciferases are described, for example, in U.S. Pat. No. (e.g., N-target-reporter-C, N-reporter-target-C, N-target 8,669,103 and U.S. Pat. No. 8,557,970. In some embodi linker-reporter-C, N-reporter-linker-target-C, etc.). In some ments, a luciferase polypeptide comprises at least 60% (e.g., embodiments, vectors (e.g., plasmids, bacmids, cosmids, >65%, >70%, >75%. >80%, >85%, >90%, >95%, >98%, viral vectors (e.g., lentivirus vectors, adeno-associated viral >99%, 100%, and any ranges therein) sequence identify with vectors (AAVs), etc.), etc.) comprising nucleic acid con SEQ ID NO: 1. In some embodiments, comprises a amino structs that encode fusion polypeptides (e.g., along with acid Substitutions at positions relative to one or more of appropriate incorporation and/or expression elements) are positions: 1, 2, 4, 6, 10, 11, 14, 15, 16, 18, 19, 20, 21, 22. provided. In some embodiments, cells (e.g., bacterial, mam 23, 24, 25, 27, 28, 31, 32, 33, 34, 36, 38, 39, 40, 42, 43, 44, malian, human, etc.) transformed or transfected (e.g., tran 45, 46, 47, 48, 49, 50, 51,54, 55, 56,58, 59, 60, 66, 67, 68, siently or stably) with nucleic acids and/or vectors encoding 69, 70, 71, 72, 74, 75, 76, 77, 86, 87, 89,90, 92,93, 94, 95, fusion polypeptides useful for assays described herein are 96, 97,98, 99, 100, 102, 104, 106, 109, 110, 111, 112, 113, provided. 115, 117, 119, 124, 125, 126, 127, 128, 129, 130, 135, 136, US 2016/0282360 A1 Sep. 29, 2016

138, 139, 142, 143, 144, 145, 146, 147, 148, 149, 150, 152, therein) but less than 100% sequence identify with SEQ ID 154, 155, 159, 158, 163, 166, 167, 168, or 169 of SEQ ID NO: 3. In some embodiments, the first polypeptide com NO: 1. In some embodiments, a luciferase exhibits one or prises a amino acid Substitutions at positions relative to one more of enhanced luminescence, enhanced signal stability, or more of positions (e.g., Q11E. G15A, F31L, G35A, and enhanced protein stability relative to a wild-type Oplo L46R, G51A, G67A, G71A, M75E, I76V, H93P. I107L, phorus luciferase. In some embodiments, comprises at least D108N, N144T, L149M, 157S (addition of S at 157 posi 60% (e.g., >65%, >70%, >75%. >80%, >85%, >90%, >95%, tion) of SEQID NO: 4 (wherein SEQID NO: 4 numbering >98%, >99%, 100%, and any ranges therein) sequence is 1-156)). In some embodiments, the complement peptide identify with SEQ ID NO: 2. In some embodiments, com comprises a amino acid Substitutions at positions relative to prises SEQ ID NO: 2. one or more of positions (e.g., G157del, T159S. C164F, 0072. In some embodiments, a target protein is fused to E165K, N166K, L168S, A169del of SEQID NO:3 (wherein a first peptide or polypeptide (e.g., that does not indepen SEQ ID NO: 3 numbering is 157-169)). dently exhibit substantial detectable activity) that forms an 0075 Depending upon the identity of the bioluminescent active reporter construct through structural complementa reporter used, an appropriate substrate will be selected, for tion with a second polypeptide or peptide (See, e.g. U.S. Ser. example, from those including, but not limited to: firefly No. 14/209,610 and U.S. Ser. No. 14/209,546; herein incor luciferin, latia luciferin, bacterial luciferin, coelenterazine, porated by reference in their entireties.). In such embodi dinoflagellate luciferin, Vargulin, and Suitable derivatives ments, only one of the two (or more) elements (peptide or thereof. In some embodiments, the substrate is a substrate polypeptide) that form the reporter construct is fused to the for an Oplophorus luciferase, e.g., NANOLUC enzyme target. The structural complement of the fused element of the from Promega Corporation (e.g., SEQ ID NO: 2). In some reporter construct is added separately to the system (e.g., cell embodiments, the Substrate comprises coelenterazine, a (e.g., added exogenously, expressed by the cell), cell lysate, coelenterazine derivative, a structural or functional equiva in vitro system, etc. In particular embodiments, a target lent of coelenterazine, a molecule Substantially equivalent to protein is fused to a first peptide or polypeptide (e.g., that coelenterazine (e.g., structurally and/or functionally), or doesn't independently exhibit a detectable activity) that molecule functionally or structurally similar to coelentera forms an active luciferase construct through structural Zine. In some embodiments, the bioluminescent reporter complementation with a second polypeptide or peptide (See, converts the coelenterazine, coelenterazine derivative, struc e.g. U.S. Ser. No. 14/209,610 and U.S. Ser. No. 14/209,546: tural or functional equivalent of coelenterazine, or Substan herein incorporated by reference in their entireties.). In such tial equivalent to coelenterazine into coelenteramide, a coel embodiments, the fusion polypeptide will not independently enteramide derivative, a structural or functional equivalent catalyze a significant amount of Substrate a high-energy of coelenteramide, or a Substantial equivalent to coelenter state product that will produce light upon return to a stable amide and releases light energy as a byproduct. state. Rather, only in the presence of the complement 0076. In some embodiments, a reporter is an epitope tag polypeptide or peptide is the active luciferase construct (See FIG. 21, top panel). In such embodiments, a labeled formed. Embodiments will typically be described as com (e.g., fluorescently labeled) antibody that recognizes and prising a target fused to a complete reporter; however, unless binds the epitope is included. In some embodiments, BRET indicated otherwise, it should be understood that the reporter occurs between the label on the antibody and the fluorescent may be formed by structural complementation of multiple dye when both are bound to the epitope-tagged target (e.g., 2, 3, or more) elements, only one of which is fused to protein. In some embodiments, the epitope is accessible to the target. the antibody when the protein is folded or unfolded. In other 0073. In some embodiments, a first peptide (e.g., fused to embodiments. Some degree of target unfolding is required the target protein) comprises at least 60% (e.g., 65%, 70%, for the antibody to access the epitope tag. In some embodi 75%. 80%, 85%, 90%, and any ranges therein) but less than ments, following a thermal denaturation step, addition of a 100% sequence identify with SEQID NO:3, and a comple detection antibody labeled with a donor fluorophore (e.g. ment polypeptide comprises at least 60% (e.g., 65%. 70%, terbium, europium, etc.) is used in a FRET assay with 75%. 80%, 85%, 90%, 95%, and any ranges therein) but less denaturation/aggregation-sensitive (e.g., environmentally than 100% sequence identify with SEQ ID NO: 4. In some sensitive dye, hydrophobic dye, etc.) dye as a FRET accep embodiments, the first peptide comprises an amino acid tor. Ligand-mediated thermal stabilization results in a loss of Substitutions at positions relative to one or more of posi FRET/TR-FRET signal and/or the requirement of more tions: G157del, T159S, C164F, E165K, N166K, L168S, denaturing conditions (e.g., higher temperature) to achieve A169del of SEQ ID NO: 3 (wherein SEQ ID NO: 3 the FRET/TR-FRET signal. numbering is 157-169). In some embodiments, the comple 0077. In some embodiments, two epitope tags (e.g. ment polypeptide comprises a amino acid Substitutions at FLAG-V5) are fused to a target protein (See FIG. 21, bottom positions relative to one or more of positions: Q11E. G15A, panel). In Such embodiments, the epitopes are accessible to F31L, G35A, L46R, G51A, G67A, G.71A, M75E, I76V, separate, differently-labeled antibodies (e.g., donor and H93P. I107L, D108N, N144T, L149M, 157S (addition of S acceptor labeled) for the respective epitopes when the target at 157 position) of SEQID NO: 4 (wherein SEQ ID NO: 4 protein is folded (FRET signal is produced). Upon partial or numbering is 1-156). complete unfolding of the target, the epitopes become 0074. In some embodiments, a first polypeptide (e.g., unavailable and the FRET signal is lost. fused to the target protein) comprises at least 60% (e.g., 0078. In some embodiments, the formation of a fusion 65%, 70%, 75%. 80%, 85%, 90%, 95%, and any ranges protein does not substantially impact or alter the stability or therein) but less than 100% sequence identify with SEQ ID function of either the target or reporter relative to the NO: 4, and a complement polypeptide comprises at least function of each individual protein. For example, for a 60% (e.g., 65%, 70%, 75%. 80%, 85%, 90%, and any ranges fusion of target enzyme and a luciferase, the formation of the US 2016/0282360 A1 Sep. 29, 2016

fusion does not substantially alter the stability of either stabilization of the reporter and enhancement of signal portion, the luminescence of the luciferase, the signal sta stability, binding of the ligand to the target can be observed bility of the luciferase, or the activity of the enzyme. as an increase in the stability of the reporter signal. Embodi 0079. In some embodiments, the target polypeptide and ments in which the signal from the reporter (e.g., luciferase) reporter polypeptide (e.g., luciferase) are connected directly at a given temperature (e.g., around the T of the protein) or without intervening amino acid sequence. For example, the temperature range is directly used as a measure of protein target polypeptide may be fused to the N-terminus or the stability are referred to herein as “direct detection.” C-terminus of the reporter. In other embodiments, the target I0081. In some embodiments, ligand binding destabilized polypeptide and reporter polypeptide (e.g., luciferase) are the target protein. In some embodiments, the reporter is connected by a linker moiety or connector sequence. In thermally stable or labile compared to the protein of interest Some embodiments, a linker provides connection and allows it is fused to. a desired amount of space/distance between the elements I0082 In some embodiments, the activity (e.g., lumines (e.g., to reduce interactions between target and reporter). cence) from a target/reporter fusion is measured at one or Although a linker is typically a peptide linker, in some more temperatures (e.g., over a range of temperatures (e.g., embodiments, other chains or polymers may be utilized. In from below the T of the target protein to above the T of Some embodiments, a connector sequence comprises or the target protein)), both in the presence and absence of a consists of one or more amino acids (e.g., a peptide or ligand for the target or a test ligand. If the ligand or test polypeptide chain of 2-50 amino acids (e.g., 2, 3, 4, 5, 6, 7, ligand binds to or interacts with the target protein under the 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, conditions assayed (e.g., in a cell, in a cell lysate, etc.), a 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, corresponding shift in the activity of the reporter is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, and any ranges therein). observed. For example, at a given temperature (e.g., near the In some embodiments, the presence of a connector sequence T of the target protein) the activity (e.g., luminescence in a fusion protein does not substantially impact or alter the (e.g., RLUS)) of the reporter (e.g., luciferase) is increased. If stability or function of either the target or reporter relative to the unfolding of the protein is analyzed at a range of the function of each individual protein. For example, for a temperatures, so as to determine a T, ligand binding and fusion of target enzyme and a luciferase, the presence of a stabilization of the target protein is apparent as a shift (e.g., connector sequence does not substantially alter the stability right shift) in the T, to a higher temperature. of either portion, the light output of the luciferase, or the I0083 Traditional thermal shift assays utilize purified activity of the enzyme. In some embodiments, a connector target protein and a dye that (1) binds nonspecifically to sequence is included in a fusion to prevent interactions hydrophobic Surfaces and (2) is quenched by water (e.g., between the target and reporter that could affect the stability Sypro Orange). In Such an assay, at lower temperatures (e.g., of activity of either or both portions. For any particular <50° C., <48° C., <46° C., <44° C., <42° C., <40° C., <38° combination of proteins in a fusion, a wide variety of C., <36° C., 34° C., <32° C., <30° C., and ranges therein connector sequences may be employed. In one embodiment, (e.g., 30-50° C., 35-45°C., etc.), depending upon the target the connector sequence is a sequence recognized by an stability) the target is stably folded, and the hydrophobic enzyme, e.g., a cleavable sequence. For instance, the con regions of the target are buried and inaccessible to the nector sequence may be one recognized by a caspase or TEV hydrophobic dye. Under these folded conditions, the dye protease, or may be a chemically-cleavable or photocleav cannot significantly bind to the target protein, and therefore able sequence. fluorescence from the dye is quenched by water in the 0080. In some embodiments, the activity (e.g., lumines aqueous environment. As temperatures are increased, the cence) of a reporter (e.g., luciferase) fused to a target protein target begins unfolding and/or aggregating, the hydrophobic is directly detected. In some embodiments, the activity (e.g., regions of the target become reveled, and the dye binds to luminescence) of a reporter construct (e.g., luciferase the target. Upon binding the target protein, the dye becomes dimer), a portion of which is fused to a target protein (e.g., unduenched and the fluorescent signal increases. The more the structural complement portion is not fused to the target stable the target, the higher the temperature required to protein), is directly detected. In some embodiments, reporter observe the fluorescence increase. Similarly, a less stable activity is measured at one or more temperatures as a target results in destabilization of the target and an increase measure of target protein stability. In some embodiments, at in fluorescent signal at a lower temperature. In Such an assay, lower temperatures (e.g. <50° C., <48° C., <46° C., <44°C., if a ligand binds to the target protein, the target protein is <42°C., <40° C., <38° C., <36° C., 34° C., <32° C., <30° stabilized, which can be observed as an increase in the C., and ranges therein (e.g., 30-50° C., 35-45° C., etc.), temperature required to generate the fluorescent signal. depending upon the target and reporter stabilities) both the I0084. The traditional TSA relies on nonspecific binding target and reporter are stably folded. As temperatures are of the dye (e.g., Sypro Orange) to the hydrophobic regions increased, and one or both of the target and reporter begin of the target protein. Therefore, if proteins other than the unfolding or aggregating, signal from the reporter decreases target protein are present in the assay, the dye will bind to (e.g., the activity of the reporter decreases as it becomes those proteins as well (e.g., as they become unfolded), unfolded or aggregated). In some embodiments, because the thereby confounding the results. For at least this reason, reporter and target are fused, a more stable the target protein traditional TSAS are performed using purified target protein results in a more stable reporter and reporter signal. Simi and cannot be performed in cells, cell lysate, or other larly, a less stable target results in destabilization of the complex environments. reporter and a loss, or reduction, of reporter signal at a lower 0085. The direct detection embodiments described above temperature. In some embodiments, if a ligand binds to the overcome the limitation of the traditional TSA by detecting target protein, the target protein is stabilized. Since, in some activity of a reporter fused to the target protein. The unfold embodiments, stabilization of the target protein results in ing of proteins not fused to the reporter (e.g., proteins US 2016/0282360 A1 Sep. 29, 2016 present in the cellular milieu or cell lysate) do not directly generate increased BRET signal (e.g., increased over back affect the signal. In another embodiment, a BRET TSA is ground)). In BRETTSA, although the hydrophobic dye will provided that makes use of the target-specificity of the bind to any proteins present in the complex sample (e.g., direct-detection embodiments described herein (e.g., only cell, cell lysate, etc.), only the target protein is fused to the the unfolding of proteins fused to a reporter are monitored reporter, therefore the BRET signal will be specific to the (e.g., even in a complex environment)), as well as the target protein. Due to the proximity constraints placed on sensitivity of the traditional TSA (e.g., the increase in BRET, significant energy transfer only occurs between the fluorescence from the dye is directly correlated with the reporter and target-bound dye. The BRET signal is made degree of unfolding of the target protein). In Such embodi possible by 1) presence of reporter (e.g., NLuc) activity and ments, a fusion of a target protein and reporter (e.g., 2) binding of the dye to the fusion protein allowing the luciferase) is generated, and fluorescence measurements are reporter to donate and excite the dye to emit at a certain taken in the presence of an environmentally-sensitive dye wavelength. The fusion of the reporter to the target provides (e.g., a dye that: (1) binds nonspecifically to hydrophobic the target specificity; the dye provides a readout of the target Surfaces, (2) has an excitation spectra that significantly protein state (e.g., folded, partially folded, unfolded, etc.). overlaps the emission spectra of the reporter, and optionally 0086. In some embodiments, BRET TSAs described (3) the fluorescence of which is quenched by water). The herein utilize fluorogenic dyes. In other embodiments, dyes activity of the dye may be quenched by water (e.g., as in a are non-fluorogenic. traditional TSA), or the dye may be active (e.g., capable of 0087. In some embodiments, BRET TSAs described emitting light at a particular wavelength in response to an herein utilize fluorescent dyes that: (1) are quenched by an excitation) whether bound to a protein or free in solution. In aqueous environment (e.g., when not bound by a target); (2) some embodiments, because the dye will be excited via interact with (e.g., bind) hydrophobic Surfaces, such a BRET from the reporter and not directly, the capacity for the hydrophobic peptide segments; and (3) exhibit enhanced or dye to fluoresce when not bound to the target (e.g., when increased fluorescence upon binding a hydrophobic Surface. bound to other proteins within a cell or cell lysate.) does not I0088 Certain fluorescent compounds exhibit only a weak affect the assay (e.g., because only target-bound dye is fluorescence emission when free in aqueous solution within the range (e.g., 1-10 nm) to receive energy transfer (Semisotnov, et al., Biopolymers 31:119, 1991; herein incor from the reporter). In some embodiments, at lower tempera porated by reference in its entirety), but fluoresce much tures (e.g., <50° C., <48°C., <46°C., <44°C., <42°C., <40° more strongly when bound to organized hydrophobic Sur C., <38°C., <36° C., 34° C., <32° C., <30° C., and ranges faces. Binding of these compounds to fully folded globular therein (e.g., 30-50° C., 35-45°C., etc.), depending upon the proteins is typically weak, since hydrophobic residues are target stability) the target is stably folded, and the hydro predominantly buried in the interior of the protein. Further phobic regions of the target are buried and inaccessible to more, binding of these compounds to random coil confor the hydrophobic dye. Under these folded conditions, signal mations (as found in fully unfolded or denatured polypep from the reporter (e.g., fused to the target) is detected, but tides) is also disfavored because in these conformations because the dye cannot significantly bind to the folded target hydrophobic residues, though exposed, are not sufficiently protein (e.g., because hydrophobic regions are no accessible well organized to Support high affinity binding of the probes. on the folded target) fluorescence from the dye as a result of The dyes, however, typically bind with higher affinity and BRET is not detected (e.g., significant direct reporter signal Stoichiometry to compact unfolded protein conformations, is observed, but BRET to the dye is not observed, is such as “molten globules, which are characterized by decreased, and/or is minimal). The proximity limitation of compactness relative to random coil unfolded States, the BRET prevents significant energy transfer from occurring presence of Substantial secondary structure, and the lack of between the reporter and unbound dye. In embodiments in a unique overall conformation. These probes may be which the dye is quenched by water, unbound dye is also not referred to herein as “environmentally-sensitive hydropho detected by direct excitation (e.g., from a light source). In bic dyes.” embodiments in which the dye maintains its activity in 0089. In some embodiments, BRET TSAs described water, while not producing a signal from BRET, the herein utilize fluorescent dyes that bind to the hydrophobic unbound dye may be detected by direct excitation (e.g., from regions of unfolding proteins, molten globules, and aggre a light Source). As temperatures are increased, the target gates. Due to the proximity limitations of BRET, significant begins unfolding and/or aggregating, the hydrophobic energy transfer from the bioluminescent report to the fluo regions of the target become revealed, the dye binds to the rescent dye only occurs when the dye is bound to the target, and the dye becomes unduenched (e.g., the quantum unfolding target protein. When the target protein is fully yield of the dye increases). Under these partially-folded or folded, the fluorescent dye does not bind the target protein, unfolded conditions, BRET occurs from the reporter to the the dye is not within the proximity of the bioluminescent bound dye (e.g., dye bound to the revealed hydrophobic reporter, and BRET does not occur. In such embodiments, portions of the target protein), and fluorescence from the the bioluminescent reporter provides the system's specificity dye, as the result of BRET, is detectable as a direct measure for the target protein while the fluorescent dye provides the of (e.g., is proportional to) target protein unfolding. The measure of protein unfolding. more stable the target, the higher the temperature required to 0090. Examples of dyes that find use in embodiments observe the increase in BRET. Similarly, a less stable target described herein include SYPRO Orange, 1-anilino-8-naph results in destabilization of the target and an increase in the thalene sulfonate (ANS), bis-1-anilino-8-naphthalene sul BRET signal at a lower temperature. In such an assay, if a fonate (bis-ANS), 6-propionyl-2-(N,N-dimethyl)-aminon ligand binds to the target protein, the target protein is aphthalene (Prodan) (Molecular Probes, Eugene, Oreg.), stabilized, which can be observed as an increase in the SYPRO Red, SYPRO Ruby, SYPRO Tangerine, and Nile temperature required to generate the BRET signal (e.g., to Red. Other suitable dyes are those listed in Table 1.

US 2016/0282360 A1 Sep. 29, 2016 15

TABLE 1-continued

Optical Property (in DMSO)

Structure MS Emission Excitation Reference #

359.7 470-480 62O CSOO75

21N 21

479.7 480 624 CSO101 als (M'.) ch, 21 N B9 O 4. ~s OEt

451.7 480 624 CSOO36 th (M'.) ch, 21 ---"N Bo

711.1 480 622 CSO1 OO al, (M'.) cu?, 21 N B9 O

2 i-->H

> *r-s-s-O

654.9 48O 622 CSOO45 l, (M'.) ch, US 2016/0282360 A1 Sep. 29, 2016 16

TABLE 1-continued

Optical Property (in DMSO)

Structure MS Emission Excitation Reference #

C6H13 776.1 478 62O CSOO48

N C6H13Y 21 N O

26i-- N S O H O 3 N-1N1 r- N-1 no-1N-1 O

C6H13 695.1 480 62O CSOO73

N Y C6H13 21 N O

26i-- N 1N1SSSO N

455.7 550 604 CSOO67 (M'.)

O 553.9 550 604 CSOO68 OS C6H13

C6H131 NGE 21 e

499.8 480 618 CSOO96 (M'.)

C6H13 N f Y GE) w N le O Br

US 2016/0282360 A1 Sep. 29, 2016 18

TABLE 1-continued

Optical Property (in DMSO)

Structure MS Emission Excitation Reference #

480 62O CSO158

N h C6H1.Y 21 N 4. Be 2

491.8 S16 702 CSO155 (M2) Nal, Y C6H 21N 21 N

N N|- a SeGE) N-1N16N SeGE)

737.2 S16 702 CSO147 (M'.) Nals C6H131 21a 21 N O

Br N ad NH

O O O

> O --~~

537.8 462 654 CSOO38

Nal, C6H1.1 21 N 9 2 N-1a-1's

537.8 not fluorescent CSOO71

CH6 Nals O 21 N O US 2016/0282360 A1 Sep. 29, 2016 19

TABLE 1-continued Optical Property in DMSO

Structure MS Emission Excitation Reference # C6H13 537.8 not fluorescent CSOO72

N Y

C6H13 O 21 N G92 N

N- SO3S 1N1)-O 523.7 CSOOOX

1N1NO O 21 N le O 2 N-1N- SO

0091. In some embodiments, combinations of dyes are turants and increasing concentrations thereof, favor protein used. In some embodiments, a combination of dyes having unfolding. Numerous protein denaturants are known in the varying binding properties is used. Such that the dye mix art and could be empirically selected for use herein. Without finds use with target proteins having different structural being limited to a particular protein denaturant, exemplary characteristics. Other dyes (e.g., dyes known in the field) protein denaturants include guanidinium thiocyanate, guani may also find use in embodiments described herein. dinium hydrochloride, arginine, Sodium dodecyl sulfate 0092. One advantage of some embodiments described (SDS), urea, or any combination thereof. In some embodi herein is the ability of the assays to be performed in complex ments, a combination of one or more protein denaturants and samples (e.g., those containing non-target proteins). In some increased temperature is employed. embodiments, samples that find use with the TSAS described herein include cells (e.g., cell culture, cell sample from a 0094. In some embodiments, the reporter signal and/or patient, etc.), tissue samples (e.g., tissue from biopsy), cell BRET signal (e.g., the result of energy transfer from biolu lysates (e.g., lysed of cells expressing a target/reporter minescent protein to environmentally-sensitive hydrophobic fusion, cell lysate expressing target/reporter fusion in vitro, dye) is detected under a single condition, and a comparison cell lysate with exogenous target/reporter fusion, etc.), com is made between the signals in the presence and absence of plex reaction mixtures (e.g., in vitro reaction mixtures ligand. In Such embodiments, the signal is typically detected comprising the components necessary for the assay as well underpartially denaturing conditions, or conditions in which as non-essential components (e.g., non-target proteins, com the unbound target protein is beginning to unfold, but it is petitor proteins, competitor ligands, etc.), simple in vitro expected that the ligand-bound target will not be unfolding reaction mixtures comprising the components necessary for or will be less unfolded (as will be apparent from the change the assay, etc.), etc. in signal (e.g., direct reporter signal or BRET signal, 0093. In some embodiments, reporter and/or BRET sig depending upon the assay)). Appropriate conditions for Such nal is detected under conditions that favor protein folding a single-point assay can be determined empirically based on and/or conditions that favor Some degree of protein unfold the stability of the particular target protein. In some embodi ing. Two means of varying conditions are temperature and ments, conditions (e.g., temperature and/or denaturant con protein denaturants. For example, lower temperatures (e.g., centration) are selected at or near (e.g., +5%, it 10%, +15%, <50° C., <48° C., <46° C., <44° C., <42°C., <40° C., <38° +20%, +25%, +30%, +35%, +40%, +45%, +50%) the melt C., <36° C., 34° C., <32° C., <30° C., and ranges therein ing temperature (T) for the target protein. The melting (e.g., 30-50° C., 35-45° C., etc.)) favor protein folding, temperature (e.g., in the presence of the intended denaturant while higher temperatures (e.g., >50° C., >55° C., >60° C., concentration) of the target protein can be determined using >650 C., >70° C., >75° C., >80° C., >850 C., >90° C., >950 any of a variety of well-known techniques, including, but C., and ranges therein (e.g., 60-90, C. 55-75° C., etc.)) not limited to: UV/visible spectrometry, nuclear magnetic favor protein unfolding, exposure of hydrophobic regions, resonance spectroscopy (NMR), circular dichroism (CD), and/or aggregation. Similarly, the presence of various dena etc. US 2016/0282360 A1 Sep. 29, 2016 20

0095. In other embodiments, the reporter signal and/or under various conditions in the presence and absence of BRET signal (e.g., the result of energy transfer from biolu ligand. In some embodiments, cells are transiently and/or minescent protein to environmentally-sensitive hydrophobic stably transformed or transfected with vector(s) (e.g., encod dye) is detected under multiple conditions or over a range of ing target/reporter fusion protein, structural complement conditions (e.g., varying temperature and/or denaturant con (e.g., NLPep and NLPoly), etc.). In some embodiments, centration), and a comparison is made between the signals in transgenic organisms are generated that code for the neces the presence and absence of ligand. In Such embodiments, sary assay components (e.g., target/reporter fusion protein, signal may be detected at a first condition in which both the structural complement (e.g., NLPep and NLPoly), etc.) for ligand-bound and inbound target are expected to be well carrying out the assays described herein. In other embodi folded. Under Such conditions, similar signals are typically ments, vectors are injected into whole organisms. A Suitable (although not always) expected in both the presence and vector may be viral (e.g., lentivirus, AAV, etc.) or non-viral absence of ligand. In some embodiments, signal is also (e.g., plasmid, bacmid, etc.). In some embodiments, in detected under one or more additional conditions (e.g., addition to encoding a target/reporter fusion, a vector further increasing temperature, increasing pressure, and/or increas comprises elements to allow/promote expression of as ing denaturant concentration) approaching and/or exceeding much. the expected T (e.g., under the denaturant concentration) of 0099. In some embodiments, other assay components not the target protein in the absence of ligand. As increasingly expressed in cells or organisms (e.g., reporter Substrate, unfavorable folding conditions are applied, a difference will ligand, environmentally-sensitive hydrophobic dye, etc.) are be observed between the signal (e.g., from a direct reporter added to the cell or organism exogenously. In embodiments signal or BRET signal, depending upon the assay) from a in which assays are not performed in vivo, the target/reporter ligand-bound sample and an unbound target sample. fusion may also be added exogenously. 0096. The number of conditions to assay may be made by 0100 Embodiments described herein provide composi weighing factors such as: desired speed of the assay, desired tions, systems, and methods that are useful in a variety of precision/accuracy of the measurements, affinity of binding fields including basic research, medical research, drug dis to be detected, etc. For example, in Some embodiments, an covery, etc. The reagents and assays described herein are not assay detects signal under a first condition in which the limited to any particular applications, and any useful appli target is well-folded, a second condition near the T of the cation should be viewed as being within the scope of the unbound target, and a third condition in which the unbound present invention. target is expected to be unfolded and/or aggregated. These conditions may be determined empirically using the meth 0101 Typical applications that make use of embodiments ods described herein or other techniques for monitoring described herein involve the monitoring/detection of pro protein folding that are known in the field. As another tein-ligand interactions. Such assays are useful for monitor example, an assay makes stepwise (e.g., every 0.1° C., every ing molecular interactions under any Suitable conditions (e.g., in vitro, in Vivo, in situ, whole animal, etc.), and find 0.2° C., every 0.5° C., every 1.0° C., every 2.0° C., every use in, for example, drug discovery, elucidating molecular 5.0° C., etc.) signal detections (e.g., from reporter and/or pathways, studying equilibrium or kinetic aspects of com BRET) over a range of temperatures (e.g., from: 10°C., 15° plex assembly, high throughput screening, etc. C., 20° C., 25°C., 30° C., 35° C., 40° C., etc.; to:50° C., 55° C., 60° C., 650 C., 70° C., 75° C., 80° C., 85°C., 90° C. 950 0102. In some embodiments, assays are used to elucidate C., etc.), and or a range of denaturant concentrations. the affinity of, or understand the interaction of, a protein of 0097. In some embodiments, a melt-curve analysis is interest and a potentially associated entity of interest (pro performed for each sample, according to the detected signals tein, nucleic acid, Small molecule, etc.) under complex at the various conditions assayed. In some embodiments, a conditions (e.g., intracellularly). visual or automated analysis of the melt-curves in the 0103 Embodiments described herein find use in drug presence and absence of ligand reveals target/ligand inter screening and/or drug development. For example, the inter action or binding. In some embodiments, a melting tem action of a small molecule drug or an entire library of Small perature (T,) is calculated for each sample, according to the molecules with target protein of interest (e.g., therapeutic detected signals at the various conditions assayed. In some target) is monitored (e.g., within a physiologically-relevant embodiments, a comparison of Ts in the presence and cell line, etc.). In some embodiments, drug screening appli absence of ligand reveals target/ligand interaction or bind cations are carried out in a high through-put format to allow 1ng for the detection of the binding of tens of thousands of 0098. In some embodiments, assays described herein are different molecules to a target, or to test the effect of those performed in cells, e.g., live intact cells, or in a lysate of molecules on the binding of a ligand. cells. In such embodiments, a nucleic acid construct encod 0104. In some embodiments, provided herein is the ing a target/reporter fusion may be transfected or trans detection of target/ligand interactions in living organisms formed into cells in order to allow expression of the fusion (e.g., bacteria, yeast, eukaryotes, mammals, primates, within cells (e.g., live intact cells, cells in which the assay human, etc.) and/or cells. In some embodiments, cells are will be performed, cells which will be lysed before the assay transiently and/or stably transformed or transfected with is performed, etc.). In some embodiments, assays described vector(s) (e.g., encoding target/reporter fusions, comple herein provide the detection of molecular interactions ment polypeptide, etc.). In some embodiments, transgenic between a target and ligand within a cell, e.g., a live intact organisms are generated that code for the necessary com cell, or in living organisms (e.g., bacteria, yeast, eukaryotes, ponents (e.g., encoding target/reporter fusions, complement mammals, primates, human, etc.). In some embodiments, a polypeptide, etc.) to carry out the assays described herein. In target/reporter fusion protein is expressed in the cell, e.g., a other embodiments, vectors are injected into whole organ live intact cell, or whole organism, and signal is detected 1SS. US 2016/0282360 A1 Sep. 29, 2016 21

EXPERIMENTAL General Protocol Cell Transfection for all Experiments: Example 1 0117 NANOLUC-target or NLPep and NLPoly-target fusion constructs were diluted into carrier DNA Materials and Methods (pGEM37F-, Promega) at a mass ratio of 1:10 or 1:100 (mass/mas) prior to forming FuGENE HD complexes Method for Expression Plasmid Construction for Full according to the manufacturer's protocol (Promega). DNA: Length NANOL UC-Target Fusions (Applies to Examples FuGENE complexes were formed at a ratio of 1:3 (ug Using Full Length NANOL UC) DNA/uIL FuGENE), then 1 part of the transfection com 0105 To produce NANOLUC fusions with protein tar plexes was then mixed with 20 parts (volume/volume) of gets of interest, pF31K Nluc (CMV/Neo and pF32 CMV/ HeLa cells (ATCC) suspended at a density of 2x10 in Neo were used to place NANOLUC at the N-terminus or DMEM (Gibco)+10% FBS (Hyclone), followed by incuba the C-terminus of the target protein (respectively) using the tion in a humidified, 37° C./5% CO, incubator for 18-24 manufacturer's protocol (Promega). cDNAS encoding the hours. following target proteins were made and were a 100% match to their respective NCBI reference sequence identifiers: Example 2 CDK2, DHFR, KDR, SRC, EZH2, HDAC1, BRD4. MAPK14, LCK, ABL, DDR1, and BTK. Direct-Luciferase-Detection Thermal Shift Assay Method for Expression Plasmid Construction for NLPep and with Full Length NANOLUC NLPoly NLPep and NLPoly Target Fusions (pep86-CDK2, CDK2-pep86, CDK2-Nluc156, CDK2-pep11S) 0118 Experiments were conducted during development 0106 For plasmid constructions, the source of CDK2 of embodiments of the present invention to demonstrate was pFN21-CDK2 (Promega FLEXI vector backbone) direct detection of the stabilization of a target protein by the received from Kazusa DNA Research Institute (Chiba, interaction with a target ligand via temperature dependent Japan). alteration of luminescent signal from a luciferase fused to 01.07 Pep86-CDK2: the target protein. 0108. This construct was built by transferring CDK2 to 0119) To ensure the NANOLUC (Nluc) luminescent sig the FLEXI vector, pF-n5K3-86-GSSG-barnase, according to nal was originating from intact cells after the cells had been Promega's recommended protocols in the FLEXI vector exposed to a temperature gradient, the integrity of the cells technical manual. and cell membranes after heating was investigated as well as 0109) CDK2-Pep86: measuring Nluc activity in the cells versus in the Superna 0110. This construct was built by transferring CDK2 to tant. HeLa cells were heated to different temperatures, the FLEXI vector, pF-c5K3-barnase-GSSG-86, according to cooled at room temperature, mixed with trypan blue, and the Promega’s recommended protocols in the FLEXI vector absolute cell numbers and dye exclusion were measured. technical manual. Parallel samples were spun down to pellet, and the cells and 0111 CDK2-Nluc156: Supernatant collected. 0112 This construct was built by first transferring CDK2 0.120. HeLa cells were transfected with CDK2-Nluc to the FLEXI vector, pF-nCA-barnase-GSSG-Nluc, accord DNA at a 1:10 DNA ratio and incubated overnight. The cells ing to Promega’s recommended protocols in the FLEXI were trypsinized using Trypsin-EDTA (0.25%) (Gibco) and vector technical manual. The Nluc portion of this vector was re-suspended in Opti-MEM without phenol red (Gibco)+1 x then modified by site-directed mutagenesis to contain a Protease Inhibitor Cocktail (Promega) which had been G51A substitution. It was then further modified by site reconstituted in DMSO (Sigma). The cells were seeded into directed mutagenesis to contain a deletion of the sequence 96-well tall-chimney PCR plate (Fisherbrand) at a density of coding for the final 13 amino acids of Nluc. 2x10 cells/well in a 100 uL/well volume, and the plates 0113 CDK2-11S: were placed into a thermal cycler (MW Research) where the 0114. This construct was built by transferring CDK2 to samples were heated individually at different temperatures the FLEXI vector, pF-c5K3-barnase-GSSG-11S, according spanning a gradient of 42-66° C. across the plate for 3 to Promega’s recommended protocols in the FLEXI vector minutes followed by cooling at 22°C. for 3 minutes. To technical manual. determine the absolute cell count and viability, 10 uI cell 0115 Each of the constructs contained a GSSG linker aliquot from each temperature was mixed with an equal between CDK2 and either NLPep86, Nluc156, or volume of 0.4% (w/v) trypan blue exclusion dye solution NLpoly11S. (Life technologies) and analyzed using the Countess Auto mated cell counter (Life Technologies). To determine where Method for Generation of Purified Detection NLPep86 and the Nluc luminescent signal was originating, the 96-well NLPoly 11S PCR plate was centrifuged to pellet the cells. The superna 0116 NLPep86 (SEQ ID NO: 5) (containing an N-ter tant and cell pellet was collected separately and placed into minal acetyl and C-terminal amide) was made synthetically a 96-well tissue culture plate (Corning). Furimazine Live by Peptide 2.0. NLpoly 11S (SEQ ID NO: 6)(NLpoly11S Cell Substrate (Promega) was added to a final concentration with C-terminal 6His tag) was isolated by transforming E. of 1x, and luminescence was measured on a BMG Clariostar coli KRX cells (Promega) with the FLEXI vector, luminometer equipped with a 450 nm BP filter. Samples pF6HisCK, and then overexpressing and purifying accord were also collected from a 96-well PCR plate that had not ing to Promega’s recommended protocol for isolating His undergone centrifugation and placed into a tissue culture tagged proteins (HISLINK Protein Purification technical plate followed by addition of furimazine substrate and manual). luminescence analysis. US 2016/0282360 A1 Sep. 29, 2016 22

0121 The data demonstrates that the cells were not lysed, CDK2, which showed a distinct melt curve in the presence even at higher temperatures, since the number of cells of DMSO (control), was evaluated. When AZD5438 (a drug remained unchanged and membranes remained intact as known to bind to CDK2) was added, clear shifts in the determined by dye exclusion (FIG. 1A-B). Furthermore, the melting curves with increases in apparent T. were detected Nluc activity was retained in the cells with the luciferase compared to DMSO controls and analyzed by luciferase signal originating from the cell samples and not the Super activity indicating specific binding and protein stabilization natant post-heat treatment (FIG. 1C). of the CDK2 by the inhibitor. 0127. HeLa cells were transfected with CDK2-Nluc or Example 3 Nluc-CDK2 DNA at a 1:10 DNA ratio and incubated overnight. The cells were trypsinized using Trypsin-EDTA Direct-Luciferase-Detection Thermal Shift Assay (0.25%) (Gibco) and re-suspended in Opti-MEM without with Full Length NANOLUC phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro 0122 Experiments were conducted during development mega) which had been reconstituted in DMSO (Sigma). The of embodiments of the present invention to demonstrate cells were seeded into 96-well tall-chimney PCR plate direct detection of the stabilization of a target protein by the (Fisherbrand) at a density of 2x10" cells/well in a 90 uL/well interaction with a target ligand via temperature dependent volume and treated with 10 uL of 10% DMSO (Sigma), 10 alteration of luminescent signal from a luciferase fused to uL/well of a 0.5 mg/ml solution of digitonin (Sigma), 10 ul the target protein. of 1 mMAZD5438 (Selleckchem) in 10% DMSO, and/or 10 0123. HeLa cells were transfected separately with the uL of 1 mM Methotrexate (Sigma) in 10% DMSO. The various Nluc-fusion DNA at a 1:10 DNA ratio and incubated plates were then incubated for 1-2 hours in a humidified, 37° overnight. The cells were trypsinized using Trypsin-EDTA C./5% CO incubator. The plates were placed into a thermal (0.25%) (Gibco) and re-suspended in Opti-MEM without cycler (MW Research) where the samples were heated phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro individually at different temperatures spanning a gradient of mega) which had been reconstituted in DMSO (Sigma). The 42-66° C. across the plate for 3 minutes followed by cooling cells were seeded into 96-well tall-chimney PCR plate at 22° C. for 3 minutes. 90 uL/well samples were then (Fisherbrand) at a density of 2x10 cells/well in a 90 uL/well transferred to a tissue culture plate (Corning), and FurimaZ volume, and either treated with 10 uL/well of a 10% DMSO ine Live Cell Substrate (to a final concentration of 1 x: solution (Sigma) or 10 uI/well of a 0.5 mg/ml solution of Promega) or NANOGLO lytic reagent (Promega) was digitonin (Sigma). The plates were placed into a thermal added. Luminescence was measured on a BMG Clariostar cycler (MW Research) where the samples were heated luminometer equipped with a 450 nm BP filter. Data was individually at different temperatures spanning a gradient of normalized by relating RLU signals to the RLU signals of 42-66° C. across the plate for 3 minutes followed by cooling the lowest temperature for the respective sample. Data was at 22° C. for 3 minutes. 90 uL/well samples were then then fitted to obtain apparent melting temperature where the transferred to a tissue culture plate (Corning), and FurimaZ protein is precipitating (T values) using the Boltzmann ine Live Cell Substrate (Promega) was added to a final Sigmoidal equation with Graphpad Prism software. concentration of 1x. Luminescence was measured on a I0128. The thermal shift induced by AZD5438 was insen BMG Clariostar luminometer equipped with a 450 nm BP sitive to Nluc positioning on the N- or C-terminal of CDK2 filter. Data was normalized by relating RLU signals to the (FIG. 3) and was demonstrated in live (FIG. 3A-B) and RLU of the lowest temperature for the respective sample. digitonin-treated cells (FIG. 3C-D) as well as in cells lysed Data was then fitted to obtain apparent melting temperature with the addition of lytic substrate (FIG. 4). where the protein is precipitating (T values) using the Boltzmann Sigmoidal equation with Graphpad Prism soft Example 5 Wae. 0.124 Protein thermal melt curves can be generated for Direct-Luciferase-Detection Thermal Shift Assay Nluc-target fusions spanning several different target classes with Full Length NANOLUC and Subcellular localization (nuclear, cytoplasmic, and I0129. Experiments were conducted during development membrane) in live, intact cells (FIG. 2A), and cells that had of embodiments of the present invention to demonstrate been treated with the detergent digitonin (FIG. 2B) with direct detection of the stabilization of a target protein by the results indicating each protein displayed distinct melting interaction with a target ligand via temperature dependent curves as analyzed by luciferase activity and reported as alteration of luminescent signal from a luciferase fused to apparent aggregation temperatures (T). the target protein. 0.130. To demonstrate that the assay can be applied to a Example 4 larger class of kinase proteins, specific melt curves and thermal shifts across a panel of kinases were obtained after Direct-Luciferase-Detection Thermal Shift Assay exposure to specific stabilizing compounds. HeLa cells, with Full Length NANOLUC expressing different kinase Nluc-target fusions with varying 0.125 Experiments were conducted during development functions and Subcellular localization, were exposed to of embodiments of the present invention to demonstrate compounds known to target each one and then exposed to direct detection of the stabilization of a target protein by the the temperature treatment. interaction with a target ligand via temperature dependent I0131 HeLa cells were transfected separately with the alteration of luminescent signal from a luciferase fused to various Nluc-target DNA at a 1:10 DNA ratio and incubated the target protein. overnight. The cells were trypsinized using Trypsin-EDTA I0126) To determine if a shift in T. after exposure to (0.25%) (Gibco) and re-suspended in Opti-MEM without stabilizing ligand can be detected, the thermal melt curve for phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro US 2016/0282360 A1 Sep. 29, 2016

mega) which had been reconstituted in DMSO (Sigma). The in 10% DMSO. Test compounds included: Staurosporine cells were seeded into 96-well tall-chimney PCR plate (LC Laboratories), Dasatinib (BioVision), Nilotinib (BioVi (Fisherbrand) at a density of 2x10" cells/well in a 80 uIL/well sion), Ponatinib (SYNKinase), AMG548 (Tocris), volume and treated with 10 u/well of a 0.5 mg/ml solution SB203580 (AdipoGen), and AZD5438 (Selleckchem). The of digitonin (Sigma), 10 uL of 1 mM AZD5438 (Selleck plates were then incubated for /2 hour in a humidified, 37° chem) in 10% DMSO, 10 uL of 0.25 mM AMG548 (Tocris) C/5% CO, incubator prior to addition of 10 uI/well of a 0.5 in 10% DMSO, 10 uL of 1 mM Dasatinib (BioVision) in mg/ml solution of digitonin (Sigma) and 10% DMSO 10% DMSO, or 10 uL of 0.5 mM Staurosporine (LC (Sigma). The plates were placed into a thermal cycler (MW Laboratories) in 10% DMSO. The plates were then incu Research) where the samples were heated individually at bated for 1 hour in a humidified, 37° C./5% CO, incubator. different temperatures spanning a gradient of 42-66° C. The plates were placed into a thermal cycler (MW Research) across the plate for 3 minutes followed by cooling at 22°C. where the samples were heated individually at different for 3 minutes. Furimazine Live Cell Substrate (Promega) temperatures spanning a gradient of 42-66° C. across the was added to a final concentration of 1x in 20 uL/well plate for 3 minutes followed by cooling at 22° C. for 3 volume. 100 uL?well was then transferred to a tissue culture minutes. Furimazine Live Cell Substrate (Promega) was plate (Corning), and luminescence was measured on a BMG added to a final concentration of 1x in 20 uL?well volume. Clariostar luminometer equipped with a 450 nm BP filter. 100 uL/well was then transferred to a tissue culture plate Data was normalized by relating RLU signals to the RLU of (Corning), and luminescence was measured on a BMG the lowest temperature for the respective sample. Data was Clariostar luminometer equipped with a 450 nm BP filter. then fitted to obtain apparent melting temperature where the Data was normalized by relating RLU signals to the RLU protein was precipitating (T values) using the Boltzmann signals of the lowest temperature for the respective sample. Sigmoidal equation with Graphpad Prism software. Data was then fitted to obtain apparent melting temperature 0.136 For the CDK2 target fusion, the known target where the protein is precipitating (T values) using the inhibitors, staurosporine, Ponatinib, and AZD5438, all Boltzmann Sigmoidal equation with Graphpad Prism soft caused a significant increase in apparent T. Dasatinib, Wae. Nilotinib, AMG548, and SB20358, which are all compounds 0.132. Obvious shifts in melting temperature, with not known to interact with CDK2, did not result in a increases in Te4°C. in all cases, were seen when the cells significant shift in apparent T. compared to DMSO con were treated with the specific binding compounds as com trols when incubated with cells expressing the CDK2-Nluc pared to DMSO controls for each kinase tested (FIGS. fusion (FIG. 6A-B). Similarly, Staurosporine, Dasatinib, 5A-F). Nilotinib, and Ponatinib, all of which target ABL1 kinase, caused a significant shift in apparent T. compared to Example 6 DMSO controls, whereas the negative ligands AMG548, SB203580, and AZD5438 did not as expected (FIG. 6C-D). Direct-Luciferase-Detection Thermal Shift Assay with Full Length NANOLUC Example 7 0.133 Experiments were conducted during development of embodiments of the present invention to demonstrate Direct-Luciferase-Detection Thermal Shift Assay direct detection of the stabilization of a target protein by the with Full Length NANOLUC interaction with a target ligand via temperature dependent 0.137 Experiments were conducted during development alteration of luminescent signal from a luciferase fused to of embodiments of the present invention to demonstrate the target protein. direct detection of the stabilization of a target protein by the 0134) To demonstrate the assays ability to determine interaction with a target ligand via temperature dependent compound target selectivity and highlight the assay’s poten alteration of luminescent signal from a luciferase fused to tial screening capabilities for proposed inhibitors, an assay the target protein. against two kinase target fusions, CDK2-Nluc and Nluc 0.138. To demonstrate the TSA approach described herein ABL1, with the same panel of kinase inhibitors known to can be applied broadly, the experiment performed in FIGS. have different selectivity against these two kinase targets 6A-D was repeated using a target protein that maintains a was performed. The assay accurately validated ligand selec nuclear Subcellular localization and originated from a drug tivity, and rank order potency, with the compounds known to target class very different from kinases. Cells were trans bind to CDK2 and/or ABL1 causing a shift in melting fected with HDAC1-Nluc fusions and tested against a panel temperature (apparent T), and those compounds known of known HDAC inhibitors with Staurosporine, a kinase not to have affinity towards CDK2 and/or ABL 1 leaving the inhibitor, serving as an additional negative control with melting temperature relatively unchanged, as compared to DMSO. the DMSO control. 0.139. HeLa cells were transfected with HDAC1-Nluc 0135. HeLa cells were transfected separately with DNA at a 1:100 DNA ratio and incubated overnight. The CDK2-Nluc or Nuc-ABL1 DNA at a 1:100 DNA ratio and cells were trypsinized using Trypsin-EDTA (0.25%) (Gibco) incubated overnight. The cells were trypsinized using and re-suspended in Opti-MEM without phenol red Trypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti (Gibco)+1x Protease Inhibitor Cocktail (Promega) which MEM without phenol red (Gibco)+1x Protease Inhibitor had been reconstituted in DMSO (Sigma). The cells were Cocktail (Promega) which had been reconstituted in DMSO seeded into 96-well tall-chimney PCR plate (Fisherbrand) at (Sigma). The cells were seeded into 96-well tall-chimney a density of 2x10 cells/well in a 90 uL/well volume and PCR plate (Fisherbrand) at a density of 2x10 cells/well in treated with 10 uL of 10% DMSO (Sigma) or 10 uL of a 1 a 90 u/well volume and treated with 10 u, of 10% DMSO mM stock solution of test compound in 10% DMSO. Test (Sigma) or 10 uI of a 1 mM stock solution of test compound compounds included: SAHA (TOCRIS), Mocetinostat (Sell US 2016/0282360 A1 Sep. 29, 2016 24 eckchem), Panibinostat (LC Laboratories), ACY 1215 (Sell normalized by relating RLU signals to the RLU of the lowest eckchem), and Staurosporine (LC Laboratories). The plates temperature for the respective sample. Data was then fitted were then incubated for /2 hour in a humidified, 37° C./5% to obtain apparent melting temperature where the protein is CO, incubator prior to addition of 10 uL/well of a 0.5 mg/ml precipitating (T values) using the Boltzmann Sigmoidal solution of digitonin (Sigma) and Furimazine Live Cell equation with Graphpad Prism software. Substrate (final concentration 1 x (Promega)). The plates 014.4 FIGS. 8A-B show detection of an increase in were placed into a thermal cycler (MW Research) where the melting temperature for the targets KDR-Nluc (FIG. 8A) samples were heated individually at different temperatures and DHFR-Nluc (FIG. 8B) as determined by NANOLUC spanning a gradient of 42-66° C. across the plate for 3 activity (RLU) after incubation with known stabilizing minutes followed by cooling at 22° C. for 3 minutes. 100 ligand in mammalian cells Subsequently harvested with the uL/well was then transferred to a tissue culture plate (Corn compound being washed out, and then Subjected to a tem ing), and luminescence was measured on a BMG ClarioStar perature. KDR is a membrane protein highlighting another luminometer equipped with a 450 nm BP filter. Data was subcellular location that the assay is able to monitor. normalized by relating RLU signals to the RLU of the lowest temperature for the respective sample. Data was then fitted Example 9 to obtain apparent melting temperature where the protein is precipitating (T values) using the Boltzmann Sigmoidal Direct-Luciferase-Detection Thermal Shift Assay equation with Graphpad Prism software. with NLPep and NLPoly Through Spontaneous 0140 All four HDAC known inhibitors (SAHA, Moce Binary Complementation tinostat, Panibinostat, and ACY 1215) produced a significant 0145 Experiments were conducted during development shift in melting temperature (apparent T) indicating of embodiments of the present invention to demonstrate ligand binding compared to the controls as analyzed by direct detection of the stabilization of a target protein by the luciferase activity (FIG. 7). interaction with a target ligand via temperature dependent alteration of luminescent signal in which the luminescent Example 8 signal results through spontaneous binary complementation from a peptide fused to the target protein with the comple Direct-Luciferase-Detection Thermal Shift Assay mentary subunit present in the detection reagent. with Full Length NANOLUC 0146 To determine if the luciferase-based thermal shift 01.41 Experiments were conducted during development assay described herein could be performed using a sponta of embodiments of the present invention to demonstrate neous binary complementation, the NLPep and NLPoly direct detection of the stabilization of a target protein by the technology (Promega: See, e.g., U.S. Pub. No. 2014/ interaction with a target ligand via temperature dependent 0348747: herein incorporated by reference in its entirety) alteration of luminescent signal from a luciferase fused to was used. the target protein. (0.147. HeLa cells were transfected with CDK2-NLpep86 0142. To determine if the assay can be performed when or NLpep86-CDK2 DNA at a 1:10 DNA ratio and incubated the ligands added are not maintained under equilibrium overnight. The cells were trypsinized using Trypsin-EDTA conditions, the assay was performed with a compound (0.25%) (Gibco) and re-suspended in Opti-MEM without washout step. This would also confirm that the target phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro engagement was happening in live cells as well as have the mega), which had been reconstituted in DMSO (Sigma). The ability to analyze binding kinetics. cells were seeded into 96-well tall-chimney PCR plate 0143 HeLa cells were transfected with KDR-Nluc or (Fisherbrand) at a density of 2x10 cells/well in a 90 uL/well DHFR-Nluc at a 1:10 DNA ratio and incubated overnight in volume and treated with 1 uL of 100% DMSO (Sigma) or 1 t75 tissue culture flasks (Corning). Media was aspirated and uL of a 10 mM stock solution of AZD5438 (Selleckchem) in replaced with media containing a final concentration of 100 10% DMSO, and 10 uL/well of a 0.5 mg/ml solution of uM BIBF-1120 (Selleckchem) for cells expressing KDR digitonin (Sigma). The plates were then incubated for 1-2 Nluc or 100 uM Methotrexate (Sigma) for cells expressing hour in a humidified, 37° C./5% CO, incubator. The plates DHFR-Nluc. DMSO at a final concentration of 1% was used were placed into a thermal cycler (MW Research) where the as control and added to each cell population. The cells were samples were heated individually at different temperatures allowed to incubate with compounds for 2 hours in a spanning a gradient of 42-66° C. across the plate for 3 humidified, 37° C./5% CO, incubator. The cells were minutes followed by cooling at 22°C. for 3 minutes. 90 trypsinized using Trypsin-EDTA (0.25%) (Gibco) and re uL/well was then transferred to a tissue culture plate (Corn suspended in Opti-MEM without phenol red (Gibco)+1 x ing), and the complementary Subunit, NLpoly 11S, was Protease Inhibitor Cocktail (Promega) which had been added to a final concentration of 1 uM with Furimazine Live reconstituted in DMSO (Sigma). Cells were seeded into Cell Substrate (final concentration 1 x (Promega)). Lumines 96-well tall-chimney PCR plate (Fisherbrand) at a density of cence was measured on a BMG Clariostar luminometer 2x10 cells/well in a 100 uL/well and placed into a thermal equipped with a 450 nm BP filter. Data was normalized by cycler (MW Research) where the samples were heated relating RLU signals to the RLU of the lowest temperature individually at different temperatures spanning a gradient of for the respective sample and Subtracting the background 42-66° C. across the plate for 3 minutes followed by cooling luminescence signal generated by NLpoly 11 S. Data was at 22° C. for 3 minutes. 90 uL?well was then transferred to then fitted to obtain apparent melting temperature where the a tissue culture plate (Corning), and Furimazine Live Cell protein is precipitating (T values) using the Boltzmann Substrate (final concentration 1 x (Promega)) was added. Sigmoidal equation with Graphpad Prism software. Luminescence was measured on a BMG Clariostar lumi 0148 FIG. 9 shows detection of an increase in melting nometer equipped with a 450 nm BP filter. Data was temperature for CDK2-NLpep86 and NLpep86-CDK2 as US 2016/0282360 A1 Sep. 29, 2016 determined by luciferase activity through spontaneous Example 11 binary complementation after incubation in the presence of the stabilizing ligand AZD5438 in digitonin-treated mam Direct-Luciferase-Detection Thermal Shift Assay malian cells Subsequently exposed to a temperature gradient with NLPep and NLPoly Through Spontaneous and addition of Substrate and complementary Subunit Binary Complementation NLpoly 11S as compared to the DMSO control. 0153 Experiments were conducted during development of embodiments of the present invention to demonstrate Example 10 direct detection of the stabilization of a target protein by the interaction with a target ligand via temperature dependent alteration of luminescent signal in which the luminescent Direct-Luciferase-Detection Thermal Shift Assay signal results through spontaneous binary complementation with NLPep and NLPoly Through Spontaneous from a peptide fused to the target protein with the comple Binary Complementation mentary subunit present in the detection reagent. 0154). HeLa cells were transfected with CDK2-NLpep86 0149 Experiments were conducted during development or CDK2-NLpoly 11S DNA at a 1:10 DNA ratio and incu of embodiments of the present invention to demonstrate bated overnight. The cells were trypsinized using Trypsin direct detection of the stabilization of a target protein by the EDTA (0.25%) (Gibco) and re-suspended in Opti-MEM interaction with a target ligand via temperature dependent without phenol red (Gibco)+1x Protease Inhibitor Cocktail alteration of luminescent signal in which the luminescent (Promega) which had been reconstituted in DMSO (Sigma). signal results through spontaneous binary complementation The cells were seeded into 96-well tall-chimney PCR plate from a peptide fused to the target protein with the comple (Fisherbrand) at a density of 2x10 cells/well in a 90 uL/well mentary subunit present in the detection reagent. volume and treated with 1 u, of a 100x stock solution in a dilution series of AZD5438 (Selleckchem) in 10% DMSO, 0150. The following experiment is similar to Example 9, and 10 ul/well of a 0.5 mg/ml solution of digitonin (Sigma). but used another pair of complementary subunits, The plates were then incubated for 1-2 hour in a humidified, NLpoly 156 (SEQID NO: 7) and NLpep86. NLpoly 156 was 37° C./5% CO incubator. The plates were placed into a fused to the target protein, and NLpep86 was placed in the thermal cycler (MW Research) where the samples were detection reagent. heated individually at different temperatures spanning a 0151. HeLa cells were transfected with CDK2-Nluc156 gradient of 42-66° C. across the plate for 3 minutes followed DNA at a 1:10 DNA ratio and incubated overnight. The cells by cooling at 22° C. for 3 minutes. 90 uL/well was then were trypsinized using Trypsin-EDTA (0.25%) (Gibco) and transferred to a tissue culture plate (Corning) and comple re-suspended in Opti-MEM without phenol red (Gibco)+1 x mentary subunit NLpoly 11S or NLpep86 was added to a Protease Inhibitor Cocktail (Promega) which had been final concentration of 1 uM with Furimazine Live Cell reconstituted in DMSO (Sigma). The cells were seeded into Substrate (final concentration 1X (Promega)). Luminescence 96-well tall-chimney PCR plate (Fisherbrand) at a density of was measured on a BMG Clariostar luminometer equipped 2x10 cells/well in a 90 uL/well volume and treated with 1 with a 450 nm BP filter. For ECs analysis, compound uL of 100% DMSO (Sigma) or 1 uL of a 10 mM stock concentration was plotted against RLU, and the data was solution of AZD5438 (Selleckchem) in 10% DMSO, and 10 transformed prior to being fitted using the sigmoidal dose uL/well of a 0.5 mg/ml solution of digitonin (Sigma). The response (variable slope) equation with Graphpad Prism plates were then incubated for 1-2 hour in a humidified, 37° software. For apparent T analysis, data was normalized C./5% CO, incubator. The plates were placed into a thermal by relating RLU signals to the RLU of the lowest tempera cycler (MW Research) where the samples were heated ture for the respective. Data was then fitted to obtain individually at different temperatures spanning a gradient of apparent melting temperature where the protein is precipi 42-66° C. across the plate for 3 minutes followed by cooling tating (T values) using the Boltzmann Sigmoidal equation at 22° C. for 3 minutes. 90 uL?well was then transferred to with Graphpad Prism software. a tissue culture plate (Corning), and the complementary 0155 FIGS. 11 and 12 demonstrates the temperature and subunit, NLpep86, was added to a final concentration of 1 stabilizing ligand concentration dependency of the uM with Furimazine Live Cell Substrate (final concentration luciferase-based thermal shift assay which is consistent with 1x (Promega)). Luminescence was measured on a BMG other reported methods of thermal shift assay. Here, the Clariostar luminometer equipped with a 450 nm BP filter. ECso of the ligand AZD5438 shifts as a function of tem Data was normalized by relating RLU signals to the RLU of perature and the apparent T shifts as a function of the lowest temperature for the respective. Data was then stabilizing ligand concentration. In this case, the targets fitted to obtain apparent melting temperature where the consisted of CDK2-NLpep86 fusions (FIGS. 11 A-C) and protein is precipitating (T values) using the Boltzmann CDK2-NLpoly 11S fusions (FIGS. 12A-B). Sigmoidal equation with Graphpad Prism software. Example 12 0152 FIGS. 10A-C demonstrates the detection of an increase in melting temperature for CDK2-NLpoly 156 as determined by luciferase activity through spontaneous BRET-Detection Thermal Shift Assay binary complementation after incubation in the presence of 0156 Experiments were conducted during development the stabilizing ligand AZD5438 in digitonin-treated mam of embodiments of the present invention to demonstrate malian cells Subsequently exposed to a temperature gradient detection of the stabilization of a target protein by the and addition of Substrate and complementary Subunit interaction with a target ligand via temperature dependent NLpep86 as compared to the DMSO control. alteration of BRET signal generated by the energy transfer US 2016/0282360 A1 Sep. 29, 2016 26 from a luciferase fused to the target protein to an environ (0160. In FIGS. 14A-B, to obtain isothermal dose mentally sensitive dye that binds to all proteins unfolding in response curves (ITDRC) in order to obtain compound rank the sample including the target protein. order affinity and apparent EC50s, the curves generated 0157 Protein thermal melting curves can be generated when the samples were subjected to the temperature gradient for purified proteins in which the extent of unfolding is to ensure compliance with the model were inspected first, measured by the gain in fluorescent signal from environ apparent T. calculated, and an appropriate temperature mentally sensitive dyes that bind to the exposed hydropho chosen for follow-up ITDRC or screening experiments. This bic Surfaces of the protein as they unfold. Taking advantage was a temperature at which a majority of the protein is of this property and adding these dyes to the luciferase unfolded or precipitated in the absence of stabilizing com based thermal shift assay described herein, the environmen pound, but at which a majority of the protein remains soluble tally-sensitive dye binds to the target protein-Nluc fusion, in the presence of a Saturating concentration of known and BRET is produced upon addition of substrate when there stabilizing compound. To demonstrate this, ITDRC for a is active Nluc available to excite the dye. The dye acts as a panel of kinase inhibitors with known different selectivity fluorescent BRET acceptor, and the transfer of energy is was established and tested for target engagement with moderated by the proximity of the two partners. While the CDK2-Nluc and LCK-Nluc fusions as determined by BRET dye will bind to all proteins unfolding in the sample, the after incubation in the presence of different concentrations luciferase fusion gives the target protein specificity; hence of the individual compounds while time of heating and any BRET signal is a direct readout on the state of the temperature were kept constant. This demonstrates the con protein of interest. centration dependence of the thermal stabilization and 0158. HeLa cells were transfected with CDK2-Nluc or allows for compound affinity signatures to be obtained. Nluc-LCK DNA at a 1:100 DNA ratio and incubated over night. The cells were trypsinized using Trypsin-EDTA Example 13 (0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro BRET-Detection Thermal Shift Assay mega) which had been reconstituted in DMSO (Sigma). The 0.161 Experiments were conducted during development cells were seeded into 96-well tall-chimney PCR plate of embodiments of the present invention to demonstrate (Fisherbrand) at a density of 2x10 cells/well in an 80 detection of the stabilization of a target protein by the uL/well volume and treated with 10 uI/well of a 0.5 mg/ml interaction with a target ligand via temperature dependent solution of digitonin (Sigma) and 10 ul of 10% DMSO alteration of BRET signal generated by the energy transfer (Sigma) or 10 uI of a 1 mM stock solution of test compound from a luciferase fused to the target protein to an environ in 10% DMSO. Test compounds included: Dasatinib (Bio mentally sensitive dye that binds to all proteins unfolding in Vision), AZD5438 (Selleckchem), Staurosporine (LC Labo the sample including the target protein. ratories), and Nilotinib (BioVision). The plates were then 0162 To demonstrate analysis of compound selectivity incubated for 1 hour in a humidified, 37° C./5% CO, using the BRET method of thermal shift, a panel of com incubator. The plates were placed into a thermal cycler (MW pounds with known selectivity against CDK2, MAPK14. Research) where the samples were heated either individually and HDAC1 was tested. It was found that the assay accu at different temperatures spanning a gradient of 42-66° C. rately detected the positive and negative ligands against across the plate for 3 minutes to obtain apparent T or the these targets. samples are all heated for 3 minutes at one constant tem (0163. HeLa cells were transfected with CDK2-Nluc, perature to obtain ITDRC, followed by cooling at 22°C. for Nluc-MAPK14, or HDAC1-Nluc DNA at a 1:100 DNA ratio 3 minutes. The Protein Thermal ShiftTM dye (Life Technolo and incubated overnight. The cells were trypsinized using gies) was added at a final concentration of 0.5x concentra Trypsin-EDTA (0.25%) (Gibco) and re-suspended in Opti tion (supplied as 1000x) in 1 uL/well volume. Furimazine MEM without phenol red (Gibco)+1x Protease Inhibitor Live Cell Substrate (Promega) was added to a final concen Cocktail (Promega) which had been reconstituted in DMSO tration of 1x in 20 uL/well volume, and a total volume of (Sigma). The cells were seeded into 96-well tall-chimney 100 uL/well was then transferred to a tissue culture plate PCR plate (Fisherbrand) at a density of 2x10 cells/well in (Corning). To measure BRET, filtered luminescence was an 90 u/well volume and treated with 10 u, of 10% DMSO measured on a BMG Clariostar luminometer equipped with (Sigma) or 10 uI of a 1 mM stock solution of test compound 450 nm BP filter (donor) and 610 nm LP filter (acceptor), in 10% DMSO. Test compounds included: Dasatinib (Bio using 0.5 s integration time. MilliBRET units (mBU) were Vision), AZD5438 (Selleckchem), Staurosporine (LC Labo calculated by using the equation: (acceptor? donor)*1000. To ratories), Nilotinib (BioVision), Ponatinib (SYNKinase), obtain apparent T values, data was fitted using the bell AMG548 (TOCRIS), SB203580 (Adipogen), SAHA (TOC shaped curve equation. To analyze ITDFC data, data was RIS), Mocetinostat (Selleckchem), Panibinostat (LC Labo transformed and fitted to obtain apparent EC50s using the ratories), and ACY 1215 (Selleckchem). The plates were sigmoidal dose-response (variable slope) equation. All then incubated for /2 hour in a humidified, 37° C./5% CO, analyses were performed with Graphpad Prism software. incubator. Immediately prior to heating, 10 u/well of a 0.5 0159 FIG. 13 demonstrates that BRET can be used to mg/ml Solution of digitonin (Sigma), Protein Thermal detect ligand binding through a change in melting tempera ShiftTM dye (Life Technologies) was added at a final con ture curves compared to DMSO controls as exampled with centration of 0.5x concentration (supplied as 1000x) in 1 CDK2-Nluc (+/-AZD5438) and LCK-Nluc (+/-Dasatinib) uL/well volume, and Furimazine Live Cell Substrate (Pro target fusions. As expected, the shape of the BRET curves mega) was added to a final concentration of 1x in 20 uL/well are bell shaped due to loss of Nluc signal with protein volume. Plates were placed into a thermal cycler (MW unfolding and increasing temperatures or dye dissociation Research) where the samples were heated individually at upon protein aggregation or both. different temperatures spanning a gradient of 42-66° C. US 2016/0282360 A1 Sep. 29, 2016 27 across the plate for 3 minutes followed by cooling at 22°C. transferred to a tissue culture plate (Corning). To measure for 3 minutes, and a total volume of 100 uL?well was then BRET, filtered luminescence was measured on a BMG transferred to a tissue culture plate (Corning). To measure Clariostar luminometer equipped with 450 nm BP filter BRET, filtered luminescence was measured on a BMG (donor) and 610 nm LP filter (acceptor), using 0.5 s inte Clariostar luminometer equipped with 450 nm BP filter gration time. MilliBRET units (mBU) were calculated by (donor) and 610 nm LP filter (acceptor), using 0.5 s inte using the equation: (acceptor? donor)*1000. Data was then gration time. MilliBRET units (mBU) were calculated by fitted to obtain apparent melting temperature where the using the equation: (acceptor? donor)*1000. Data was ana protein is precipitating (T values) using the Boltzmann lyzed up to 62° C., and the data fitted to obtain apparent Sigmoidal equation with Graphpad Prism software. melting temperature where the protein is precipitating (T. (0168 FIGS. 17A-B show detection of an increase in values) using the Boltzmann Sigmoidal equation with melting temperature for CDK2-Nluc as determined by Graphpad Prism software. BRET after incubation with stabilizing ligand (stauro 0164 FIG. 15 shows detection of an increase in melting sporine) in mammalian cells Subsequently exposed to digi temperatures (Stabilizing ligand) or no change in melting tonin and a temperature gradient using three different envi temperatures (non-binding) for cytoplasmic target fusions ronmentally sensitive acceptor dyes reporting on protein CDK2-Nluc and Nluc-MAPK14 as determined by BRET folding status as BRET acceptor dyes. B is acceptor dye after incubation with a panel of compounds, thus displaying dose dependent, but that there is no change in the apparent compound selectivity in mammalian cells Subsequently melting temperature (T). exposed to digitonin and a temperature gradient. FIGS. 16A-F show detection of an increase in melting tempera Example 15 tures (stabilizing ligand) or no change in melting tempera tures (non-binding) for the nuclear target fusion HDAC1 BRET-Detection Thermal Shift Assay Nluc as determined by BRET after incubation with a panel 0169 Experiments were conducted during development of compounds in mammalian cells Subsequently exposed to of embodiments of the present invention to demonstrate digitonin and a temperature gradient. detection of the stabilization of a target protein by the interaction with a target ligand via temperature dependent Example 14 alteration of BRET signal generated by the energy transfer from a luciferase fused to the target protein to an environ BRET-Detection Thermal Shift Assay mentally sensitive dye that binds to all proteins unfolding in 0.165 Experiments were conducted during development the sample including the target protein. of embodiments of the present invention to demonstrate 0170 To demonstrate that the acceptor dye can be added detection of the stabilization of a target protein by the either before or after the heating step in the luciferase-based interaction with a target ligand via temperature dependent thermal shift assay using BRET analysis, two different alteration of BRET signal generated by the energy transfer environmentally sensitive dyes were used to determine from a luciferase fused to the target protein to an environ folded protein status and serve as BRET acceptor dyes. The mentally sensitive dye that binds to all proteins unfolding in dyes included in the Protein Thermal ShiftTM Dye (Life the sample including the target protein. Technologies) and the PROTEOSTAT dye (Enzo), which 0166 To demonstrate that many dyes are suitable for the have differing properties in regards to protein status binding. luciferase-based thermal shift assay analyzed by BRET The PROTEOSTAT dye binds to protein aggregates rather described herein, several different dyes were tested. Dye than unfolding proteins, whereas the Protein Thermal examples included: Protein Thermal ShiftTM Dye, SYPRO ShiftTM dye binds to proteins as they unfold, but dissociates Orange protein gel stain, and SYPRO Red protein gel stain. as the proteins aggregate. (FIG. 18). (0167. HeLa cells were transfected with CDK2-Nluc at a (0171 HeLa cells were transfected with CDK2-Nluc 1:100 DNA ratio and incubated overnight. The cells were DNA at a 1:10 DNA ratio and incubated overnight. The cells trypsinized using Trypsin-EDTA (0.25%) (Gibco) and re were trypsinized using Trypsin-EDTA (0.25%) (Gibco) and suspended in Opti-MEM without phenol red (Gibco)+1 x re-suspended in Opti-MEM without phenol red (Gibco)+1 x Protease Inhibitor Cocktail (Promega) which had been Protease Inhibitor Cocktail (Promega) which had been reconstituted in DMSO (Sigma). The cells were seeded into reconstituted in DMSO (Sigma). The cells were seeded into 96-well tall-chimney PCR plate (Fisherbrand) at a density of 96-well tall-chimney PCR plate (Fisherbrand) at a density of 2x10 cells/well in an 90 uL/well volume and treated with 10 2x10 cells/well in a 90 uL/well volume and treated with 10 uL of 10% DMSO (Sigma) or 10 uL of a 1 mM stock uL/well of a 0.5 mg/ml solution of digitonin (Sigma) in 10% solution of Staurosporine (LC Laboratories) in 10% DMSO. DMSO (Sigma) and 1 uL of 100% DMSO (Sigma) or 1 uL The plates were then incubated for /2 hour in a humidified, of a 10 mM stock solution of Staurosporine (LC Laborato 37° C./5% CO incubator. Immediately prior to heating, 10 ries) in 100% DMSO. The plates were then incubated for 2 uL/well of a 0.5 mg/ml Solution of digitonin (Sigma), hour in a humidified, 37° C./5% CO incubator. The plates Furimazine Live Cell Substrate (Promega) to a final con were placed into a thermal cycler (MW Research) where the centration of 1x in 20 uL/well volume, and 1 ul of each dye samples were heated either individually at different tem dilution was added. Dyes included the SYPRO Orange, peratures spanning a gradient of 42-66° C. across the plate SYPRO Red, and PROTEIN THERMAL SHIFT Dye (all for 3 minutes followed by cooling at 22°C. for 3 minutes. from Life Tech). Plates were placed into a thermal cycler The Protein Thermal ShiftTM Dye (Life Technologies) and (MW Research) where the samples were heated individually the PROTEOSTAT dye (Enzo) were added at a final con at different temperatures spanning a gradient of 42-66° C. centration of 1 x (supplied as 1000x) in 1 uI/well volume across the plate for 3 minutes followed by cooling at 22°C. either before or after the heating step. 90 ul/well was for 3 minutes, and a total volume of 100 uL?well was then transferred to a tissue culture plate (Corning), and FurimaZ US 2016/0282360 A1 Sep. 29, 2016 28 ine Live Cell Substrate (Promega) was added to a final Example 17 concentration of 1x in 20 uL? well volume. To measure BRET, filtered luminescence was measured on a BMG Other Proposed Proximity Based Reporter Methods Clariostar luminometer equipped with 450 nm BP filter for Detection of Ligand Binding in a Thermal Shift (donor) and 610 nm LP filter (acceptor), using 0.5 s inte Assay gration time. MilliBRET units (mBU) were calculated by using the equation: (acceptor? donor)*1000. Data was fitted (0175 Based on the successful use of a direct luciferase using the bell shaped curve equation with Graphpad Prism fusion or BRET to detect ligand-mediated thermal stabili software. (FIG. 18) Zation, other proximity-based reporter chemistries are understood to be useful. FIG. 21 gives schematic examples Example 16 of other proximity based reporter methods that may be Suitable for analysis of target engagement using thermal BRET-Detection Thermal Shift Assay shift. For example, an epitope tag is attached to the protein of interest and following a thermal denaturation step, addi 0172 Experiments were conducted during development tion of a detection antibody labeled with a donor fluorophore of embodiments of the present invention to demonstrate detection of the stabilization of a target protein by the (e.g., terbium, europium, etc.) is used in a FRET assay with interaction with a target ligand via temperature dependent an denaturation/aggregation-sensitive dye as a FRET accep alteration of BRET signal generated by the energy transfer tor. Ligand-mediated thermal stabilization results in a loss of from a luciferase fused to the target protein to an environ FRET/TR-FRET signal. Another potential iteration is using mentally sensitive dye that binds to all proteins unfolding in a combination of labeled antibodies (e.g. donor-labeled the sample including the target protein. anti-FLAG and acceptor labeled anti-V5) and a tandem (0173 HeLa cells were transfected with CDK2-Nluc or epitope (e.g. FLAG-V5) tethered to the target protein are HDAC1-Nluc at a 1:100 DNA ratio and incubated over used as a detection system. When the target is stabilized by night. The cells were trypsinized using Trypsin-EDTA binding of a ligand, the epitope is presented and both (0.25%) (Gibco) and re-suspended in Opti-MEM without antibodies bind, generating a proximity-based signal (e.g. phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro FRET. TR-FRET). Upon thermal denaturation, the epitopes mega) which had been reconstituted in DMSO (Sigma). The are unavailable to the antibody pair, resulting in a loss of cells were seeded into 96-well tall-chimney PCR plate proximity-based signal. Various detection chemistries can be (Fisherbrand) at a density of 2x10 cells/well in an 80 applied for these prophetic examples (e.g. TR-FRET prox uL? well volume and treated with 10 u, of 10% DMSO or 10 imity ligation, singlet oxygen transfer/alphaScreen, etc.). uL of a 1 mM stock solution of AZD5438 (for CDK2-Nluc) or Mocetinostat (for HDAC1-Nluc) in 10% DMSO. The plates were then incubated for /2 hour in a humidified, 37° Example 18 C./5% CO incubator. Immediately prior to heating, 10 uL/well of a 0.5 mg/ml Solution of digitonin (Sigma), Chemical Synthesis Description Furimazine Live Cell Substrate (Promega) to a final con centration of 1x in 20 uL?well volume, and 1 ul of the 0176 A series of styryl dye derivatives were synthesized Protein Thermal ShiftTM Dye (Life Technologies) for a final and screened in BRET detection thermal shift assay. The dye concentration of 0.5x was added. Plates were placed into a derivatives CS000, CS0004, CSO007, CSO008, CS0013, thermal cycler (MW Research) where the samples were CS0010, CS0017, CS0018, CS0020, CS0024, CS0028, heated individually at different temperatures spanning a CS0038, CS0067, CS0068, CS0075, CS0081, CS0085, gradient of 42-66° C. across the plate for 3 minutes. Some CS0086, CS0087, CS0096, and CSO155 and are generally plates were also then further cooled at 22°C. for 3 minutes. synthesized analogously to CSO 101 via a condensation A total volume of 100 uL?well was then transferred to a reaction between various aryl aldehydes and pyridinium tissue culture plate (Corning). To measure BRET, filtered betaines or N-alkyl pyridinium, which has been described in luminescence was measured on a BMG Clariostar luminom literature (1' reaction in Scheme 1) (Hassner, A.; Birnbaum, eter equipped with 450 nm BP filter (donor) and 610 nm LP D.: Loew, L. M. J. Org. Chem. 1984,49, 2546-2551.; herein filter (acceptor), using 0.5 s integration time. MilliBRET incorporated by reference in its entirety). As depicted in units (mBU) were calculated by using the equation: (accep Scheme 1, CSO101 was hydrolyzed under acidic condition tor? donor)*1000. Data was then fitted to obtain apparent to afford CS0036. The subsequent amide coupling and the melting temperature where the protein is precipitating (T. acidic deprotection afforded CS0100 and CS0045 respec values) using the Boltzmann Sigmoidal equation with tively. CS0048 was obtained via an amide coupling between Graphpad Prism software. CS0045 and 3-amino-1-propanesulfonic acid. CS0043 and CS0147 was synthesized analogously to CS0045. CS0073 0.174 FIGS. 19 and 20A-B demonstrate data obtained was obtained via a N-alkylation reaction between CS0043 when the assay was run with and without the cooling step and neat 1.3 propane sultone (Scheme 2). CSO117 was that follows the 3 minutes heating gradient step using two synthesized via a Heck reaction between the para-bromo different targets, CDK2-Nluc (FIG. 19) and HDAC1-Nluc aniline 5 and para-vinyl pyridine 6 (Scheme 3)." N-alky (FIGS. 20A-B). A clear shift in apparent T. was found lation of CS0117 afforded CS0121 (Scheme 3). whether a cooling step was included in the protocol or not CSO112, CSO158, CSO071 and CSO072 were synthesized analogously to as analyzed by both, direct luciferase readout and BRET. CSO121.

US 2016/0282360 A1 Sep. 29, 2016 30

Scheme 2

CH-1 N GE 1n 1a O 21 N O HN SO C (4) He 2 N-N-1-N-CO2H TSTU, DIPEA GE) DMF-HO CSOO36

l, O CH-1 N \%O N 2 N O O H --- 1N1a G 120° C., 3 h GE) N SO CSOO43 C6H13 CH-1 N 2 N O N-N- O 2 GE) N-so1-1s. CSOO73

-continued Scheme 3 C6H13 / \ GN-CH (CF).5CF C6H13 / CH-1 M e CSO121 Br (5) (E)-4-(4-(Dihexylamino)styryl)-1-(4-ethoxy-4- Oxobutyl)pyridin-1-ium bromide (CSO101) N 0177. To a solution of 4-(dihexylamino)benzaldehyde (1, 144 mg. 0.5 mmol. 1.0 equiv) in EtOH (4 mL) at RT, - N NEt3, 110°C.,48h piperidine (4.3 mg 0.05 mmol. 0.1 equiv) and 1-(4-ethoxy 4-oxobutyl)-4-methylpyridin-1-ium bromide (2,144 mg. 0.5 (6) mmol. 1.0 equiv) was added in one portion. The mixture was Stirred at 90° C. for 15 h and cooled down at RT for 30 min. The desired product precipitated from the solution and was purified by silica gel chromatography (CH.Cl/MeOH, 1/9). C6H13 F F F F F F LRMS: observed for M-Br", 479.7. CF CH1:1 F F F F (E)-1-(3-Carboxypropyl)-4-(4-(dihexylamino)styryl) (7) pyridin-1-ium chloride (CS0036) Hs eN CHCN, reflux (0178. To a solution of CS0101 (324 mg., 0.58 mmol, 1.0 CSO 117 equiv) in 1,4-dioxane (4 mL) at RT, 6 NHCl (aq, 4 mL, 24 mmol. 41 equiv) was added. The mixture was stirred at 100° US 2016/0282360 A1 Sep. 29, 2016

C. for 15 h and concentrated in vacuo to afford the crude. aniline (5. 340 mg, 1 mmol. 1.0 equiv) and para-vinyl The desired product was purified by Silica gel chromatog pyridine (6,115.7 mg, 1.1 mmol. 1.1 equiv) was added. The raphy (CHC1/MeoH, 1/9). LRMS: observed for M-Cl", mixture was heated up to 120° C. and stirred for 48 h under 451.7. N. The reaction was cooled down and concentrated to afford the crude. The desired product was purified by silica (E)-4-(4-(Dihexylamino)styryl)-1-(2,2-dimethyl-4, gel chromatography (Heptane/EtOAc, 6/4). LRMS: 17-dioxo-3,7,10,13-tetraoxa-16-azaicosan-20-yl) observed for M+H, 365.6. pyridin-1-ium chloride (CSO100) (E)-4-(4-(dihexylamino)styryl)-1-(3,3,4,4,5,5,6,6,7, (0179 To a solution of CS0036 (70 mg, 0.13 mmol, 1.0 7,8,8,8-tridecafluorooctyl)pyridin-1-iumiodide equiv) in DMF (1.5 mL) at RT, TSTU (40 mg, 0.13 mmol. 1.0 equiv) and NEt (60 uL, 0.43 mmol. 2.5 equiv) was (CSO121) added. The solution was stirred at RT for 1 h. To the reaction 0184. To a solution of CS0117 (70 mg, 0.19 mmol, 1.0 mixture, tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) equiv) in CHCN (3 mL), 1,1,1,2,2,3,3,4,4,5,5,6,6-trideca propanoate (3, 43.8 mg, 0.16 mmol. 1.2 equiv) in DMF (1.5 fluoro-8-iodooctane (7, 108 mg 0.23 mmol. 1.2 equiv) was mL) was added. The reaction was stirred at RT for 15 h. added. The reaction was heated to reflux for 15 h. LC-MS LC-MS analysis indicated full conversion of the starting indicated full conversion to the desired product. The mixture material. The solvent was removed in vacuo to afford the was concentrated in vacuo and purified by Silica gel chro crude and the desired product was purified by silica gel matography (CHC1/MeCH, 1/9). LRMS: observed for chromatography (CH.Cl/MeOH, 1/9). LRMS: observed for M-II", 711.8. M-C1, 711.1. Example 19 (E)-1-(1-Carboxy-13-oxo-3,6,9-trioxa-12-azahexa decan-16-yl)-4-(4-(dihexylamino)Styryl)pyridin-1- BRET-Detection Thermal Shift Assay ium chloride (CS0045) 0185. Experiments were conducted during development 0180. The starting material CS0100 (40 mg, 0.5 mmol, of embodiments of the present invention to demonstrate 1.0 equiv) was dissolved in 4N HCl in dioxane (2 mL, 8 detection of the stabilization of a target protein by the mmol. 16 equiv). The mixture was stirred at 100° C. for 15 interaction with a target ligand via temperature dependent h and concentrated in vacuo to afford the crude. The desired alteration of BRET signal generated by the energy transfer product was purified by silica gel chromatography (CH2Cl2/ from a luciferase fused to the target protein to an environ MeOH, 1/9). LRMS: observed for M-Cl", 65.4.9. mentally sensitive dye that binds to all proteins unfolding in (E)-21-(4-(4-(Dilhexylamino)styryl)pyridin-1-ium-1- the sample including the target protein. yl)-5,18-dioxo-8,11,14-trioxa-4,17-diazahenicos 0186 To demonstrate analysis of compound selectivity using the BRET method of thermal shift, a panel of com ane-1-sulfonate (CS0048) pounds with known selectivity against CDK2 and MAPKI4 0181. To a solution of CS0045 (60 mg, 0.09 mmol, 1.0 was tested. It was found that the assay accurately detected equiv) in DMF (1.5 mL) at RT, TSTU (33 mg, 0.11 mmol, the positive and negative ligands against these targets. 1.2 equiv) and NE t (51 uL, 0.37 mmol. 4 equiv) was added. 0187. HeLa cells were transfected with CDK2-Nluc or The solution was stirred at RT for 1 h. To the reaction Nluc-MAPKI4 DNA at a 1:10 DNA ratio and incubated mixture, 3-amino-1-propanesulfonic acid (4, 19.1 mg, 0.14 overnight. The cells were trypsinized using Trypsin-EDTA mmol. 1.5 equiv) in HO (1 mL) was added. The reaction (0.25%) (Gibco) and re-suspended in Opti-MEM without was stirred at RT for 15 h. LC-MS analysis indicated full phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro conversion of the starting material. The solvent was mega), which had been reconstituted in DMSO (Sigma). The removed in vacuo to afford the crude and the desired product cells were seeded into wells of a 96-well, tall-chimney PCR was purified by silica gel chromatography (CH.Cl/MeOH, plate (Fisherbrand) at a density of 2x10 cells/well in an 90 1/9). LRMS: observed for M+H, 776.1. uL? well volume and treated with 10 u, of 10% DMSO (Sigma) or 10 uI of a 1 mM stock solution of the test (E)-3-(4-(4-(4-(Dilhexylamino)styryl)pyridin-1-ium compound Staurosporine (LC Laboratories) in 10% DMSO. 1-yl)-N-(3-sulfopropyl)butanamido)propane-1-sul The plates were then incubated for /2 hour in a humidified, fonate (CS0073) 37° C./5% CO incubator. Immediately prior to heating, 10 0182 CS0043 was mixed with 1.3 propane sultone (1 uL/well of a 0.5 mg/ml Solution of digitonin (Sigma) and mL) and heated at 120° C. for 3 h. The mixture was cooled dye derivative was added at a final concentration of 10 uM down to RT and resuspended in hot MeOH (20 mL), filtered or in a dose response as indicated in B and C of each figure in 1 uL/well volume. Plates were placed into a thermal over Celite. Concentration in vacuo afforded the crude, cycler (MW Research) where the samples were heated which was purified by silica gel chromatography (CH2Cl2/ individually at different temperatures spanning a gradient of MeOH, 1/9). LRMS: observed for M+H", 695.1. 42-66° C. across the plate for 3 minutes followed by cooling (E)-N,N-Dihexyl-4-(2-(pyridin-4-yl)Vinyl)aniline at 22° C. for 3 minutes, and a total volume of 100 uL/well was then transferred to a tissue culture plate (Coming). (CSO117) Furimazine Live Cell Substrate (Promega) was added to a 0183) To a flask charged with Pd(OAc) (11.2 mg, 50 uL. final concentration of 1x in 20 uL?well volume. To measure 0.05 equiv) and tri-(o-tolyl) phosphine (45.6 mg, 0.14 mmol. BRET, filtered luminescence was measured on a BMG 0.15 equiv), NEt (5 mL) was added. The mixture was Clariostar luminometer equipped with 450 nm BP filter stirred under N for 20 min. To the mixture, para-bromo (donor) and 610 nm LP filter (acceptor), using 0.5 s inte US 2016/0282360 A1 Sep. 29, 2016 32 gration 5 time. MilliBRET units (mBU) were calculated by cells. These assays are performed under live cell or lytic using the equation: (acceptor? donor)* 1000. conditions. Assays in which the peptide reporters are used 0188 Data was analyzed up to 62°C., and the data fitted (high affinity NLpoly and NLpoly) require that the comple to obtain apparent melting temperature where the protein is mentary peptide (i.e., NLpep) be present for reconstitution precipitating (Tagg values) using the Boltzmann Sigmoidal of active reporter luciferase and signal. In some embodi equation with Graphpad Prism software. ments, this is done lytically using a reagent that contains the (0189 FIGS. 23-34 demonstrate: (A) that BRET using complementary NLpep or through using mammalian cells dyes synthesized in Example 18 detects ligand binding that expresses the NLpep complementary to the NLpep used through a change in relative BRET compared to DMSO for the genomic editing of the target of interest. controls; (B) the fold change in BRET signal at 56°C. over the background BRET signal at 42°C. for cells transfected with CDK2-Nluc target that were treated with DMSO and varying dye concentrations; and (C) the fold change in SEQUENCES BRET signal at 52° C. over the background BRET signal at SEQ ID NO : 1 (Wild-type Ogluc) - 42° C. for cells transfected with Nluc-MAPK14 target that mftladfvgdwgdtagyngdovledgglsslfgalgvsvtpickvvlsge were treated with DMSO and varying dye concentrations nglkadihvilipyeglsgfc.mgliemifkvvypvddhhfkiilhygtlvi dgvtpnmidyfgrpypgiavfdgkgitvtgtlWingnkiyderlinpdgsl Example 20 lfrvtingvtgwrilcenila 0190. The properties of environmentally sensitive dyes for thermal shift assays include having again in fluorescence SEQ ID NO: 2 (NANOLUC) - upon binding to proteins that are unfolding due to thermal mvftled fivgdwrqtagynildovledggvsslfqnligvsvtpigrivlsg insult. FIGS. 35-37 demonstrate the fluorescent spectra for englkidihvilipyeglsgdomggiekifkvvypvddhhfkvillhygtlv some of the synthesized dyes (described in Example 18) idgvtpnmidyfgrpyegiavfdgkkitvtgtlWingnki iderlinpdgs when added to cells lysates that had been transfected with ll frvtingvtgwrilcerila Nluc-target protein, and then either heated at 42°C. (base line), 56° C. (for CDK2-Nluc target), or 52° C. (for Nluc SEQ ID NO: 3 - MAPK14 target). The higher temperature results in again in gvtgWrlcerila fluorescence thus showing the change in dye binding and SEO ID NO: 4 - fluorescent properties indicating that it is a useful dye for mvftladfvgdwgqtagynoldovledgglls slfqalgvsvtpickvvlsg this application. englkadihvilipyeglsgfgmgliemifkvvypvddhhfkiilhygtlv (0191 HeLa cells were transfected with CDK2-Nluc or idgvtpnmidyfgrpypgiavfdgkoitvtgtlWingnkiyderlinpdgs Nluc-MAPKI4 DNA at a 1:10 DNA ratio and incubated overnight. The cells were trypsinized using Trypsin-EDTA llfirwtin (0.25%) (Gibco) and re-suspended in Opti-MEM without phenol red (Gibco)+1x Protease Inhibitor Cocktail (Pro SEQ ID NO: 5 (NLpep86) - mega), which had been reconstituted in DMSO (Sigma). The vsgwrlfkkis cells were seeded into wells of a 96-well, tall-chimney PCR plate (Fisherbrand) at a density of 2x104 cells/well in an 90 SEQ ID NO: 6 (NLpoly11S) - uL/well volume and treated with 10 uL of 10x dye solution mvftled fivgdweqtaaynildovledggvssillonlaysvtpigrivrsg and heated at 42° C., 52° C., or 56° C. for 3 minutes enalkidihvilipyeglsadqmaqileevfikvvypvddhhfkvilpygtlv followed by cooling at 25°C. for 2 minutes. Samples were idgvtpnmlnyfgrpyegiavfdgkkitvtgtlWingnki iderlitpdgs transferred to a tissue culture plate and analyzed in fluores mlfrvtins cent spectral mode on a Clariostar. SEQ ID NO: 7 (NLpoly 156) - Example 21 mvftled fivgdwrqtagynildovledggvsslfqnligvsvtpigrivlsg enalkidihvilipyeglsgdomggiekifkvvypvddhhfkvillhygtlv Target Genome Editing idgvtpnmidyfgrpyegiavfdgkkitvtgtlWingnki iderlinpdgs llfirwtin 0.192 Targeted genome editing technologies to introduce reporter tags to a target protein of interest are used to evaluate ligand binding induced thermal stabilization at endogenous levels of protein expression in the relevant 0193 All publications and patents provided herein are cellular context. Targeted genome editing technologies incorporated by reference in their entireties. Various modi include, but are not limited to, clustered regularly-inter fications and variations of the described compositions and spaced short palindromic repeats (CRISPR/Cas system), methods of the invention will be apparent to those skilled in Zinc finger nucleases (ZFN), transcription-activator-like the art without departing from the scope and spirit of the effector-based nucleases (TALENs), and engineered mega invention. Although the invention has been described in nucleases re-engineered homing endonucleases. Targeted connection with specific preferred embodiments, it should protein fusions are made, in some embodiments, with a high be understood that the invention as claimed should not be affinity NLpoly, NLpoly, or NANOLUC luciferase to a unduly limited to Such specific embodiments. Indeed, vari protein of interest. These tags are fused to the N- or ous modifications of the described modes for carrying out C-terminal of the protein of interest or internally within the the invention that are obvious to those skilled in the relevant target protein of interest. Assays are performed in both fields are intended to be within the scope of the present selected clonal cell lines as well as in unselected pools of invention. US 2016/0282360 A1 Sep. 29, 2016 33

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 7

<210s, SEQ ID NO 1 &211s LENGTH: 170 212. TYPE: PRT <213> ORGANISM: Oplophorus gracilirostris

<4 OOs, SEQUENCE: 1 Met Phe Thr Lieu Ala Asp Phe Val Gly Asp Trp Gln Gln Thr Ala Gly 1. 5 1O 15 Tyr Asn Glin Asp Glin Val Lieu. Glu Glin Gly Gly Lieu. Ser Ser Lieu. Phe 2O 25 3O Glin Ala Leu Gly Val Ser Val Thr Pro Ile Gln Lys Val Val Leu Ser 35 4 O 45 Gly Glu Asn Gly Lieu Lys Ala Asp Ile His Val Ile Ile Pro Tyr Glu SO 55 6 O Gly Leu Ser Gly Phe Glin Met Gly Lieu. Ile Glu Met Ile Phe Llys Val 65 70 7s 8O Val Tyr Pro Val Asp Asp His His Phe Lys Ile Ile Lieu. His Tyr Gly 85 90 95 Thr Lieu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly 1OO 105 11 O Arg Pro Tyr Pro Gly Ile Ala Val Phe Asp Gly Lys Glin Ile Thr Val 115 12 O 125 Thr Gly. Thir Lieu. Trp Asn Gly Asn Lys Ile Tyr Asp Glu Arg Lieu. Ile 13 O 135 14 O Asn Pro Asp Gly Ser Lieu Lleu Phe Arg Val Thir Ile Asin Gly Val Thr 145 150 155 160 Gly Trp Arg Lieu. Cys Glu Asn. Ile Lieu Ala 1.65 17O

<210s, SEQ ID NO 2 &211s LENGTH: 171 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic enzyeme

<4 OOs, SEQUENCE: 2 Met Val Phe Thr Lieu. Glu Asp Phe Val Gly Asp Trp Arg Glin Thr Ala 1. 5 1O 15 Gly Tyr Asn Lieu. Asp Glin Val Lieu. Glu Glin Gly Gly Val Ser Ser Lieu. 2O 25 3O Phe Glin Asn Lieu. Gly Val Ser Val Thr Pro Ile Glin Arg Ile Val Lieu. 35 4 O 45 Ser Gly Glu Asn Gly Lieu Lys Ile Asp Ile His Val Ile Ile Pro Tyr SO 55 6 O Glu Gly Lieu. Ser Gly Asp Gln Met Gly Glin Ile Glu Lys Ile Phe Lys 65 70 7s 8O Val Val Tyr Pro Val Asp Asp His His Phe Llys Val Ile Lieu. His Tyr 85 90 95 Gly Thr Lieu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 1OO 105 11 O Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Llys Lys Ile Thr 115 12 O 125 US 2016/0282360 A1 Sep. 29, 2016 34

- Continued

Wall Thir Gly Thr Lieu. Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Lieu. 13 O 135 14 O

Ile Asn Pro Asp Gly Ser Lieu Leu Phe Arg Val Thr Ile Asin Gly Val 145 150 155 160

Thir Gly Trp Arg Lieu. Cys Glu Arg Ile Lieu Ala 1.65

SEQ ID NO 3 LENGTH: 13 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic enzyeme

<4 OOs, SEQUENCE: 3 Gly Val Thr Gly Trp Arg Lieu. Cys Glu Arg Ile Lieu Ala 1. 5

<210s, SEQ ID NO 4 &211s LENGTH: 158 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic enzyeme

<4 OOs, SEQUENCE: 4 Met Val Phe Thr Lieu Ala Asp Phe Val Gly Asp Trp Gln Gln Thr Ala 1. 5 1O 15

Gly Tyr Asn Glin Asp Glin Val Lieu. Glu Glin Gly Gly Lieu. Ser Ser Luell 2O 25

Phe Glin Ala Leu Gly Val Ser Val Thr Pro Ile Gln Lys Wall Wall Luell 35 4 O 45

Ser Gly Glu Asn Gly Lieu Lys Ala Asp Ile His Wall Ile Ile Pro Tyr SO 55 6 O

Glu Gly Lieu Ser Gly Phe Gln Met Gly Luell Ile Glu Met Ile Phe Lys 65 70 7s

Val Val Tyr Pro Val Asp Asp His His Phe Lys Ile Ile Luell His Tyr 85 90 95

Gly Thr Lieu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Phe 1OO 105 11 O

Gly Arg Pro Tyr Pro Gly Ile Ala Val Phe Asp Gly Lys Glin Ile Thir 115 12 O 125

Val Thr Gly. Thir Lieu. Trp Asn Gly Asn Ile Tyr Asp Glu Arg Luell 13 O 135 14 O

Ile Asin Pro Asp Gly Ser Lieu. Lieu. Phe Arg Wall. Thir Ile Asn 145 150 155

<210s, SEQ ID NO 5 &211s LENGTH: 11 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic enzyeme

<4 OOs, SEQUENCE: 5 Val Ser Gly Trp Arg Lieu. Phe Llys Lys Ile Ser 1. 5 US 2016/0282360 A1 Sep. 29, 2016 35

- Continued

<210s, SEQ ID NO 6 &211s LENGTH: 159 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic enzyeme

<4 OOs, SEQUENCE: 6 Met Val Phe Thr Lieu. Glu Asp Phe Val Gly Asp Trp Glu Glin Thr Ala 1. 5 1O 15 Ala Tyr Asn Lieu. Asp Glin Val Lieu. Glu Glin Gly Gly Val Ser Ser Lieu 2O 25 3O Lieu. Glin Asn Lieu Ala Val Ser Val Thr Pro Ile Glin Arg Ile Val Arg 35 4 O 45 Ser Gly Glu Asn Ala Lieu Lys Ile Asp Ile His Val Ile Ile Pro Tyr SO 55 6 O Glu Gly Lieu. Ser Ala Asp Gln Met Ala Glin Ile Glu Glu Val Phe Lys 65 70 7s 8O Val Val Tyr Pro Val Asp Asp His His Phe Llys Val Ile Leu Pro Tyr 85 90 95

Gly Thr Lieu Val Ile Asp Gly Val Thr Pro Asn Met Lieu. Asn Tyr Phe 1OO 105 11 O

Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Llys Lys Ile Thr 115 12 O 125

Val Thr Gly. Thir Lieu. Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Lieu 13 O 135 14 O

Ile Thr Pro Asp Gly Ser Met Leu Phe Arg Val Thr Ile Asin Ser 145 150 155

<210s, SEQ ID NO 7 &211s LENGTH: 158 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic enzyeme

<4 OO > SEQUENCE: 7 Met Val Phe Thr Lieu. Glu Asp Phe Val Gly Asp Trp Arg Glin Thr Ala 1. 5 1O 15

Gly Tyr Asn Lieu. Asp Glin Val Lieu. Glu Glin Gly Gly Val Ser Ser Lieu. 2O 25 3O

Phe Glin Asn Lieu. Gly Val Ser Val Thr Pro Ile Glin Arg Ile Val Lieu. 35 4 O 45 Ser Gly Glu Asn Ala Lieu Lys Ile Asp Ile His Val Ile Ile Pro Tyr SO 55 6 O

Glu Gly Lieu. Ser Gly Asp Gln Met Gly Glin Ile Glu Lys Ile Phe Lys 65 70 7s 8O Val Val Tyr Pro Val Asp Asp His His Phe Llys Val Ile Lieu. His Tyr 85 90 95

Gly Thr Lieu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 1OO 105 11 O

Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Llys Lys Ile Thr 115 12 O 125