US 2007.0128662A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0128662 A1 Isacoff et al. (43) Pub. Date: Jun. 7, 2007 (54) PHOTOREACTIVE REGULATOR OF Publication Classification PROTEIN FUNCTION AND METHODS OF USE THEREOF (51) Int. Cl. GOIN 33/53 (2006.01) (76) Inventors: Ehud Y. Isacoff, Berkeley, CA (US); C07K I4/95 (2006.01) Richard H. Kramer, Oakland, CA C7H 2L/04 (2006.01) (US); Dirk Trauner, San Francisco, CA CI2P 2/06 (2006.01) (US); Matthew R. Banghart, Oakland, CI2N 15/74 (2006.01) CA (US); Matthew Volgraf, Oakland, CI2N I/2 (2006.01) CA (US); Pablo Ignacio Gorostiza A6II 38/16 (2006.01) Langa, Barcelona (ES) (52) U.S. Cl. ...................... 435/7.1; 435/69.1; 435/252.3: 435/471; 530/350, 536/23.5; Correspondence Address: 514f12 BOZICEVIC, FIELD & FRANCIS LLP 1900 UNIVERSITY AVENUE SUTE 200 (57) ABSTRACT EAST PALO ALTO, CA 94.303 (US) The present invention provides a synthetic regulator of Appl. No.: 11/601,591 protein function, which regulator is a light-sensitive regu (21) lator. The present invention further provides a light-regu (22) Filed: Nov. 17, 2006 lated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject Related U.S. Application Data light-regulated polypeptide. The present invention further provides methods of modulating protein function, involving (60) Provisional application No. 60/737,935, filed on Nov. use of light. The present invention further provides methods 18, 2005. of identifying agents that modulate protein function. Patent Application Publication Jun. 7, 2007 Sheet 1 of 34 US 2007/O128662 A1 F.G. 1 a MAL-AZO-GRA Patent Application Publication Jun. 7, 2007 Sheet 2 of 34 US 2007/0128662 A1 FIG 2 3. b 350 30 Ontrol Control Control 2 O Block UV Vis. eO 250 C S. g 200 C O 150 100 O 2 4 8 6 10 Time (min) C d 72 ZDZZZZZTAZZZZZ M 1000 200 ZZ Wis. Dark 800 OUV 150 ZVis. 2 2 OUV 9- 600 Soo E wa 95 400 50 O ( ) 200 O O O 2 4 6 8 10 2 14 16 O 50 100 150 200 250 300 Time (min) Time (s) Patent Application Publication Jun. 7, 2007 Sheet 3 of 34 US 2007/0128662 A1 3. b 500 360 340 380 400 420 500 0.25 100 0.20 tranS 3 a. 80 is 0.15 CC -S 9 60 3 0.10 40 -O s a 0.05 cis O 20 0.00 O 250 300 350 400 450 500 550 O 5 10 15 20 Wavelength (nm) Time (min) C d 1.0 1.O 2 30.85 0.85-Y s O O D S 0.6 06 P s W. Y cus E.N 0.4 0.4.S25 s O 5 E 5 s Z 0.0-0 0.0 O.O 0.2 0.4 0.6 0.8 10 320 360 400 440 480 520 560 600 Time (min) Wavelength (nm) FIG. 3 Patent Application Publication Jun. 7, 2007 Sheet 4 of 34 US 2007/0128662 A1 3. I S&T DXX X& mV 500 390 nt nm. 10 S b mV 50 mS C 7 6 500 nm 9 5 O 390 nm s on 4 5 is 3 2S 2 Z MAL-AZO-QA MAL-AZO-QA Shaker + Shaker FIG. 4 Patent Application Publication Jun. 7, 2007 Sheet 5 of 34 US 2007/O128662 A1 FIG. 5A Patent Application Publication Jun. 7, 2007 Sheet 6 of 34 US 2007/O128662 A1 -OOC1)-1 COO 2: (2S,4R)-4-allylglutamate H O 6 H NH -OOc1N1 coo 3: Tether model F.G. 6B AZObenzene Maleimide N O TranS y - C's,N Ki C 1N1as '''3 O H 4: MAG OOC COO 380m 500 nm Agonist O N // O O N O H on Cis OOC HN *-n-so -OOc'NH; Patent Application Publication Jun. 7, 2007 Sheet 7 of 34 US 2007/O128662 A1 FIG. 6C -OOc1N1 coo Patent Application Publication Jun. 7, 2007 Sheet 8 of 34 US 2007/O128662 A1 O.J.'SDI uluu9 Patent Application Publication Jun. 7, 2007 Sheet 9 of 34 US 2007/O128662 A1 H NH 1NH ''O Hera,b NSNs O 2 HN HN 5: Azodianiline 6 O l Ho' C de f-h o-cooh s o-cooE Hair o?-cooE -- H Boc BOC 7: L-Pyroglutamic acid 8 9 H N-N NC 5 H i-k H.N- N C 10 H o-cooE BOC H (ii, C NC5N ge O N HOOC1N1COOH 4: MAGHC Patent Application Publication Jun. 7, 2007 Sheet 10 of 34 US 2007/O128662 A1 FIG 9A glu 300 M 6OO 500 s 400 3OO FIG. 9B tether model (nM) glu 300 um 3 1 O.3 0.10.01 Patent Application Publication Jun. 7, 2007 Sheet 11 of 34 US 2007/O128662 A1 FIG 10 O gyu. NH ooc1\1COO nic Q H O 3: Tether model '''d) H NH 3 Patent Application Publication Jun. 7, 2007 Sheet 12 of 34 US 2007/O128662 A1 A 51.00 al 0.75 w UDs 0,50 2 0.25 600 E500 Sooo 400 300 400 SOO 600 3OO excitation wavelength (nm) D 3S 1.00 s 0.75 E to so ge s N se gas 9Sis via 2000 3oo Too r so" so excitation wavelength (nm) 05 - excitation wavelength (nm) Patent Application Publication Jun. 7, 2007 Sheet 13 of 34 US 2007/O128662 A1 FIG. 12 A 2ootooooooo time (min) B 200 pA 2 min Patent Application Publication Jun. 7, 2007 Sheet 14 of 34 US 2007/0128662 A1 FIG. 13 A B 500 nm. 380 nm O O. /1BDy O Glu O)."A G e C gluReimsoniii. D glu glu 300M glu MAG 100nM glu 300uM + MAG 100nM 300uM R" 36M A \A".is 380/500nm - 380/500mm 10 min Patent Application Publication Jun. 7, 2007 Sheet 15 of 34 US 2007/O128662 A1 FIG. 14 300 uM glu 1 nA 1 OS 600 Patent Application Publication Jun. 7, 2007 Sheet 16 of 34 US 2007/0128662 A1 miOOZmioOLQmioO£ W uopenueouoso emoee | XONG] esuodsel empeau Patent Application Publication Jun. 7, 2007 Sheet 17 of 34 US 2007/0128662 A1 FIG. 16 &83§8. NZ MAG-1 MAG-2 photoswitch length D NYNY MAG-1 MAG-2 MAG-1 MAG-2 photoswitch length photoswitch length Patent Application Publication Jun. 7, 2007 Sheet 18 of 34 US 2007/0128662 A1 H N1 -- N(4. -/ O H Z cºmºaegae, Z k O o? Cy <!--N C Patent Application Publication Jun. 7, 2007 Sheet 19 of 34 US 2007/O128662 A1 s 3 S. s SuluoMSoloud 9, 3 e US Ozzy S e or a v ( e. vo so O O. O. O. fuuoAASOoud 9. (vod) ueluno (9 Patent Application Publication Jun. 7, 2007 Sheet 20 of 34 US 2007/O128662 A1 8 s C 0 to r (N. c. vie C C C C C. f5uguo MSoloud 9, () Patent Application Publication Jun. 7, 2007 Sheet 21 of 34 US 2007/O128662 A1 FG. 20 b O DDO O". OO ... OOO OO |- S Patent Application Publication Jun. 7, 2007 Sheet 22 of 34 US 2007/0128662 A1 FIG 21 15 min 80 60 min Patent Application Publication Jun. 7, 2007 Sheet 25 of 34 US 2007/0128662 A1 (ZH) / e3e LoMS 3 C O v ve - ve s 88 9. s epndue pezieuou Patent Application Publication Jun. 7, 2007 Sheet 26 of 34 US 2007/O128662 A1 mm,.~~~~); E. C) Patent Application Publication Jun. 7, 2007 Sheet 27 of 34 US 2007/O128662 A1 FIG. 26 21 Hz. 4oo . 400 is t 5 SH somy E 400 is logou Isusual so SOO be as a D SSHz 4 O - also III Bl E. OO . Patent Application Publication Jun. 7, 2007 Sheet 28 of 34 US 2007/0128662 A1 FIG. 27 2 - -20 D E-40 > -60 -80 to500 E b 20 mV 10 sec < 500 Patent Application Publication Jun. 7, 2007 Sheet 29 of 34 US 2007/O128662 A1 TipTTT 5 (uu). Patent Application Publication Jun. 7, 2007 Sheet 30 of 34 US 2007/0128662 A1 Patent Application Publication Jun. 7, 2007 Sheet 31 of 34 US 2007/0128662 A1 FIG. 30 Patent Application Publication Jun. 7, 2007 Sheet 32 of 34 US 2007/0128662 A1 FIG 31 Patent Application Publication Jun. 7, 2007 Sheet 33 of 34 US 2007/O128662 A1 FIG. 32 500 nm 380 nm 10 sec 500 m 380 nm 2 mSec Patent Application Publication Jun. 7, 2007 Sheet 34 of 34 US 2007/0128662 A1 US 2007/0128662 A1 Jun. 7, 2007 PHOTOREACTIVE REGULATOR OF PROTEIN 0010 FIGS. 3A-D depict absorbance and action spectra FUNCTION AND METHODS OF USE THEREOF of MAL-AZO-QA. 0011 FIGS. 4A-C depict expression of light-activated CROSS-REFERENCE channels confers light-sensitivity on hippocampal pyramidal 0001) This application claims the benefit of U.S. Provi UOS. sional Patent Application No. 60/737,935, filed Nov. 18, 0012 FIGS. 5A-C schematically depict a design of an 2005, which application is incorporated herein by reference allosteric photoswitch. in its entirety. 0013 FIGS. 6A-D depict structures and fit of photo switched agonist and iGluR6 LBD. (a) Chemical structure of STATEMENT REGARDING FEDERALLY the iGluR6 agonists (2S,4R)-4-methylglutamate 1, (2S,4R)- SPONSORED RESEARCH 4-allyl glutamate 2 and tether model 3. 0002 The U.S. government may have certain rights in 0014 FIGS. 7A-E depict calcium imaging of iGluR6 this invention, pursuant to grant no.