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(12) United States Patent (10) Patent No.: US 9,097,707 B2 Isacoffet Al US009097707B2 (12) United States Patent (10) Patent No.: US 9,097,707 B2 Isacoffet al. (45) Date of Patent: Aug. 4, 2015 (54) PHOTOREACTIVE REGULATOR OF 2003. O181531 A1 9, 2003 Sherris et al. PROTEIN FUNCTION AND METHODS OF 2005/0272677 A1 12/2005 Friesen et al. 2006/0252677 A1* 11/2006 Ohara et al. .................... 514/12 USE THEREOF 2007,0191411 A1 8, 2007 Smith (71) Applicant: The Regents of the University of 2009/0170901 A1* 7/2009 Borza et al. ................... 514,330 California, Oakland, CA (US) FOREIGN PATENT DOCUMENTS (72) Inventors: Ehud Y. Isacoff, Berkeley, CA (US); WO WO O2/O61415 8, 2002 Richard H. Kramer, Oakland, CA WO WO 2007/024290 3, 2007 (US); Dirk Trauner, Munich (DE): OTHER PUBLICATIONS Matthew R. Banghart, Oakland, CA (US); Matthew Volgraf, Oakland, CA Ichinose et al. (2003) Ca2+-independent, but voltage- and activity (US); Pablo Ignacio Gorostiza Langa, dependent regulation of the NMDA receptor outward K+ current in Barcelona (ES); Katharine Borges, mouse cortical neurons, vol. 551, pp. 403-417.* Centerport, NY (US) Colledge etal. (2003) Ubiquitination Regulates PSD-95 Degradation and AMPA Receptor Surface Expression, Neuron, vol. 40, No. 3, pp. (73) Assignee: The Regents of the University of 595-6O7.* California, Oakland, CA (US) Allen, C. "A Molecular Light Switch Turns Off Neural Activity”. - 2004, Nat. Neurosci., 2004, vol. 7, No. 12, p. 1291. (*) Notice: Subject to any disclaimer the term of this Banghartet al., “Light Activated Ion Channels for Remote Control of patent is extended or adjusted under 35 Neuronal Firing”, 2004, Nature Neuroscience, vol. 7, No. 12, pp. U.S.C. 154(b) by 118 days. 1381-1386. Behrendt, et al., “Photomodulation of Conformational State. Synthe (21) Appl. No.: 13/671,908 sis of Cyclic Peptides with Backbone-Azobenzene Moieties”, 1999, J. Pept. Sci., vol. 5, No. 11, pp. 519-529. (22) Filed: Nov. 8, 2012 Buster et al., “Na-Nuclear Megnetic Resonance Investigation of Gramicidin-Induced Ion Transport Through Membranes Under (65) Prior Publication Data Equilibrium Conditions”, 1988, Biophys.J., vol. 53, No. 2, pp. 145 152. US 2013/O137113 A1 May 30, 2013 Chambers et al. “Light-induced Depolarization of Neurons. Using a Modified Shaker (K+) Channel and a Molecular Photoswitch', 2006, Related U.S. Application Data J. Neurophysiol. vol. 96, No. 5, pp. 2792-2796. Essen... et al. “Ino-Channel Engineering”. 2008, Annual Reports (60) Continuation of application No. 13/364,105, filed O Section C. vol. 104, pp. 165-168. Feb. 1. 2012, now. Pat. No. 8,309,350, which is a Evans, et al., "Acetylcholine-Activated Single Channel Currents in division of application No. 1 1/601,591, filed on Nov. Cultured. Aplysia Neurons', 1989, Biophy, J., vol. 55, pp. 553a, 17, 2006, now Pat. No. 8,114,843. abstract No. W-Pos257. Fortin, et al., “Photochemical Control of Endogenous Ion Channels (60) Provisional application No. 60/737,935, filed on Nov. and Cellular Excitability”, 2008, Nat. Methods, vol. 5, No. 4, pp. 18, 2005. 331-338 (51) Int. Cl Grigoriev, et al., “Differences in Ion Permeability of an Artificial CO 7C 29/56 2006.O1 Bilayer Membrane Caused by Ampullosporin and Bergofungin, New ( .01) 15-Membered Peptaibol-Type Antibiotics', 1997, Bioelectrochem. GOIN33/53 (2006.01) Bioeng, vol. 44, pp. 155-158. C07K 4/705 (2006.01) Continued A61 K38/00 (2006.01) (Continued) (52) U.S. Cl. CPC .......... G0IN33/53 (2013.01); C07K 14/7.0571 Primary Examiner — Manjunath Rao (2013.01); A61 K38/00 (2013.01); G0IN Assistant Examiner — Samuel Liu 2500/10 (2013.01) (74) Attorney, Agent, or Firm — Bozicevic, Field & Francis (58) Field of Classification Search LLP: Paula A. Borden CPC .................................................... GO1N 24f088 See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention provides a synthetic regulator of pro tein function, which regulator is a light-sensitive regulator. U.S. PATENT DOCUMENTS The present invention further provides a light-regulated polypeptide that includes a Subject synthetic regulator. Also 4,589,882 A 5/1986 Urry 5.437,982 A 8, 1995 Catterall provided are cells and membranes comprising a subject light 5.998.588 A 12/1999 Hoffman et al. regulated polypeptide. The present invention further provides 6,376,655 B1 4/2002 Berg et al. methods of modulating protein function, involving use of 7,034,014 B2 4/2006 Hostetler et al. light. The present invention further provides methods of iden 7,420,044 B1 9/2008 Harrington et al. 2001/0053849 A1 12/2001 Kreek et al. tifying agents that modulate protein function. 2002/0001842 A1 1/2002 Chapman 2003. O166840 A1 9/2003 Urry et al. 10 Claims, 34 Drawing Sheets US 9,097,707 B2 Page 2 (56) References Cited Sigma, BES Sodium Salt, 2009, www.sigmaaldrich.com/catalog/ productdetail.do?N4=B2891%7CSIGMA%N5=SEARCH OTHER PUBLICATIONS CONCAT PNO%7CBRAND KEY%F=SPEC, p. 1. Thompson, et al., “Flashy Science: Controlling Neural Function with Heidmann et al., “Fast Kinetic Studies on the Allosteric Interactions Light”, 2005, J. Neurosci., vol. 25, No. 45, pp. 10358-10365. Between Acetylcholine Receptor and Local Anesthetic Binding Sites”, 1979, Eur, J. Biochem., vol. 94, pp. 281-206. Urman, et al., “The Constrained Amino Acid Beta-ACC Confers Kramer, et al. “Photochemical Tools for Remote Control of Ion Potency and Selectivity to Integrin Ligands', 2007, Agnew Chem Int. Channels in Excitable Cells', 2005, Nat. Chem. Biol., vol. 1, No. 7. Ed. Engl., vol. 46.No. 1, pp. 3976-3978. pp. 360-365. Wang, et al., “A Stable Mixed Disulfide Between Thioredoxin Kim, et al., “Synthesis and Characterization of Photochromic Liquid Reductase and its Substrate. Thioredoxin: Preparation and Charac Crystalline Polymer Beads”, 2005, Mol. Cryst. Liq. Cryst., vol. 443, terization', 1996, Biochemistry, vol. 35. No. 15, pp. 4812–4819. pp. 127-135. Wikipedia, "Opioid Receptor', 2010, en.wikipedia.org/wiki/ Lester, et al., “A Covalently Bound Photisomerizable Agonist”, 1980, opioid Receptor, pp. 1-5. J. Gen. Physiol., vol. 75, pp. 207-232. Lien, et al., “Photomodulated Blocking of Gramicidin Ion Chan Wikipedia, "Metabotropic Receptor', 2010, en.wikipedia.org/wiki/ nels', 1996, J. Am. Chem. Soc., vol. 118, pp. 12222-12223. metabotropic receptor, pp. 1-2. O'Leary, et al., “Mutational Analysis of Ligand-Induced Activation Zhu, et al., “Analysis of the Roles of RGD-Binding Integrins, Alpha of the Torpedo Acetylcholine Receptor', 1992, J. Biol. Chem... vol. (4)/Alpha (9) Integrins, and CD9 in the Interaction of the Fertilin Beta 267, No. 12, pp. 8360-8365. (ADAM2) Disintegrin Domain with the Mouse Egg Membrane'. Powl, et al., “Lipid Interactions with Bacterial Channels: Fluores 2002, Biol. Reprod., vol. 66, No. 4, pp. 1193-1202. cence Studies', 2005, Biochem. Soc. Trans., vol. 33, part 5, pp. 905-909. * cited by examiner U.S. Patent Aug. 4, 2015 Sheet 1 of 34 US 9,097,707 B2 U.S. Patent Aug. 4, 2015 Sheet 3 of 34 US 9,097,707 B2 CD C CD C. Cd s (Wod) sue no 09G00909?700709909Z 8090z0 eou edioSqW O Jueng pezieu ON U.S. Patent Aug. 4, 2015 Sheet 4 of 34 US 9,097,707 B2 C 7 6 8 ZZ 500 nm : 5 D 390 nm 4 is 3 2 O1 %2 MA-AZO-QAMAL-AZO-OA Shaker + Shaker FIG. 4 U.S. Patent Aug. 4, 2015 Sheet 5 of 34 US 9,097,707 B2 !--- (O ses S$37N-7Y U.S. Patent Aug. 4, 2015 Sheet 6 of 34 US 9,097,707 B2 Me NH -OOC1)-1 COO 1: (2S,4R)-4-methylglutamate NH, -OOC11 COO 2: (2S,4R)-4-ally glutamate H N-1N O H NH, -OOC? 1 COO 3: Tether model FIG. 6A AZObenzene Maleimide y Fi?O O DYor NsN O O H 4: MAG -OOCN1 COO Agonist 380 rm 500 nm U.S. Patent Aug. 4, 2015 Sheet 7 of 34 US 9,097,707 B2 Cys 439 Me NH -OOC11GOO F.G. 6C U.S. Patent Aug. 4, 2015 Sheet 9 of 34 US 9,097,707 B2 H NH N O a,b SN O'NH.O HN Crs - HN 5: Azodianitine 6 O l s' o-cooH --C o-cooet de - o-cooet f - BOC Boc 7: L-Pyroglutamic acid 8 9. O Cry"N i-k H.N-N C“N O -- one H on-rcoOEt Boc H O O O N O'ri8 H (C.N N O N HOOC1N1-COOHNHHC O 4: MAGHC FIG. 8 U.S. Patent Aug. 4, 2015 Sheet 10 of 34 US 9,097,707 B2 glu 300 uM 500 pA V 20s 6OO e 500 s 400 3OO FIG. 9A tether model (mM) glu 300 uM 3 1 0.3 O. 1 0.01 300 pA 20 s U.S. Patent Aug. 4, 2015 Sheet 11 of 34 US 9,097,707 B2 A B H s O O toN- Cr's MAG o HOOC?n-1 S NH; COO O s Crgy O H O H NH3 Nrn N OOC1\1 COO \ o O nic O H O 3: Tether mode N-NH o H S NH, 2, MAG -OOC-Y-COO F.G. 1 O U.S. Patent Aug. 4, 2015 Sheet 13 of 34 US 9,097,707 B2 A 20 40 60 80 100 120 140 160 time (min) B 200 pA 2 min 380 500 dark 500 FIG. 12 U.S. Patent Aug. 4, 2015 Sheet 14 of 34 US 9,097,707 B2 soon 38O B O Osgu /1BOYO AG O / Yo VL4 CN-2O C glu 300M MAG 100nM MAGOON | AETOURoca CD 380/500rmCoPD 380/500mm D glu glu 300M glu MAG 100nM glu 300M + MAG 100nM 300M noglu 300M s: - - 380/500mm as 38O1500m i I Orrin FIG.
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