Evan O'Brien

All Publications

• A µ-opioid receptor modulator that works cooperatively with naloxone. Nature O'Brien, E. S., Rangari, V. A., El Daibani, A., Eans, S. O., Hammond, H. R., White, E., Wang, H., Shiimura, Y., Krishna Kumar, K., Jiang, Q., Appourchaux, K., Huang, W., Zhang, C., Kennedy, B. J., Mathiesen, J. M., Che, T., McLaughlin, J. P., Majumdar, S., Kobilka, B. K. 2024

  Abstract

  The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.

  View details for DOI 10.1038/s41586-024-07587-7

  View details for PubMedID 38961287

  View details for PubMedCentralID 5689219

• Author Correction: Stepwise activation of a metabotropic glutamate receptor. Nature Krishna Kumar, K., Wang, H., Habrian, C., Latorraca, N. R., Xu, J., O'Brien, E. S., Zhang, C., Montabana, E., Koehl, A., Marqusee, S., Isacoff, E. Y., Kobilka, B. K. 2024

  View details for DOI 10.1038/s41586-024-07470-5

  View details for PubMedID 38702522

• Stepwise activation of a metabotropic glutamate receptor. Nature Krishna Kumar, K., Wang, H., Habrian, C., Latorraca, N. R., Xu, J., O'Brien, E. S., Zhang, C., Montabana, E., Koehl, A., Marqusee, S., Isacoff, E. Y., Kobilka, B. K. 2024

  Abstract

  Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

  View details for DOI 10.1038/s41586-024-07327-x

  View details for PubMedID 38632403

  View details for PubMedCentralID 6709600

• Ligand efficacy modulates conformational dynamics of the -opioid receptor. Nature Zhao, J., Elgeti, M., O'Brien, E. S., Sar, C. P., Ei Daibani, A., Heng, J., Sun, X., White, E., Che, T., Hubbell, W. L., Kobilka, B. K., Chen, C. 2024

  Abstract

  The -opioid receptor (OR) is an important target for pain management1 and molecular understanding of drug action on OR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of OR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of beta-arrestin-1 with the muOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.

  View details for DOI 10.1038/s41586-024-07295-2

  View details for PubMedID 38600384

• Large library docking for cannabinoid-1 receptor agonists with reduced side effects. bioRxiv : the preprint server for biology Tummino, T. A., Iliopoulos-Tsoutsouvas, C., Braz, J. M., O'Brien, E. S., Stein, R. M., Craik, V., Tran, N. K., Ganapathy, S., Liu, F., Shiimura, Y., Tong, F., Ho, T. C., Radchenko, D. S., Moroz, Y. S., Rosado, S. R., Bhardwaj, K., Benitez, J., Liu, Y., Kandasamy, H., Normand, C., Semache, M., Sabbagh, L., Glenn, I., Irwin, J. J., Kumar, K. K., Makriyannis, A., Basbaum, A. I., Shoichet, B. K. 2024

  Abstract

  Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 muM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

  View details for DOI 10.1101/2023.02.27.530254

  View details for PubMedID 38328157

• Step-wise activation of a Family C GPCR. bioRxiv : the preprint server for biology Kumar, K. K., Wang, H., Habrian, C., Latorraca, N. R., Xu, J., O'Brien, E. S., Zhang, C., Montabana, E., Koehl, A., Marqusee, S., Isacoff, E. Y., Kobilka, B. K. 2023

  Abstract

  Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

  View details for DOI 10.1101/2023.08.29.555158

  View details for PubMedID 37693614

  View details for PubMedCentralID PMC10491200

• Conformational dynamics of the μ-opioid receptor determine ligand intrinsic efficacy. bioRxiv : the preprint server for biology Zhao, J., Elgeti, M., O'Brien, E. S., Sár, C. P., Ei Daibani, A., Heng, J., Sun, X., Che, T., Hubbell, W. L., Kobilka, B. K., Chen, C. 2023

  Abstract

  The μ-opioid receptor (μOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the μOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi.

  View details for DOI 10.1101/2023.04.28.538657

  View details for PubMedID 37163120

  View details for PubMedCentralID PMC10168371

• Negative allosteric modulation of the glucagon receptor by RAMP2. Cell Krishna Kumar, K., O'Brien, E. S., Habrian, C. H., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. 2023; 186 (7): 1465-1477.e18

  Abstract

  Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

  View details for DOI 10.1016/j.cell.2023.02.028

  View details for PubMedID 37001505

• Negative allosteric modulation of the glucagon receptor by RAMP2 O'Brien, E. S., Kumar, K., Habrian, C., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. CELL PRESS. 2023: 161A

  View details for Web of Science ID 000989629700786

• Negative allosteric modulation of the glucagon receptor by RAMP2. Biophysical journal O'Brien, E. S., Krishna Kumar, K., Habrian, C., Latorraca, N. R., Wang, H., Tuneew, I., Montabana, E., Marqusee, S., Hilger, D., Isacoff, E. Y., Mathiesen, J. M., Kobilka, B. K. 2023; 122 (3S1): 161a

  View details for DOI 10.1016/j.bpj.2022.11.1023

  View details for PubMedID 36782752

• Structural insights into differences in G protein activation by family A and family B GPCRs. Science (New York, N.Y.) Hilger, D. n., Kumar, K. K., Hu, H. n., Pedersen, M. F., O'Brien, E. S., Giehm, L. n., Jennings, C. n., Eskici, G. n., Inoue, A. n., Lerch, M. n., Mathiesen, J. M., Skiniotis, G. n., Kobilka, B. K. 2020; 369 (6503)

  Abstract

  Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play important roles in carbohydrate metabolism. Recent structures of family B GPCR-Gs protein complexes reveal a disruption in the α-helix of transmembrane segment 6 (TM6) not observed in family A GPCRs. To investigate the functional impact of this structural difference, we compared the structure and function of the glucagon receptor (GCGR; family B) with the β2 adrenergic receptor (β2AR; family A). We determined the structure of the GCGR-Gs complex by means of cryo-electron microscopy at 3.1-angstrom resolution. This structure shows the distinct break in TM6. Guanosine triphosphate (GTP) turnover, guanosine diphosphate release, GTP binding, and G protein dissociation studies revealed much slower rates for G protein activation by the GCGR compared with the β2AR. Fluorescence and double electron-electron resonance studies suggest that this difference is due to the inability of agonist alone to induce a detectable outward movement of the cytoplasmic end of TM6.

  View details for DOI 10.1126/science.aba3373

  View details for PubMedID 32732395

• Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy. Angewandte Chemie (International ed. in English) O'Brien, E. S., Fuglestad, B. n., Lessen, H. J., Stetz, M. A., Lin, D. W., Marques, B. S., Gupta, K. n., Fleming, K. G., Wand, J. J. 2020

  Abstract

  For a variety of reasons, the internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side chain dynamics of the 7-transmembrane helix protein sensory rhodopsin II and the beta-barrel bacterial outer membrane channel protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Though of quite different topologies, both proteins are found to have a similar and striking distribution of methyl-bearing amino acid side chain motion that is independent of membrane mimetic. The methyl-bearing side chains of both proteins are, on average, more dynamic in the ps-ns time regime than any soluble protein characterized to date. Approximately one third of methyl-bearing side chains in both proteins exhibit extreme rotameric averaging on this timescale. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect that normally stabilizes the folded state of water-soluble proteins. Furthermore, the large reservoir of conformational entropy that is observed provides the potential to greatly influence the thermodynamics underlying a plethora of membrane protein functions including ligand binding, allostery and signaling.

  View details for DOI 10.1002/anie.202003527

  View details for PubMedID 32277554