GPCRs are the subjects of intense drug discovery efforts. Orthosteric drugs, which target the natural hormone binding site of GPCRs, are the most common therapeutics. However orthosteric drugs often have problems with subtype selectivity, as orthosteric binding pockets are highly conserved among GPCRs of the same subfamily, for examples the nine adrenergic receptors in human. Allosteric drugs are more likely to be subtype selective because they bind to less conserved regions. Recently, many GPCR structures bound with a variety of allosteric modulators have been reported. Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive state. Before the publication of this paper, only one active GPCR bound to a small molecule positive allosteric modulator (PAM) has been reported, namely, the structure of M2 muscarinic acetylcholine receptor bound with LY2119620, which was solved by Brian Kobilka’s research group at Stanford in 2013 (Kruse et al., 2013).
The Beta2 adrenergic receptor (β2AR) belongs to the GPCR family and plays essential roles in cardiovascular and respiratory physiology. Orthosteric agonists for the β2AR are commonly used to treat asthma and chronic obstructive lung disease. In 2017, Prof. Brian Kobilka’s group at Tsinghua University and Prof. Robert Lefkowitz’s group at Duke University jointly reported the first crystal structure of the β2AR bound to an intracellular negative allosteric modulator Cmpd-15(Liu et al., 2017). Recently, Prof. Robert Lefkowitz’s group discovered Cmpd-6 as a positive allosteric modulator for the β2AR through screening DNA encoded libraries (Ahn et al., 2018). Cmpd-6 exhibits robust positive cooperativity with orthosteric agonists and transducers like G protein and arrestin.
Prof. Brian Kobilka’s research group and Prof. Robert Lefkowitz’s research group established a collaboration to determine the complex structure of the active state β2AR bound with Cmpd-6. However the initial trials failed, no electron density was observed for Cmpd-6. The researchers reasoned that it was due to Cmpd-6’s micromolar affinity and poor solubility. Prof. Robert Lefkowitz’s group further optimized the compound by adding a free carboxylic acid group to its terminal amide site (previously used for conjugating its DNA tag) to get an analog (Cmpd-6FA) with better solubility. Dr. Xiangyu Liu and colleagues in Prof. Brian Kobilka’s group found that the compounds were more soluble in solutions with high concentration of PEG400 and successfully crystallized active state β2AR and solved the structure at a resolution of 3.2 Å. Cmpd-6FA bound to a pocket formed by transmembrane (TM) helices 2, 3, 4 and intracellular loop 2 (ICL2). (Figure 1).
Ahn, S., Pani, B., Kahsai, A.W., Olsen, E.K., Husemoen, G., Vestergaard, M., Jin, L., Zhao, S., Wingler, L.M., Rambarat, P.K., et al. (2018). Small-Molecule Positive Allosteric Modulators of the beta2-Adrenoceptor Isolated from DNA-Encoded Libraries. Mol Pharmacol 94, 850-861.
Kruse, A.C., Ring, A.M., Manglik, A., Hu, J., Hu, K., Eitel, K., Hubner, H., Pardon, E., Valant, C., Sexton, P.M., et al. (2013). Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101-106.
Liu, X., Ahn, S., Kahsai, A.W., Meng, K.C., Latorraca, N.R., Pani, B., Venkatakrishnan, A.J., Masoudi, A., Weis, W.I., Dror, R.O., et al. (2017). Mechanism of intracellular allosteric beta2AR antagonist revealed by X-ray crystal structure. Nature 548, 480-484.
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