Pharmtastic

I am going to reproduce some of the term papers that I wrote for the spring semester in school. Hopefully I can pull myself back to writing more posts this year especially over the summer now that I have some breathing space from school.

(PS. Unfortunately, I could not post any of the images/figures I used in the actual paper here for obvious reasons. If you see any mention of them in the text just ignore them and I am hoping that the content still makes sense to you guys.)

Abstract:

Gamma amino butyric acid (GABA) is one of the major inhibitory neurotransmitters found in the mammalian central nervous system. Until recently, GABA was known to function by binding to a ligand-gated chloride ion channel known as GABA A receptors, thus producing its inhibitory effects. GABA B receptors are newly discovered metabotropic subtype of GABA receptors and belong to the Class C family of G-protein coupled receptors (GPCRs). There are the first and only GPCRs that have been identified to show obligate oligomerization. They have been identified to modulate the release of a number of key neurotransmitters in the CNS by indirectly activating ion-channels and therefore are considered to be a key member in a diverse set of physiological pathways. Therefore, understanding these proteins could help in the on-going drug discovery efforts to treat conditions such as pathological pain, muscle spasms, drug craving, anxiety, schizophrenia, depression and autism.

Furthermore, investigating the structural and functional aspects of these proteins can reveal answers to some of the fundamental questions of GPCR oligomerization. They can provide an insight into the integral molecular mechanisms involved in neurotransmitter recognition and signal transduction in receptor oligomers and can unravel the unexpected level of pharmacological diversity. In this review, I will address the key structural features and molecular mechanisms that make these receptors unique and how the knowledge gained from studying these receptors can be generalized to all other G-protein coupled receptors.

Introduction:

G-protein coupled receptor (GPCR) superfamily comprises of more than 800 protein and represent the largest family of integral membrane proteins in the human genome¹. They are characterized by 7 transmembrane-spanning helices (H1-H7), which are interconnected by 3 extracellular loops (ECL1-3) and 3 intracellular loops (ICL1-3). Based on a comprehensive phylogenetic analysis, GRAFS classification categorizes the mammalian GPCR repertoire into five main families; Glutamate (Class C), Rhodopsin (Class A), Adhesion (Class B), Frizzled (Class F), and Secretin (Class B).

GPCRs mediate a large number of vital cellular responses and therefore are of extremely importance as a drug target and for translational research. Currently about 50% of all small molecule drugs approved by the FDA target these receptors and are one of the most targeted families of proteins in the human body³. They are therefore of enormous pharmacotherapeutic importanceand it is vital that we better understand these complex proteins and how they function in order to develop better pharmacological agents.

Histroy of GPCR oligomerization:

Traditionally, the function of ligand binding and signal transduction was assumed to be through monomeric receptor units and through interactions with a single guanine binding nucleotide protein once activated by the ligand. But in 1978-82, Dr. Nigel Birdsall from the National Institute of Medical research, London raised the earliest questions about receptor-receptor interactions. His hypothesis that receptors interact with each other was based on his observations in muscarinic receptor membrane preparations when treated with beta-adrenergic receptor ligands⁴. Seeman and collaborators further confirmed the existence of dopamine receptor dimers by positron emission tomography (PET) in human brain⁵. Other biochemical studies, such as photo-affinity labeling assays of the muscarinic receptor, and radiation quenching studies of beta-2 adrenergic, gonadotropin and adenosine A1 receptors further provided evidence of the existence of GPCR oligomers⁶. The crosslinking of the glucagon receptors²², and hydrodynamic properties of cardiac muscarinic receptors,further supported this idea. But unfortunately, these early studies did not gain much interest from the scientific community.

In 1993, Dr. Wess et al. confirmed the intermolecular interactions between muscarinic receptors using chimeric muscarinic/adrenergic receptors overexpressed in CHO cells⁷. They observed that the chimeric receptors were either not expressed alone or showed no ligand binding and functional activity. But when the two chimeras were co-expressed, they exhibited ligand binding and functional signaling to both muscarinic and adrenergic ligands. They also proposed a hypothetical model for receptor dimerization, which resulted in much excitement in the field of receptor pharmacology. For the first time, the importance of oligomerization in ligand affinity and functional activity was being discussed. Until then, only the hetero-dimerization of two nonfunctional (Chimeric) receptors had been confirmed. But in 1999, Dr. Lakshmi Devi and collaborators identified the presence of hetero dimers of two fully functional opioid receptors, κ and δ. They also characterized the changes in their ligand affinities and observed the presence of receptor synergism⁸.

Over the past two decades, tremendous research has been done on GPCR dimerization and one of the most well studied receptors is the GABA receptor. These studies have been able to paint a vivid picture of the interactions between receptors and their implications in their activity and function. In this critical review, I plan to address the progress made in the field of GPCR oligomerization using GABA receptor as the template.

GABA receptor:

            Gamma amino butyric acid (GABA), an inhibitory neurotransmitter found mostly in the central nervous system (CNS), binds to the membrane proteins known as GABA receptors. Over the years, a number of different classes of GABA receptors have been identified and are currently broadly classified into three groups – GABA A, GABA B and GABA C receptors. GABA A and C receptors are ionotropic receptors, whereas the GABA B receptors are of the metabotropic nature.

GABA A and C receptors are chloride-ion channels and each receptor is composed of 5 hetero-oligomeric subunits that are arranged into a cylindrical channel with the central pore of chloride ion entry¹⁰. Each subunit is made up of 4 transmembrane domains and is folded such that the 5 2nd transmembrane domains form the wall of the ion pore. The GABA binding domain has been identified at the outer surface of the receptor. The allosteric binding sites for drugs such as Benzodiazepines and Barbiturates that mediate through the GABA receptors have also been identified at the interface between subunits. To date, there are 16 human GABA A receptor subunits (α1-6, β1-4, γ1-4, δ, ε) and two human GABA C receptor subunits (ρ1 and ρ2) have been identified⁹. 

            GABA B receptors are seven transmembrane metabotropic G-protein coupled receptors and belong to the family C GPCR. Family C GPCRs are characterized by the unusually long N-terminal loop of transmembrane domain connected with with a large Venus Flytrap domain (VFT) through a Cysteine rich domain (CRD)¹⁰. GABA B receptors mediate the second messenger systems - phospholipase C and adenylate cyclase, through the guanine-nucleotide binding proteins (G-proteins) and thus activate K⁺ and Ca²⁺ ion channels. Unlike the GABA A receptors that produce rapid inhibitory signals through chloride influx through the ion channel, the GABA B receptors produce slow, prolonged inhibitory signals. They are localized at both the pre-synaptic and post-synaptic neurons and function to modulate the release of neurotransmitters and key post-synaptic cell signals respectively⁹.

Dimerization of GABA B receptor:

To date, three subtypes of GABA B receptors have been cloned and are termed as GABA B1a, GABA B1b and GABA B2. The GABA B receptors resemble the metabotropic glutamate receptors (mGluR) and were found to be functionally active only as heterodimers of either GABA B1a or GABA B1b subunit when combined with the GABA B2 subunit. It has ben identified that GABA_B1 contains the GABA binding site¹¹, while GABA_B2 is responsible for G_i/o protein activation^12.

Due to the close sequence homology among the Class C GPCR family of receptors, a number of homology models have been constructed for the GABA B receptor based on the published crystal structure of the mGluR1. The structure as shown below consists of two key domains – Venous Flytrap domain and Heptahelical transmembrane domain similar to the other Class C GPCRs. Interestingly, unlike the other Class C GPCRs, the Cysteine-rich domain connecting these two domains is absent in GABA B receptors.

The Venous Flytrap domain (VFT) is large hydrophobic bi-lobed structure found extending from the surface of the cell membrane. The ligand has been identified to be binding at the cleft between the two lobes of the domain and when bound it stabilizes its closed structure. It has also been observed that the lobe 1 of each monomer interacts with each other and form constitutive dimers¹³. It has also been shown that the absence of this interaction results in the loss of activity of the GABA B receptor¹⁴. Mutational studies have shown that the key residues in lobe 1 involved in Agonist binding are Ser246 and Asp 471. The key residues for antagonist binding are Ser246, Ser265, Tyr266, Phe463 and Tyr470. Although most of the key residues for ligand binding are mostly present in the lobe 1 some residues such as the Tyr366 in lobe 2 are also considered important. Most of these residues are absent in the VFT domain of the GABA B2 and therefore this monomer is not involved in ligand binding¹⁵.

The Heptahelical transmembrane domain (HTD), similar to other GPCRs, consists of 7 alpha helices that are linked by 3 intracellular loops (ICL 1-3) and 3 extracellular loops (ECL 1-3), which are less than 30 residues in length¹⁶. The two C-termini are considered to be in the form of a coiled-coil and are not involved in signaling but involved in receptor trafficking and also in holding the two monomers together¹⁷. The Class 3 GPCRs have a low sequence homology to the Class A GPCRs whose 3D structural features have been well studied. Of the 19 conserved residues in Class C receptors, only 7 are found in the rhodopsin-family of GPCRs. But there still a few key residues and interactions similar to the ones seen in rhodopsin-like receptors. GABA B1 receptor has a PKxR and GABA B2 has a PKxI motif similar to the NPxxP motif in the TM7 of Family A receptors¹⁸. The disulfide bond that connects the top of TM3 with the ECL2 is also conserved in Family C receptors¹⁶. The highly conserved DRY region in Family A receptors is present only in GABA B2 receptor which may explain why only the B2 monomer is involved in G-protein signilaing¹⁸. Also the C-terminal end forms an 8th helix that is related to G-protein coupling similar to the Class A family of GPCRs. But one of the key differences between the GABA B receptor and Rhodopsin-like GPCRs is the absence of ligand binding at the extracellular domains of the seven transmembrane helices. However, a conserved binding pocket has still been identified and could be of potential use to develop allosteric drugs targeting this site to bind and modulate receptor activity¹⁹.

Mechanism of activation of GABA B dimers:

            The ligand binding at the VFT cleft is followed by a series of conformational changes that are translated to the intracellular surface from the VFT through the heptahelical tansmenbrane domain resulting in the activation of the G-protein. The exact mechanism of the transduction of these signals from the extracellular domains to the G-protein is still unclear. But the mGluR1 crystal structure provides us some insight into what must be the mechanism.

            It has identified that the receptor dimer in the resting state exists such that both the lobe 2 VFT domains are far away from each other. Once bound by the ligand at the cleft, the lobes are stabilized into a closed conformation and move to the active state. In this state, the lobe 2 of the GABA B1 VFT domain move closer and make contact with the lobe 2 of the GABA B2 VFT domain¹⁹. Rondard et al. confirmed this that by observing a decrease in receptor activity in response to orthosteric ligands, when a N-glycan wedge was introduced to the lobe 2¹⁴. It can be hypothesized that signal is translated from the VFT domains to the transmembrane domains in a direct interaction as the GABA B receptors lack the cysteine-rich domain. Owing to the lack of a crystal structure of the membrane-bound domain, the exact mechanism of signal transduction across the membrane to the G-protein is still a mystery. But based on the phylogenetic studies, it can be predicted that similar to the Class A GPCRs, the GABA B transmembrane domains show conformational changes in the helices 3, 5 and 6 and thus causing changes in the interactions with the G-protein at the ICL 2 and 3.

            Apart from the orthosteric binding pocket in the cleft of the VFT domain, a number of allosteric sites have also been identified and are considered to be of interest for drug discovery and development¹⁹. Currently only one drug – Baclofen, an anti-spasmodic agent is known to interact at the orthosteric site at GABA B1. It is a GABA derivative and currently in clinical trials for the indication of alcoholism treatment. Recently, a positive allosteric modulator - CGP7930 has been discovered to increase both potency and efficacy of GABA. It has been shown to bind to the 7-transmembrane domain and enhance the binding to the Go protein. A model has been proposed that the binding of the agonist at the VFT site causes a increase in the size of the cleft between the two transmembrane domain and CGP7930 is expected to further widen the cleft by binding between the 2 heptahelical domain²⁰. Other positive allosteric inhibitors that have been discovered and synthesized are GS39783 and analogs of rac-BHFF²¹. Unfortunately, none of these ligands have been clinical viable due to their low potency and unfavorable pharmacokinetics.

Role of dimerization in GABA-B function:

The GABA B receptor is the first GPCR identified to be an obligatory heterodimer composed of two subunits, GABA B1 and GABA B2. The role of GABA-B receptor in the central nervous system synaptic transmission has been well studied over the past two decades. The activated GABA-B receptors have been identified to reduce transmission at excitatory and inhibitory synapses, as a result of an increase in K+ conductance through adenylyl cyclase or through inwardly rectifying potassium channels (GIRKs), or decrease the voltage-dependent Ca2+ currents. The GB1 monomer binds to the ligand and GB2 monomer has been identified to interact with the G-protein and does not bind to a ligand. In general, this system can therefore be considered as an excellent model to study the functional significance of GPCR dimerization.

Dr. Jean-Philippe Pin’s group has demonstrated that GABA B receptors can also exist as stable tetramers²³. They also established that the interaction between heterodimers to form tetramers does not result just from overexpression in in-vivo cell cultures and that they exist as tetramers even in the human brain. They further showed that the tetramers exhibited a lower G-protein coupling efficacy than the heterodimers produced by mutations at the contact area. GABA B receptor heterodimer GB1–GB2 could be compared with a monomeric class A receptors in that higher oligomers show reduced G-protein efficacy. But unlike the Class A receptors where this phenomenon was due the steric forces not allowing the interaction of two G-proteins at once, in case of GABA-B receptor it is hypothesized that the it is due to the fact that only one of the dimer in each tetramer can reach the active conformation at one instance.

The exact physiological significance for this inclination to form tetramers despite being less efficient than dimers is unclear. But we could speculate that the higher oligomer formation may be a receptor storage mechanism, as in the case of Rhodopsin and that they form dimers when needed for activation. Also tetramers provide better stability during receptor translocation, desensitization/internalization and turnover. They could also provide a method to alter the strength of signaling by segregating the pre-synaptic and post-synaptic receptors accordingly²³.

Clinical implications of GABA-B dimierization:

            GABA B receptors have been observed in high density in the thalamic nuclei, the molecular layer of the cerebellum, the cerebral cortex, the interpeduncular nucleus, and the dorsal horn of the spinal cord²⁴. Other studies have shown that GABA B1a receptors are also present in peripheral tissues such the adrenals, pituitary, spleen, and prostate, whereas GABA B1b receptors have been found in the rat kidney and liver. Both the pre- and post-synaptic receptors influence the release and effects of a number of key neurotransmitters such as dopamine, nor-adrenaline, adenosine, glycine, glutamate and serotonin. Thus, the GABA_B receptor agonists display a number of pharmacological effects, including anti-spasticity, antitussive action, bronchiolar relaxation, alteration of micturition, gastrointestinal motility, epileptogenesis, cognition, gastric acid secretion, and hormone release, drug craving suppression, anti-nociception, induction of hypotension and brown fat thermogenesis²⁵. Therefore, there is tremendous potential for developing drugs that could help treat a number of pathological conditions.          

            Furthermore, obligatory oligomerization of receptors opens new avenues to drug discovery. A number of new techniques such as dimer ligands, or ligands disrupting or enhancing oligomerization have become popular among pharmaceutical and biotech companies²⁶. STX209, is an oral selective GABA-B receptor agonist developed by Seaside Therapeutics, Inc. is currently being studied in a Phase 2b study in autism spectrum disorders and two Phase 3 studies in fragile X syndrome²⁷.

Conclusion:

GABA B receptor is unique in that it is the only Family C GPCR known to show obligatory dimerization and signaling through transactivation. This makes GABA B receptor an ideal model for studying GPCR oligomerization. In recent years, allosteric modulation has become an attractive approach and theoretically can overcome some undesirable side effects, tolerance and provide subtype selectivity easily unlike orthosteric ligands. Apart from the orthosteric site at the VFT domain, a large number of binding pockets have been identified on these receptors, which makes it an attractive target for allosteric drugs. A number of drug discovery projects focusing on the development of allosteric modulators, targeting Class C receptors, are currently underway both in the pharmaceutical industry and academia. Until now, very few drugs have been successful in clinical studies and it calls for further investigation of the precise structural dynamics and allosteric modulation mechanisms. Further studies using advanced functional assays, such as BRET (bioluminescence resonance energy transfer) or FRET (fluorescence resonance energy transfer) could reveal details about conformational changes during dimer or oligomer formation. Understanding the mechanisms of dimerization could be important in the development and screening of drugs that act through not only Class C receptors but also give vital information about all GPCR dimers. The changes in ligand-binding and signaling properties due to hetero-dimerization could also open doors to novel pharmacological interventions.

References:

1. Wilson, S. et al. Orphan G‐protein‐coupled receptors: the next generation of drug targets? British Journal of Pharmacology 125, 1387–1392 (1998).
   
2. Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63: 1256–1272.

3. Overington, J. P., Al-Lazikani, B. & Hopkins, A. L. How many drug targets are there? Nature Reviews Drug Discovery 5, 993 (2006).
4. Birdsall, N. J. M. Can different receptors interact directly with each other? Trends in Neurosciences 5, 137–138 (1982).
5. Seeman, P. et al. The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography. European Journal of Pharmacology: Molecular Pharmacology 227, 139–146 (1992).
6. González-Maeso, J. GPCR oligomers in pharmacology and signaling. Molecular Brain 4, 20 (2011).
7. Maggio, R., Vogel, Z. & Wess, J. Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular ‘cross-talk’ between G-protein-linked receptors. Proc. Natl. Acad. Sci. U.S.A. 90, 3103–3107 (1993).
8. Jordan, B. A. & Devi, L. A. G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399, 697–700 (1999).
9. Chebib, M. & Johnston, G. A. The ‘ABC’ of GABA receptors: a brief review. Clin. Exp. Pharmacol. Physiol. 26, 937–940 (1999).
10. Bräuner-Osborne, H., Wellendorph, P. & Jensen, A. A. Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors. Curr Drug Targets 8, 169–184 (2007).
11. Galvez, T. et al. Mutagenesis and Modeling of the GABAB Receptor Extracellular Domain Support a Venus Flytrap Mechanism for Ligand Binding. J. Biol. Chem. 274, 13362–13369 (1999).
12. Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. Function of GB1 and GB2 Subunits in G Protein Coupling of GABAB Receptors. PNAS 98, 14649–14654 (2001).
13. Liu, J. et al. Molecular Determinants Involved in the Allosteric Control of Agonist Affinity in the GABAB Receptor by the GABAB2 Subunit. J. Biol. Chem. 279, 15824–15830 (2004).
14. Rondard, P. et al. Functioning of the dimeric GABAB receptor extracellular domain revealed by glycan wedge scanning. The EMBO Journal 27, 1321–1332 (2008).
15. Bowery, N. G. Historical Perspective and Emergence of the GABAB Receptor. GABABReceptor Pharmacology A Tribute to Norman Bowery Volume 58, 1–18 (2010).
16. Pin, J.-P., Galvez, T. & Prézeau, L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacology & Therapeutics 98, 325–354 (2003).
17. Calver, A. R. et al. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling. J. Neurosci. 21, 1203–1210 (2001).
18. Thomas Blackburn GABAb Receptor Pharmacology: A Tribute to Norman Bowery. 
19. Chun, L., Zhang, W. & Liu, J. Structure and ligand recognition of class C GPCRs. Acta Pharmacologica Sinica 33, 312–323 (2012).
20. Urwyler, S. et al. Positive allosteric modulation of native and recombinant gamma-aminobutyric acid(B) receptors by 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501. Mol. Pharmacol. 60, 963–971 (2001).
21. Malherbe, P. et al. Characterization of (R,S)‐5,7‐di‐tert‐butyl‐3‐hydroxy‐3‐trifluoromethyl‐3H‐benzofuran‐2‐one as a positive allosteric modulator of GABAB receptors. British Journal of Pharmacology 154, 797–811 (2008).
22. Herberg, J. T., Codina, J., Rich, K. A., Rojas, F. J. & Iyengar, R. The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor. J. Biol. Chem. 259, 9285–9294 (1984).
23. Comps-Agrar, L. et al. The oligomeric state sets GABAB receptor signalling efficacy. The EMBO Journal 30, 2336–2349 (2011).
24. Bettler, B. & Tiao, J. Y.-H. Molecular diversity, trafficking and subcellular localization of GABAB receptors. Pharmacology & Therapeutics 110, 533–543 (2006).
25. Bowery, N. GABAB receptors and their significance in mammalian pharmacology. Trends in Pharmacological Sciences 10, 401–407 (1989).
26. George, S. R., O’Dowd, B. F. & Lee, S. P. G-Protein-coupled receptor oligomerization and its potential for drug discovery. Nature Reviews Drug Discovery 1, 808 (2002).
27. Seaside Therapeutics Announces Issuance of Key Patent for Lead Autism Candidate STX209. MarketWatch at <http://www.marketwatch.com/story/seaside-therapeutics-announces-issuance-of-key-patent-for-lead-autism-candidate-stx209-2012-03-27>

Time to rehash my fall term paper for the Behavioral Pharmacology course. -

Abstract: Our understanding of the neurophysiological and psychological mechanisms of placebo analgesic effects has expanded considerably over the last 20 years owing to the developments in brain mapping and imaging techniques. We have now identified a number of neural circuits in the brain involved in the modulation of pain perception based on emotional and cognitive factors. Numerous studies have shed light on the role of specific receptors and neurotransmitters involved in these circuits and how they regulate each other in different areas of the brain resulting in modulation of placebo analgesia. We also now understand the importance of environmental factors, learning, memory, emotional state, gender, personality traits, pre-notions and non-specific factors in pain interpretation. The identification of the clinical implications of placebo-nocebo effects will enable us to design better clinical studies and provide absolute data for new analgesic drugs. It is important to understand the ethical consequences of implementing psychosocial strategies based on placebo effects in conjunction with traditional treatments to improve pharmacotherapy.  Further studies in this field could provide new treatment strategies in disease conditions like chronic idiopathic pain, Parkinson’s disease and Alzheimer’s disease.

Introduction – What is Pain?
The origin and mechanism of pain has fascinated various philosophers and scientists over a number of centuries. Charles Darwin described pain as a ‘homeostatic emotion,’ which is essential for the survival of a species1. Over the years, our understanding of this phenomenon has improved remarkably. Today, the International Association for the Study of Pain (IASP) defines Pain as – “An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”2. From the evolutionary and behavioral stand point, pain reflex is a part of the body’s defense mechanism that alerts the individual from harmful stimuli and prevents from reinjuring a wound.

The general mechanism of pain reflex has been well studied over the years. Noxious stimuli of various modalities are sensed by a specialized set of nerve fibers (unmyelinated C fibers and thinly myelinated A fibers). These nerve fibers with the help of Purinergic channels and receptors convert the physiochemical stimuli into electrical signals (Action potential). These sensory inputs are then integrated at the spinal dorsal horn and transmitted to specific areas of the brain through numerous neural pathways. The thalamus and limbic areas are believed to mediate the emotional and aversive components of pain. The cortex is identified to perceive the pain and accordingly relay back motor signals through the efferent nerve fibers to the spinal cord, which enables the withdrawal from the noxious stimuli. This general mechanism may not be true in case of chronic pain, which is usually idiopathic and serves no clear biological purpose.

Molecular physiology of Nociception:
The most important advances in pain and analgesia research have come through our understanding of basic molecular neurophysiology involving specific endogenous neurotransmitters and receptors. The peripheral sensory network mainly involves glutamate acting on ionotropic and metabotropic glutamate receptors, Substance P acting on TrkA tyrosine kinase receptors and Lectin analogues acting on P2X3 receptors1. The phosphorylation of Ionotropic glutamate receptors (NMDA and AMPA receptors) lead to biophysical changes and results in central sensitization, which is characterized by hyperalgesia, allodynia and chronic pain. This central sensitization can also be modulated by the metabotropic glutamate receptors and also through dis-inhibition of GABAergic signals3. Cannabinoid receptors have also been discovered to show anti-nociception action by inhibiting signals from periphery, through their interaction with opioid receptors and PAG, a neural analgesic substrate. Numerous other receptors and proteins are also currently being discovered that have implications in pain signal relay along the spinal chord to the brain.

The nociceptive processing in the supra-spinal sites is mainly carried out at the thalamic relay nuclei. They play a key role in pain modulation and further relay of signals to the cerebral cortex. It has been discovered that few descending inhibitory systems combine together at the brainstem rostral ventro-medial medulla (RVM) and modulate the spinal transmission1. These modulatory signals are controlled through the endogenous opioid system and are of evolutionary significance as they enable the organism to ignore pain in critical situations of stress and recovery. This relay system between the frontal cortex, limbic systems and thalamus enables cognitive and emotional control respectively over nociception and therefore plays a major part in placebo-based analgesia4,5.

Placebo and Nocebo effects:
Placebo effects are the psychobiological effects in the brain and/or body that occur following an inert treatment or procedure that has no direct pharmacological actions6. When these observed effects are harmful or undesirable they are termed as Nocebo effects. Numerous studies have confirmed that modulation of pain perception can be controlled by ‘expectation’ alone and that both placebo and nocebo effects can change when pre-conditioned6,7. Brain imaging and radiotracer studies have identified that all subtypes (μ, κ, and δ) of Opioid receptors are involved in placebo analgesic effects and that Cholecystokinin (CCK) system opposes this effect (therefore are usually involved in nocebo effects)7. These opioid-based effects were primarily discovered in specific brain regions - prefrontal cortex, Pre-aquaductal grey area and amygdala1.

Other systems have also been identified to be involved in placebo analgesia. Nucleus accumbens is a major area of the brain that is involved in placebo analgesia and features numerous dopamine neurons. It is considered that Dopaminergic responses to placebo interventions interact with downstream opioid signaling8. Another study has identified that central Serotonergic and Noradrenergic neurons also modulate the descending pain modulatory circuitry that mediates analgesia induced by opioids, which explains the analgesic effects of certain tricyclic antidepressants9.

Behavioral and Clinical Implications of placebo analgesia:
It has been confirmed that placebos have better effect than ‘no treatment’, in case of subjective continuous outcomes and for treatment of pain10.  We now understand that positive and negative expectations can be used to manipulate pain perception and thereby behavior11. It has been observed that under the expectation of high pain, the anxiety mechanisms are stimulated in the hippocampus and brain stem regions and thereby stimulating CCK systems resulting in nocebo effects12. Also both cognitive and emotional systems are involved in the modulation of pain perception through various mechanisms such as learning, memory, reward, beliefs, personality, psychological traits etc. The most well studied mechanisms involved in placebo analgesia are expectancy and conditioning. Pavlovnian conditioning which involves pairing an unconditioned stimulus (drug) with a conditioned stimulus (shape, color, flavor, taste, of the pill or route of administration of the drug) leads to a conditioned response (analgesia), even when only the conditioned stimulus is administered. The magnitude of the effect depends on the duration of the acquisition phase and the effect of the conditioning procedure13.

Reward expectation has also been shown to effect placebo analgesia perception through dopaminergic mechanisms14. This could have significant implications in clinical trial patient selection. The expectation created based on past experience and learned memory also influence perception of drug effect and is called cognitive reappraisal. This explains why red placebo pills are more likely to act as stimulants compared with blue placebo pills as red is associated with ‘danger’ or ‘hot’. Also more expensive placebo treatments produce higher analgesic effects than less expensive ones15. Personality traits also influence placebo analgesic effects through dopaminergic pathways and using this data, patients can be selected based on expected placebo analgesia16.

The influence of the study design, verbal suggestions and other environmental factors can indirectly affect the perception of pain due to placebo analgesia. It has been identified that the pharmacodynamics and efficacy of the drug can also be altered based on Expectation18. In a ‘hidden-open’ placebo-nocebo intervention study, patients perceived pain relief only after informing them that the drug infusion had began even though they had already begun the administered. Similarly, the patients stopped perceiving analgesia upon informing them that drug was stopped even though it wasn’t and the neuroimaging data confirmed this effect. The placebo/nocebo effect due to the Expectation was able to overcome the actual pharmacodynamics effect of the drug.18 

Placebo analgesia study models:
While designing studies for placebo-analgesia, the confounding factors such as natural history, personal biases and co-interventions should be taken into consideration and minimized so that the absolute pharmacological effect of the drug can be determined. Most analgesia studies use standardized pain intensity rating systems to obtained psychological details from patients directly. The common designs used are - Balanced placebo design, Double blind versus deceptive design, Open-hidden paradigm and Brain mapping using PET, fMRI and ECG17.

Balanced placebo design involves the comparison of the responses in patients to the informed amount of drug with the informed amount of placebo and between the actual doses of drug and actual doses of placebo. This design enables the study of the effects of verbal suggestions on placebo analgesia.

Double blind versus deceptive design compares the therapeutic outcome of a double-blind administration of an active drug with a deceptive one. The option of getting either a placebo or drug, during the double blind study, results in less placebo analgesia than when administering only the placebo, but disguised as a potent drug, during the ‘deception’ study.

Open-hidden paradigm is used to identify the placebo effect in context to specific instructions. The administration of active drug is hidden from patient initially and changes in the analgesia perception once informed are studies. Similarly, the cessation of the administration of the drug is hidden and informed later to observe changes in perception. This design has been used in studying placebo analgesia in case of anxiety, Alzheimer’s disease and Parkinson’s disease.

Brain mapping techniques enable the identification of the brain area and circuits involved in placebo analgesia under different physiological and pathological conditions. There are some drawbacks of using brain mapping techniques in that the brain patterns that predict placebo analgesia might differ among individuals as this does not take into account the affective information (Information on which aspects of pain that were actually judged by the patient). Data obtained from Pain intensity ratings includes this factor and therefore may differ from conclusions obtained from brain images.

Importance of placebo analgesia:
Most analgesic drugs show different efficacy and potency in different patients due to variability in the patients’ prior beliefs, personality traits, other environmental factors and study design factors19. Unexplained variability due to placebo analgesia makes it a challenge to control non-pharmacological responses in clinical trials and capitalize on them in clinical care. Clear understanding of placebo analgesia should provide new insight into development of efficient screening procedures for pain medication.

Placebo analgesia could also be harnessed either as a solitary treatment strategy or in conjunction with pharmacologically active drugs to improve the end-effects in patients. Better understanding of the effects of physician-patient interactions, clinical practices, verbal suggestions, patient expectations and classical conditioning techniques may pave way for new strategies for placebo-based analgesic therapy. Numerous brain-mapping studies have established that placebo effects of analgesia can be clinically implemented owing to its considerable magnitude and long duration of action. But, further large-scale clinical studies establishing these concepts are required for the wide-scale use of deception or non-deception-based strategies for placebo analgesia in the clinical environment.

Conclusion:
Although the primary molecular neurophysiology of placebo analgesia is complicated and involves a variety of receptor systems, it is irrefutable that it plays a major role in the efficacy of a number of drug classes such as Tricyclic antidepressants, opioid analgesics, etc. Comprehensive understanding of these mechanisms is crucial for the development of not just new drugs but also better treatment strategies. The use of placebo analgesic effects as a treatment using deception models are strictly prohibited due to obvious ethical reasons19. A recent study of the influence of placebo effects in IBS patients, at Harvard Medical School, highlights a new argument that placebo treatment can work even without deception20. Although further clinical studies are required to confirm this phenomenon and understand the underlying mechanism, it can potentially open new doors for the use of clinical placebo analgesia based therapies. For now, we can try to put into practice our knowledge of the positive effects of expectation and belief in the form of better clinical practices and patient care along with improving the patient-physician interactions.

References:
1.    Kuner, R. Central mechanisms of pathological pain. Nat Med 16, 1258-1266 (2010).
2.    Part III: Pain Terms, A Current List with Definitions and Notes on Usage (pp 209-214) Classification of Chronic Pain, Second Edition, IASP Task Force on Taxonomy, edited by H. Merskey and N. Bogduk, IASP Press, Seattle, ©1994.
3.    Fields, H.L. & Levine, J.D. Pain—Mechanisms and Management. West J Med 141, 347-357 (1984).
4.    Basbaum, A.I., Bautista, D.M., Scherrer, G & Julius, D. Cellular and Molecular Mechanisms of Pain. Cell 139, 267-284 (2009).
5.    Benedetti, F., Mayberg, H.S., Wager, T.D., Stohler, C.S. & Zubieta, J.-K. Neurobiological Mechanisms of the Placebo Effect. The Journal of Neuroscience 25, 10390 -10402 (2005).
6.    Benedetti, F. Mechanisms of placebo and placebo-related effects across diseases and treatments. Annu. Rev. Pharmacol. Toxicol. 48, 33-60 (2008).
7.    Bingel, U., Colloca, L. & Vase, L. Mechanisms and Clinical Implications of the Placebo Effect: Is There a Potential for the Elderly? A Mini-Review. Gerontology 57, 354-363 (2011).
8.    Scott, D.J. et al. Individual Differences in Reward Responding Explain Placebo-Induced Expectations and Effects. Neuron 55, 325-336 (2007).
9.    Porreca, F., Ossipov, M.H. & Gebhart, G.F. Chronic pain and medullary descending facilitation. Trends Neurosci. 25, 319-325 (2002).
10.    Review: placebo is better than no treatment for subjective continuous outcomes and for treatment of pain. Evidence Based Medicine 7, 11 (2002).
11.    Price, D.D. et al. An analysis of factors that contribute to the magnitude of placebo analgesia in an experimental paradigm. Pain 83, 147-156 (1999).
12.    Benedetti, F. Cholecystokinin Type A and Type B Receptors and Their Modulation of Opioid Analgesia. Physiology 12, 263 -268 (1997).
13.    Siegel S. 2002. Explanatory mechanisms for placebo effects: Pavlovian conditioning. In The Science of the Placebo: Toward an Interdisciplinary Research Agenda, ed. HA Guess, A Kleinman, JW Kusek, LW Engel, pp. 133–57, London: BMJ Books
14.    de la Fuente-Fernández, R. The placebo-reward hypothesis: dopamine and the placebo effect. Parkinsonism Relat. Disord. 15 Suppl 3, S72-74 (2009).
15.    Blackwell, B., Bloomfield, S.S. & Buncher, C.R. Demonstration to medical students of placebo responses and non-drug factors. Lancet 1, 1279-1282 (1972).
16.    Schweinhardt, P., Seminowicz, D.A., Jaeger, E., Duncan, G.H. & Bushnell, M.C. The Anatomy of the Mesolimbic Reward System: A Link between Personality and the Placebo Analgesic Response. The Journal of Neuroscience 29, 4882 -4887 (2009).
17.    Colloca, L., Benedetti, F. & Porro, C.A. Experimental designs and brain mapping approaches for studying the placebo analgesic effect. Eur. J. Appl. Physiol. 102, 371-380 (2008).
18.    Finniss, D.G., Kaptchuk, T.J., Miller, F. & Benedetti, F. Biological, clinical, and ethical advances of placebo effects. The Lancet 375, 686-695 (2010).
19.    Price, D.D., Finniss, D.G. & Benedetti, F. A Comprehensive Review of the Placebo Effect: Recent Advances and Current Thought. Annual Review of Psychology 59, 565-590 (2008).
20.    Kaptchuk, T.J. et al. Placebos without Deception: A Randomized Controlled Trial in Irritable Bowel Syndrome. PLoS ONE 5, e15591 (2010).

To any of the skeptics out there, regarding the application of well-known concepts of open-source and crowd-source (from the computer science industry) in drug discovery should watch this TEDTalk by Jay Bradner from the Dana Farber Cancer Research Institute at Harvard University.

The crystal structure of the beta2-adrenergic receptor coupled to the Gs G-protein was published on Nature (online) couple of months back and now it has made it to the cover of last week’s Nature (Magazine). Apart from the fact that it might be “the” study that provides crucial insights into the functioning of the ever-present GPCRs, the volume and quality of work that went into this study is just mind-boggling. The way they vanquish every obstacle that they face along the way using some ingenious techniques is worth appreciating. As Dr. Thue W. Schwartz points out in the introductory/review article -

A complete  review of the biochemical methods used to prepare the material that eventually yielded crystals for Rasmussen and co-workers’ study would occupy an entire semester-long course in graduate school (and maybe it should).

But another article that was also published in the same week which I found to be more interesting (if at all possible) was - Conformational changes in the G protein Gs induced by the beta2-adrenergic receptor. They use the peptide amide hydrogen deuterium exchange mas spectroscopy (DXMS) to understand the dynamic changes that occur during receptor-G protein binding and the subsequent GDP-GTP exchange. The basic concept is that the positions of the complex that shows most Hydrogen-Deuterium exchange, are considered to the most dynamic and therefore considered as vital conformational changes required for G-protein activation. The main conclusions from the study were -

• The C-terminus of Gα subunit penetrates into the cytoplasmic core of the agonist-bound receptor.
• The N-terminal of Gα subunit interacts with the second intracellular loop (ICL 2) and connects the receptor to the P-loop (which holds the nucleotide) through the β1-strand. 
  
• The β1-strand conformational change disrupts a hydrogen bond network and causes changes in the P-loop holding the GDP. This interaction results in the GDP release.
  
• The further hypothesis is that this hydrogen bond disruption also leads to an open (and empty) conformation of the Gα and thus allowing the entry of GTP and the activation of the G protein.

The remarkable number GPCR studies published over the past few months from Dr. Kobilka’s lab deserve a standing ovation. Each of these studies sheds a new light on the working of (one of) nature’s unique and therapeutically vital receptor and I can’t wait for their next paper.

Today I walked blindly to a talk organized by my lab and discovered that Cannabinoid receptors have recently been suggested to be actively involved in the Insulin signaling pathway. The lecture was by Dr. Rohit Kulkarni from the Joslin Diabetes Center at Harvard Medical school. His lab has been working on the Insulin signaling pathway and they stumbled upon this discovery - Cannabinoid receptors are actively involved in the insulin pathway. They recently published this work - Cannabinoids inhibit insulin receptor signaling in pancreatic beta cells. The paper talks about the general pattern of the involvement of CB1 receptors in the inhibition of insulin signaling pathway by the changes in the mRNA levels of the signaling proteins. It is a previously known fact that CB1 receptors are found on the Beta cells from the Islets of Langerhans and was suggested that they maybe involved in Glucose metabolism. This study clearly shows that CB1 endogenous ligands 2-AG or Anandamide showed an inhibition in the Insulin pathway protein levels and also caused a decrease in the beta-cell size by a considerable margin. This obviously brings up the idea that selective CB1 antagonists maybe used to block this inhibitory effect and essentially increase insulin signaling. We will probably learn more about this soon.

But the more interesting point to consider as one of my colleague pondered, is the mind-boggling number of disease states that have so far been identified involving the cannabinoid receptors/system. It therefore raises the question of how a ligand/drug can be designed to be effective for just one specific system or pathway. This obviously then brings us to the quintessential question of GPCR signaling pathway - How do some drugs/compounds activate only certain pathways and not others?

Of course we can guess that based on the difference in the ligand interaction with the receptor at the binding site and its influence on the G-protein or any other downstream protein (Beta-Arrestin, etc.) results in the varying patterns of pathway activation/inhibition. Now that the GPCR-gs protein structure has been resolved (Crystal structure of the beta2 adrenergic receptor-Gs protein complex) this question could be answered with some real data soon.

I finally got around to reading the entire paper from Nature about the crystal structure of H1 receptor. I will stick to talking about the receptor expression and purification which are more related to the topics of my interest. This GPCR was expressed in the yeast system unlike previously crystallized GPCRs (which were from insect cell-lines). Another interesting approach is the use of T4 lysozyme in place of the 3rd intracellular loop. This technique has already worked for the Beta-adrenergic receptor developed by Kobilka’s lab in Stanford, which raises the obvious question - Is this effect of stabilization (due to the introduction of T4 lysozyme) a universal phenomenon in case of all GPCRs. We will probably get an answer to this question in a couple of years when I am sure a bunch of GPCRs would have been crystallized. I was also extremely glad to see a seminal GPCR paper coming from Kyoto University, Japan.

I’m back to blogging after a long time. Its been a couple of busy months having just started working as a Lab tech in the biochemistry lab. I hope to chip in atleast a couple of posts in a week this month onwards. The first post going to be on the latest GPCR crystal structure paper in Nature from last week’s edition. Adios!

jerrybrito:

Myth #1 – Introverts don’t like to talk.
This is not true. Introverts just don’t talk unless they have something to say. They hate small talk. Get an introvert talking about something they are interested in, and they won’t shut up for days.

Myth #2 – Introverts are shy.
Shyness has nothing to…

(Source: carlkingcreative.com)

Its time to recycle a paper I wrote for my Pharmcology course last semester. (P.S. Never write a receptor-based drug discovery paper for a clinical aspect-oriented Pharmacology course.)    

Introduction:

Since the discovery of the adrenergic system of receptors, there have been numerous attempts to develop drugs that can modulate their effects. The earlier attempts were mostly synthetic approaches based on the structures of endogenous ligands like epinephrine and norepinephrine. Over the years our understanding of the ligand binding sites and the general structure of the receptor has improved considerably and therefore development of drugs for specific receptors is possible now. The x-ray crystallography structure of b^­₂ adrenergic receptor was resolved recently and this provides a very good insight into the structure of the receptor and its differences from other GPCRs.

G-protein coupled receptors (GPCRs):

These are seven transmembrane receptors associated with heterotrimeric guanine nucleotide-binding proteins (G-proteins) that mediate a number of cellular responses to hormones and neurotransmitters. There are more than 800 different G-protein coupled receptors identified so far and a number of them are potential drug targets for a wide range of diseases.

Multiple phylogenetic analyses of GPCRs have revealed that they can be commonly divided into five families on the basis of their sequences and structural similarities. GRAFS classification system - Rhodopsin (Family A), Secretin (Family B), Glutamate (Family C), Adhesion (Family D) and Frizzled/Taste2 (Family E).

The general structure of GPCRs includes the characteristic seven membrane-spanning a-helical segments connected by alternating 3 intracellular loops and 3 extracellular loops. The ligand binds to the binding site on the extracellular surface resulting in conformational changes in the final intracellular loop. This conformational change allows for the binding of specific G-protein present on the inner surface of the membrane to the intracellular loop followed by the activation of the G-protein (replacement of GDP for GTP). The subunits of the activated G-protein can then trigger a cascade of intracellular signaling pathways that result in the necessary cellular responses. Many GPCRs can activate multiple signaling pathways by coupling with different G-proteins and different ligands.

According to the widely accepted, Ternary Complex Model, the receptor exists in two states – R state and R* state. The receptor stays in the R state in the absence of the agonist and in R* state (the activated state) in the presence of the agonist. However, recent studies have shown that a ‘basal activity’ or ‘constitutive activity’ exists even in the absence of a ligand. It is hypothesized that an equilibrium exists between the active state and the inactive state in the absence of a ligand and this equilibrium is shifted to active state (R*) on binding to an agonist. Studies show that mutations at certain positions in the transmembrane segments result in increased basal activity which shows some proof for this hypothesis, although we do not have a complete understanding of these conformations.  

Thus according to this modified model, there are four possible types of ligands –

Full Agonists, Partial agonists, Antagonists and Inverse agonists. Full agonists exhibit maximal receptor stimulation whereas partial agonists are unable to elicit full activity. Neutral antagonists have no effect on receptor activation and Inverse agonists decrease the baseline receptor activity or ‘constitutive activity’.

Numerous biophysical studies suggest that a specific ligand stablizes a distinct receptor conformation and has distinct efficacies for different signaling pathways. This implies that a ligand can act as an agonist for one signaling pathway and simultaneously act as an antagonist to a different pathway on binding to the same receptor. 

GPCRs are extensively targeted for the treatment of numerous diseases and therefore resolving their structures is vital for the development of novel and subtype-specific drugs. Owing to their structural flexibility and instability, only 6 GPCR structures have been resolved so far - b^­₁, b^­₂ adrenergic receptors, Adenosine receptor, Rhodopsin receptor, Dopamine (D3) receptor and Chemokine receptor (CXCR4).

b^­₂ adrenergic receptors:

b^­₂ Adrenergic receptors (b^­₂-AR) are a part of the Rhodopsin family and are a good example for ligand-binding GPCRs. b^­₂-AR are located almost throughout the body and are mainly implicated in cardiovascular and bronchial therapy. They are also clinically important in glaucoma treatment, smooth muscle relaxation (especially uterine relaxation, GIT relaxation and ciliary relaxation).

b^­₂-AR interacts with both the stimulatory G-protein (G_s) and the inhibitory G-protein (G_i) and also MAPK. They can also activate MAPK through the b^­-Arrestin pathway (Figure 1). The G­­_s pathway activates the Adenylyl cyclase, which produces cAMP. The cAMP release results in smooth muscle relaxation and thus has implications in the treatment of asthma, inhibition of uterine contraction in premature labor and in glaucoma. The G_i pathway is regulated by PKA-mediated receptor phosphorylation and occurs only when the G_s pathway is blocked. b^­-arrestins are mainly considered to be regulatory proteins that cause receptor desensitization and internalization. But studies show that when certain inverse agonists bind to the receptor, b^­-arrestins can cause MAPK activation. So, it was found that individual ligands favor different pathways due to their varying affinities to each conformation of the seven transmembrane helices.

b^­₂-AR-specific drugs are required in order to prevent unwanted side effects especially cardiovascular effects through b^­₂-ARs. The most commonly used b^­₂-AR specific drugs are – salbutamol, salmeterol, terbutaline etc. A number of agonists and antagonists for β-adrenergic receptors have been synthesized and tested for almost 50 years, and about 155 adrenergic drugs have been developed. Of these agents about 32 are considered specific to b^­₂-AR and have been widely used clinically.

Crystal structure of b^­₂ adrenergic receptor:

X-ray crystallography has been an important tool to understand the structures of cellular proteins and receptors. Bovine rhodopsin was the first GPCR structure to be resolved by X-ray crystallography in the year 2000. But due to the low natural abundance and structural flexibility or instability of most GPCRs, no significant progress was made in solving other structures. Ligand-binding GPCRs such as b^­₂-AR show large number of conformational changes after ligand binding and therefore are very difficult to crystallize. Therefore, using targeted protein-engineering methods, mutants were developed to increase stability and crystallization of b^­₂-AR. The following modifications were made –

·       Intracellular loop 3 (ICL3) is considered to interact with the G-protein after the ligand binds to the receptor. This loop shows large physical movements thereby causing difficulties during crystallization. Therefore, ICL3 loop was truncated and replaced with the T4 Lysosome sequence that could restrict large movements (Figure A).

·       The b^­₂-AR-T4L mutants were cloned in Sf9 insect cells to improve yields.

·       This mutant receptor was crystallized in the presence of Carazolol, a b^­₂-AR-specific partial inverse agonist. This ligand favors the inactive conformation and proved to improve stability of the receptor.

But the resulting structure may have been compromised due to the limited movement of the seven transmembrane segments. Therefore, another method was developed using a monoclonal antibody (Mab5) that recognizes that native ICL3 (Figure A). The Fab segment of the antibody binds to the ICL3 segment forming the b^­₂-AR–Fab complex. This did not alter the agonist-induced conformational changes, and ligand-binding affinities of the wild-type b^­₂-AR. On comparing the two structures, they were found to be almost similar except for the positions of the extracellular loops and the higher basal activity seen in b₂-AR-T4L receptors. Therefore, either structure can be used as a template for In-silico screening studies.

In 2008, another b₂-AR crystal structure was resolved but this time while binding to Timolol (a partial inverse agonist) along with 2 molecules of cholesterol. This structure provided additional details about the binding sites of cholesterol molecules to the GPCR and also its effect on receptor stability when present in the plasma membrane. It could also provide insights into the trafficking mechanisms of b₂-AR through cholesterol sequestration.

More recently in Jan 2011, the same lab from Stanford has published the structures of an agonist-bound b₂-AR in both high affinity (bound to the G-protein) and low affinity (in the absence of G-protein) states. The b₂-AR when bound with the G_s protein shows higher agonist affinity. Therefore, to resolve this high-affinity state structure, a camelid antibody fragment called Nanobody (Nb) was used. The Nb showed G_s-like binding properties and stabilized the high-affinity state during crystallization. The crystal structure of the b₂-AR-T4L in the presence of an agonist (BI-167107) was obtained when stabilized with Nb80. For resolving the unstable low-affinity structure, an agonist (Procaterol) was covalently bound using a linker. These structures could reveal vital details about the differences in conformation between the antagonist- and agonist-bound GPCR.

In silico screening for drugs:

Virtual screening in general involves the assessment of a large database of compounds and the identification of potential ligands for specific target. There are two main types of methodologies – Ligand-based screening and Receptor-based screening.

a)    Ligand-based screening involves the identification of new ligands using the 2D structures of previously known ligands. It produces very few hits as only related compounds from the same or similar classes can be obtained. Since only the 2D structures are being considered, therefore, inaccurate hits maybe obtained. Another ligand-based technique is the use of a 3D structure of the pharmacophore as the template through which some of the problems of 2D structure screening can be avoided.

b)    Receptor-based screening or Structure-based drug discovery involves the use of computational docking modules to assess a large database against an X-ray crystal structure of the receptor. Most molecular docking studies include the process of assessing the various conformations of the ligand against the rigid target. Based on the binding force calculations and potential potency values, each ligand is scored and ranked. Recently developed docking software like GLIDE, DOCK, AUTODOCK, etc also take into consideration the flexibility of both the ligand and the receptor (template). Thus using these novel modules, better screening of compound databases can be performed.

Requirement of Structure-based drug discovery (SBDD):

Currently, all of the agents that are being used clinically were developed using ligand-based analoging techniques and not using the SBDD studies. Structure-based drug discovery techniques could potentially enable us to optimize selectivity, duration of action as well as to combine β₂-agonist activity with desirable ancillary actions (e.g. anti-oxidant activity, calcium channel block, etc.). It also provides us with the possibility to design molecules that interact with multiple subtypes but show subtype selectivity. The most important advantage of SBDD is the potential to find newer classes of ligands whose pharmacophore is different from the regular catecholamine motif. These compounds could not be considered as possibilities during earlier studies.

Virtual screening results and potential drugs:

So far, a couple of studies have been published that used the Carazolol-bound b₂-AR structure for small molecule screening. One study from Stanford University used the DOCK program to screen about 972,608 molecules from the ZINC database and individual ranks were assigned to each ligand. From the top 500, 25 compounds from 4 classes were selected and studied using radioligand-binding experiments. These were then compared with the known adrenergic ligands (8063 molecules) from the WOMBAT database. Most compounds were found to be similar to known adrenergic agents; although, few were novel chemotypes that have not been explored before. But the most interesting result observed from these studies was that all the compounds from the screen were either antagonists or inverse agonists and none were agonists.

Similarly in the second study from Lundbeck research, the GLIDE module was used to screen 400,000 compounds from proprietary database and about 4 million compounds from commercial databases. The physically available ones of the top 150 compounds from each database were selected and studied. These included a number of known antagonists such as carvedilol; thus validating the X-ray structure of the receptor and the in-silico screening program.

Recent studies in heart failure have shown that although agonists enhance acute effects, chronic use results in decreased signaling owing to desensitization mechanisms. In case of antagonists, they can potentially increase signaling on chronic use but cause severe side effects in acute conditions. In contrast, inverse agonists inhibit basal signaling initially and with chronic use up-regulate the receptors thus can be used as effective therapeutics.

The above studies provide a large list of inverse agonists of different affinities, pharmacokinetics and toxicity profiles which can be further studied for developing into drug candidates. It is clear that structure-based drug discovery using X-ray crystal structures are effective in the discovery of novel subtype specific drugs.

Problems faced in X-ray structure-based drug discovery:

The X-ray structures provide considerable advantages relative to the rhodopsin-based homology models using which a number of SBDD was being carried out earlier. But we still face a number of problems for precise determination of active ligands. One of the biggest obstacles with SBDD is the static nature of the X-ray crystal structures. It has been observed that the binding pocket of avian β₁-AR bound to cyanopindolol and human β₂-AR bound to carazolol are identical owing to a high conservation of binding-site contact residues. However, subtype-specific binding affinities can be observed for both β­₁- and β­₂-AR. These differences are due to the subtype-specific conformational preferences in distant residues, which in turn influence the amino acid spatial positions at the binding site.

Thus the static template of the receptor can only provide the data with respect to that particular conformation which is being used as the template. The above virtual screening studies using the carazolol-bound β₂-AR crystal structure as a template identified number of new β₂-AR ligands showing high affinities; however, most of the compounds exhibited inverse agonistic or antagonistic activity. Agonists could not be obtained from the screen using an inverse-agonist bound receptor template.

Conclusion:

The GPCRs are currently the most widely targeted proteins for therapeutics and the X-ray crystal structures of these complex receptors has given the opportunity to study them in extreme detail and potentially develop novel, more potent drugs for existing and newer disease conditions. The primary intent for resolving the X-ray crystal structure of Beta-2 AR was to study and understand the structural differences and also the conformational mechanisms of signal transduction. But these structures have also been proven to be an excellent resource for drug discovery research.

Docking studies have revealed that most of the highly ranked ligands are inverse agonists or antagonists when screened against the inactive state of the receptor. The recently published agonist-bound structures may have to be used for obtaining potential potent agonists. A recent study has shown that by virtually modifying the binding site parameters of the inactive state receptor, the screen can also identify agonists and partial agonists. Such improvements allow us to potentially use any one of these structures to discover a wider range of subtype-specific and potent ligands.

Other future directions could include the use of dynamic images from other biophysical methods along with the information from static X-ray structures. To study the conformational changes and the rates of interconversion between these states we need to develop other time-dependent biophysical methods. Developments in fluorescence and NMR spectroscopy may help us to understand GPCR dynamics. Using such novel methods, more effective virtual drug discovery efforts can be undertaken and potentially develop novel, potent drugs for less cost and in less time.

References:

1.     Audet M and Bouvier M (2008) Insights into signaling from the β2-adrenergic receptor structure. Nat. Chem. Biol. 4, 397.

2.     Bond RA and IJzerman AP (2006) Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Science 27, 92–96

3.      Fredriksson R, Lagerstrom MC, Lundin LG and Schioth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272.

4.      Hanson MA, Cherezov V, Roth CB, Griffith MT, Jaakola VP, Chien EYT, Velasquez J, Kuhn P, and Stevens RC (2007) A Specific Cholesterol Binding Site Is Established by the 2.8 Å Structure of the Human β₂-Adrenergic Receptor. Science 318, pp. 1258–1265.

5.     Kolb P, Rosenbaum DM, Irwin JJ, Fung JJ, Kobilka BK, Shoichet BK. (2009) Structure-based discovery of β₂-adrenergic receptor ligands. Proc. Natl. Acad. Sci. U.S.A. 106 (16): 6843-6848.

6.     Rasmussen SGF, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VRP, Sanishvili R, Fischetti RF, Schertler GFX, Weis WI and Kobilka BK (2007) Crystal structure of the human β₂ adrenergic G-protein-coupled receptor. Nature 450, 383–387.

7.      Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC and Kobilka BK (2007) GPCR engineering yields high-resolution structural insights into β₂-adrenergic receptor function. Science 318, 1266–1273

8.     Rosenbaum DM, Rasmussen SG and Kobilka BK (2009) The structure and function of G-protein-coupled receptors. Nature 459, 356.

9.     Rubenstein LA, Zauhar RJ and Lanzara RG, (2006) Molecular dynamics of a biophysical model for beta2-adrenergic and G protein-coupled receptor activation, J Mol Graph Model 25 (4), pp. 396–409.

10.   Sabio M^a,b, Jones K^b and Topiol S^a,b (2008) Use of the X-ray structure of the _β2-adrenergic receptor for drug discovery. 18:1598–1602^.aPart 2: Identification of active compounds. Bioorg Med Chem Lett 18:5391–5395^.b