Photoresponsive Hybrid Compounds

Luca Agnetta , Michael Decker , in Design of Hybrid Molecules for Drug Development, 2017

11.5.1.2 Ligand-gated Ion Channels

LGICs are regulated by small molecules. In order to provide light sensitivity, the simplest and most intuitive way is to look at those endogenous ligands. Among different LGICs iGluRs are outstanding, mediating the majority of excitatory synaptic transmission in the central nervous system (CNS). They are key receptors in synaptic plasticity, substantial for memory and learning. Glutamate represents the endogenous ligand of iGluRs and the most abundant neurotransmitter. Its importance and application as photochromic compound was already shown before. IGluRs are subdivided into different classes. The AMPA receptors, responsible for the fast synaptic transmission, are named after the synthetic glutamate analogue α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (Fig. 11-15), which can, as an agonist, selectively activate these receptors. In 2012 Trauner and coworkers developed a photochromic hybrid for the AMPA receptor. 59 The design was based on the potent and highly selective BnTetAMPA (benzyl-tertazolyl-AMPA). Using the azo-extension approach the azobenzene moiety was merged with the AMPA structure providing azobenzene-tetrazolyl-AMPA (ATA). Investigations employing cortical mouse neurons showed reversible generation of APs. In the dark, neuronal firing is triggered effectively, whereas during illumination with green-blue light quickly deactivates firing. In a follow-up study in 2016, ATA found application in restoring light sensitivity in blind retinae, such as AAQ and DENAQ, not as channel blocker but rather as the first photochromic agonist. 60

Figure 11-15. AMPA and a (photoswitchable) derivative.

N-methyl-D-aspartate (NMDA) receptors, also belonging to the family of iGluRs, are expressed throughout the brain in nerve cells and are important for synaptic plasticity control, memory, and learning. As such, selective agonists might play a significant clinical role in the treatment of neurological dysfunction as Alzheimer's, Parkinson's, and Huntington's diseases. DiGregorio, Trauner, and coworkers synthesized a photochromic glutamate analogue selectively activating NMDARs. 61 Following the experience gained with GluAzo and ATA the new compound was designed as an azobenzene-triazole conjugated glutamate (ATG). In contrast to ATA, ATG is inactive in the dark-adapted trans-form. Irradiation with 370   nm quickly converts it into the active cis-form, representing the first cis-agonist. Therefore the activity of ATG can be precisely regulated upon illumination on a millisecond scale. This behavior is highly advantageous because nerve cell damage that stems from excessive stimulation is prevented. Fig. 11-16 illustrates light-controlled AP firing in cortical neurons. Another example of controlling LGICs in a light-dependent manner is AzoCholine. 62 It was designed to resemble MG624, a α7 nicotinic acetylcholine receptor (nAChR) antagonist, using the azologization approach and replacing the stilbene group with azobenzene. Binding of trans-AzoCholine on the neuronal-type pentameric ion channel resulted in currents twice as large compared to acetylcholine, detected by patch-clamp electrophysiology (Fig. 11-16). However, 360   nm irradiation reversed this process and deactivated α7 nACh receptors. Finally, AzoCholine showed light-dependent perturbation of behavior in nematodes (Fig. 11-17).

Figure 11-16. (A) Photochemical reaction of ATG. (B) Light-dependent AP firing in cortical neuron.

Figure 11-17. (A) Azologization of MG624 leading to the design of AzoCholine. (B) Light-controlled activation of nAChR with a photochromic ligand.

Among numerous pioneering research works, Trauner and coworkers were the first to apply photopharmacology to ATP-sensitive potassium channels (KATP). 49 These are hetero-octameric proteins comprised of four sulfonylurea receptor subunits (SUR1) along with four Kir6 components, creating a channel that allows potassium ion efflux. The SUR1 units monitor the energy balance within the cell by sensing intracellular levels of ATP and in response opening or closing the inward rectifying potassium channel. In pancreatic beta cells, high levels of glucose leads to increased production of ATP, which in turns binds to the KATP channel. This results in KATP closure causing depolarization of the membrane and opening of calcium channels, which trigger insulin secretion. Hence, light activation of KATP channel may offer a useful research tool for diabetes. Glimepiride, as a sulfonylurea binding to the SUR1 component, is approved for the treatment of type 2 diabetes mellitus (T2DM). It was used as a template for the design and synthesis of JB253 and a redshifted derivative JB558, both photoswitchable glimepiride analogues, by extending its aromatic core to a (heterocyclic) azobenzene (Fig. 11-18). With incorporation of the chromophore, JB253 was readily converted to the cis-state applying blue light, while the trans-state occurred rapidly in the dark through thermal relaxation. JB558 possesses bathochromic-shifted absorption maximum and is cis-converted with yellow-green light (λ=520   nm). 63 It was reported that pancreatic beta cell function and insulin release can be regulated upon illumination using these photochromic sulfonylureas.

Figure 11-18. (A) Glimepiride and photoswitchable variations. (B) Light-induced binding of photoswitchable sulfonylureas on SUR1 causing insulin release.

Recently, the family of G-protein coupled inwardly rectifying potassium channels (GIRK) channels have emerged as a potential target for photopharmacology. GIRK channels are downstream effectors of G-GPCRs and are activated upon binging of Gβγ subunit becoming permeable for potassium ions. This results in hyperpolarization of the cell membrane, reducing the activity of excitable cells. They are expressed in the pancreas, heart, and brain and play a significant role in cardiac output, coordination of movement, and cognition. With the discovery of the first potent and selective activators of GIRK channels ML297 and VU0259369, the necessary foundation was laid for the design and synthesis of light-operated GIRK channel opener (LOGO) by employing the azo-extension approach. As the first photochromic potassium channel opener, LOGO5 was found to enable the optical control of GIRK channels in the trans-configuration and is inactivated with UV light, causing isomerization to cis-LOGO5. The potency of trans-LOGO5 is comparable to VU0259369 (Fig. 11-19). In vitro, this phenomenon is used for silencing AP firing in dissociated hippocampal neurons. In vivo, the motility of zebrafish larvae can be controlled in a light-dependent fashion. 64

Figure 11-19. Light-operated GIRK channel opener 5 (LOGO5) inspired by ML297 and VU259369 activators.

Lastly, a methodology was found in 2013 to stimulate transient receptor potential (TRP) channels with light. 65 TRP channels are found throughout the body of mammals in almost every cell type and are mainly localized in the cell membrane. They mediate the perception of pain, temperature, pressure, and noxious and pungent chemicals. The study focused on the vanilloid receptor 1 (TRPV1) activated by a variety of chemical stimuli such as capsaicin (CAP), spider toxins, allicin, and physical triggers such as voltage, heat, and low pH but not by light. It acts as an intracellular calcium channel but is also permeable for sodium and potassium to a small extent. The pungent component of hot chili peppers, CAP, is known as an agonist while capsazepine (CPZ), BCTC, and thio-BCTC are specific antagonists with analgesic effects (Fig. 11-20). Bearing aromatic rings extendable to azobenzene, these small molecules represented the basis for the design of photoswitchable derivatives, namely, azo-capsazepine (AC) and azo-BCTC (ABCTC). In the course of in vitro investigations of their light-controlled activity, AC-4 was found to be trans-antagonist upon voltage activation of TRPV1, while cis-AC-4 inhibits CAP-induced TRPV1 current. ABCTC showed antagonist behavior only as cis-isomer.

Figure 11-20. Ligands for TRPV1 channel and photoswitchable derivatives.

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Introduction to enzymes, receptors and the action of small molecule drugs

Stanley M. Roberts , Alasdair J. Gibb , in Introduction to Biological and Small Molecule Drug Research and Development, 2013

1.3.4.1 Nicotinic AChR structure: a ligand-gated ion channel

Of the ligand-gated ion channels, by far the best studied is the nicotinic AChR (Figure 1.26). Most studies have utilized the AChR from the electric organ of the Californian ray, Torpedo californica which is an exceedingly rich source of these receptors. By utilizing α-bungarotoxin (a component of cobra snake venom with extremely high affinity for the nicotinic AChR), it was possible in the late 1970s and early 1980s to isolate and characterize this receptor. The Torpedo AChR was found to be composed of four different protein subunits in a stoichiometry α2βγδ arranged in a pseudosymmetrical fashion around a central ion channel pore (Figure 1.26). Each subunit crosses the cell membrane four times giving four transmembrane (TM) domains which are numbered from the amino terminus of the protein. Since five subunits make up each receptor, there are 20 TM domains per receptor (Figure 1.26). The amino acid residues in TM2 of each subunit line the central ion channel and determine its conductance properties.

FIGURE 1.26. Schematic diagram of the structure of the nicotinic AChR. This general structure is thought to hold for all pentameric ligand-gated receptors. The receptor is composed of five subunits each with the general structure shown in (a). (a) Schematic arrangement of a nicotinic AChR α-subunit. The four transmembrane domains (TM1–TM4) are shown crossing the cell membrane. The large extracellular domain at the amino terminus of the α-subunit is where the main determinants of agonist and antagonist binding are located. (b) Arrangement of the AChR subunits when viewed from above the cell membrane. Each subunit is arranged so that the transmembrane segment TM2 lines the central ion channel of the receptor (c) Schematic of the three-dimensional structure of the nicotinic AChR as it sits in the cell membrane.

 Source: (a) and (b) adapted from Huganir and Greengard (1990) with permission.

ACh, tubocurarine and other nicotinic receptor ligands were found to compete with α-bungarotoxin for its binding site on the α-subunit. The α-subunit was found to be unique in having two cysteine residues at amino acid positions 192 and 193. Subsequently, molecular genetic techniques were used to isolate and sequence multiple subtypes of neuronal (found in the CNS and in autonomic ganglia) nicotinic receptor subunits and all α-subunits were found to contain two cysteine residues at positions analogous to 192 and 193. These cysteine residues are located before the first TM domain in the extracellular part of the protein and the agonist-binding site is thought to be close by, perhaps in a shallow cleft between the α- and adjacent subunits.

The neuronal nicotinic receptors are different from muscle receptors in a number of ways, including the fact that they have a higher sensitivity to nicotine and are not sensitive to α-bungarotoxin. In contrast with the α-, β-, γ- and δ-subunits of electric organ and muscle AChRs, neuronal nicotinic receptor subunits are divided into α- and β-subunits. The exact stoichiometry of neuronal nicotinic receptors is unknown but is probably α2β3. Because nine different α-subunits and five different β-subunits have been identified, the possibilities for variation in neuronal nicotinic receptor structure are substantial. A similar situation has arisen with the receptors for the inhibitory amino acid γ-aminobutyric acid (GABA) and with the excitatory amino acid glutamate receptors of the kainate, 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid and N-methyl-d-aspartate type. Multiple subtypes of the subunits that make up the glutamate receptors and the GABA receptors (Table 1.10) have been cloned. In each case, 10 or more different subunits (based on amino acid sequence) are known.

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Analgesics

Ruben Vardanyan , Victor Hruby , in Synthesis of Best-Seller Drugs, 2016

Modulators of Nicotinic Acetylcholine Receptors

The analgesic properties of nicotine have generated attempts to develop compounds targeting nicotinic acetylcholine receptors (nAChRs). Acetylcholine mediates effects through both the nAChR (ligand-gated ion channels) and the G-protein–coupled muscarinic receptors. Only nAChR agonists have been reported as possible analgesics, although nAChR antagonists could also have an analgesic action. The effects of receptor agonists, termed cholinomimetics in analgesia, are well established. nAChR agonists exhibit antinociceptive, antihyperalgesic, and antiallodynic effects. These compounds successfully inhibit pain in different preclinical and clinical pain models without acting through an opioid mechanism, although suggesting a definite therapeutic potential. Various problems associated with the use of nAChR agonists as analgesics have been identified. Several nAChR agonist compounds, like tebanicline (ABT-594) (3.3.89), sofinicline (ABT-894) (3.3.90) alkaloid epibatidine (3.3,91) and its analogues A-85380 (3.3.92), SIB-1663 (3.3.93) , ABT-202 (3.3.94), and ABT-366833 (3.3.95). (Fig. 3.40.), have been proposed as analgesics, but in general, efforts to create new analgesics targeting the cholinergic system have been largely unsuccessful. Tebanicline, in particular, has unacceptable gastrointestinal side effects. Other compounds have many undesirable effects, including addictive properties [170-175].

Fig. 3.40. Modulators of nAChRs with analgesic activity.

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Voltage-Gated Channels

Edward C. Conley , in Ion Channel Factsbook: Voltage-Gated Channels, 1999

Book references:

Brown, A.M., Drewe, J.A., Hartmann, H.A., Taglialatela, M., Debiasi, M., Soman, K. and Kirsch, G.E. (1993) The potassium pore and its regulation. In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 74–80. Annals of the New York Academy of Sciences, vol. 707, New York.

Chandy, K.G. and Gutman, G.A. (1994) Voltage-gated K+ channel genes. In Ligand-and Voltage-gated Ion Channels (ed. R.A. North). Handbook of Receptors and Channels. CRC Press, Boca Raton.

Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn. Sinauer Associates, Sunderland, MA.

Lewin, B. (1994) Genes V. Oxford University Press, Oxford and New York.

Li, M., Isacoff, E., Jan, Y.N. and Jan, L.Y. (1993) Assembly of potassium channels. In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 51–9. Annals of the New York Academy of Sciences, vol. 707, New York.

Saito, M., Zhao, M.L. and Wu, C.-F. (1993) In Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission and Muscle Contraction (eds T. Yoshioka, K. Mikoshiba and H. Higashida), pp. 392–5. Annals of the New York Academy of Sciences, vol. 707, New York.

Spooner, P.M., Brown, A.M., Catterall, W.A., Kaczorowski, G.J. and Strauss, G.J. (eds) Ion Channels in the Cardiovascular System. Futura, New York.

Stefani, E., Toro, L., Perozo, E. and Bezanilla, F. (1994) Gating currents of cloned Shaker K+ channels. In Handbook of Membrane Channels (ed. C. Perrachia), pp. 199–210. Academic Press, San Diego.

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Strategy and Drug Research

C.A. Briggs , M. Gopalakrishnan , in Comprehensive Medicinal Chemistry II, 2007

2.22.1 Introduction

Ion channels expressed in the plasma membrane have been classified broadly as voltage-gated ion channels (VGICs; see 2.21 Ion Channels – Voltage Gated) and ligand-gated ion channels (LGICs). In case of the latter, the ligand is generally considered to be an extracellular messenger such as a neurotransmitter. Often, the same transmitter acts upon an array of G protein-coupled receptors (GPCRs) as well as LGIC, for example, acetylcholine acting via the muscarinic GPCR as well as nicotinic acetylcholine receptor (nAChR) LGIC. A few notable exceptions include glycine which, to date, is known to act on LGIC only and neuropeptides and histamine which appear to act exclusively via GPCRs. The boundaries between LGIC and VGIC become less marked when one considers intracellular ligands, for example Ca2+/calmodulin or G protein-regulated channels, or the fact that some channels may be regulated by both ligand and voltage. In this chapter, however, we will consider the term 'ligand' to refer to extracellular messengers that are meant, physiologically, to exert predominant control over the opening or closing of the ion channel. Typically, this would comprise neurotransmitter receptor-associated LGIC hosting an ion channel domain that is integral to the transmembrane region and an extracellular ligand binding domain. The chemical signal to which an LGIC responds depends on the specificity of ligand binding whereas the nature of the electrical signal generated depends on both selectivity of the ion channel and the electrochemical gradient across the membrane. Generally, cation-selective channels generate a net depolarizing or excitatory signal and anion-selective channels generate a net hyperpolarizing or inhibitory signal.

LGIC activated by extracellular ligands may be divided into five families. Three LGIC families that have initially been recognized based upon protein sequence and predicted receptor structure are (1) the Cys-loop superfamily (nAChRs, 5-hydroxytryptamine 5HT3 receptors, γ-amino-butyric acid (GABAA/C) receptors, glycine receptor (GlyR), and a newly discovered Zn2+-gated channel, (2) the glutamate (Glu)-gated family, and (3) the ATP (purinergic)-gated family. Two additional LGIC that are increasingly being recognized and whose better-known roles lie outside the synapse are (1) the transient receptor potential (TRP) channel family where members have been shown to respond to specific extracellular messengers, and (2) the acid-sensitive ion channel (ASIC) family whose members are gated by proton interaction. TRP and ASIC generally appear to function as sensory channels, but some family members are also expressed in central neural pathways, including brain, where the roles remain to be further elucidated. Many LGIC are expressed not only in neurons and muscle, but also in nonneuronal, 'nonexcitable' cells such as glia, lymphocytes, keratinocytes, endothelial cells, pancreatic cells, sperm, and others. Thus, LGIC undoubtedly serve many roles apart from neurotransmission, but these roles are only beginning to be elucidated. This chapter will present an overview of the properties of various LGIC. For more detailed information and discussion, the reader is referred to recent review articles cited in the following topical sections. Topography models of LGIC are depicted in Figure 1, and a general summary of functional and pharmacological properties are presented in Table 1.

Figure 1. Topography models of ligand-gated ion channels (LGICs). Schematic representation of transmembrane topology of LGIC – Cys-loop (nAChR, 5HT3R, GABAA, and GlyR), glutamate (NMDA, AMPA, and kainate), ATP (P2X), ASIC, and TRP members. N and C indicate the N- and C-terminus, respectively. The heterooligomeric (trimeric, tetrameric, or pentameric) organization is also depicted.

Table 1. Ligand-gated ion channels: functional and pharmacological distinctions

Transmitter class Receptor subtype Ion selectivity a Agonists Antagonists Modulators
ACh nicotinic α1β1γδ / muscle, Torpedo Cation; low PCa2+ Nicotine, decamethonium, succinylcholine Pancuronium, vecuronium, α-bungarotoxin, α-conotoxin MI Physostigmine, galantamine, proadifen, steroids, e.g., promgestone, cholesterol
α3β4 / ganglionic nAChR Nicotine, lobeline, DMPP Hexamethonium, trimetaphan
α4β2 Cation; moderate PCa2+ Nicotine, cytisine, isproniciline, ABT-418, SIB-1508Y Dihydro-β-erythroidine
α6 Epibatidine, A-85380, cytisine α-Conotoxin MII
α7 Choline, PNU-282987 Methyllycaconitine PNU-120596, 5-hydroxy indole
α9, α9α10 Cation; high PCa2+ ACh α-Conotoxin PeIA, nicotine, strychnine
Serotonin 5HT3A Cation; moderate PCa2+ 2-methyl-5-hydroxytryptamine, phenylbiguanide, m-chlorophenylbiguanide, SR 57227A Ondansetron; Granisetron; MDL72222 Ethanol, anesthetics (volatile)
GABA GABAA GABAA α1β2γ2 Muscimol >GABA, THIP (selective for GABAA versus GABAC) Bicuculline Benzodiazepines, barbiturates, neuroactive steroids, ethanol
GABAA α4, α6 Anion (Cl−) Relatively insensitive to benzodiazepines
GABA GABAC ρ1, ρ2, ρ3 GABA >muscimol, (+)-CAMP TPMPA, CGP36742, 3-ACPBuPA Glycine potentiates, Zn2+ inhibits, loreclezole and (+)-ROD188 potentiate GABAA, inhibit GABAC; μM neurosteroids potentiate GABAC versus nM for GABAA
Glycine Anion Glycine, β-alanine, taurine Strychnine, nipecotic acid, ginkgolide B, tropisetron Positive ivermectin, ethanol and volatile anesthetics Negative progesterone, pregnenolone (inhibition)
Zn2+ ZAC Cation Zn2+ d-Tubocurarine
Glutamate b GluR1 Cation, moderate PCa2+ (S)-AMPA, (S)-ACPA, (S)-5-fluorowillardiine ATPO, GYKI 53655, GYKI 52466, SYM-2206, Spider toxins (iGluR subunit and Q/R editing dependent), CNQX, DNQX Cyclothiazide, PEPA, AMPAkines
AMPA-sensitive Cation and low Cl− (0.1 PCl−/PNa+)
GluR2 Q/R RNA editing reduces conductance and PCa2+/PNa+ c
Glutamate kainate-sensitive Generic GluR5-GluR6 Spider toxins (Q/R dependent), CNQX/DNQX (also block AMPA-sensitive iGluR) Concanavalin A
GluR5 Cation (S)-5-iodowillardiine, (S)-ATPA, LY339434 LY382884, LY294486, NS-102, SYM-2081 (desensitizer)
GluR6 Cation, Q/R RNA editing reduces PCa2+/PNa+ and increases PCl−/PNa+ NS-102, SYM-2081 (desensitizer)
GluR6/KA 5-Iodowillardiine (weak)
Glutamate NMDA-sensitive NR1 (glycine coagonist site) Kynurenic acid derivatives, quinoxalinediones, phthalazinediones, benzazepinediones
NR1/NR2 generic Cation; high PCa2+ NMDA, aspartate Positive: arachidonic acid, PACAP
Coagonist: glycine Mg2+, AP5, AP7; MK801 Negative: dynorphin, H+, Zn2+
NR1/NR2A Cation; moderate–high PCa2+, low PCl− 10-fold less sensitive to glycine site antagonists High Zn2+ and dynorphin sensitivity
NR1/NR2B Cation; high PCa2+ CGP 61594 glycine site antagonist, Ifenprodil, Ro 25-6981, CP 101,606
NR1/NR2C Cation; moderate PCa2+ Low Zn2+ sensitivity
NR1/NR2D
Unknown NR3A Cation Glycine alone is agonist
NR3B
ATP (purines) P2X1 Cation, high PCa2+, (PCa2+/Na+ ∼3.9) BzATP, α,β-MeATP Ip5I, TNP-ATP, suramin analogs, PPADS analogs
P2X1/5 Cation, moderate PCa2+ α-MeSATP, α,β-MeATP Weak sensitivity to TNP-ATP
P2X2 Cation, moderate PCa2+, large pore formation 2-MeSATP, ATPγS (insensitive to α,β-MeATP) PPADS Zn2+ potentiates, H+ potentiates
P2X2/3 Cation, moderate PCa2+ BzATP, α,β-MeATP, little response to Ap5A TNP-ATP, A-317491 H+ potentiates
P2X3 Cation, moderate PCa2+ BzATP, α,β-MeATP, Ap5A TNP-ATP, A-317491, suramin, PPADS Zn2+ potentiates, H+ inhibits
P2X4 Cation, large pore formation BzATP (Insensitive to suramin, PPADS) Zn2+; ivermectin
P2X5 Cation ATP (insensitive to α,β-MeATP) (Insensitive to suramin, PPADS)
P2X6 α,β-MeATP
P2X7 Cation, large pore formation Calmidazolium; KN-62
Sensory stimuli TRPA1 Mustard oil, cinnamon oil
TRPM8 Cation, moderate PCa2+ (PCa2+/Na+ 1–3) Menthol, Icilin
TRPV1 Cation, high PCa2+ (PCa2+/Na+ ∼10), PMg2+ (PMg2+/Na+ ∼5) Capsaicin, resiniferatoxin, anandamide, olvanil, SDZ-249665 Capsazepine, Iodo-resiniferatoxin, NDT-9525276, ruthenium red "Agonists', protons, temperature, may act in concert as modulators
Endogenous: anandaminde, 12-HPETE, leukotriene B4, N-arachidonoyl dopamine
TRPV3 Camphor
Proton ASIC1a Cation; Ca2+ Proton PcTx1 Zn2+ (nM inhibition), FMRFamide (potentiation)
ASIC1b Cation Proton FMRFamide (potentiation)
ASIC2a Cation, low PCa2+ Proton Zn2+ (μM potentiation)
ASIC2b Cation Proton
ASIC3 Cation Proton APETx2; Gd3+ FMRFamide, neuropeptide SF, neuropeptide FF d
ASIC4 Proton
a
Calcium permeability (PCa2+): low PCa2+: PCa2+/Na+ ≤1; moderate PCa2+: PCa2+/Na+ 1–3; high PCa2+: PCa2+/Na+ 3–20.
b
Glutamate LGIC are subject to RNA editing and alternative splicing at sites in or near the channel region. 145
c
RNA-edited forms tend to be predominant in the adult (for further discussion, see text).
d
Lower concentrations of neuropeptide FF appear selective for ASIC2a/3 heteromeric receptors. 228

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Sensing technology based on olfactory receptors

Hidefumi Mitsuno , ... Ryohei Kanzaki , in Chemical, Gas, and Biosensors for Internet of Things and Related Applications, 2019

4.3 Summary

In this chapter, we summarized the mechanisms of olfaction, especially signal transduction by ORs, in living organisms and introduced proposed sensing technologies based on their ORs. Since the mid-1990s, numerous sensing technologies using ORs have been actively researched and developed, although the studies that were introduced in this chapter represent only a subset of these technologies. Application of ORs may enable the development of innovative odorant sensor platforms that can detect target odorants with high selectivity and selectivity beyond those of conventional odor-sensing systems. Although a number of effective technologies are considered to reach sufficiently high level to allow practical use, few odorant sensors based on ORs are currently available owing to the limitations of biological materials such as problems of, for example, stability or long-term usage. Therefore improvement of such factors would be very important for the realization of practical odor sensors using ORs in the future.

We have introduced sensing technologies based on "mammalian ORs" and "insect ORs" separately. Since mammalian ORs are GPCRs and insect ORs are ligand-gated ion channels, there are the advantages and disadvantages in sensing technologies using each type of ORs in accordance with their original characteristics. For example, one of the advantages of GPCRs is to be capable of signal amplification by the metabotropic signaling pathway. Therefore it might be possible to develop more sensitive odor-sensing systems based on cells expressing mammalian ORs. Nevertheless, it would be difficult to completely reconstruct the complicated signaling pathway, which starts from the GPCRs, in heterologous cells. On the other hand, one of the advantages of ligand-gated ion channels is to be capable of quickly inducing ion influx after the interaction with ligands. Therefore use of insect ORs might lead to development of odor-sensing systems with faster response time, that is, real-time sensing systems. Thus utilization of the advantages of mammalian ORs or insect ORs would enable us to develop various types of artificial sensing systems suitable for our intended purposes.

In addition, the sensing technologies described in this chapter are applicable to numerous ORs from living organisms inhabiting various environments worldwide. The use of optimized ORs for target odorants would lead to the development of specific odorant sensor elements that detect various kinds of desired target odorants for designated applications. However, as the performance of the odorant sensing elements (e.g., sensitivity, selectivity, and specificity) is dependent on the intrinsic characteristics of the expressed ORs, it is crucial to clarify the relationships between odorants and ORs to facilitate the selection of optimized ORs. In insects, over 100 ORs have been characterized from various species. Among these, the fruit fly, D. melanogaster, has been utilized in the scientific field of olfaction as a model organism and its ORs have been comprehensively investigated by numerous researchers using various functional analysis methods, such as the fruit fly in vivo expression system (termed the "empty neuron system") or heterologous cell-expression systems. Focusing on these data, the team led by Prof. Giovanni Galizia has developed a database of odor responses (DoOR; http://neuro.uni-konstanz.de/DoOR/default.html), wherein information regarding specific odorant responses for different receptors can be obtained [94,95]. As this database expands, it will facilitate the selection of sets of ORs that are specific to target odorants and are optimized for the discrimination of defined odors. Through conjugating such information with the sensing technologies described in this chapter, it is expected that a new era will soon be attained wherein odorant sensors can be provided for the detection of desired target odorants in various situations.

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Serotonin receptor imaging by 18F-PET

Thierry Billard , ... Mathieu Verdurand , in Fluorine in Life Sciences: Pharmaceuticals, Medicinal Diagnostics, and Agrochemicals, 2019

1 Introduction

The serotonergic system plays a key modulatory role in many central nervous system (CNS) functions [1,2]. The serotonin (5-HT) receptors are phylogenetically one of the oldest systems, one of the most diverse groups of brain receptors in the human genome, and are differentially expressed in the central and peripheral nervous system (PNS), gut, blood, and cardiovascular system [1,2].

The 5-HT neurons are a relatively small population of specialized brain cells whose cell bodies are located in the raphe nuclei of the pons and upper medulla and that produce a dense network of neuronal projections [3,4]. These innervate the entire forebrain with rostral projections to the cortex, hippocampus, basal ganglia, limbic system, hypothalamus, and caudal projections to the cerebellum, medulla, and spinal cord [5]. Currently, 14 5-HT receptors have been described structurally and pharmacologically [6]. These 14 subtypes are divided into seven classes (5-HT1 to 5-HT7) according to structural and functional characteristics [7]. With the exception of the 5-HT3 receptor, a ligand-gated ion channel, all belong to the transmembrane G-protein-coupled receptor (GPCR) family. The levels of 5-HT within the synapse are controlled by the serotonin reuptake transporter, SERT. The role of SERT is to terminate monoamine serotonergic neurotransmission by removing 5-HT from the synapse.

Despite the relatively small number of 5-HT neurons in the brain, the system plays a key modulatory role in many CNS functions [8,9]. Multiple serotoninergic receptor subtypes and region-specific innervations result in a complex pattern of modulatory control over various physiological, emotional, and cognitive processes. At autoreceptors (receptors located on 5-HT neurons), 5-HT can control its own release. On heteroreceptors, 5-HT can control the release of virtually all the other major neurotransmitters in the brain. As a result of a long evolutionary history, serotonin plays a variety of roles in normal physiology, including developmental, cardiovascular, gastrointestinal, and endocrine function, sensory perception, and behaviors such as aggression, appetite, sex, sleep, mood, cognition, and memory [10]. Pathophysiologically, serotoninergic dysfunction is implicated in psychiatric disorders, such as depression, anxiety, and schizophrenia, and neurological disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD) [11–13].

The development of noninvasive brain-imaging techniques such as positron emission tomography (PET) allows researchers to study the serotonergic system in the human brain. PET imaging uses radioligands (or radiotracers), defined as radiolabeled biochemical substances, that can bind to molecular targets like brain receptors. PET radiotracers can be regarded as key molecular imaging tools, studying the function and neurochemistry of rodents, nonhuman primates, and humans [14]. PET is an important tool for elucidating the mechanisms underlying brain diseases and pharmacological processes, enabling longitudinal studies and facilitating translation between basic and clinical research. PET imaging is not only useful for clinical diagnosis and for preclinical and clinical research, but also for drug discovery and development. Indeed, developing suitable radioligands for quantitative PET imaging allows assessing if these "drug candidates" can engage their "target" [15]. The two principal outcome measures are receptor density and binding affinity of the radioligand to the receptor of interest (or receptor occupancy). PET ligands are injected in very small amounts called a "tracer dose" (less than 5% of receptors must be occupied) so that no pharmacological effects are triggered. Despite this "tracer dose," radioligands are still able to image molecular targets present in nanomolar to picomolar (nM to pM) quantities in the brain, thanks to radiolabeling with high specific radioactivity, making PET imaging a very sensitive technique. A prospective radioligand must comply with several basic criteria to be effective for PET imaging of brain receptors [16,17]: high selectivity (at least 20–100-fold) and high affinity (nM range) for the receptor, low or moderate lipophilicity (logD   =   1−3 for blood–brain barrier [BBB] penetration and to avoid excessive nonspecific binding), low metabolism (or noninterfering radioactive metabolites), and amenability to labeling with a positron-emitting isotope. Among the positron-emitting isotopes, oxygen-15 (T1/2  =   2.037   min), nitrogen-13 (T1/2  = 9.96   min), carbon-11 (T1/2  =   20.34   min), and fluorine-18 (T1/2  =   109.77   min) are the common nuclei produced by means of a cyclotron, reported for neuroimaging with PET. In this chapter, we will focus on fluorine-18 labeled radioligands that target serotonin receptors. Radiosyntheses with fluorine-18 have not represented the primary strategy for radiolabeling purposes, as inserting a carbon-11 atom in chemical structures is theoretically easier. While it is true that labeling with carbon-11 is easier, the greater advantage of carbon-11 is that the resulting radiotracer is identical to the unlabeled analog (i.e., the efficacy of the drug is not changed by chemical modification with a different isotope). However, fluorine-18 presents the major advantage of having a longer half-life than carbon-11 and thus can be shipped to greater distances from the production cyclotron.

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Natural Compounds (Small Molecules) as Potential and Real Drugs of Alzheimer's Disease

Lucie Cahlíková , ... Lubomír Opletal , in Studies in Natural Products Chemistry, 2014

Change in ACh Metabolism (AChE and BuChE Influence)

ACh has an exclusive role as a neurotransmitter, not only because it is involved in neurotransmission and memory formation but also intervenes in the production and secretion of other neurotransmitters (e.g., glutamate, glycine, and dopamine), in the CNS. One of the theories of this pathophysiology—cholinergic theory—which was published 40 years ago [80] is still discussed and elaborated.

ACh, which is formed in the presynaptic area of neuron by action of ChAT (EC 2.3.1.6), is applied on various receptors in the body, in the brain; however, it plays an essential role of neurotransmitter [81] significantly forming memory and transmission of memory information on two main types of receptors—muscarinic acetylcholine receptor (mAChR; activation of G-proteins), nicotinic acetylcholine receptor (nAChR; form of ligand-gated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction), or more precisely their subtypes. In the synaptic cleft, ACh is degraded by AChE (and under pathological conditions in AD and dementia with Lewy bodies also BuChE) to choline and acetic acid. In AD, presynaptic section of acetylcholinergic neuron is affected—entry of precursors into neurons, ACh synthesis, and release. Attention, stimulus detection, perceptual processing, and information encoding are allowed by stimulation of the cholinergic system. Although the memory consolidation is impaired by cholinergic activation, it is not clear whether information retrieval may be improved [82]. ACh receptors are found throughout the CNS in a huge amount, especially in the cortex, thalamus, hippocampus, and various nuclei in the basal forebrain. Forebrain cholinergic systems are essential for cognitive processes [83]. It has been proved that entorhinal cortex (EC) is one of the first locations of degeneration [84]. EC acts as a hub in a large network for memory and navigation. This is the main interface between the hippocampus and neocortex. Combination of EC–hippocampus plays an important role in the existence of an autobiographical/declarative/episodic memory, but especially in spatial memory, including its formation and consolidation. This cortex is the first of the regions of disability at the onset of AD [83]. There is a link between cholinergic activation and APP metabolism: lesions of cholinergic nuclei show a rapid increase of cortical APP and CFS. Decrease of cholinergic transmission in AD leads to amyloidogenic metabolism and contributes to cognitive dysfunctions [85].

The level of ACh is regulated by ChEs (AChE and BuChE). These serine esterases are present in various tissues in the human body, in which they fulfill the role of a hydrolytic enzyme. AChE (EC 3.1.1.7) cleaves ACh to the basic components and maintains a metabolic balance. Cleavage of ACh in the CNS is normally an important factor for the regeneration of neuron. The second enzyme, which appears to be concentrated in the brain of AD patients, is BuChE (EC 3.1.1.8). It is present in the CNS in other regions than AChE, in particular in endothelial cells, neurons, and glia, and it has been proved that it is synthesized in the brain [86]. BuChE is located in neurons, glia, neuritic plaques, and tangles. When decreasing the activity of AChE, BuChE may replace it [85]. Human brain and liver BuChE and hydrophilic plasmatic G4 BuChE have an identical amino acid sequence.

Both AChE and BuChE, present mainly in the form of tetramer G4, but also dimer G2 and monomer G1, which are secreted to the tissues as hydrophilic form, occur in the tissues. The major amount of AChE in the CNS is amphiphilic, containing both hydrophilic and hydrophobic regions [86,87]. In the human brain, there are two structural types of AChE: type G4 (highly prevalent) and monomer G1 (in minor amount). Proportion of G1 is significantly increased in AD. In this case, BuChE pathologically generated in mobilized neuroglia forming inflammatory edge of Aβ plaque is also involved in the degradation of ACh. Both enzymes have the different ability to hydrolyze substrates. These differences are probably caused by changes in the arrangement of amino acids in the aromatic cavity. Altered expression of AChE in the brain of patients with AD suggests that AChE activity increases at the periphery of amyloid plaque (around the Aβ plaques) and Aβ may actually affect the levels of AChE [87]. It has been found that different forms of AChE in the brain and cerebrospinal fluid of patients with AD are changed in connection with abnormal glycosylation [88]. The role of AChE in neurodegenerative diseases is relatively well known, the role of BuChE is still not completely clarified. This pseudocholinesterase does not have natural substrates in organism [87].

ChEs fulfill other physiological roles than just degradation of ACh, especially noncholinergic trophic functions. In the brain, therefore, they are present even in regions other than the cholinergic terminals. They can modify APP metabolism and lead to increased formation of Aβ. AChE induces formation of Aβ fibrils; hydrophobic peripheral anionic site of the enzyme is responsible for this activity.

It appeared that cholinergic transmission performed other new functions. It can modulate various aspects of immune function, both innate and adaptive. Cholinergic transmission influences immune cell proliferation, cytokine production, differentiation of T-helper cells, and antigen presentation. These effects are mediated by cholinergic mAChR and nAChR and other cholinergic components present in immune cells, for example, α7 nAChR has the ability to induce anti-inflammatory activity [89]. This is probably one of the reasons why acetylcholinesterase inhibitors (AChEIs) act far broader than just to the inhibition of AChE.

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Classical Targets in Drug Discovery

Benjamin E. Blass , in Basic Principles of Drug Discovery and Development, 2015

Ion Channels

Although living organisms are often viewed as complex machines run by chemical reactions, there are many critical functions in living organisms that cannot be accomplished solely by chemical means. In many cases, the generation of an electrical impulse or voltage gradient is required. Ion channels, transmembrane protein assemblies that regulate the flow of ions across biological barriers, play a major role in this process. Nerve impulse transmission, 48 muscle contraction, 49 and cardiovascular function, especially heart rate and rhythm, 50 all depend on the exquisitely balanced flow of ions created by a network of ion channels opening and closing in a coordinated fashion. T-cell activation in an immune response, 51 hormonal secretions (e.g., insulin), cellular proliferation (e.g., lymphocytes, cancer cells 52 ), and even cell volume regulation 53 are all impacted by various ion channels. These proteins also play a major role in preventing cellular depolarization by counter balancing the impact of Na+-coupled transporters (e.g., glucose transporter, amino acid transporters) and Ca+-signaling events. 54 Modulation of ion channel activity has provided a number of important drugs and lethal toxins (Figure 3.28). Improper ion channel function has been implicated in a number of important disease states (channelopathies, Table 3.1) such as cystic fibrosis, 55 epilepsy, 56 and long QT syndrome.

Figure 3.28. (a) Norvasc® (Amlodipine), an antihypertensive agent that blocks calcium channels. (b) Amiodarone, an antiarrhythmic agent that blocks potassium channels. (c) Novocaine, a local anesthetic that blocks sodium channels. (d) Glipizide, an antidiabetic that blocks potassium channels in the pancreatic β-cells. (e) Phenytoin, an antiepileptic that blocks sodium channels. (f) Tetrodotoxin, a pufferfish toxin that blocks sodium channels, that is 100 times more lethal than cyanide.

Table 3.1. Disease States Associated with Ion Channels

Disease Channel Gene
Arrhythmia Nav1.5 SCN5A
Arrhythmia Kv1.5 KCNA5
Cystic fibrosis CFTR CFTR
Diabetes mellitus Kir6.2 KCNJ11
Epilepsy KCNQ2 KCNQ2
Epilepsy Nav1.2 SCN2A
Episodic Ataxia Kv1.1 KCNA1
Erythromelalgia Nav1.7 SCN9A
Migraine Cav2.1 CACNA1A
Fibromyalgia Nav1.7 SCN9A
Long QT syndrome hERG KCNH2
Malignant hyperthermia Cav1.1 CACNA1S
Neuropathic pain TrpV1 TRPV1
Osteoporosis ClC-7 CLCN7
Timothy syndrome Cav1.2 CACNA2

Modern efforts to understand the function of ion channels and the role of electrical currents in biological processes predate the age of modern drug discovery. The concept of "bioelectricity" was, in fact, explored as early the mid-1840s by Carlo Matteucci 57 and Emil du Bois-Reymond, 58 who separately studied the impact of electrical currents on nerve and muscle tissue. Their experiments provided support for the role of electricity in biological processes, but did not provide an understanding of how living tissue could support electrical conductance. Hermann von Helmholtz provided additional insight into the role of electricity in living organisms in 1850 by demonstrating that the electrical signals traveled far slower in living tissue than they did in metal wires. His experiments indicated that simple conductance was not a viable explanation and suggested that an underlying chemical process might be involved. 59

Nearly 50 years later, in 1902, Julius Bernstein introduced the "membrane theory" as a general explanation for bioelectrical events in living organism. The concept of semipermeable membranes was relatively new at the time, and Bernstein hypothesized that nerves and muscle were surrounded by semipermeable membranes. He further suggested that the electrical gradients across cellular barriers were the result of differences in ion concentrations on the inside and outside of cells created by the selective movement of ions across the cellular barrier. Bernstein referred to this effect as the formation of an "electrical double layer," but it is more often referred to as a "membrane potential" or "membrane voltage." 60 While his basic premise proved to be correct, a full understanding of the true nature of ion channel would remain a mystery for most of the twentieth century. In fact, at the dawn of the biotechnology revolution, Armstrong and his colleagues summed up the situation by writing "The ionic channels of nerve membrane and the gates that control ion movement through them are widely supposed to be composed of protein, but there is surprisingly little evidence on the question." 61

The biotechnological tools and computer technology that became available in the last 30 years of the twentieth century provided the tools necessary to finally unravel the mystery of ion channels. Recombinant technologies, transfection methods, and advances in electronics allowed Erwin Neher and Bert Sakmann to develop the "patch clamp" method for studying ion channels directly. Prior to the advent of these technologies, ion channels experiments were limited to an analysis of macroscopic currents of a naturally occurring cell. Biotechnology provided the tools necessary to create cell lines that expressed a single type of ion channel. Neher and Sakmann's "patch clamp" technique placed a salt water containing micropipette against the surface of a single cell and measured the electrical currents generated by the flow of ions through an ion channel in much the same manner as electrical current are measured across a wire (Figure 3.29). Electrical signal amplification technologies made it possible to study the action of a single ion channel in a cell, providing for the first time a direct measurement of ion channel activity. 62 Neher and Sakmann were awarded the Nobel Prize in physiology or medicine in 1991 in recognition of the importance of their work. 63 Modernized version of this method 64 remains the gold standard for the study of ion channels and is one of only a few technologies available for the direct study of individual proteins.

Figure 3.29. A basic patch clamp system consists of a micropipette with an opening on the order of 1 μm pressed against the surface of a cell. The inside of the micropipette covers a limited number of ion channels, and a seal with high electrical resistance ("gigaohm seal") is created by suction on the surface of the cell. An electrode, salt solution inside the micropipette, and the appropriate electrical amplification and monitoring systems can then be employed to either maintain a constant voltage while monitoring current or maintain a constant current while monitoring changes in membrane potential in the presence of test compounds.

Structural details of ion channels also began to emerge as a result of the technological advances that occurred at the end of the twentieth century. Protein sequences that encoded various ion channels were determined, and advances in molecular modeling were employed to predict the structural feature that would allow a transmembrane protein to transport a charged species through a lipophilic barrier. While various structural models were proposed, direct crystallographic evidence of the structural details of ion channels did not become available until 1998. Roderick Mackinnon's X-ray crystal structure of potassium channel designated KcsA from the soil bacteria Streptomyces lividans (Figure 3.30) provided the first complete view of an intact ion channel, an effort which won him the Nobel Prize in chemistry in 2003. 65a,65b As of 2013, the RCSB Protein Data Bank contains over 3100 crystal structures categorized as ion channels.

Figure 3.30. (a) Side view of X-ray crystal structure of Streptomyces lividans KcsA potassium channel. (b) Top view of X-ray crystal structure of Streptomyces lividans KcsA potassium channel.

 RCSB 1BL8.

Over 300 ion channels have been identified to date, and while their individual structures are designed to meet the need of their specific function, there are a number of common features that can be described. Much like the GPCRs, ion channels are integral membrane proteins comprised of a series of transmembrane domains that are linked by extracellular and intracellular loops. The majority of ion channels are multiunit assemblies, and functional channels can only be formed when multiple, compatible protein structure come together to form the active channel. Subunit homogeneity is not required, however, and this can lead to subtle differences in function. In many cases, the pore section of the channel is only wide enough for passage of a single ion at a time and passage is restricted to a single ion type. The selectivity of channels for a specific ion type is driven by the structural features of the proteins that makes up the channel. There are a number of sodium channels that cannot facilitate the movement of potassium ions across a membrane. Conversely, even though sodium ions are significantly smaller than potassium ions, there are potassium channels that do not accommodate sodium ions. To date, ion channels have been identified that support the flow of sodium, calcium, potassium, chloride, and hydrogen ions.

Gating Mechanisms

Another key feature of ion channels is the mechanism through which they are activated, also referred to as a "gating mechanism." In general, ion channels remain closed in the absence of an external stimulus. The action of a stimulating event or agent causes conformational changes in the proteins, opening the "gate" and allowing the flow of ions across a biological barrier. When the stimulus is removed, the channel reverts back to its closed state, stopping the flow of ions. The gating of a channel can be dependent on the presence of a ligand, environmental pH, temperature, or membrane voltage differences. Ion channels that are mechanosensitive (altered by mechanical deformations of a membrane such as tension and curvature changes) and light sensitive have also been identified. Ligand-gated and voltage-gated channels are the most extensively studied types of channels, and an examination of their modes of action can serve as a basis for understanding the nature of the remaining types of gating mechanisms.

Ligand-Gated Channels

Ligand-gated channels are activated when a ligand interacts with a specific binding site on the channel. When the ligand is removed or displaced, the channel closes, terminating the flow of ions (Figure 3.31). The nicotinic acetylcholine receptor (nAChR), a key player in neurotransmission, is a prototypical example. It is comprised of five 290 kDa subunits that arrange symmetrically to form a central pore, and each subunit contains four transmembrane domains that contribute to the overall structure of the channel. In the absence of a ligand such as acetylcholine, the pore is closed to traffic. When acetylcholine interacts with the binding site on the extracellular surface of the cell, however, the proteins undergo conformational changes that cause the channel to open, allowing the flow of ions through the membrane, creating an electrical signal. Removal of the agonist leads to a rearrangement of the proteins back to their closed state, terminating the signal.

Figure 3.31. Ligand-gated channels are closed in the absence of a ligand (red). Binding of the ligand to channel leads to conformational changes that cause the channel to open, allowing migration of suitable ions through the channel. Removal of the ligand causes the channel to close, stopping ion flow. Ligand-gated channels can be activated by synthetic ligands or blocked with antagonists (compounds that bind to the ligand-binding site, but do not lead to channel opening). Direct blockade of the channel is also possible.

Modulation of ligand-gated channel activity can be accomplished in a number of ways. Activation of the channel can be accomplished with compounds that mimic the natural ligand. Nicotine, for example, is an agonist of nAChR, and its activity at this ligand-gated channel is at least partially responsible for activation of reward system of the brain by tobacco products. 66 The smoking-cessation medication Chantix® (Varenicline) is a partial agonist of nAChR, and provides a lower level of channel activity upon binding than nicotine. 67 It has been successfully employed to decrease the cravings and the pleasurable effects of nicotine, as it competes with nicotine for the same binding site on nAChR (Figure 3.32). 68

Figure 3.32. The acetylcholine-binding protein (AChBP) has been employed as a model system for the nicotinic acetylcholine receptor (nAChR). The crystal structure shows a ligand bound to the acetylcholine-binding sites. (a) Top view. (b) Side view. Nicotine and Chantix® (Varenicline) both bind to nAChR, but differential receptor responses provide an opportunity for therapeutic intervention in nicotine addiction.

 RCSB 2XNT.

Blocking activity of a ligand-gated channel is also possible. Compounds that compete for the natural ligands binding site, but do not cause the conformational changes associated with ligand binding will prevent opening of the channel, acting as functional antagonists. Similarly, compounds that bind to an allosteric site and either stabilize the closed form of the channel or cause conformational changes that prevent binding of the natural ligand also act as functional antagonists. The α-neurotoxins, for example, are a family of peptides from snake venom that are antagonists of postsynaptic nAChR located in neuromuscular synapses (Figure 3.33). These relatively small proteins (60–75 amino acid residues) tightly bind to nAChR in skeletal muscle, preventing acetylcholine-mediated neurotransmission through the opening of nAChR, causing paralysis in snake bite victims. 69 Of course, it is also possible to block the channel itself. In this case, the presence of an ligand opens the channel, but ion flow is prevented and the associated cellular response does not occur.

Figure 3.33. (a) Crystal structure of the extracellular domain of the nicotinic acetylcholine receptor 1 subunit (green) bound to α-bungarotoxin (red), from the venom of the snake Bungarus multicinctus, at 1.9 Å resolution. (b) Close-up of the binding-site interactions.

 RCSB 2QC1.

Allosteric activation of ligand-gated ion channels is also possible. The γ-aminobutyric acid type A receptor (GABAAR), for example, is a ligand-gated chloride channel that plays a critical role in the central nervous system. Activation of GABAAR by the endogenous ligand γ-aminobutyric acid (GABA, Figure 3.34(a)), an inhibitory neurotransmitter, opens the chloride channel of GABAAR, which leads to hyperpolarization of neurons, inhibiting neurotransmission. 70 The presence of benzodiazepines and barbiturates increases the activity of GABAAR. When compounds such as phenobarbital 71 and lorazepam 72 (Figure 3.34) bind to their respective allosteric sites on GABAAR, they cause a conformational change in its structure generating a configuration with significantly higher affinity for GABA. This, in turn, increases the frequency of the opening of the associated chloride channel, increasing chloride transfer across the membrane, hyperpolarizing the associated neuron.

Figure 3.34. (a) γ-aminobutyric acid (GABA). (b) Phenobarbital, a barbiturate. (c) Lorazepam, a benzodiazepine.

Voltage-Gated Channels

The voltage-gated channels represent another major class of ion channels. Unlike ligand-gated channels, voltage-gated channels have no natural ligand. They open and close as a result of changes in membrane potential produced as electrical currents move through biological systems. Voltage-sensing domains allow these channels to be exquisitely sensitive to changes in membrane potential, making them ideally suited for the propagation of nerve impulse through axons, muscle contraction, and cardiac function. The opening and subsequent closing of ion channels give rise to action potentials (Figures 3.35 and 3.36), the rapid rise and fall of the cellular membrane potential, which gives rise to the aforementioned functions. Mechanistically, voltage-gated channels are closed when the membrane electrical potential is at its resting potential. It is worth noting at this point that different types of voltage-gated channels will have different resting potentials, and thus will activate at different membrane potentials. If an electrical impulse (or other stimulus) causes the membrane potential to rise above the membrane threshold for activation, the channel will open via a series of conformational changes, causing a rapid change in membrane polarization via ion flow across the membrane. This leads to hyperpolarization of the cellular membrane, triggering inactivation of the voltage-gated channel through another series of conformational changes, stopping ion flow. Once the voltage-gated channel is inactivated by membrane hyperpolarization, it will not respond to another stimulus until the membrane potential has been "reset" to the resting potential, typically by the action of another voltage-gated channel with a different set of activation and deactivation parameters. This time period is referred to as a refractory period. Once the membrane potential has been reset, the voltage-gated channel reverts to its original conformation, ready for the next stimulus. 73

Figure 3.35. In the resting state, voltage-gated channels are closed. When the membrane potential reaches the proper level, conformational changes cause the channel to open, allowing the flow of ions across the membrane. This quickly leads to a hyperpolarized state, which induces another set of conformational changes that inactivate the channel. The channel cannot reopen until the resting potential is restored and its conformation shifts back to the closed resting potential state.

Figure 3.36. An electrical diagram of voltage-gated ion channel action over time, also referred to as an action potential, provides another view of channel activity. A stimulus must rise above the gating threshold in order to induce channel opening. Rapid depolarization caused by ion flow through the channel leads to hyperpolarization and closing of the inactivation gate. The inactivation gate remains closed until the membrane potential is reset by the action of opposing forces. Stimulation of the channel will not evoke a response until this "refractory period" has ended and the resting potential is restored.

Since voltage-gated channels do not have a natural ligand, modulation of their activity by replacing a natural ligand with either an agonist or antagonist is not an option. It is, however, possible to manipulate their activity in other ways. Blocking the open channel directly (Figure 3.37(a)) is perhaps the most obvious route to suppressing channel activity. Flecainide (Figure 3.37(e)), for example, blocks Nav1.5, a voltage-gated sodium channel that plays a major role in cardiac function, and is useful for the treatment of arrhythmia and the prevention of tachycardia. 74 Similarly, the scorpion venom Margatoxin (Figure 3.37(f)) blocks the Kv1.3 channel, a voltage-gated potassium channel found in a variety of cell types, including neuronal cells. Kv1.3 channel blockade alters the membrane potential of neuronal cells, leading to changes in the time required for action potential conduction and nerve transmission. The same channel is also present in T-lymphocytes, and Kv1.3 blockade can induce immunosuppression by decreasing T-cell proliferation. 75

Figure 3.37. (a) Direct blockade of the open configuration of the channel by a drug (red) prevents ion flow through the pore. (b) Stabilization of the closed form of the channel by a compound (red) effectively increases the activation threshold, decreasing channel activity. (c) The hyperpolarized state of a voltage-gated channel can be stabilized by a drug (red), maintaining the position of the inactivation gate, slowing conformational changes required to reach the closed resting state. (d) Interaction of a drug (red) with the open channel can stabilize the open configuration, leading to increased ion flow across a cellular barrier. (e) Flecainide, a Nav1.5 blocker and antiarrhythmic agent. (f) Margatoxin, a 39-amino acid peptide found in the venom of Centruroides margaritatus (the Central American Bark Scorpion) and Kv1.3 channel blocker. (g) Retigabine, a Kv7.2 and Kv7.3 channel opener and antiseizure agent.

Alternative methods of modulating voltage-gated ion channel activity depend on the interaction of compounds with the protein at sites other than the pore region. This could be considered a form of allosteric modulation, as the "active site" of an ion channel is the pore through which ions move. As previously mentioned, the opening, hyperpolarization, and subsequent resetting of voltage-gated channel are accompanied by conformational changes, so compounds that interfere with these changes will have an impact on functional activity of the channel. Compounds that stabilize the closed resting potential conformation (Figure 3.37(b)), for example, will prevent channel activation, blocking activity. In a similar manner, compounds that stabilize the inactivated hyperpolarized state of the channel will prevent resetting of the channel after hyperpolarization (Figure 3.37(c)), preventing further channel activity. Conversely, compounds that stabilize the open conformation of a voltage-gated channel will increase ion channel activity (Figure 3.37(d)). Retigabine (Figure 3.37(g)), an anticonvulsant useful for the treatment of epilepsy and seizures, for example, stabilizes the open forms of voltage-gated potassium channels Kv7.2 and Kv7.3, leading to increased potassium flow and seizure suppression. 76

Other Gating Mechanisms

Ligand gating and voltage gating are perhaps the most well-studied gating mechanisms, but there are other gating mechanisms that play important roles in both normal and disease states. Temperature-gated channels open and close based on distinct thermal thresholds and form the basis for the sensation of hot and cold. 77a,77b Similarly, mechanosensitive ion channels are activated by mechanical deformations of membranes such as increased tension or changes in curvature and play a part in the sensation of touch. 78 pH gating has also been observed, 79a,79b and in fact the KcsA potassium channel from Streptomyces lividans crystallized by Mackinnon in 1998 is pH-gated. Irrespective of the gating mechanism, however, the opening and closing of channels is intimately connected to conformational changes in the protein, and methods for modulating activity of these gating mechanisms are similar to those described for ligand- and voltage-gated channels.

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Origin(s) of biological chirality

Gyula Pályi , in Biological Chirality, 2020

Oxygenic metabolism and consequences

The identification of isoprenoids in ∼2.7 Gyr-old rocks was a very important result. Beyond the significance for biological chirality, it was regarded also as a sign of oxygenic metabolism by cyanobacteria, especially because of the presence of sterane derivatives. This interpretation shifted the start of oxygenic photosynthesis in the biosphere (Bekker et al., 2004; Fischer and Pietruszka, 2010) back into the past by more than ∼200 Myr (Eigenbrode and Freeman, 2006; Kopp et al., 2005; Planavsky et al., 2014). Later it turned out, that the initial evolution (Lyons, 2014) toward oxygen-based, sterane-producing metabolism started (or, could have been started) with very low oxygen concentrations (Anbar et al., 2007; Farquhar et al., 2011; Rasmussen et al., 2008; Waldbauer et al., 2011) or in limited O2-rich regions of the Earth (Riding et al., 2014). 2-Methylhopanoids were believed to be very characteristic biomarkers of cyanobacteria (Summons et al., 1999), however, later this marker was found to be produced also by other microorganisms (Newman et al., 2016; Rashby et al., 2007; Ricci et al., 2014, 2015 Ricci et al., 2014 Ricci et al., 2015 ; Welander et al., 2010). In the last decade some other similar molecular fossil ensembles (Craig et al., 2013; Eigenbrode et al., 2008; Satkoski et al., 2015 Waldbauer et al., 2009) have been found from the late Archean (2.72–2.56   Gyr   B.P.), indicating that the change of metabolism was fairly general as well as the presence of "survived" chiral excesses (primarily: hopanes and steranes). In a sophisticated δ13C isotopic study the biogenic origin of depositional kerogen (main source of hopanes and steranes) was proved (Eigenbrode and Freeman, 2006). One of the reasons of the richness of data on hopanoids may derive from the fact, that this class of isoprenoids appear to be the most abundant organic material on Earth (∼109  kg carbon, nearly the same quantity as the total biomass of all extant living beings today: Bucher and Grapes, 2011; Fago et al., 1991; Ourisson, 1994; Ourisson and Albrecht, 1992). In present-day living organisms hopanoids and steroids play important biological functions in ligand-gated ion channels (Barrantes and Fantini, 2016) or in membrane architecture (Sáenz et al., 2015). It should be, however, pointed out repeatedly that the ideas about the function and specificity of these biomarkers in billion-years-ago lived organisms is based mainly upon observations of present-day life (e.g., Newman et al., 2016; Rasmussen et al., 2008; Rohmer, 2008; Summons and Jahnke, 1992; Wille et al., 2007).

It should also be mentioned, that various isotope ratio-based studies gave results which were different from the biomarker ages for oxygenic photosynthesis (Bekker et al., 2004; Buick, 1992; Cowe et al., 2013; Des Marais, 2000; Frei et al., 2009; Holland, 2004; Kasting, 1993; Nisbet et al., 2007; Ourisson et al., 1987; Planavsky et al., 2014; Rasmussen and Buick, 1999; Rasmussen et al., 2008; Satkoski et al., 2015/2017; Schirrmeister et al., 2016; Summons et al., 2006; Vogelin et al., 2010; Wille et al., 2007; Xiong et al., 2000). Opinions show some divergence also in this field (c.f also French et al., 2015; Fischer et al., 2016; Kamber et al., 2014; Lyons et al., 2014; Rohmer, 2010; Sessions et al., 2009; Shih, 2015; Shih et al., 2017; Ward et al., 2016). The enrichment of oxygen in the terrestrial atmosphere and oceans to levels which are close to present-day observed concentrations (as Great Oxygenation Event, GOE) cca. 2.5   Gyr   B. P., or somewhat less, around 2.35 Gyr (see e.g., Kasting, 2001; Luo et al., 2016). This dating was step-by-step shifted to 3.2   Gyr   B. P. by the above cited isotopic studies (Satkoski et al., 2015; Schirrmeister et al., 2016). This date is fairly near to the actually accepted date of the origin(s) of life. It is believed, that sterane derivatives become more important (abundant) only with the start of photochemical oxygen based metabolism (GOE), but a kind of "coevolution" of these compound classes appears to be also a reasonable scenario (Barrantes and Fantini, 2016). Methylhopanes were interpreted also as molecular signals of early (late Archean) aerobiosis (Eigenbrode and Freeman, 2006; Eigenbrode et al., 2008). Another approach to this important problem suggests, that dissolved O2 in oceanic seawater was present in higher concentration prior to atmospheric enrichment of oxygen (Waldbauer et al., 2011). This could explain the early appearance of sterane derivatives in the early Precambrian molecular fossil record and let accepted the connection of these biomarkers to oxygenic metabolism.

The key element in the uncertainty about the beginning of the oxygenic photochemical metabolism is the divergence of Cyanobacteria from other microorganisms. This problem has been approached by various methods, including geo- and geobiochemistry (Brocks et al., 1999, Butterfield, 2015; Cowe et al., 2013; Fischer et al., 2016; Johnson et al., 2014; Kopp et al., 2005; Lazcano and Miller, 1994; Riding et al., 2014; Rosing and Frei, 2004; Ward et al., 2016). The deviation of Cyanobacteria from their ancestors spans, in the relevant literature, about one billion years. This picture became even more complicated by the discovery of a closely related sister phylum, termed Melainabacteria (Di Renzi et al., 2013; Soo et al., 2014), which belongs to the same group of phyla but which are nonphotosynthetic microorganisms. A very recent genomic, "molecular clock" study concluded, that aerobic respiration of the Cyanobacteria phylum was acquired at least twice and this happened after the oxygenic phototrophy (Shih et al., 2017). These results put into different light the earlier considerations about the biomarker (hopanoids, steranes) based considerations, at least from biological viewpoint. However, we do not think that the message of the diastereomeric/chiral structures of the eldest fossil molecules should be reconsidered, especially because the dating of these organic materials was based (also) on different, isotopic, age determination.

It appears that an important consequence of the appearance of oxygenic metabolism is that it has (or, could have) triggered the evolution of animals (Mills and Canefield, 2014).

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