Annual Review of Pharmacology and Toxicology - Volume 60, 2020

“Ion Channels and Neuropharmacology: From the Past to the Future” is the main theme of articles in Volume 60 of the Annual Review of Pharmacology and Toxicology. Reviews in this volume discuss a wide spectrum of therapeutically relevant ion channels and GPCRs with a particular emphasis on structural studies that elucidate drug binding sites and mechanisms of action. The regulation of ion channels by second messengers, including Ca²⁺ and cyclic AMP, and lipid mediators is also highly relevant to several of the ion channels discussed, including KCNQ channels, HCN channels, L-type Ca²⁺ channels, and AMPA receptors, as well as the aquaporin channels. Molecular identification of exactly where drugs bind in the structure not only elucidates their mechanism of action but also aids future structure-based drug discovery efforts to focus on relevant pharmacophores. The ion channels discussed here are targets for multiple nervous system diseases, including epilepsy and neuropathic pain. This theme complements several previous themes, including “New Therapeutic Targets,” “New Approaches for Studying Drug and Toxicant Action: Applications to Drug Discovery and Development,” and “New Methods and Novel Therapeutic Approaches in Pharmacology and Toxicology.”

Here, I recount some adventures that I and my colleagues have had over some 60 years since 1957 studying the effects of drugs and neurotransmitters on neuronal excitability and ion channel function, largely, but not exclusively, using sympathetic neurons as test objects. Studies include effects of centrally active drugs on sympathetic transmission; neuronal action and neuroglial uptake of GABA in the ganglia and brain; the action of muscarinic agonists on sympathetic neurons; the action of bradykinin on neuroblastoma-derived cells; and the identification of M-current as a target for muscarinic action, including experiments to determine its distribution, molecular composition, neurotransmitter sensitivity, and intracellular regulation by phospholipids and their hydrolysis products. Techniques used include electrophysiological recording (extracellular, intracellular microelectrode, whole-cell, and single-channel patch-clamp), autoradiography, messenger RNA and complementary DNA expression, antibody injection, antisense knockdown, and membrane-targeted lipidated peptides. I finish with some recollections about my scientific career, funding, and changes in laboratory life and pharmacology research over the past 60 years.

Ion channels and G protein–coupled receptors (GPCRs) are regulated by lipids in their membrane environment. Structural studies combined with biophysical and molecular simulation investigations reveal interaction sites for specific lipids on membrane protein structures. For K channels, PIP2 plays a key role in regulating Kv and Kir channels. Likewise, several recent cryo-EM structures of TRP channels have revealed bound lipids, including PIP2 and cholesterol. Among the pentameric ligand-gated ion channel family, structural and biophysical studies suggest the M4 TM helix may act as a lipid sensor, e.g., forming part of the binding sites for neurosteroids on the GABAA receptor. Structures of GPCRs have revealed multiple cholesterol sites, which may modulate both receptor dynamics and receptor oligomerization. PIP2 also interacts with GPCRs and may modulate their interactions with G proteins. Overall, it is evident that multiple lipid binding sites exist on channels and receptors that modulate their function allosterically and are potential druggable sites.

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein–coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR–G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR–G protein selectivity, how accessory membrane proteins alter receptor–ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

G protein–coupled receptors (GPCRs) mediate the effects of numerous hormones and neurotransmitters and are major pharmacological targets. Classical studies with crude cell lysates or membrane preparations have identified the main biochemical steps involved in GPCR signaling. Moreover, recent studies on purified proteins have provided astounding details at the atomic level of the 3-D structures of receptors in multiple conformations, including in complex with G proteins and β-arrestins. However, several fundamental questions remain regarding the highly specific effects and rapid nature of GPCR signaling. Recent developments in single-molecule microscopy are providing important contributions to answering these questions. Overall, single-molecule studies have revealed unexpected levels of complexity, with receptors existing in different conformations and dynamically interacting among themselves, their signaling partners, and structural elements of the plasma membrane to produce highly localized signals in space and time. These findings may provide a new basis to develop innovative strategies to modulate GPCR function for pharmacological purposes.

Recent advances in our understanding of the structure and function of class B G protein–coupled receptors (GPCRs) provide multiple opportunities for targeted development of allosteric modulators. Given the pleiotropic signaling patterns emanating from these receptors in response to a variety of natural agonist ligands, modulators have the potential to sculpt the responses to meet distinct needs of different groups of patients. In this review, we provide insights into how this family of GPCRs differs from the rest of the superfamily, how orthosteric agonists bind and activate these receptors, the potential for allosteric modulators to interact with various regions of these targets, and the allosteric influence of endogenous proteins on the pharmacology of these receptors, all of which are important considerations when developing new therapies.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that critically modulate neuronal activity. Four HCN subunits (HCN1–4) have been cloned, each having a unique expression profile and distinctive effects on neuronal excitability within the brain. Consistent with this, the expression and function of these subunits are altered in diverse ways in neurological disorders. Here, we review current knowledge on the structure and distribution of the individual HCN channel isoforms, their effects on neuronal activity under physiological conditions, and how their expression and function are altered in neurological disorders, particularly epilepsy, neuropathic pain, and affective disorders. We discuss the suitability of HCN channels as therapeutic targets and how drugs might be strategically designed to specifically act on particular isoforms. We conclude that medicines that target individual HCN isoforms and/or their auxiliary subunit, TRIP8b, may provide valuable means of treating distinct neurological conditions.

Voltage-gated sodium and calcium channels are evolutionarily related transmembrane signaling proteins that initiate action potentials, neurotransmission, excitation-contraction coupling, and other physiological processes. Genetic or acquired dysfunction of these proteins causes numerous diseases, termed channelopathies, and sodium and calcium channels are the molecular targets for several major classes of drugs. Recent advances in the structural biology of these proteins using X-ray crystallography and cryo-electron microscopy have given new insights into the molecular basis for their function and pharmacology. Here we review this recent literature and integrate findings on sodium and calcium channels to reveal the structural basis for their voltage-dependent activation, fast and slow inactivation, ion conductance and selectivity, and complex pharmacology at the atomic level. We conclude with the theme that new understanding of the diseases and therapeutics of these channels will be derived from application of the emerging structural principles from these recent structural analyses.

Formation of signaling complexes is crucial for the orchestration of fast, efficient, and specific signal transduction. Pharmacological disruption of defined signaling complexes has the potential for specific intervention in selected regulatory pathways without affecting organism-wide disruption of parallel pathways. Signaling by epinephrine and norepinephrine through α and β adrenergic receptors acts on many signaling pathways in many cell types. Here, we initially provide an overview of the signaling complexes formed between the paradigmatic β2 adrenergic receptor and two of its most important targets, the L-type Ca²⁺ channel CaV1.2 and the AMPA-type glutamate receptor. Importantly, both complexes contain the trimeric Gs protein, adenylyl cyclase, and the cAMP-dependent protein kinase, PKA. We then discuss the functional implications of the formation of these complexes, how those complexes can be specifically disrupted, and how such disruption could be utilized in the pharmacological treatment of disease.

The ability to regulate water movement is vital for the survival of cells and organisms. In addition to passively crossing lipid bilayers by diffusion, water transport is also driven across cell membranes by osmotic gradients through aquaporin water channels. There are 13 aquaporins in human tissues, and of these, aquaporin-2 (AQP2) is the most highly regulated water channel in the kidney: The expression and trafficking of AQP2 respond to body volume status and plasma osmolality via the antidiuretic hormone, vasopressin (VP). Dysfunctional VP signaling in renal epithelial cells contributes to disorders of water balance, and research initially focused on regulating the major cAMP/PKA pathway to normalize urine concentrating ability. With the discovery of novel and more complex signaling networks that regulate AQP2 trafficking, promising therapeutic targets have since been identified. Several strategies based on data from preclinical studies may ultimately translate to the care of patients with defective water homeostasis.

The mechanically activated Piezo channels, including Piezo1 and Piezo2 in mammals, function as key mechanotransducers for converting mechanical force into electrochemical signals. This review highlights key evidence for the potential of Piezo channel drug discovery. First, both mouse and human genetic studies have unequivocally demonstrated the prominent role of Piezo channels in various mammalian physiologies and pathophysiologies, validating their potential as novel therapeutic targets. Second, the cryo-electron microscopy structure of the 2,547-residue mouse Piezo1 trimer has been determined, providing a solid foundation for studying its structure-function relationship and drug action mechanisms and conducting virtual drug screening. Third, Piezo1 chemical activators, named Yoda1 and Jedi1/2, have been identified through high-throughput screening assays, demonstrating the drugability of Piezo channels. However, the pharmacology of Piezo channels is in its infancy. By establishing an integrated drug discovery platform, we may hopefully discover and develop a fleet of Jedi masters for battling Piezo-related human diseases.

The three small-conductance calcium-activated potassium (KCa2) channels and the related intermediate-conductance KCa3.1 channel are voltage-independent K⁺ channels that mediate calcium-induced membrane hyperpolarization. When intracellular calcium increases in the channel vicinity, it calcifies the flexible N lobe of the channel-bound calmodulin, which then swings over to the S4-S5 linker and opens the channel. KCa2 and KCa3.1 channels are highly druggable and offer multiple binding sites for venom peptides and small-molecule blockers as well as for positive- and negative-gating modulators. In this review, we briefly summarize the physiological role of KCa channels and then discuss the pharmacophores and the mechanism of action of the most commonly used peptidic and small-molecule KCa2 and KCa3.1 modulators. Finally, we describe the progress that has been made in advancing KCa3.1 blockers and KCa2.2 negative- and positive-gating modulators toward the clinic for neurological and cardiovascular diseases and discuss the remaining challenges.

Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

Neuropathic pain (NeP) can result from sources as varied as nerve compression, channelopathies, autoimmune disease, and incision. By identifying the neurobiological changes that underlie the pain state, it will be clinically possible to exploit mechanism-based therapeutics for maximum analgesic effect as diagnostic accuracy is optimized. Obtaining sufficient knowledge regarding the neuroadaptive alterations that occur in a particular NeP state will result in improved patient analgesia and a mechanism-based, as opposed to a disease-based, therapeutic approach to facilitate target identification. This will rely on comprehensive disease pathology insight; our knowledge is vastly improving due to continued forward and back translational preclinical and clinical research efforts. Here we discuss the clinical aspects of neuropathy and currently used drugs whose mechanisms of action are outlined alongside their clinical use. Finally, we consider sensory phenotypes, patient clusters, and predicting the efficacy of an analgesic for neuropathy.

High and rising prescription drug costs have become a preoccupying policy problem in the United States. Notwithstanding broad, bipartisan interest in finding effective policy solutions, several aspects of the drug affordability problem make it an uncommonly difficult one to solve. This article reviews the moral, market, and political factors contributing to the difficulty. Among the moral problems is lack of agreement about how to weigh the fundamental tradeoff involved in regulating drug prices—affordability versus incentives for innovation—and about what constitutes a fair price. Market-related factors include the lack of price transparency and a myriad of perverse incentives in the system through which prescription drugs are supplied to patients. Finally, current policy choices are constrained by past political compromises, and an atmosphere of scandal focusing on egregious instances of price gouging has made rational deliberation about fixes to deeper problems in the system difficult.

Cerebral edema, a common and often fatal companion to most forms of acute central nervous system disease, has been recognized since the time of ancient Egypt. Unfortunately, our therapeutic armamentarium remains limited, in part due to historic limitations in our understanding of cerebral edema pathophysiology. Recent advancements have led to a number of clinical trials for novel therapeutics that could fundamentally alter the treatment of cerebral edema. In this review, we discuss these agents, their targets, and the data supporting their use, with a focus on agents that have progressed to clinical trials.

Pharmacogenetics is a key component of precision medicine. Genetic variation in drug metabolism enzymes can lead to variable exposure to drugs and metabolites, potentially leading to inefficacy and drug toxicity. Although the evidence for pharmacogenetic associations in children is not as extensive as for adults, there are several drugs across diverse therapeutic areas with robust pediatric data indicating important, and relatively common, drug–gene interactions. Guidelines to assist gene-based dose optimization are available for codeine, thiopurine drugs, selective serotonin reuptake inhibitors, atomoxetine, tacrolimus, and voriconazole. For each of these drugs, there is an opportunity to clinically implement precision medicine approaches with children for whom genetic test results are known or are obtained at the time of prescribing. For many more drugs that are commonly used in pediatric patients, additional investigation is needed to determine the genetic factors influencing appropriate dose.

The promise of drug repurposing is to accelerate the translation of knowledge to treatment of human disease, bypassing common challenges associated with drug development to be more time- and cost-efficient. Repurposing has an increased chance of success due to the previous validation of drug safety and allows for the incorporation of omics. Hypothesis-generating omics processes inform drug repurposing decision-making methods on drug efficacy and toxicity. This review summarizes drug repurposing strategies and methodologies in the context of the following omics fields: genomics, epigenomics, transcriptomics, proteomics, metabolomics, microbiomics, phenomics, pregomics, and personomics. While each omics field has specific strengths and limitations, incorporating omics into the drug repurposing landscape is integral to its success.

The most common applications of artificial intelligence (AI) in drug treatment have to do with matching patients to their optimal drug or combination of drugs, predicting drug-target or drug-drug interactions, and optimizing treatment protocols. This review outlines some of the recently developed AI methods aiding the drug treatment and administration process. Selection of the best drug(s) for a patient typically requires the integration of patient data, such as genetics or proteomics, with drug data, like compound chemical descriptors, to score the therapeutic efficacy of drugs. The prediction of drug interactions often relies on similarity metrics, assuming that drugs with similar structures or targets will have comparable behavior or may interfere with each other. Optimizing the dosage schedule for administration of drugs is performed using mathematical models to interpret pharmacokinetic and pharmacodynamic data. The recently developed and powerful models for each of these tasks are addressed, explained, and analyzed here.

Retinoic acid–related orphan receptor γt (RORγt) functions as a ligand-dependent transcription factor that regulates multiple proinflammatory genes and plays a critical role in several inflammatory and autoimmune diseases. Various endogenous and synthetic RORγ (inverse) agonists have been identified that regulate RORγ transcriptional activity, including many cholesterol intermediates and oxysterols. Changes in cholesterol biosynthesis and metabolism can therefore have a significant impact on the generation of oxysterol RORγ ligands and, consequently, can control RORγt activity and inflammation. These observations contribute to a growing literature that connects cholesterol metabolism to the regulation of immune responses and autoimmune disease. Loss of RORγ function in knockout mice and in mice treated with RORγ inverse agonists results in reduced production of proinflammatory cytokines, such as IL-17A/F, and increased resistance to autoimmune disease in several experimental rodent models. Thus, RORγt inverse agonists might provide an attractive therapeutic approach to treat a variety of autoimmune diseases.