Structural basis of selective TRPM7 inhibition by the anticancer agent CCT128930.

TRP channels are implicated in various diseases, but high structural similarity between them makes selective pharmacological modulation challenging. Here, we study the molecular mechanism underlying specific inhibition of the TRPM7 channel, which is essential for cancer cell proliferation, by the anticancer agent CCT128930 (CCT). Using cryo-EM, functional analysis, and MD simulations, we show that CCT binds to a vanilloid-like (VL) site, stabilizing TRPM7 in the closed non-conducting state. Similar to other allosteric inhibitors of TRPM7, NS8593 and VER155008, binding of CCT is accompanied by displacement of a lipid that resides in the VL site in the apo condition. Moreover, we demonstrate the principal role of several residues in the VL site enabling CCT to inhibit TRPM7 without impacting the homologous TRPM6 channel. Hence, our results uncover the central role of the VL site for the selective interaction of TRPM7 with small molecules that can be explored in future drug design.

Dissecting the biophysics and biology of intrinsically disordered proteins.

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer.

Parkinson's disease, epilepsy, and amyotrophic lateral sclerosis-emerging role of AMPA and kainate subtypes of ionotropic glutamate receptors.

Ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission and are implicated in various neurological disorders. In this review, we discuss the role of the two fastest iGluRs subtypes, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, in the pathogenesis and treatment of Parkinson's disease, epilepsy, and amyotrophic lateral sclerosis. Although both AMPA and kainate receptors represent promising therapeutic targets for the treatment of these diseases, many of their antagonists show adverse side effects. Further studies of factors affecting the selective subunit expression and trafficking of AMPA and kainate receptors, and a reasonable approach to their regulation by the recently identified novel compounds remain promising directions for pharmacological research.

Molecular pharmacology of the onco-TRP channel TRPV6.

TRPV6, a representative of the vanilloid subfamily of TRP channels, serves as the principal calcium uptake channel in the gut. Dysregulation of TRPV6 results in disturbed calcium homeostasis leading to a variety of human diseases, including many forms of cancer. Inhibitors of this oncochannel are therefore particularly needed. In this review, we provide an overview of recent advances in structural pharmacology that uncovered the molecular mechanisms of TRPV6 inhibition by a variety of small molecules, including synthetic and natural, plant-derived compounds as well as some prospective and clinically approved drugs.

Molecular pathway and structural mechanism of human oncochannel TRPV6 inhibition by the phytocannabinoid tetrahydrocannabivarin.

The calcium-selective oncochannel TRPV6 is an important driver of cell proliferation in human cancers. Despite increasing interest of pharmacological research in developing synthetic inhibitors of TRPV6, natural compounds acting at this channel have been largely neglected. On the other hand, pharmacokinetics of natural small-molecule antagonists optimized by nature throughout evolution endows these compounds with a medicinal potential to serve as potent and safe next-generation anti-cancer drugs. Here we report the structure of human TRPV6 in complex with tetrahydrocannabivarin (THCV), a natural cannabinoid inhibitor extracted from Cannabis sativa. We use cryo-electron microscopy combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to identify THCV binding sites in the portals that connect the membrane environment surrounding the protein to the central cavity of the channel pore and to characterize the allosteric mechanism of TRPV6 inhibition. We also propose the molecular pathway taken by THCV to reach its binding site. Our study provides a foundation for the development of new TRPV6-targeting drugs.

Modulation of GluA2-γ5 synaptic complex desensitization, polyamine block and antiepileptic perampanel inhibition by auxiliary subunit cornichon-2.

Synaptic complexes of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) with auxiliary subunits mediate most excitatory neurotransmission and can be targeted to treat neuropsychiatric and neurological disorders, including epilepsy. Here we present cryogenic-electron microscopy structures of rat GluA2 AMPAR complexes with inhibitory mouse γ5 and potentiating human cornichon-2 (CNIH2) auxiliary subunits. CNIH2 appears to destabilize the desensitized state of the complex by reducing the separation of the upper lobes in ligand-binding domain dimers. At the same time, CNIH2 stabilizes binding of polyamine spermidine to the selectivity filter of the closed ion channel. Nevertheless, CNIH2, and to a lesser extent γ5, attenuate polyamine block of the open channel and reduce the potency of the antiepileptic drug perampanel that inhibits the synaptic complex allosterically by binding to sites in the ion channel extracellular collar. These findings illustrate the fine-tuning of synaptic complex structure and function in an auxiliary subunit-dependent manner, which is critical for the study of brain region-specific neurotransmission and design of therapeutics for disease treatment.

Structure of human TRPV4 in complex with GTPase RhoA.

Transient receptor potential (TRP) channel TRPV4 is a polymodal cellular sensor that responds to moderate heat, cell swelling, shear stress, and small-molecule ligands. It is involved in thermogenesis, regulation of vascular tone, bone homeostasis, renal and pulmonary functions. TRPV4 is implicated in neuromuscular and skeletal disorders, pulmonary edema, and cancers, and represents an important drug target. The cytoskeletal remodeling GTPase RhoA has been shown to suppress TRPV4 activity. Here, we present a structure of the human TRPV4-RhoA complex that shows RhoA interaction with the membrane-facing surface of the TRPV4 ankyrin repeat domains. The contact interface reveals residues that are mutated in neuropathies, providing an insight into the disease pathogenesis. We also identify the binding sites of the TRPV4 agonist 4α-PDD and the inhibitor HC-067047 at the base of the S1-S4 bundle, and show that agonist binding leads to pore opening, while channel inhibition involves a π-to-α transition in the pore-forming helix S6. Our structures elucidate the interaction interface between hTRPV4 and RhoA, as well as residues at this interface that are involved in TRPV4 disease-causing mutations. They shed light on TRPV4 activation and inhibition and provide a template for the design of future therapeutics for treatment of TRPV4-related diseases.

Structural mechanisms of TRPM7 activation and inhibition.

The transient receptor potential channel TRPM7 is a master regulator of the organismal balance of divalent cations that plays an essential role in embryonic development, immune responses, cell mobility, proliferation, and differentiation. TRPM7 is implicated in neuronal and cardiovascular disorders, tumor progression and has emerged as a new drug target. Here we use cryo-EM, functional analysis, and molecular dynamics simulations to uncover two distinct structural mechanisms of TRPM7 activation by a gain-of-function mutation and by the agonist naltriben, which show different conformational dynamics and domain involvement. We identify a binding site for highly potent and selective inhibitors and show that they act by stabilizing the TRPM7 closed state. The discovered structural mechanisms provide foundations for understanding the molecular basis of TRPM7 channelopathies and drug development.

Structural mechanism of human oncochannel TRPV6 inhibition by the natural phytoestrogen genistein.

Calcium-selective oncochannel TRPV6 is the major driver of cell proliferation in human cancers. While significant effort has been invested in the development of synthetic TRPV6 inhibitors, natural channel blockers have been largely neglected. Here we report the structure of human TRPV6 in complex with the plant-derived phytoestrogen genistein, extracted from Styphnolobium japonicum, that was shown to inhibit cell invasion and metastasis in cancer clinical trials. Despite the pharmacological value, the molecular mechanism of TRPV6 inhibition by genistein has remained enigmatic. We use cryo-EM combined with electrophysiology, calcium imaging, mutagenesis, and molecular dynamics simulations to show that genistein binds in the intracellular half of the TRPV6 pore and acts as an ion channel blocker and gating modifier. Genistein binding to the open channel causes pore closure and a two-fold symmetrical conformational rearrangement in the S4-S5 and S6-TRP helix regions. The unprecedented mechanism of TRPV6 inhibition by genistein uncovers new possibilities in structure-based drug design.

Human TRPV1 structure and inhibition by the analgesic SB-366791.

Pain therapy has remained conceptually stagnant since the opioid crisis, which highlighted the dangers of treating pain with opioids. An alternative addiction-free strategy to conventional painkiller-based treatment is targeting receptors at the origin of the pain pathway, such as transient receptor potential (TRP) ion channels. Thus, a founding member of the vanilloid subfamily of TRP channels, TRPV1, represents one of the most sought-after pain therapy targets. The need for selective TRPV1 inhibitors extends beyond pain treatment, to other diseases associated with this channel, including psychiatric disorders. Here we report the cryo-electron microscopy structures of human TRPV1 in the apo state and in complex with the TRPV1-specific nanomolar-affinity analgesic antagonist SB-366791. SB-366791 binds to the vanilloid site and acts as an allosteric hTRPV1 inhibitor. SB-366791 binding site is supported by mutagenesis combined with electrophysiological recordings and can be further explored to design new drugs targeting TRPV1 in disease conditions.

Positive and negative allosteric modulation of GluK2 kainate receptors by BPAM344 and antiepileptic perampanel.

Kainate receptors (KARs) are a subtype of ionotropic glutamate receptors that control synaptic transmission in the central nervous system and are implicated in neurological, psychiatric, and neurodevelopmental disorders. Understanding the regulation of KAR function by small molecules is essential for exploring these receptors as drug targets. Here, we present cryoelectron microscopy (cryo-EM) structures of KAR GluK2 in complex with the positive allosteric modulator BPAM344, competitive antagonist DNQX, and negative allosteric modulator, antiepileptic drug perampanel. Our structures show that two BPAM344 molecules bind per ligand-binding domain dimer interface. In the absence of an agonist or in the presence of DNQX, BPAM344 stabilizes GluK2 in the closed state. The closed state is also stabilized by perampanel, which binds to the ion channel extracellular collar sites located in two out of four GluK2 subunits. The molecular mechanisms of positive and negative allosteric modulation of KAR provide a guide for developing new therapeutic strategies.

Ligand-Binding Sites in Vanilloid-Subtype TRP Channels.

Vanilloid-subfamily TRP channels TRPV1-6 play important roles in various physiological processes and are implicated in numerous human diseases. Advances in structural biology, particularly the "resolution revolution" in cryo-EM, have led to breakthroughs in molecular characterization of TRPV channels. Structures with continuously improving resolution uncover atomic details of TRPV channel interactions with small molecules and protein-binding partners. Here, we provide a classification of structurally characterized binding sites in TRPV channels and discuss the progress that has been made by structural biology combined with mutagenesis, functional recordings, and molecular dynamics simulations toward understanding of the molecular mechanisms of ligand action. Given the similarity in structural architecture of TRP channels, 16 unique sites identified in TRPV channels may be shared between TRP channel subfamilies, although the chemical identity of a particular ligand will likely depend on the local amino-acid composition. The characterized binding sites and molecular mechanisms of ligand action create a diversity of druggable targets to aid in the design of new molecules for tuning TRP channel function in disease conditions.

Structural snapshots of the mechanism of TRPV2 channel activation by small-molecule agonists.

Transient receptor potential (TRP) channels are polymodal sensors that play critical roles in various physiological processes in living organisms. These cation-permeable channels respond to a variety of physical and chemical stimuli, including cold and hot temperatures, acidic pH, and mechanical stress, often determining a sensory frontier of defense against hostile environments. Vanilloid (V) subfamily is the most studied category of TRP channels that includes six closely related members: highly calcium-selective TRPV5-6 and non-selective TRPV1-4. A remarkable feature of TRPV1-4 is their ability to sense heat, which makes them temperature-sensitive TRP channels or thermo-TRPs. TRPV channels are associated with a multitude of human diseases, including cancers, chronic pain, cardiovascular, neurological and nociceptive disorders. Despite the great clinical interest, pharmacology of TRPV channels remains largely undeveloped because of insufficient knowledge about the mechanisms of their regulation. For instance, activation of TRPV channels by small molecules or heat remains poorly understood. Numerous identified TRPV channel agonists, while effective in physiological experiments, appear limited in their ability to act in the conditions of structural biology experiments. In this regard, the recent study by Pumroy et al. [1] makes a significant contribution towards our understanding of TRPV2 structural dynamics that leads to opening of this channel in physiological conditions.

Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine.

Skin diseases are common human illnesses that occur in all cultures, at all ages, and affect between 30% and 70% of individuals globally. TRPV3 is a cation-permeable TRP channel predominantly expressed in skin keratinocytes, implicated in cutaneous sensation and associated with numerous skin diseases. TRPV3 is inhibited by the local anesthetic dyclonine, traditionally used for topical applications to relieve pain and itch. However, the structural basis of TRPV3 inhibition by dyclonine has remained elusive. Here we present a cryo-EM structure of a TRPV3-dyclonine complex that reveals binding of the inhibitor in the portals which connect the membrane environment surrounding the channel to the central cavity of the channel pore. We propose a mechanism of TRPV3 inhibition in which dyclonine molecules stick out into the channel pore, creating a barrier for ion conductance. The allosteric binding site of dyclonine can serve as a template for the design of new TRPV3-targeting drugs.

Opening of glutamate receptor channel to subconductance levels.

Ionotropic glutamate receptors (iGluRs) are tetrameric ligand-gated ion channels that open their pores in response to binding of the agonist glutamate1-3. An ionic current through a single iGluR channel shows up to four discrete conductance levels (O1-O4)4-6. Higher conductance levels have been associated with an increased number of agonist molecules bound to four individual ligand-binding domains (LBDs)6-10. Here we determine structures of a synaptic complex of AMPA-subtype iGluR and the auxiliary subunit γ2 in non-desensitizing conditions with various occupancy of the LBDs by glutamate. We show that glutamate binds to LBDs of subunits B and D only after it is already bound to at least the same number of LBDs that belong to subunits A and C. Our structures combined with single-channel recordings, molecular dynamics simulations and machine-learning analysis suggest that channel opening requires agonist binding to at least two LBDs. Conversely, agonist binding to all four LBDs does not guarantee maximal channel conductance and favours subconductance states O1 and O2, with O3 and O4 being rare and not captured structurally. The lack of subunit independence and low efficiency coupling of glutamate binding to channel opening underlie the gating of synaptic complexes to submaximal conductance levels, which provide a potential for upregulation of synaptic activity.

Structural basis of AMPA receptor inhibition by trans-4-butylcyclohexane carboxylic acid.

BACKGROUND AND PURPOSE: AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted target for anti-epileptic drugs. Trans-4-butylcyclohexane carboxylic acid (4-BCCA) has emerged as a new promising anti-epileptic drug in several in vitro and in vivo seizure models, but the mechanism of its action remained unknown. The purpose of this study is to characterize structure and dynamics of 4-BCCA interaction with AMPA receptors. EXPERIMENTAL APPROACH: We studied the molecular mechanism of AMPA receptor inhibition by 4-BCCA using a combination of X-ray crystallography, mutagenesis, electrophysiological assays, and molecular dynamics simulations. KEY RESULTS: We identified 4-BCCA binding sites in the transmembrane domain (TMD) of AMPA receptor, at the lateral portals formed by transmembrane segments M1-M4. At this binding site, 4-BCCA is very dynamic, assumes multiple poses, and can enter the ion channel pore. CONCLUSION AND IMPLICATIONS: 4-BCCA represents a low-affinity inhibitor of AMPA receptors that acts at the TMD sites distinct from non-competitive inhibitors, such as the anti-epileptic drug perampanel and the ion channel blockers. Further studies might examine the possibsility of synergistic use of these inhibitors in treatment of epilepsy and a wide range of neurological disorders and gliomas. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels.

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.

Structural mechanisms of TRPV6 inhibition by ruthenium red and econazole.

TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel's periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel's selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.

Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L.

AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission. Their surface expression, trafficking, gating, and pharmacology are regulated by auxiliary subunits. Of the two types of TARP auxiliary subunits, type I TARPs assume activating roles, while type II TARPs serve suppressive functions. We present cryo-EM structures of GluA2 AMPAR in complex with type II TARP γ5, which reduces steady-state currents, increases single-channel conductance, and slows recovery from desensitization. Regulation of AMPAR function depends on its ligand-binding domain (LBD) interaction with the γ5 head domain. GluA2-γ5 complex shows maximum stoichiometry of two TARPs per AMPAR tetramer, being different from type I TARPs but reminiscent of the auxiliary subunit GSG1L. Desensitization of both GluA2-GSG1L and GluA2-γ5 complexes is accompanied by rupture of LBD dimer interface, while GluA2-γ5 but not GluA2-GSG1L LBD dimers remain two-fold symmetric. Different structural architectures and desensitization mechanisms of complexes with auxiliary subunits endow AMPARs with broad functional capabilities.