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.

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.

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 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.

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 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 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.

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.

Structural mechanism of TRPV3 channel inhibition by the plant-derived coumarin osthole.

TRPV3, a representative of the vanilloid subfamily of TRP channels, is predominantly expressed in skin keratinocytes and has been implicated in cutaneous sensation and associated with numerous skin pathologies and cancers. TRPV3 is inhibited by the natural coumarin derivative osthole, an active ingredient of Cnidium monnieri, which has been used in traditional Chinese medicine for the treatment of a variety of human diseases. However, the structural basis of channel inhibition by osthole has remained elusive. Here we present cryo-EM structures of TRPV3 in complex with osthole, revealing two types of osthole binding sites in the transmembrane region of TRPV3 that coincide with the binding sites of agonist 2-APB. Osthole binding converts the channel pore into a previously unidentified conformation with a widely open selectivity filter and closed intracellular gate. Our structures provide insight into competitive inhibition of TRPV3 by osthole and can serve as a template for the design of osthole chemistry-inspired drugs targeting TRPV3-associated diseases.

Structure of the Arabidopsis thaliana glutamate receptor-like channel GLR3.4.

Glutamate receptor-like channels (GLRs) play vital roles in various physiological processes in plants, such as wound response, stomatal aperture control, seed germination, root development, innate immune response, pollen tube growth, and morphogenesis. Despite the importance of GLRs, knowledge about their molecular organization is limited. Here we use X-ray crystallography and single-particle cryo-EM to solve structures of the Arabidopsis thaliana GLR3.4. Our structures reveal the tetrameric assembly of GLR3.4 subunits into a three-layer domain architecture, reminiscent of animal ionotropic glutamate receptors (iGluRs). However, the non-swapped arrangement between layers of GLR3.4 domains, binding of glutathione through S-glutathionylation of cysteine C205 inside the amino-terminal domain clamshell, unique symmetry, inter-domain interfaces, and ligand specificity distinguish GLR3.4 from representatives of the iGluR family and suggest distinct features of the GLR gating mechanism. Our work elaborates on the principles of GLR architecture and symmetry and provides a molecular template for deciphering GLR-dependent signaling mechanisms in plants.

GEM-IL: A highly responsive fluorescent lactate indicator.

Lactate metabolism has been shown to have increasingly important implications in cellular functions as well as in the development and pathophysiology of disease. The various roles as a signaling molecule and metabolite have led to interest in establishing a new method to detect lactate changes in live cells. Here we report our development of a genetically encoded metabolic indicator specifically for probing lactate (GEM-IL) based on superfolder fluorescent proteins and mutagenesis. With improvements in its design, specificity, and sensitivity, GEM-IL allows new applications compared with the previous lactate indicators, Laconic and Green Lindoblum. We demonstrate the functionality of GEM-IL to detect differences in lactate changes in human oncogenic neural progenitor cells and mouse primary ventricular myocytes. The development and application of GEM-IL show promise for enhancing our understanding of lactate dynamics and roles.

Inactivation-mimicking block of the epithelial calcium channel TRPV6.

Epithelial calcium channel TRPV6 plays vital roles in calcium homeostasis, and its dysregulation is implicated in multifactorial diseases, including cancers. Here, we study the molecular mechanism of selective nanomolar-affinity TRPV6 inhibition by (4-phenylcyclohexyl)piperazine derivatives (PCHPDs). We use x-ray crystallography and cryo-electron microscopy to solve the inhibitor-bound structures of TRPV6 and identify two types of inhibitor binding sites in the transmembrane region: (i) modulatory sites between the S1-S4 and pore domains normally occupied by lipids and (ii) the main site in the ion channel pore. Our structural data combined with mutagenesis, functional and computational approaches suggest that PCHPDs plug the open pore of TRPV6 and convert the channel into a nonconducting state, mimicking the action of calmodulin, which causes inactivation of TRPV6 channels under physiological conditions. This mechanism of inhibition explains the high selectivity and potency of PCHPDs and opens up unexplored avenues for the design of future-generation biomimetic drugs.

Hodgkin-Huxley-Katz Prize Lecture: Genetic and pharmacological control of glutamate receptor channel through a highly conserved gating motif.

Glutamate receptors are essential ligand-gated ion channels in the central nervous system that mediate excitatory synaptic transmission in response to the release of glutamate from presynaptic terminals. The structural and biophysical basis underlying the function of these receptors has been studied for decades by a wide range of approaches. However recent structural, pharmacological and genetic studies have provided new insight into the regions of this protein that are critical determinants of receptor function. Lack of variation in specific areas of the protein amino acid sequences in the human population has defined three regions in each receptor subunit that are under selective pressure, which has focused research efforts and driven new hypotheses. In addition, these three closely positioned elements reside near a cavity that is shown by multiple studies to be a likely site of action for allosteric modulators, one of which is currently in use as an FDA-approved anticonvulsant. These structural elements are capable of controlling gating of the pore, and appear to permit some modulators bound within the cavity to also alter permeation properties. This creates a new precedent whereby features of the channel pore can be modulated by exogenous drugs that bind outside the pore. The convergence of structural, genetic, biophysical and pharmacological approaches is a powerful means to gain insight into the complex biological processes defined by neurotransmitter receptor function.

Cytoplasmic Inter-Subunit Interface Controls Use-Dependence of Thermal Activation of TRPV3 Channel.

The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.

Expression, Purification, and Crystallization of the Transient Receptor Potential Channel TRPV6.

Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.

Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6.

Calcium (Ca2+) plays a major role in numerous physiological processes. Ca2+ homeostasis is tightly controlled by ion channels, the aberrant regulation of which results in various diseases including cancers. Calmodulin (CaM)-mediated Ca2+-induced inactivation is an ion channel regulatory mechanism that protects cells against the toxic effects of Ca2+ overload. We used cryo-electron microscopy to capture the epithelial calcium channel TRPV6 (transient receptor potential vanilloid subfamily member 6) inactivated by CaM. The TRPV6-CaM complex exhibits 1:1 stoichiometry; one TRPV6 tetramer binds both CaM lobes, which adopt a distinct head-to-tail arrangement. The CaM carboxyl-terminal lobe plugs the channel through a unique cation-π interaction by inserting the side chain of lysine K115 into a tetra-tryptophan cage at the pore's intracellular entrance. We propose a mechanism of CaM-mediated Ca2+-induced inactivation that can be explored for therapeutic design.

Structural bases of TRP channel TRPV6 allosteric modulation by 2-APB.

Transient receptor potential (TRP) channels are involved in various physiological processes, including sensory transduction. The TRP channel TRPV6 mediates calcium uptake in epithelia and its expression is dramatically increased in numerous types of cancer. TRPV6 inhibitors suppress tumor growth, but the molecular mechanism of inhibition remains unknown. Here, we present crystal and cryo-EM structures of human and rat TRPV6 bound to 2-aminoethoxydiphenyl borate (2-APB), a TRPV6 inhibitor and modulator of numerous TRP channels. 2-APB binds to TRPV6 in a pocket formed by the cytoplasmic half of the S1-S4 transmembrane helix bundle. Comparing human wild-type and high-affinity mutant Y467A structures, we show that 2-APB induces TRPV6 channel closure by modulating protein-lipid interactions. Mutagenesis and functional analyses suggest that the identified 2-APB binding site might be present in other members of vanilloid subfamily TRP channels. Our findings reveal a mechanism of ion channel allosteric modulation that can be exploited for therapeutic design.

Ion Permeation Mechanism in Epithelial Calcium Channel TRVP6.

Calcium is the most abundant metal in the human body that plays vital roles as a cellular electrolyte as well as the smallest and most frequently used signaling molecule. Calcium uptake in epithelial tissues is mediated by tetrameric calcium-selective transient receptor potential (TRP) channels TRPV6 that are implicated in a variety of human diseases, including numerous forms of cancer. We used TRPV6 crystal structures as templates for molecular dynamics simulations to identify ion binding sites and to study the permeation mechanism of calcium and other ions through TRPV6 channels. We found that at low Ca2+ concentrations, a single calcium ion binds at the selectivity filter narrow constriction formed by aspartates D541 and allows Na+ permeation. In the presence of ions, no water binds to or crosses the pore constriction. At high Ca2+ concentrations, calcium permeates the pore according to the knock-off mechanism that includes formation of a short-lived transition state with three calcium ions bound near D541. For Ba2+, the transition state lives longer and the knock-off permeation occurs slower. Gd3+ binds at D541 tightly, blocks the channel and prevents Na+ from permeating the pore. Our results provide structural foundations for understanding permeation and block in tetrameric calcium-selective ion channels.