Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents.

Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin (CaM). Molecular details of both CaM and drug binding have remained elusive so far. Here, we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and CaM. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand-binding site can be transmitted to the central ion-conducting pore of TRPC4. CaM binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel CaM-induced regulatory mechanism of canonical TRP channels.

Water Thermodynamics of Peptide Toxin Binding Sites on Ion Channels.

Peptide toxins isolated from venomous creatures, long prized as research tools due to their innate potency for ion channels, are emerging as drugs as well. However, it remains challenging to understand why peptide toxins bind with high potency to ion channels, to identify residues that are key for activity, and to improve their affinities via mutagenesis. We use WaterMap, a molecular dynamics simulation-based method, to gain computational insight into these three questions by calculating the locations and thermodynamic properties of water molecules in the peptide toxin binding sites of five ion channels. These include an acid-sensing ion channel, voltage-gated potassium channel, sodium channel in activated and deactivated states, transient-receptor potential channel, and a nicotinic receptor whose structures were recently determined by crystallography and cryo-electron microscopy (cryo-EM). All channels had water sites in the peptide toxin binding site, and an average of 75% of these sites were stable (low-energy), and 25% were unstable (medium or high energy). For the sodium channel, more unstable water sites were present in the deactivated state structure than the activated. Additionally, for each channel, unstable water sites coincided with the positions of peptide toxin residues that previous mutagenesis experiments had shown were important for activity. Finally, for the sodium channel in the deactivated state, unstable water sites were present in the peptide toxin binding pocket but did not overlap with the peptide toxin, suggesting that future experimental efforts could focus on targeting these sites to optimize potency.

Involvement of a Na+-coupled Oligopeptide Transport System for ß-amyloid Peptide (Aß1-42) in Brain Cells.

PURPOSE: A Na+-coupled transport system in mammalian cells is responsible for the uptake of oligopeptides consisting of 5 or more amino acids. Here we investigated if this transport system is expressed in brain cells and transports the 42-amino-acid ß-amyloid peptide (Aß1-42). METHODS: The human and mouse neuronal cell lines SK-N-SH and HT22, human microglial cell line HMC-3, and human blood-brain barrier endothelial cell line hCMEC/D3 were used to monitor the uptake of [3H]-deltorphin II (a heptapeptide) and fluorescence-labeled Aß1-42. RESULTS: All four cell lines exhibited Na+-coupled uptake of deltorphin II. Aß1-42 competed with deltorphin II for the uptake. Uptake of fluorescence-labeled Aß1-42 was detectable in these cell lines, and the uptake was Na+-dependent and inhibitable by deltorphin II. The Na+-coupled uptake disappeared at high concentrations of Aß1-42 due to oligomerization of the peptide. Exposure of the cells to excess iron abolished the uptake. In hCMEC/D3 cells cultured on Transwell filters, the uptake was localized preferentially to the abluminal membrane. CONCLUSION: A Na+-coupled transport system mediates the uptake of Aß1-42 monomers in neuronal and microglial cells. The same system is also responsible for the uptake of Aß1-42 from brain into blood-brain barrier endothelial cells. These findings have relevance to Alzheimer's disease.

NaStEP, an essential protein for self-incompatibility in Nicotiana, performs a dual activity as a proteinase inhibitor and as a voltage-dependent channel blocker.

The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.

Centipede Venom Peptides Acting on Ion Channels.

Centipedes are among the oldest venomous arthropods that use their venom to subdue the prey. The major components of centipede venom are a variety of low-molecular-weight peptide toxins that have evolved to target voltage-gated ion channels to interfere with the central system of prey and produce pain or paralysis for efficient hunting. Peptide toxins usually contain several intramolecular disulfide bonds, which confer chemical, thermal and biological stability. In addition, centipede peptides generally have novel structures and high potency and specificity and therefore hold great promise both as diagnostic tools and in the treatment of human disease. Here, we review the centipede peptide toxins with reported effects on ion channels, including Nav, Kv, Cav and the nonselective cation channel polymodal transient receptor potential vanilloid 1 (TRPV1).

Mechanosensitive ion channel inhibitors promote the stiffening of the plasma membrane of mouse sensory neurons.

The mechanosensitivity of cells depends on the lipid-protein interactions of the plasma membrane. Affectations in the lipid region of the plasma membrane affect the transduction of mechanical forces, and any molecule that modifies the biophysical integrity of the lipid bilayer can alter the mechanical activity of the proteins inside the membrane. To understand whether inhibitors of mechanically activated ion channels affect the mechanical properties of the plasma membrane, we evaluated the rigidity of the membrane of sensory neurons of the DRG of mice using a variant of the scanning ion conductance microscopy method, which allows us to calculate the Young's modulus of individual cells before and after the perfusion of different doses of Gd3+, ruthenium red and GsMTx-4. Our results suggest that these molecules compromise the membrane by increasing the Young's modulus value, which indicates that the membrane becomes more rigid; these compounds act through different mechanisms and by a non-specific manner, each one shows a certain preference for specific cell subpopulations, depending on their cell size and their reactivity to isolectin B4. Our results support the idea that the biophysical properties that result from the interactions that arise in the membranes are part of the mechanotransduction process.

Molecular determinants of chemical modulation of two-pore domain potassium channels.

The K+ ion channels comprising the two-pore domain (K2P) family have specific biophysical roles in generating the critical regulatory K+ current. Ion flow through K2P channels and, implicitly, channel regulation is mediated by diverse metabolic and physical inputs such as mechanical stimulation, interaction with lipids or endogenous regulators, intra- or extracellular pH, and phosphorylation, while their function can be finely tuned by chemical compounds. In the latter category, some drug-channel interactions can lead to side effects or have clinical action, while identifying novel chemical modulators of K2Ps is an area of intense research. Due to their cellular and therapeutic importance, much attention was turned to these channels in recent years and several experimental approaches have pinpointed the molecular determinants of K2P chemical modulation. Given their unique structural features and properties, chemical modulators act on K2P channels in multiple and diverse ways. In this review, the particularities of K2P modulation by chemical compounds, such as binding modality, affinity, or position, are identified, synthesized, and linked to structural and functional properties in order to refer to how activators and blockers modify channel function and vice versa, focusing on specificity related to protein structure (and its modification) and cross-linking information among different subfamilies.

Membrane Stabilizer Medications in the Treatment of Chronic Neuropathic Pain: a Comprehensive Review.

PURPOSE OF REVIEW: Neuropathic pain is often debilitating, severely limiting the daily lives of patients who are affected. Typically, neuropathic pain is difficult to manage and, as a result, leads to progression into a chronic condition that is, in many instances, refractory to medical management. RECENT FINDINGS: Gabapentinoids, belonging to the calcium channel blocking class of drugs, have shown good efficacy in the management of chronic pain and are thus commonly utilized as first-line therapy. Various sodium channel blocking drugs, belonging to the categories of anticonvulsants and local anesthetics, have demonstrated varying degrees of efficacy in the in the treatment of neurogenic pain. Though there is limited medical literature as to efficacy of any one drug, individualized multimodal therapy can provide significant analgesia to patients with chronic neuropathic pain.

Clinical Probes and Endogenous Biomarkers as Substrates for Transporter Drug-Drug Interaction Evaluation: Perspectives From the International Transporter Consortium.

Drug transporters can govern the absorption, distribution, metabolism, and excretion of substrate drugs and endogenous substances. Investigations to examine their potential impact to pharmacokinetic (PK) drug-drug interactions (DDIs) are an integral part of the risk assessment in drug development. To evaluate a new molecular entity as a potential perpetrator of transporters, use of well characterized and/or clinically relevant probe substrates with good selectivity and sensitivity are critical for robust clinical DDI assessment that could inform DDI management strategy in the product labeling. The availability of endogenous biomarkers to monitor transporter-mediated DDIs in early phases of clinical investigations would greatly benefit downstream clinical plans. This article reviews the state-of-the-art in transporter clinical probe drugs and emerging biomarkers, including current challenges and limitations, delineates methods and workflows to identify and validate novel endogenous biomarkers to support clinical DDI evaluations, and proposes how these probe drugs or biomarkers could be used in drug development.

Transporters in Drug Development: 2018 ITC Recommendations for Transporters of Emerging Clinical Importance.

This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug-induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).

Molecular Modeling of Drug-Transporter Interactions-An International Transporter Consortium Perspective.

Membrane transporters play diverse roles in the pharmacokinetics and pharmacodynamics of small-molecule drugs. Understanding the mechanisms of drug-transporter interactions at the molecular level is, therefore, essential for the design of drugs with optimal therapeutic effects. This white paper examines recent progress, applications, and challenges of molecular modeling of membrane transporters, including modeling techniques that are centered on the structures of transporter ligands, and those focusing on the structures of the transporters. The goals of this article are to illustrate current best practices and future opportunities in using molecular modeling techniques to understand and predict transporter-mediated effects on drug disposition and efficacy.Membrane transporters from the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies regulate the cellular uptake, efflux, and homeostasis of many essential nutrients and significantly impact the pharmacokinetics of drugs; further, they may provide targets for novel therapeutics as well as facilitate prodrug approaches. Because of their often broad substrate selectivity they are also implicated in many undesirable and sometimes life-threatening drug-drug interactions (DDIs).5,6.

Membrane Transport at an Organelle Interface in the Early Secretory Pathway: Take Your Coat Off and Stay a While: Evolution of the metazoan early secretory pathway.

Most metazoan organisms have evolved a mildly acidified and calcium diminished sorting hub in the early secretory pathway commonly referred to as the Endoplasmic Reticulum-Golgi intermediate compartment (ERGIC). These membranous vesicular-tubular clusters are found tightly juxtaposed to ER subdomains that are competent for the production of COPII-coated transport carriers. In contrast to many unicellular systems, metazoan COPII carriers largely transit just a few hundred nanometers to the ERGIC, prior to COPI-dependent transport on to the cis-Golgi. The mechanisms underlying formation and maintenance of ERGIC membranes are poorly defined. However, recent evidence suggests an important role for Trk-fused gene (TFG) in regulating the integrity of the ER/ERGIC interface. Moreover, in the absence of cytoskeletal elements to scaffold tracks on which COPII carriers might move, TFG appears to promote anterograde cargo transport by locally tethering COPII carriers adjacent to ERGIC membranes. This action, regulated in part by the intrinsically disordered domain of TFG, provides sufficient time for COPII coat disassembly prior to heterotypic membrane fusion and cargo delivery to the ERGIC.

Disease-Associated Changes in Drug Transporters May Impact the Pharmacokinetics and/or Toxicity of Drugs: A White Paper From the International Transporter Consortium.

Drug transporters are critically important for the absorption, distribution, metabolism, and excretion (ADME) of many drugs and endogenous compounds. Therefore, disruption of these pathways by inhibition, induction, genetic polymorphisms, or disease can have profound effects on overall physiology, drug pharmacokinetics, drug efficacy, and toxicity. This white paper provides a review of changes in transporter function associated with acute and chronic disease states, describes regulatory pathways affecting transporter expression, and identifies opportunities to advance the field.

Mutation in KCNE1 associated to early repolarization syndrome by modulation of slowly activating delayed rectifier K+ current.

BACKGROUND: Recent studies have revealed that mutation in KCNE1, ß-subunits of cardiac potassium channel, involved in ventricular fibrillation. Whereas its role in early repolarization syndrome (ERS) is less well understood. OBJECTIVE: To study whether mutant in KCNE1 is associated with ERS and explore the possible underlying molecular mechanisms. METHODS: Whole genome from four unrelated families with ERS was amplified and sequenced. Wild-type (WT) KCNE1 and/or KCNE1-S38G (S38G) were expressed in HEK293 cells with KCNQ1. Functional studies included whole-cell patch-clamp, western blot and immunofluorescence were performed to reveal the possible underlying mechanisms. RESULTS: The co-expression of KCNE1-S38G and KCNQ1 decreased tail current density of IKs but had little effect in modulation channel kinetics of IKs. Compared with KCNE1-WT, the expression and membrane location of KCNE1-S38G decreased. Co-expression of KCNE1-WT and KCNE1-S38G partially rescued the function of IKs channel. CONCLUSIONS: The S38G mutation induced a loss-of-function of IKs due to decreasing of KCNE1 protein expression and defecting in KCNE1 protein membrane trafficking. Our findings suggested that KCNE1 may be one of the possible modulatory genes associated to ERS.

Pharmacological Targeting of Neuronal Kv7.2/3 Channels: A Focus on Chemotypes and Receptor Sites.

BACKGROUND: The Kv7 (KCNQ) subfamily of voltage-gated potassium channels consists of 5 members (Kv7.1-5) each showing characteristic tissue distribution and physiological roles. Given their functional heterogeneity, Kv7 channels represent important pharmacological targets for the development of new drugs for neuronal, cardiovascular and metabolic diseases. OBJECTIVE: In the present manuscript, we focus on describing the pharmacological relevance and potential therapeutic applications of drugs acting on neuronally-expressed Kv7.2/3 channels, placing particular emphasis on the different chemotypes, and highlighting their pharmacodynamic and, whenever possible, pharmacokinetic peculiarities. METHODS: The present work is based on an in-depth search of the currently available scientific literature, and on our own experience and knowledge in the field of neuronal Kv7 channel pharmacology. Space limitations impeded to describe the full pharmacological potential of Kv7 channels; thus, we have chosen to focus on neuronal channels composed of Kv7.2 and Kv7.3 subunits, and to mainly concentrate on their involvement in epilepsy. RESULTS: An astonishing heterogeneity in the molecular scaffolds exploitable to develop Kv7.2/3 modulators is evident, with important structural/functional peculiarities of distinct compound classes. CONCLUSION: In the present work we have attempted to show the current status and growing potential of the Kv7 pharmacology field. We anticipate a bright future for the field, and express our hopes that the efforts herein reviewed will result in an improved treatment of hyperexcitability (or any other) diseases.

Entropy and Polarity Control the Partition and Transportation of Drug-like Molecules in Biological Membrane.

Partition and transportation of drug in the plasma membrane of a mammalian cell are the prerequisite for its function on target protein. Therefore, comprehensive understanding of the physicochemical properties and mechanism behind these complex phenomena is crucial in pharmaceutical research. By using the state-of-art molecular simulations with polarization effect implicitly or explicitly included, we studied the permeation behavior of 2-aminoethoxydiphenyl borate (2-APB), a broad-spectrum modulator for a number of membrane proteins. We showed that the protonation state and therefore the polarity of the drug is critical for its partition, and that the drug is likely to switch between different protonation states along its permeation pathway. By changing the degrees of freedom, protonation further affects the thermodynamic of the permeation pathway of 2-APB, leading to different entropic contributions. A survey on 54 analog structures with similar backbone to 2-APB showed that delicate balance between entropy and polarity plays an important role in drugs' potency.

N-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation.

K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

Decrease in Long-Chain Acylcarnitine Tissue Content Determines the Duration of and Correlates with the Cardioprotective Effect of Methyl-GBB.

Ischaemia in the heart is accompanied by the accumulation of long-chain acylcarnitines (LCACs) which is one of the multiple factors that contribute to the ischaemia-reperfusion damage development. Long-term pre-treatment that decreases carnitine and LCAC contents also reduces ischaemia-reperfusion (IR) damage; however, the duration of the post-treatment effects is not known. The aim of the study was to assess the post-treatment effects of the carnitine transport (OCTN2) inhibitor, methyl-GBB, on LCAC content and the duration of its cardioprotective effect. Male Wistar rats received methyl-GBB (5 mg/kg for 28 days), and the anti-infarction effects on Langendorff-perfused hearts and the acylcarnitine profile in cardiac tissues were measured up to 28 days following the end of the treatment. Methyl-GBB pre-treatment for 28 days decreased LCAC heart tissue content by 87%, and the infarct size was decreased by 57%. Fourteen days post-treatment, the LCAC content was still decreased by 69%, and the infarct size was decreased by 32% compared to Control. A significant Pearson correlation (r = 0.48, p = 0.026) was found between infarct size and LCAC tissue content in the methyl-GBB-treated rat hearts. The addition of 2 mM carnitine to isolated heart perfusate significantly diminished the methyl-GBB-induced decrease in LCACs and infarct size. In conclusion, the anti-infarction effect of methyl-GBB continues for at least 2 weeks post-treatment. No less than a 70% decrease in LCAC content is required to protect ischaemic heart tissues, and the decrease in LCAC levels defines the duration of the post-treatment cardioprotective effect of the OCTN2 inhibitor, methyl-GBB.