RNA sequences that direct selective ADAR editing from a SELEX library bearing 8-azanebularine.

Adenosine Deaminases Acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA (dsRNA). ADARs' ability to recognize and edit dsRNA is dependent on local sequence context surrounding the edited adenosine and the length of the duplex. A deeper understanding of how editing efficiency is affected by mismatches, loops, and bulges around the editing site would aid in the development of therapeutic gRNAs for ADAR-mediated site-directed RNA editing (SDRE). Here, a SELEX (systematic evolution of ligands by exponential enrichment) approach was employed to identify dsRNA substrates that bind to the deaminase domain of human ADAR2 (hADAR2d) with high affinity. A library of single-stranded RNAs was hybridized with a fixed-sequence target strand containing the nucleoside analog 8-azanebularine that mimics the adenosine deamination transition state. The presence of this nucleoside analog in the library biased the screen to identify hit sequences compatible with adenosine deamination at the site of 8-azanebularine modification. SELEX also identified non-duplex structural elements that supported editing at the target site while inhibiting editing at bystander sites.

Purine nucleosides replace cAMP in allosteric regulation of PKA in trypanosomatid pathogens.

Cyclic nucleotide binding domains (CNB) confer allosteric regulation by cAMP or cGMP to many signaling proteins, including PKA and PKG. PKA of phylogenetically distant Trypanosoma is the first exception as it is cyclic nucleotide-independent and responsive to nucleoside analogues (Bachmaier et al., 2019). Here, we show that natural nucleosides inosine, guanosine and adenosine are nanomolar affinity CNB ligands and activators of PKA orthologs of the important tropical pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. The sequence and structural determinants of binding affinity, -specificity and kinase activation of PKAR were established by structure-activity relationship (SAR) analysis, co-crystal structures and mutagenesis. Substitution of two to three amino acids in the binding sites is sufficient for conversion of CNB domains from nucleoside to cyclic nucleotide specificity. In addition, a trypanosomatid-specific C-terminal helix (αD) is required for high affinity binding to CNB-B. The αD helix functions as a lid of the binding site that shields ligands from solvent. Selectivity of guanosine for CNB-B and of adenosine for CNB-A results in synergistic kinase activation at low nanomolar concentration. PKA pulldown from rapid lysis establishes guanosine as the predominant ligand in vivo in T. brucei bloodstream forms, whereas guanosine and adenosine seem to synergize in the procyclic developmental stage in the insect vector. We discuss the versatile use of CNB domains in evolution and recruitment of PKA for novel nucleoside-mediated signaling.

Identification of reaction sites and chlorinated products of purine bases and nucleosides during chlorination: a computational study.

Hypochlorous acid (HOCl) released from activated leukocytes plays a significant role in the human immune system, but is also implicated in numerous diseases due to its inappropriate production. Chlorinated nucleobases induce genetic changes that potentially enable and stimulate carcinogenesis, and thus have attracted considerable attention. However, their multiple halogenation sites pose challenges to identify them. As a good complement to experiments, quantum chemical computation was used to uncover chlorination sites and chlorinated products in this study. The results indicate that anion salt forms of all purine compounds play significant roles in chlorination except for adenosine. The kinetic reactivity order of all reaction sites in terms of the estimated apparent rate constant kobs-est (in M-1 s-1) is heterocyclic NH/N (102-107) > exocyclic NH2 (10-2-10) > heterocyclic C8 (10-5-10-1), but the order is reversed for thermodynamics. Combining kinetics and thermodynamics, the numerical simulation results show that N9 is the most reactive site for purine bases to form the main initial chlorinated product, while for purine nucleosides N1 and exocyclic N2/N6 are the most reactive sites to produce the main products controlled by kinetics and thermodynamics, respectively, and C8 is a possible site to generate the minor product. The formation mechanisms of biomarker 8-Cl- and 8-oxo-purine derivatives were also investigated. Additionally, the structure-kinetic reactivity relationship study reveals a good correlation between lg kobs-est and APT charge in all purine compounds compared to FED2 (HOMO), which proves again that the electrostatic interaction plays a key role. The results are helpful to further understand the reactivity of various reaction sites in aromatic compounds during chlorination.

Synthesis and Antiproliferative Evaluation of d-Glucuronamide-Based Nucleosides and (Triazolyl)methyl Amide-Linked Pseudodisaccharide Nucleosides.

The synthesis and antiproliferative evaluation of novel d-glucopyranuronamide-containing nucleosides is described. Based on our previously reported anticancer d-glucuronamide-based nucleosides, new analogues comprising N/O-dodecyl or N-propargyl substituents at the glucuronamide unit and anomerically-N-linked 2-acetamido-6-chloropurine, 6-chloropurine or 4-(6-chloropurinyl)methyl triazole motifs were synthesized in 4-6 steps starting from acetonide-protected glucofuranurono-6,3-lactone. The methodologies were based on the access to N-substituted glycopyranuronamide precursors, namely 1,2-O-acetyl derivatives or glucuronoamidyl azides for further nucleobase N-glycosylation or 1,3-dipolar cycloaddition with N9 - and N7 -propargyl-6-chloropurines, respectively. N-Propargyl glucuronamide-based N9 -purine nucleosides were converted into (triazolyl)methyl amide-6,6-linked pseudodisaccharide nucleosides via cycloaddition with methyl 6-azido-glucopyranoside. A CuI/Amberlyst A-21 catalytic system employed in the cycloaddition reactions also effected conversion into 6-dimethylaminopurine nucleosides. Antiproliferative evaluation in chronic myeloid leukemia (K562) and breast cancer (MCF-7) cells revealed significant effects exhibited by the synthesized monododecylated purine-containing nucleosides. A N-propargyl 3-O-dodecyl glucuronamide derivative comprising a N9 -ß-linked 6-chloropurine moiety was the most active compound against MCF-7 cells (GI50 =11.9 µM) while a related α-(purinyl)methyltriazole nucleoside comprising a N7 -linked 6-chloropurine moiety exhibited the highest activity against K562 cells (GI50 =8.0 µM). Flow cytometry and immunoblotting analysis of apoptosis-related proteins in K562 cells treated with the N-propargyl 3-O-dodecyl glucuronamide-based N9 -linked 6-chloropurine nucleoside indicated that it acts via apoptosis induction.

Probing ligand selectivity in pathogens.

Why does protein kinase A respond to purine nucleosides in certain pathogens, but not to the cyclic nucleotides that activate this kinase in most other organisms?

A general and efficient approach to synthesize the phosphoramidites of 5'-18O labeled purine nucleosides.

5'-18O labeled RNA oligos are important probes to investigate the mechanism of 2'-O-transphosphorylation reactions. Here we describe a general and efficient synthetic approach to the phosphoramidite derivatives of 5'-18O labeled nucleosides starting from the corresponding commercially available 5'-O-DMT protected nucleosides. Using this method, we prepared 5'-18O-guanosine phosphoramidite in 8 steps (13.2% overall yield), 5'-18O-adenosine phosphoramidite in 9 steps (10.1% overall yield) and 5'-18O-2'-deoxyguanosine phosphoramidite in 6 steps (12.8% overall yield). These 5'-18O labeled phosphoramidites can be incorporated into RNA oligos by solid phase synthesis for determination of heavy atom isotope effects in RNA 2'-O-transphosphorylation reactions.

Thiopurines Analogues with Additional Ring: Synthesis, Spectroscopic Properties, and Anticancer Potency.

Purine scaffolds constitute a starting point for the synthesis of numerous chemotherapeutics used in treating cancer, viruses, parasites, as well as bacterial and fungal infections. In this work, we synthesized a group of guanosine analogues containing an additional five-membered ring and a sulfur atom at the C-9 position. The spectral, photophysical, and biological properties of the synthesized compounds were investigated. The spectroscopic studies revealed that a combination of the thiocarbonyl chromophore and the tricyclic structure of guanine analogues shifts the absorption region above 350 nm, allowing for selective excitation when present in biological systems. Unfortunately, due to the low fluorescence quantum yield, this process cannot be used to monitor the presence of these compounds in cells. The synthesized compounds were evaluated for their effect on the viability of human cervical carcinoma (HeLa) and mouse fibroblast (NIH/3T3) cells. It was found that all of them display anticancer activity. In vitro studies were preceded by in silico ADME and PASS analyses, which confirmed that the designed compounds are promising candidates for anticancer agents.

Enzymatic Synthesis of 2-Chloropurine Arabinonucleosides with Chiral Amino Acid Amides at the C6 Position and an Evaluation of Antiproliferative Activity In Vitro.

A number of purine arabinosides containing chiral amino acid amides at the C6 position of the purine were synthesized using a transglycosylation reaction with recombinant E. coli nucleoside phosphorylases. Arsenolysis of 2-chloropurine ribosides with chiral amino acid amides at C6 was used for the enzymatic synthesis, and the reaction equilibrium shifted towards the synthesis of arabinonucleosides. The synthesized nucleosides were shown to be resistant to the action of E. coli adenosine deaminase. The antiproliferative activity of the synthesized nucleosides was studied on human acute myeloid leukemia cell line U937. Among all the compounds, the serine derivative exhibited an activity level (IC50 = 16 µM) close to that of Nelarabine (IC50 = 3 µM) and was evaluated as active.

Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes.

Adenosine deaminases acting on RNA (ADARs) are RNA editing enzymes that catalyze the hydrolytic deamination of adenosine (A) to inosine (I) in dsRNA. In humans, two catalytically active ADARs, ADAR1 and ADAR2, perform this A-to-I editing event. The growing field of nucleotide base editing has highlighted ADARs as promising therapeutic agents while multiple studies have also identified ADAR1's role in cancer progression. However, the potential for site-directed RNA editing as well as the rational design of inhibitors is being hindered by the lack of detailed molecular understanding of RNA recognition by ADAR1. Here, we designed short RNA duplexes containing the nucleoside analog, 8-azanebularine (8-azaN), to gain insight into molecular recognition by the human ADAR1 catalytic domain. From gel shift and in vitro deamination experiments, we validate ADAR1 catalytic domain's duplex secondary structure requirement and present a minimum duplex length for binding (14 bp, with 5 bp 5' and 8 bp 3' to editing site). These findings concur with predicted RNA-binding contacts from a previous structural model of the ADAR1 catalytic domain. Finally, we establish that neither 8-azaN as a free nucleoside nor a ssRNA bearing 8-azaN inhibits ADAR1 and demonstrate that the 8-azaN-modified RNA duplexes selectively inhibit ADAR1 and not the closely related ADAR2 enzyme.

Purine Nucleosides Interfere with c-di-AMP Levels and Act as Adjuvants To Re-Sensitize MRSA To ß-Lactam Antibiotics.

The purine-derived signaling molecules c-di-AMP and (p)ppGpp control mecA/PBP2a-mediated ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) raise the possibility that purine availability can control antibiotic susceptibility. Consistent with this, exogenous guanosine and xanthosine, which are fluxed through the GTP branch of purine biosynthesis, were shown to significantly reduce MRSA ß-lactam resistance. In contrast, adenosine (fluxed to ATP) significantly increased oxacillin resistance, whereas inosine (which can be fluxed to ATP and GTP via hypoxanthine) only marginally increased oxacillin susceptibility. Furthermore, mutations that interfere with de novo purine synthesis (pur operon), transport (NupG, PbuG, PbuX) and the salvage pathway (DeoD2, Hpt) increased ß-lactam resistance in MRSA strain JE2. Increased resistance of a nupG mutant was not significantly reversed by guanosine, indicating that NupG is required for guanosine transport, which is required to reduce ß-lactam resistance. Suppressor mutants resistant to oxacillin/guanosine combinations contained several purine salvage pathway mutations, including nupG and hpt. Guanosine significantly increased cell size and reduced levels of c-di-AMP, while inactivation of GdpP, the c-di-AMP phosphodiesterase negated the impact of guanosine on ß-lactam susceptibility. PBP2a expression was unaffected in nupG or deoD2 mutants, suggesting that guanosine-induced ß-lactam susceptibility may result from dysfunctional c-di-AMP-dependent osmoregulation. These data reveal the therapeutic potential of purine nucleosides, as ß-lactam adjuvants that interfere with the normal activation of c-di-AMP are required for high-level ß-lactam resistance in MRSA. IMPORTANCE The clinical burden of infections caused by antimicrobial resistant (AMR) pathogens is a leading threat to public health. Maintaining the effectiveness of existing antimicrobial drugs or finding ways to reintroduce drugs to which resistance is widespread is an important part of efforts to address the AMR crisis. Predominantly, the safest and most effective class of antibiotics are the ß-lactams, which are no longer effective against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report that the purine nucleosides guanosine and xanthosine have potent activity as adjuvants that can resensitize MRSA to oxacillin and other ß-lactam antibiotics. Mechanistically, exposure of MRSA to these nucleosides significantly reduced the levels of the cyclic dinucleotide c-di-AMP, which is required for ß-lactam resistance. Drugs derived from nucleotides are widely used in the treatment of cancer and viral infections highlighting the clinical potential of using purine nucleosides to restore or enhance the therapeutic effectiveness of ß-lactams against MRSA and potentially other AMR pathogens.

Synthesis of Nucleosides and Deoxynucleosides via Gold(I)-Catalyzed N-Glycosylation of Glycosyl (Z)-Ynenoates.

Nucleoside analogues are widely used as anticancer and antiviral drugs. Here, we develop a highly efficient gold(I)-catalyzed N-glycosylation approach for versatile synthesis of various types of nucleosides and deoxynucleosides with glycosyl (Z)-ynenoates as donors. The wide scope of the N-glycosylation approach was demonstrated by the synthesis of 31 pyrimidine nucleosides and 8 purine nucleosides. Remarkably, the gold(I)-catalyzed N-glycosylation of pyranosyl (Z)-ynenoates with purines was found to be very effective for regioselective synthesis of pyranosyl N9 purine nucleosides. Based on the catalytic N-glycosylation approach, convenient synthesis of two 5'-deoxynucleosides drugs (capecitabine and galocitabine), four 2'-deoxynucleoside drugs (floxuridine, trifluridine, decitabine and cladribine), four 3',5'-dideoxynucleoside analogues, and four 2',5'-dideoxynucleoside analogues was achieved in a collective manner.

Adamantane-Substituted Purine Nucleosides: Synthesis, Host-Guest Complexes with ß-Cyclodextrin and Biological Activity.

Purine nucleosides represent an interesting group of nitrogen heterocycles, showing a wide range of biological effects. In this study, we designed and synthesized a series of 6,9-disubstituted and 2,6,9-trisubstituted purine ribonucleosides via consecutive nucleophilic aromatic substitution, glycosylation, and deprotection of the ribofuranose unit. We prepared eight new purine nucleosides bearing unique adamantylated aromatic amines at position 6. Additionally, the ability of the synthesized purine nucleosides to form stable host-guest complexes with ß-cyclodextrin (ß-CD) was confirmed using nuclear magnetic resonance (NMR) and mass spectrometry (ESI-MS) experiments. The in vitro antiproliferative activity of purine nucleosides and their equimolar mixtures with ß-CD was tested against two types of human tumor cell line. Six adamantane-based purine nucleosides showed an antiproliferative activity in the micromolar range. Moreover, their effect was only slightly suppressed by the presence of ß-CD, which was probably due to the competitive binding of the corresponding purine nucleoside inside the ß-CD cavity.

6-Thioguanosine Monophosphate Prodrugs Display Enhanced Performance against Thiopurine-Resistant Leukemia and Breast Cancer Cells.

Thiopurines are in widespread clinical use for the treatment of immunological disorders and certain cancers. However, treatment failure due to resistance or adverse drug reactions are common, asking for new therapeutic strategies. We investigated the potential of 6-thioguanosine monophosphate (6sGMP) prodrugs to overcome resistance to 6-thioguanine. We successfully developed synthetic routes toward diverse 6sGMP prodrugs, tested their proliferation inhibitory potential in different cell lines, and examined their mode of action. Our results show that 4-acetyloxybenzyl- and cycloSaligenyl-derivatized 6sGMP prodrugs are effective antiproliferative compounds in cells that are resistant to thiopurines. We find that resistance is related to the expression of salvage pathway enzyme HGPRT. Using TUC-seq DUAL, we demonstrate the intracellular conversion of 6sGMP prodrugs into bioactive 6sGTPs. Thus, our study offers a promising strategy for thiopurine therapy by using 6sGMP prodrugs, and it suggests TUC-seq DUAL as a simple and fast method to measure the success of thiopurine therapy.

Preliminary Evidence of the Potent and Selective Adenosine A2B Receptor Antagonist PSB-603 in Reducing Obesity and Some of Its Associated Metabolic Disorders in Mice.

The adenosine A2A and A2B receptors are promising therapeutic targets in the treatment of obesity and diabetes since the agonists and antagonists of these receptors have the potential to positively affect metabolic disorders. The present study investigated the link between body weight reduction, glucose homeostasis, and anti-inflammatory activity induced by a highly potent and specific adenosine A2B receptor antagonist, compound PSB-603. Mice were fed a high-fat diet for 14 weeks, and after 12 weeks, they were treated for 14 days intraperitoneally with the test compound. The A1/A2A/A2B receptor antagonist theophylline was used as a reference. Following two weeks of treatment, different biochemical parameters were determined, including total cholesterol, triglycerides, glucose, TNF-α, and IL-6 blood levels, as well as glucose and insulin tolerance. To avoid false positive results, mouse locomotor and spontaneous activities were assessed. Both theophylline and PSB-603 significantly reduced body weight in obese mice. Both compounds had no effects on glucose levels in the obese state; however, PSB-603, contrary to theophylline, significantly reduced triglycerides and total cholesterol blood levels. Thus, our observations showed that selective A2B adenosine receptor blockade has a more favourable effect on the lipid profile than nonselective inhibition.

Synthesis of Polycyclic Hetero-Fused 7-Deazapurine Heterocycles and Nucleosides through C-H Dibenzothiophenation and Negishi Coupling.

A new approach for synthesizing polycyclic heterofused 7-deazapurine heterocycles and the corresponding nucleosides was developed based on C-H functionalization of diverse (hetero)aromatics with dibenzothiophene-S-oxide followed by the Negishi cross-cooupling with bis(4,6-dichloropyrimidin-5-yl)zinc. This cross-coupling afforded a series of (het)aryl-pyrimidines that were converted to fused deazapurine heterocycles through azidation and thermal cyclization. The fused heterocycles were glycosylated to the corresponding 2'-deoxy- and ribonucleosides, and a series of derivatives were prepared by nucleophilic substitutions at position 4. Four series of new polycyclic thieno-fused 7-deazapurine nucleosides were synthesized using this strategy. Most of the deoxyribonucleosides showed good cytotoxic activity, especially for the CCRF-CEM cell line. Phenyl- and thienyl-substituted thieno-fused 7-deazapurine nucleosides were fluorescent, and the former one was converted to 2'-deoxyribonucleoside triphosphate for enzymatic synthesis of labeled oligonucleotides.

Direct Minisci-Type C-H Amidation of Purine Bases.

A method for the C-H carboxyamidation of purines has been developed that is capable of directly installing primary, secondary, and tertiary amides. Previous Minisci-type investigations on purines were limited to alkylations and arylations. Herein, we present the first method for the direct C-H amidation of a wide range of purines: xanthine, guanine, and adenine structures, including guanosine- and adenosine-type nucleosides. The Minisci-type reaction is also metal-free, cheap, operationally simple, scalable, and applicable to late-stage functionalizations of biologically important molecules.

Alpha-ketoglutarate ameliorates abdominal aortic aneurysm via inhibiting PXDN/HOCL/ERK signaling pathways.

Abdominal aortic aneurysm (AAA) represents the serious vascular degenerative disorder, which causes high incidence and mortality. Alpha-ketoglutarate (AKG), a crucial metabolite in the tricarboxylic acid (TCA) cycle, has been reported to exert significant actions on the oxidative stress and inflammation. However, its role in AAA still remains elusive. Herein, we examined the effects of AKG on the formation of AAA. The study established an elastase-induced mouse abdominal aortic aneurysms model as well as a TNF-α-mediated vascular smooth muscle cells (VSMCs) model, respectively. We displayed that AKG pre-treatment remarkably prevented aneurysmal dilation assessed by diameter and volume and reduced aortic rupture. In addition, it was also observed that AKG treatment suppressed the development of AAA by attenuating the macrophage infiltration, elastin degradation and collagen fibers remodeling. In vitro, AKG potently decreased TNF-α-induced inflammatory cytokines overproduction, more apoptotic cells and excessive superoxide. Mechanistically, we discovered that upregulation of vpo1 in AAA was significantly suppressed by AKG treatment. By exploring the RNA-seq data, we found that AKG ameliorates AAA mostly though inhibiting oxidative stress and the inflammatory response. PXDN overexpression neutralized the inhibitory effects of AKG on ROS generation and inflammatory reaction in MOVAS. Furthermore, AKG treatment suppressed the expression of p-ERK1/2, 3-Cl Tyr in vivo and in vitro. ERK activator disrupted the protective of AKG on TNF-α-induced VSMCs phenotypic switch. Conclusively, AKG can serve as a beneficial therapy for AAA through regulating PXDN/HOCL/ERK signaling pathways.

c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3ß axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

Quantitation of Purine in Urine by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Inborn errors of purine metabolism, either deficiencies of synthesis or catabolism pathways, lead to a wide spectrum of clinical presentations: urolithiasis (adenine phosphoribosyltransferase), primary immune deficiency (adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency), severe intellectual disability, and other neurological symptoms (Lesch-Nyhan disease, adenylosuccinase deficiency, and molybdenum cofactor deficiency). A rapid quantitative purine assay was developed using UPLC-MS/MS to determine purine nucleoside and base concentrations in urine. Taking advantages of ultra-performance liquid chromatography, we achieved satisfactory analyte separation and recovery with a polar T3 column in a short run time with no requirement of time-consuming sample preparation or derivatization. This targeted assay is intended for diagnosis and management of purine diseases, newborn screening follow-up of SCID, and evaluation of autism spectrum disorders.

Pharmacological agents selectively acting on the channel moieties of TRPM6 and TRPM7.

The transient receptor potential cation channel, subfamily M, members 6 and 7 (TRPM6 and TRPM7) are homologous membrane proteins encompassing cation channel units fused to cytosolic serine/threonine-protein kinase domains. Clinical studies and experiments with animal disease models suggested that selective inhibition of TRPM6 and TRPM7 currents might be beneficial for subjects with immune and cardiovascular disorders, tumours and other pathologies, but the suitable pharmacological toolkit remains underdeveloped. The present study identified small synthetic molecules acting specifically on the channel moieties of TRPM6 and TRPM7. Using electrophysiological analysis in conjunction with Ca2+ imaging, we show that iloperidone and ifenprodil inhibit the channel activity of recombinant TRPM6 with IC50 values of 0.73 and 3.33 µM, respectively, without an impact on the TRPM7 channel. We also found that VER155008 suppresses the TRPM7 channel with an IC50 value of 0.11 µM but does not affect TRPM6. Finally, the effects of iloperidone and VER155008 were found to be suitable for blocking native endogenous TRPM6 and TRPM7 in a collection of mouse and human cell models. Hence, the identification of iloperidone, ifenprodil, and VER155008 allows for the first time to selectively manipulate TRPM6 and TRPM7 currents.