TCP Binding Research Articles

Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

Interaction between biochar-dissolved organic matter and chlorophenols during biochar adsorption.

Biochar (BC) has been widely applied in the remediation of chlorophenols (CPs) from contaminated sites in which the role and mechanisms of BC dissolved organic matter (BDOM), as a crucial component of BC, with CPs are largely unknown and thus need to be investigated. In this study, DOM was derived from peanut hulls (PDOM) and corn stalks (CDOM) as BC sources, and the interactions between PDOM/CDOM and 2,4,6-trichlorophenol (TCP) were analysed using excitation-emission matrix spectroscopy (EEM) in combination with multiple models. EEM combined with fluorescence region integration (EEM-FRI) indicated that humic-like materials were the major materials of both PDOM and CDOM (percentage fluorescence response Ri,n > 60%), and CDOM contained more protein- and fulvic-like materials than PDOM. Based on EEM in combination with parallel factor analysis (EEM-PARAFAC), 4 components were obtained, and the percentage decrease in maximum fluorescence intensities (Fmax) showed that the main components interacting with TCP in PDOM/CDOM were protein- and fulvic-like components (> 25%). Moreover, the modified Stern-Volmer model was used to calculate the stability constants (Log KTCP) of PDOM/CDOM and TCP for the first time, and the mechanism of static quenching was dominant for interacting with TCP in PDOM (Log KTCP: 4.36-4.65) and CDOM (Log KTCP: 3.53-4.73). Furthermore, the sequential TCP binding of fluorescent components in BDOM generally followed the order of protein-like → short-wavelength fulvic-like → long-wavelength fulvic-like → humic-like components. These findings will provide a basis for screening biochar as a functional material for CP remediation applications and for understanding the environmental chemical behaviour of leached DOM during biochar application.

TCP15 interacts with GOLDEN2-LIKE 1 to control cotyledon opening in Arabidopsis.

After germination, exposure to light promotes the opening and expansion of the cotyledons and the development of the photosynthetic apparatus in a process called de-etiolation. This process is crucial for seedling establishment and photoautotrophic growth. TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are important developmental regulators of plant responses to internal and external signals that are grouped into two main classes. In this study, we identified GOLDEN2-LIKE 1 (GLK1), a key transcriptional regulator of photomorphogenesis, as a protein partner of class I TCPs during light-induced cotyledon opening and expansion in Arabidopsis. The class I TCP TCP15 and GLK1 are mutually required for cotyledon opening and the induction of SAUR and EXPANSIN genes, involved in cell expansion. TCP15 also participates in the expression of photosynthesis-associated genes regulated by GLK1, like LHCB1.4 and LHCB2.2. Furthermore, GLK1 and TCP15 bind to the same promoter regions of different target genes containing either GLK or TCP binding motifs and binding of TCP15 is affected in a GLK1-deficient background, suggesting that a complex between TCP15 and GLK1 participates in the induction of these genes. We postulate that GLK1 helps to recruit TCP15 for the modulation of cell expansion genes in cotyledons and that the functional interaction between these transcription factors may serve to coordinate the expression of cell expansion genes with that of genes involved in the development of the photosynthetic apparatus.

Functional Antagonism of WRI1 and TCP20 Modulates GH3.3 Expression to Maintain Auxin Homeostasis in Roots.

Auxin is a well-studied phytohormone, vital for diverse plant developmental processes. The GH3 genes are one of the major auxin responsive genes, whose expression changes lead to modulation of plant development and auxin homeostasis. However, the transcriptional regulation of these GH3 genes remains largely unknown. WRI1 is an essential transcriptional regulator governing plant fatty acid biosynthesis. Recently, we identified that the expression of GH3.3 is increased in the roots of wri1-1 mutant. Nevertheless, in this study we found that AtWRI1 did not activate or repress the promoter of GH3.3 (proGH3.3) despite of its binding to proGH3.3. Cross-family transcription factor interactions play pivotal roles in plant gene regulatory networks. To explore the molecular mechanism by which WRI1 controls GH3.3 expression, we screened an Arabidopsis transcription factor library and identified TCP20 as a novel AtWRI1-interacting regulator. The interaction between AtWRI1 and TCP20 was further verified by several approaches. Importantly, we found that TCP20 directly regulates GH3.3 expression via binding to TCP binding element. Furthermore, AtWRI1 repressed the TCP20-mediated transactivation of proGH3.3. EMSAs demonstrated that AtWRI1 antagonized TCP20 from binding to proGH3.3. Collectively, we provide new insights that WRI1 attenuates GH3.3 expression through interaction with TCP20, highlighting a new mechanism that contributes to fine-tuning auxin homeostasis.

N-Substituted-3-alkoxy-derivatives of dextromethorphan are functional NMDA receptor antagonists in vivo: Evidence from an NMDA-induced seizure model in rats

Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = −0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = −0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

Heteroexpression of Osa-miR319b improved switchgrass biomass yield and feedstock quality by repression of PvPCF5

BackgroundSwitchgrass (Panicum virgatum L.), a C4 perennial grass, has been recognized as one of the most potentially important lignocellulose biofuel crops. MicroRNA319 (miR319) plays a key role in plant development, abiotic resistance, and cell wall biosynthesis by repressing expression of its target TCP genes. We hypothesized miR319–TCP pathway could play important roles in switchgrass feedstock characteristics for biofuel production, and produced switchgrass transgenic plants overexpressing miR319 (by ectopic expressing Osa-MIR319b gene), blocking miR319 (by overexpressing a target mimicry of miR319/MIM319) and repression of miR319 target gene PvPCF5. Plant phenotype, biomass yield, and feedstock quality of transgenic plants were analyzed.ResultsOverexpression of miR319 in switchgrass promoted leaf elongation and expansion of transgenic plants, increased plant height, stem diameter, and resulted in a significant increase in plant biomass yield. Transgenic plants overexpressing of miR319 reduced lignin content, showed significantly higher enzymatic hydrolysis efficiency compared to the wild type plant. However, opposite results were observed in the MIM319 plants. Furthermore, suppression of miR319 target gene PvPCF5 activity also reduced lignin content, increased lignin monomer S/G ratio and the proportion of β-O-4 linkages, while significantly improving the sugar production per plant. Quantitative real-time (qRT-PCR) analysis indicated that expression of PvMYB58/63B and PvHCT with predicted TCP binding sites in their promoter regions was negatively regulated by miR319–PvPCF5 module.ConclusionsMiR319–PvPCF5 module plays positive roles in regulating biomass yield and quality of switchgrass. It can be utilized as a candidate molecular tool in regulating biomass yield and feedstock quality. The finding could also be transferred to other grasses for forage quality improvement through genetic manipulation.

Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development.

Leaves grow by distinct phases controlled by gene regulatory networks including many transcription factors. Arabidopsis thaliana homeobox 12 (ATHB12) promotes leaf growth especially during the cell expansion phase. In this study, we identify TCP13, a member of the TCP transcription factor family, as an upstream inhibitor of ATHB12. Yeast one-hybrid screening using a 1.2-kb upstream region of ATHB12 resulted in the isolation of TCP13 as well as other transcription factors. Transgenic plants constitutively expressing TCP13 displays a significant reduction in leaf cell size especially during the cell expansion period, while repression of TCP13 and its paralogs (TCP5 and TCP17) result in enlarged leaf cells, indicating that TCP13 and its paralogs inhibit leaf development, mainly at the cell expansion phase. Its expression pattern during leaf expansion phase is opposite to ATHB12 expression. Consistently, the expression of ATHB12 and its downstream genes decreases when TCP13 was overexpressed, and increases when the expression of TCP13 and its paralogs is repressed. In chromatin immunoprecipitation assays using TCP13-GFP plants, a fragment of the ATHB12 upstream region that contains the consensus sequence for TCP binding is strongly enriched. Taken together, these findings indicate that TCP13 and its paralogs inhibit leaf growth by repressing ATHB12 expression.

Direct Regulation of the EFR-Dependent Immune Response by Arabidopsis TCP Transcription Factors.

One layer of the innate immune system allows plants to recognize pathogen-associated molecular patterns (PAMPS), activating a defense response known as PAMP-triggered immunity (PTI). Maintaining an active immune response, however, comes at the cost of plant growth and development; accordingly, optimization of the balance between defense and development is critical to plant fitness. The TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor family consists of well-characterized transcriptional regulators of plant development and morphogenesis. The three closely related class I TCP transcription factors TCP8, TCP14, and TCP15 have also been implicated in the regulation of effector-triggered immunity, but there has been no previous characterization of PTI-related phenotypes. To identify TCP targets involved in PTI, we screened a PAMP-induced gene promoter library in a yeast one-hybrid assay and identified interactions of these three TCPs with the EF-Tu RECEPTOR (EFR) promoter. The direct interactions between TCP8 and EFR were confirmed to require an intact TCP binding site in planta. A tcp8 tcp14 tcp15 triple mutant was impaired in EFR-dependent PTI and exhibited reduced levels of PATHOGENESIS-RELATED PROTEIN 2 and induction of EFR expression after elicitation with elf18 but also increased production of reactive oxygen species relative to Col-0. Our data support an increasingly complex role for TCPs at the nexus of plant development and defense.

Functional dissection of the ARGONAUTE7 promoter.

ARGONAUTES are the central effector proteins of RNA silencing which bind target transcripts in a small RNA‐guided manner. Arabidopsis thaliana has 10 ARGONAUTE (AGO) genes, with specialized roles in RNA‐directed DNA methylation, post‐transcriptional gene silencing, and antiviral defense. To better understand specialization among AGO genes at the level of transcriptional regulation we tested a library of 1497 transcription factors for binding to the promoters of AGO1,AGO10, and AGO7 using yeast 1‐hybrid assays. A ranked list of candidate DNA‐binding TFs revealed binding of the AGO7 promoter by a number of proteins in two families: the miR156‐regulated SPL family and the miR319‐regulated TCP family, both of which have roles in developmental timing and leaf morphology. Possible functions for SPL and TCP binding are unclear: we showed that these binding sites are not required for the polar expression pattern of AGO7, nor for the function of AGO7 in leaf shape. Normal AGO7 transcription levels and function appear to depend instead on an adjacent 124‐bp region. Progress in understanding the structure of this promoter may aid efforts to understand how the conserved AGO7‐triggered TAS3 pathway functions in timing and polarity.

TCP Transcription Factors Interact With NPR1 and Contribute Redundantly to Systemic Acquired Resistance.

In Arabidopsis, TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factors (TF) play critical functions in developmental processes. Recent studies suggest they also function in plant immunity, but whether they play an important role in systemic acquired resistance (SAR) is still unknown. NON-EXPRESSER OF PR GENES 1 (NPR1), as an essential transcriptional regulatory node in SAR, exerts its regulatory role in downstream genes expression through interaction with TFs. In this work, we provide biochemical and genetic evidence that TCP8, TCP14, and TCP15 are involved in the SAR signaling pathway. TCP8, TCP14, and TCP15 physically interacted with NPR1 in yeast two-hybrid assays, and these interactions were further confirmed in vivo. SAR against the infection of virulent strain Pseudomonas syringae pv. maculicola (Psm) ES4326 in the triple T-DNA insertion mutant tcp8-1 tcp14-5 tcp15-3 was partially compromised compared with Columbia 0 (Col-0) wild type plants. The induction of SAR marker genes PR1, PR2, and PR5 in local and systemic leaves was dramatically decreased in the tcp8-1 tcp14-5 tcp15-3 mutant compared with that in Col-0 after local treatment with Psm ES4326 carrying avrRpt2. Results from yeast one-hybrid and chromatin immunoprecipitation (ChIP) assays demonstrated that TCP15 can bind to a conserved TCP binding motif, GCGGGAC, within the promoter of PR5, and this binding was enhanced by NPR1. Results from RT-qPCR assays showed that TCP15 promotes the expression of PR5 in response to salicylic acid induction. Taken together, these data reveal that TCP8, TCP14, and TCP15 physically interact with NPR1 and function redundantly to establish SAR, that TCP15 promotes the expression of PR5 through directly binding a TCP binding site within the promoter of PR5, and that this binding is enhanced by NPR1.

Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata.

Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K d=2960±940μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCPinterior, suggesting that the other site, TCPexterior, remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCPinterior binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.

TCP transcription factors are critical for the coordinated regulation of isochorismate synthase 1 expression in Arabidopsis thaliana.

Salicylic acid (SA) plays an important role in various aspects of plant development and responses to stresses. To elucidate the sophisticated regulatory mechanism of SA synthesis and signaling, we used a yeast one-hybrid system to screen for regulators of isochorismate synthase 1 (ICS1), a gene encoding the key enzyme in SA biosynthesis in Arabidopsis thaliana. A TCP family transcription factor AtTCP8 was initially identified as a candidate regulator of ICS1. The regulation of ICS1 by TCP proteins is supported by the presence of a typical TCP binding site in the ICS1 promoter. The binding of TCP8 to this site was confirmed by in vitro and in vivo assays. Expression patterns of TCP8 and its corresponding gene TCP9 largely overlapped with ICS1 under pathogen attack. A significant reduction in the expression of ICS1 during immune responses was observed in the tcp8 tcp9 double mutant. We also detected strong interactions between TCP8 and SAR deficient 1 (SARD1), WRKY family transcription factor 28 (WRKY28), NAC (NAM/ATAF1,ATAF2/CUC2) family transcription factor 019 (NAC019), as well as among TCP8, TCP9 and TCP20, suggesting a complex coordinated regulatory mechanism underlying ICS1 expression. Our results collectively demonstrate that TCP proteins are involved in the orchestrated regulation of ICS1 expression, with TCP8 and TCP9 being verified as major representatives.

Non-competitive Inhibition of Nicotinic Acetylcholine Receptors by Ladybird Beetle Alkaloids.

Ladybird beetles (Family Coccinellidae) secrete an alkaloid rich venom from their leg joints that protects them from predators. Coccinellines, the major venom constituents, are alkaloids composed of three fused piperidine rings that share a common nitrogen atom. Although many coccinellines have been isolated and chemically characterized, their pharmacological properties are essentially unknown. Using radioligand binding and functional assays we investigated the actions of several coccinellines on skeletal muscle and α7 nicotinic acetylcholine receptors (nAChRs). The alkaloids were shown to displace the specific binding of tritiated piperidyl-N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([(3)H]-TCP), which has been shown to bind deep within the ion channel of the electric fish (Torpedo) muscle nAChR. The stereoisomers precoccinelline and hippodamine (whose nitrogens are predicted to be ionized at physiological pH) and their respective analogs N-methyl-precoccinelline and N-methyl-hippodamine (whose quaternary nitrogens are permanently charged) displayed similar IC50s for inhibition of [(3)H]-TCP binding. However, the corresponding precoccinelline and hippodamine N-oxides, coccinelline and convergine (which have an electronegative oxygen bonded to an electropositive nitrogen) displayed significantly higher binding IC50s. Finally, exochomine, a dimeric coccinelline containing the hippodamine structure, displayed the highest IC50 (lowest affinity) for displacing specific [(3)H]-TCP binding. The presence of a desensitizing concentration (10(-3) M) of carbachol (CCh) had little or no effect on the affinity of the Torpedo nAChR for the three coccinellines tested. High concentrations of the coccinellid alkaloids did not affect binding of [(3)H]-cytisine to Torpedo receptor ACh binding sites. Inhibition of the alpha7 nAChR with pre-equilibrated precoccinelline was insurmountable with respect to ACh concentration. We conclude that the coccinellines bind to one or more allosteric sites rather than to the ACh binding sites, and inhibit nAChR responses to ACh through a non-competitive mechanism. Future chemical and pharmacological investigations of other ladybird beetle alkaloids are likely to reveal other interesting alkaloids affecting ligand-gated receptors.

Complexes of Dual-Function Hemoglobin/Dehaloperoxidase with Substrate 2,4,6-Trichlorophenol Are Inhibitory and Indicate Binding of Halophenol to Compound I

The hemoglobin of sea worm Amphitrite ornata, which for historical reasons is abbreviated as DHP for dehaloperoxidase, has two physiological functions: it binds dioxygen in the ferrous state and dehalogenates halophenols, such as 2,4,6-trichlorophenol (TCP), using hydrogen peroxide as the oxidant in the ferric state. The crystal structures of three DHP variants (Y34N, Y34N/S91G, and L100F) with TCP bound show two mutually exclusive modes of substrate binding. One of them, the internal site, is deep inside the distal pocket with the phenolic OH moiety forming a hydrogen bond to the water molecule coordinated to the heme Fe. In this complex, the distal histidine is predominantly located in the closed position and also forms a hydrogen bond to the phenolic hydroxide. The second mode of TCP binding is external, at the heme edge, with the halophenol molecule forming a lid covering the entrance to the distal cavity. The distal histidine is in the open position and forms a hydrogen bond to the OH group of TCP, which also hydrogen bonds to the hydroxyl of Tyr38. The distance between the Cl4 atom of TCP and the heme Fe is 3.9 Å (nonbonding). In both complexes, TCP molecules prevent the approach of hydrogen peroxide to the heme, indicating that the complexes are inhibitory and implying that the substrates must bind in an ordered fashion: hydrogen peroxide first and TCP second. Kinetic studies confirmed the inhibition of DHP by high concentrations of TCP. The external binding mode may resemble the interaction of TCP with Compound I, the catalytic intermediate to which halophenols bind. The measured values of the apparent Km for TCP were in the range of 0.3-0.8 mM, much lower than the concentrations required to observe TCP binding in crystals. This indicates that during catalysis TCP binds to Compound I. Mutant F21W, which likely has the internal TCP binding site blocked, has ~7% of the activity of wild-type DHP.

Lipophilicity as a determinant of binding of procaine analogs to rat α3β4 nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (nAChRs) have been studied in detail with regard to their interaction with therapeutic and drug addiction-related compounds. Using a structure-activity approach, we have examined the relationship among the molecular features of a set of eight para-R-substituted N,N-[(dimethylamino)ethyl] benzoate hydrochlorides, structurally related to procaine and their affinity for the α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Affinity values (log[1/IC50]) of these compounds for the α(3)β(4) nAChR were determined by their competition with [(3)H]TCP binding. Log(1/IC50) values were analyzed considering different hydrophobic and electronic parameters and those related to molar refractivity. These have been experimentally determined or were taken from published literature. In accordance with literature observations, the generated cross-validated quantitative structure-activity relationship (QSAR) equations indicated a significant contribution of hydrophobic term to binding affinity of procaine analogs to the receptor and predicted affinity values for several local anesthetics (LAs) sets taken from the literature. The predicted values by using the QSAR model correlated well with the published values both for neuronal and for electroplaque nAChRs. Our work also reveals the general structure features of LAs that are important for interaction with nAChRs as well as the structural modifications that could be made to enhance binding affinity.

Structure–activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca 2+ influx in human embryonic muscle AChRs with the following potency sequence (IC 50 in μM): (±)-18-methylaminocoronaridine (5.9 ± 0.3) ∼ (±)-18-methoxycoronaridine (18-MC) (6.8 ± 0.8) > (−)-ibogaine (17 ± 3) ∼ (+)-catharanthine (20 ± 1) > (±)-albifloranine (46 ± 13), (b) bind to the [ 3H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [ 3H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [ 3H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC 50) that correlate very well with the p K i values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 Å 3 for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization.

Interaction of 18-Methoxycoronaridine with Nicotinic Acetylcholine Receptors in Different Conformational States

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca2+ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [3H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [3H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6’) and valine (position 13’) rings, and (c) inhibits [3H]TCP, [3H]ibogaine, and [3H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.Support: Science Foundation Arizona

Catharanthine alkaloids are noncompetitive antagonists of muscle-type nicotinic acetylcholine receptors

We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (±)-epibatidine-induced Ca 2+ influx in TE671-hα1β1γδ cells with similar potencies (IC 50 = 17–25 μM), (b) inhibit [ 3H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [ 3H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [ 3H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6′), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20′), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel.

Interaction of fluoxetine and paroxetine with muscle nicotinic acetylcholine receptors

The interaction of serotonin selective reuptake inhibitors (SSRIs) (i.e., fluoxetine and paroxetine) with muscle nicotinic acetylcholine receptors (AChRs) in different conformational states was compared to that for dizocilpine by using functional and structural methods. The results established that: (a) SSRIs inhibit (±)‐epibatidine‐induced Ca2+ influx with higher potencies than dizocilpine, (b) SSRIs inhibit [3H]TCP binding to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [3H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where fluoxetine and dizocilpine in the neutral state interact with the valine (position 13′) and leucine (position 9′) rings. Protonated fluoxetine has two potential binding sites, one located at or near the outer ring (position 20′), and another that overlaps the domain for the neutral molecules and for protonated dizocilpine. The high proportion of protonated fluoxetine and dizocilpine at pH 7.4 (&gt;95%) suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel, in a site shared with PCP. Support: Science Foundation Arizona.

Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization.