Keywords:Rhinella marina, toad,N-methyltransferase, enzyme kinetics, indolethylamine, substrate acceptance profiling, psychedelic, drug discovery, serotonin receptors, metabolic stability

Transcriptome mining reveals novel cane toad NMT candidates

As mammalian INMTs are known toN-methylate tryptamine and serotonin substrates (34), human (Homo sapien; HsINMT) and European rabbit (Oryctolagus cuniculus; OcINMT) variants were used to query a published cane toad transcriptome (35) in search of putative NMT candidates. A total of 33 coding DNA sequences (CDS) annotating as putative INMT or nicotinamide NMT (NNMT) were identified. Candidates annotating as NNMT were included in the candidate pool since NNMTs share a high degree of structural and sequence identity with INMTs (36) and act on similar small molecules. Of the 33 CDS, alignment with functionally established INMTs and NNMTs revealed seven full-length cane toad candidates. These seven candidates shared 30 to 50% sequence identity with INMTs, and phylogenetic analysis revealed support for the clustering of two candidates (Rm25608, Rm8745) with INMTs and NNMTs, distinct from a separate cluster containing Rm54673 and Rm35569 (Figs. S2–S4). Querying the cane toad transcriptome with PcPsiM (Psilocybe cyanescens N-methyltransferase) did not yield any hits, and attempts to align PcPsiM with INMT, NNMT, or cane toad candidates were not successful likely owing to very low (<10%) sequence identity between fungal PcPsiM and animal sequences.

Rm35569 is RmNMT, the first enzyme from a toad implicated in alkaloid biosynthesis

The seven full-length cane toad candidates were synthesized as codon-optimized DNA sequences, subcloned to an expression vector in-frame with vector-encoded His₆ tags, and expressed inE. coli. The His-tagged target recombinant protein (∼25–30 kDa) was confirmed using immunoblot analysis of crude bacterial lysate (Fig. S5). To determine if any of these candidates performedN-methylation on indolethylamine-type metabolites, serotonin and tryptamine, respectively, were batch-fed toE. coli cultures following induction of recombinant protein expression under standardin vivo assay conditions. Following incubation with the substrate, the microbial broth was analyzed for the presence of secondary, tertiary, and quaternary indolethylamines (i.e., single, double, and triple (quaternary)N-methylation products, respectively) using high-resolution LC-HESI-LTQ-Orbitrap-XL instrumentation. One candidate (Rm35569, expressed using plasmid pRm35569) was capable of accepting both serotonin and tryptamine, yielding both single (N-) and double (N,N-) methylation products (Figs. 2 andS6). Quaternary indolethylamines were not detected and the remaining six cane toad candidates failed to produce anyN-methylation products. Owing to itsN-methylation activity, Rm35569 was renamed RmNMT and further investigated to determine whether or not other indolethylamine-accumulating toads possess a similar NMT and whether frogs, which in contrast with toads, do not produce methylated indolethylamines, possess such an enzyme. RmNMT was used to query National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov) databases for candidate toad and frog sequences. The search yielded an additional sequence (Bb82723) from common toad (Bufo bufo), which like cane toad accumulates bufotenin and related methylated indolethylamines. Two frog sequences were identified: one from High Himalaya frog (Nanorana parkeri) (Np27127) and another from common coquí frog (Eleutherodactylus coqui) (EcEc64034). Phylogenetic analysis of these three new sequences supported clustering with RmNMT and Rm54673, distinct from mammalian INMT sequences (Fig. S2). These additional copy DNAs were synthesized, subcloned to expression vectors used to transformE. coli, and subjected to thein vivo assays for activity with tryptamine and serotonin. Immunoblot analysis showed abundant frog proteins (Np27127 and Ec64034) but a lower abundance of toad Bb82723 protein (Fig. S5). Bb82723 from common toad also showed NMT activity (Fig. S7); thus, Bb82723 was renamed BbNMT. In contrast, the two frog candidates yielded no reaction products despite their strong similarity with toad sequences. RmNMT shares 85%, 90%, and 95% similarity with Np27127, Ec64034, and BbNMT, respectively (Fig. S4B).

Kinetic analysis of RmNMT and other NMTs

Phylogenetic analysis revealed clustering of RmNMT and BbNMT together with the inactive frog sequences in a clade distinct from mammalian INMTs. Since RmNMT and BbNMT are highly similar (95% amino acid similarity,Fig. S4B) and recombinant RmNMT appeared more abundant inE. coli protein extracts (Fig. S5), RmNMT was selected for further characterization. Recombinant RmNMT was purified (Fig. S8) and subjected to kinetics analysis (Table S1 andFig. S9) in comparison with previously published values for HsINMT and OcINMT (Table S1). Previous studies showed the occurrence of multiple HsINMT isoforms, although published kinetic data are similar among the different human variants. This ‘similarity’ among different HsINMTs is especially evident upon comparison with RmNMT, whereby all HsINMT variants displayK_m andV_max values that differ from RmNMT values by several orders of magnitude (12,37) (Table S1). TheK_m of RmNMT was consistently lower (in cases where comparison was possible), and theV_max was 1134-fold greater compared with native HsINMT alloenzyme 254C (37). Furthermore, the k_cat of RmNMT with tryptamine as the substrate (5.2 × 10⁻⁴ s⁻¹) was 1711-fold greater than the k_cat of HsINMT (3.0 × 10⁻⁷ s⁻¹), implying a much higher turnover number per second. To allow more direct comparison, we tested a mammalian variant ourselves alongside RmNMT. OcINMT was selected as the variant among mammalian INMTs exhibiting the most desirable kinetic parameters (Table 1). In addition, we also tested the fungalP. cyanescens PsiM (PcPsiM) enzyme. Although PsiM appears restricted to accepting 4-O-phosphorylated indolethylamine substrates (38) its ability to facilitate psilocybin biosynthesis in microorganisms at titers near or exceeding 1 g/L (8,9) demonstrates that it is an effective enzyme in certain contexts.

OcINMT (GenBankNP_001075512) and PcPsiM (GenBank A0A286LEZ7) were produced inE. coli using identical procedures as for RmNMT. Immunoblot analysis revealed that comparable quantities of each protein (i.e., RmNMT, OcINMT, and PcPsiM) were obtained following purification (Fig. S10). Each purified enzyme was subjected to standardin vitro assays using tryptamine,N-methyltryptamine, serotonin, andN-methylserotonin as potential substrates (Table 1). No activity was detected for PcPsiM, whereas OcINMT was unable toN,N-dimethylate tryptamine to DMT, instead ending with the formation ofN-methyltryptamine (13). However, if providedN-methyltryptamine, OcINMT produced a small amount of DMT. OcINMT did not accept serotonin, although it converted a small quantity ofN-methylserotonin to bufotenin. Overall, RmNMT activity far exceeded that of OcINMT. The toad enzyme was able to fullyN-methylate both tryptamine and serotonin yielding DMT and bufotenin, respectively, and showed activities up to 845-fold greater than OcINMT withN-methylserotonin as the substrate.

Use of RmNMT to produce purifiable quantities of novel indolethylamine derivatives

With the aim of generating diverse derivatives for pharmacological screening purposes, RmNMT was deployed in the bioproduction of variousN,N-dimethylated indolethylamines. The capacity of RmNMT to yieldN-methyl andN,N-dimethyl products was evaluated in transformedE. coli cultures batch-fed 16 different indolethylamine substrates possessing a primary amine moiety (Fig. 3 andSupporting Information S1). The substrates displayed structural variations including ring hydroxylation, halogenation, methylation, alkylation, and methoxylation, representing a plurality of combinations at various positions within the core tryptamine structure, although structural variation was largely focused on the aromatic indole moiety. Key exceptions were tryptophan and 5-hydroxytryptophan, which were tested as substrates owing to a recent report of a tryptophanN-methyltransferase from the ‘magic mushroom’ speciesPsilocybe serbica (39). Notably,N-methylated tryptophan derivatives are restricted from undergoing decarboxylation (7) and, thus, would not be predicted as intermediates in bufotenin biosynthesis (Fig. 1). RmNMT accepted 12 of 16 tested substrates to varying degrees (Fig. 3), although the enzyme was unable toN-methylate tryptophan or 5-hydroxytryptophan and displayed little or no activity with 2,7-disubstituted indolethylamines. Nevertheless, 2-substituted and 7-substituted compounds were accepted; for example, 2-methyltryptamine and 7-fluorotryptamine were converted toN,N-dimethyl product at titers of 43 mg/L and 22 mg/L, respectively. In 2 of 16 cases, the assay revealed relatively high titers for both single (N-) and double (N,N-) methylation product (i.e., 5-butyltryptamine and 4-hydroxytryptamine substrates). In most cases, however, largelyN,N-dimethylated products were observed suggesting little if any product inhibition, which is an established feature of mammalian INMTs (13). In 5 of 16 cases, the anticipated product was novel. Querying PubChem (pubchem.ncbi.nlm.nih.gov), SciFinder (cas.org), and REAxys (reaxys.com) databases for the 5-butyl-DMT product revealed no hits. However, only three of the five anticipated novel products were produced by RmNMT since two key substrates (i.e., 2,7-dimethyltryptamine, 7-trifluoromethyl-2-methyltryptamine) were not accepted. In most cases, RmNMT turned over sufficient material enabling the recovery of milligram quantities at 95% (w/w) purity (Fig. S11). In general, purification was an option when titers exceeded 10 mg/L. Below this titer, larger culture volumes would be required, which was not pursued in this work. In total, 11 of 16 conversions yielded product titers at >10 mg/L, of which six were scaled up for purification, which yielded between 5 and 37.5 mg of 95% pure product from 0.6 L of culture broth. NMR characterization was performed to assign all six pure compounds, and original spectra are provided for the one novel product that was isolated, 6-trifluoromethoxy-DMT (Supporting Information S2).

N,N-Dimethylation reduces competitive binding at 5-HT_1A receptor

As first-pass screening tools,in vitro receptor-binding assays were used for head-to-head comparisons of primary amine and correspondingN,N-dimethylated indolethylamines. Further comparisons were possible between nonsubstituted compounds (tryptamine, DMT) and those substituted at positions 4, 5, 6, and 2 of the indolic ring. The 5-HT_1A receptor was selected owing to its established association with mental health disorders (22,23,24), whereas the 5-HT_2A receptor is an emerging target in psychiatric medicine, as it binds potentially therapeutic psychedelic indolethylamines (25,40). The consistently higher binding affinity of primary amine ligands at the 5-HT_1A receptor compared with theirN,N-dimethyl counterparts was immediately apparent, withK_i ratios (N,N-dimethyl:primary amine counterparts) in the range from 1.9 to 5.1 (Table 2 andFig. S12). In contrast, these dramatic differences were not observed in 5-HT_2A receptor ligand competition assays (Table 2 andFig. S13). Generally, compounds free of ring substituents (e.g., tryptamine, DMT) or those bearing functional groups at positions 4 or 5 bound more tightly to the 5-HT_1A receptor than 6- or 2-substituted ligands (Table 2). For example,K_i values only exceeded 100 nM for 6- or 2-substituted indolethylamines, reaching micromolar levels for 6-trifluoromethoxy-DMT, 2-methyltryptamine, and 2-methyl-DMT. Clear positional effects did not emerge for 5-HT_2A receptor assays; however, poor binding was observed for 6-trifluoromethoxylated and 2-methylated ligands. Of particular interest were the effects of 6-substitution on the indolethylamine core, as certain 6-substituted derivatives are purportedly nonhallucinogenic yet still therapeutically useful (41,42). Literature regarding impact of 6-fluorination on the binding of indolethylamines to the 5-HT_2A receptor have shown enhanced affinity; for example, in comparing 6-fluoro-DMT (K_i 866 nM) to DMT (K_i 2323 nM) (15) or comparing 6-fluoro-psilocin (K_i 13 nM) to psilocin (K_i 25 nM) (43). Congruent with these studies, results herein suggest tighter binding for 6-fluoro-DMT (K_i 511 nM) compared with DMT (K_i 1513 nM) (Table 2). In contrast with 6-fluorination, previous work has demonstrated that 6-methoxylation can reduce binding at the 5-HT_2A receptor (e.g., K_i values of 7300 nM and 1200 nM for 6-methoxy-DMT and DMT, respectively) (44). Similarly, results herein reveal diminished affinity of 6-trifluoromethoxy-DMT compared to DMT (K_i values of 10,681 nM and 1513 nM, respectively;Table 2).

N,N-dimethylation enhances metabolic stability, with exception of 6-substituted derivatives

Human liver microsome (HLM) fractions are rich in catabolic enzymes known to act on indolethylamine-type molecules and as such represent a standard tool to estimate hepatic intrinsic clearance of MAO-substrate drugs (26). Beyond MAO, other liver enzymes known to metabolize indolethylamine-type molecules include CYPs, aldehyde oxidase, and UDP-glucuronosyltransferases (28,29,30,31,32,33). The most pervasive result of the HLM analysis was the enhanced stability ofN,N-dimethylated indolethylamines compared with their primary amine counterparts (Fig. 4). This trend was consistent except for 6-substituted derivatives (Fig. 4,G andH), wherein each compound appeared to degrade at a similarly rapid rate. BeyondN,N-dimethylation, metabolic stability also appeared augmented by the position of the hydroxyl in psilocin and 4-hydroxytryptamine. For example, serotonin (5-hydroxytryptamine) degraded more rapidly than 4-hydroxytryptamine, whereas bufotenin (5-hydroxy-DMT) degraded faster than psilocin (4-hydroxy-DMT) (Fig. 3,A andC). Addition of a 2-methyl moiety afforded some stability, as 2-methyltryptamine and 2-methyl-DMT (Fig. 4I) showed greater stability compared with tryptamine and DMT, respectively (Fig. 4B).

Alkaloid biosynthesis in toads

Toads accumulate an array of bioactive specialized metabolites, including steroidal compounds along with guanidine, indole, piperidine, tricyclic, and decahydroquinoline alkaloids (2). Recent metabolic biochemistry efforts have focused on the biosynthesis of toxic bufadienolides, which has resulted in the discovery of novel enzymes, including a short-chain dehydrogenase/reductase epimerase (45). Whereas toads accumulate indolethylamines such as bufotenin and DMT, frogs appear devoid of these metabolites, in agreement with the lack ofN-methylation activity among frog enzymes despite extensive (90%) amino acid sequence similarity with RmNMT (Fig. S4B). Support for a phylogenetic cluster containing both active toad enzyme (RmNMT, BbNMT) and inactive frog proteins distinct from the mammalian INMT clade suggests that NMT activity arose independently in amphibians and mammals. Furthermore, low sequence identity (<10%) of PsiM to any animal NMT suggests yet another independent evolutionary trajectory for theN-methylation of indolethylamines in fungi. Use of RmNMT sequence to query NCBI nonredundant protein database with BLASTP function (https://blast.ncbi.nlm.nih.gov/Blast.cgi) yielded uncharacterized or predicted proteins annotating as NNMT. Mammalian NNMT was originally identified as the catalyst responsible for methylation of NAM (vitamin B3) and has recently been implicated in a wider array of metabolic disorders (46). NNMT is found in organisms ranging from higher mammals to microbes (47) and thus is anticipated in toads. An outstanding question is whether RmNMT or other candidate sequence(s) tested herein accept NAM substrate and which role(s) these proteins playin vivo. Few cane toad functional genomics tools exist, hampering directin vivo investigation although a draft genome is available (48) and genetic manipulation has been discussed (49). While RmNMT readily accepts both tryptamine and serotonin substrates (Table S1), only 5-hydroxylated products such as bufotenin, cyclized dehydrobufotenin, and the likely downstream metabolite bufothionine occur in cane toad (Fig. S1) (2). The lack of DMT in cane toad could reflect general or localized inaccessibility to tryptamine as a substrate. In toad species known to accumulate DMT (e.g., Colorado river toad;Incilius alvarius), it occurs as an arguably minor metabolite compared with 5-hydroxylated or 5-methoxylated compounds such as 5-methoxy-DMT (50). The low levels of DMT in many toads could alternatively reflect a preference of RmNMT for an endogenous substrate other than tryptamine (or to a lesser extent, serotonin), which outcompetes other reactants at the active site. For example, it is possible that RmNMT acts directly on cyclized indolethylamines en route to the production of dehydrobufotenin or bufothionine (Fig. S1). Cyclized indolethylamines were not assayed as potential substrates in this work. Future efforts to elucidate biochemical pathways in toads should include an expanded evaluation of potential substrates.

RmNMT is a relatively efficient catalyst compared with INMTs

RmNMT catalyzed two sequentialN-methylations yielding tertiary amines, although it did not perform a third methylation to generate quaternary amines. Production of quaternary amines is also not a feature of INMT enzymes. While INMTs have yet to be isolated from plants, citrus species are known to accumulate primary, secondary, tertiary (e.g., DMT), and quaternary tryptamines (51). In fact, quaternary amines are dominantly abundant in plants such as bergamot, suggesting the presence of indolethylamine NMT(s) favoring additionalN-methylation reactions. Other sources of DMT includePsychotria viridis (52), an ingredient of ayahuasca. Development and mining of broader plant-based resources could yield additional indolethylamine-modifying catalysts with preferred or alternative kinetic properties. Kinetic characterization of RmNMT showed no indication of product inhibition, which is a feature of INMT enzymes (13). The lack of product inhibition contributed to a 30 to 100% turnover rate of primary indolethylamines to correspondingN,N-dimethyl products inE. coli cultures (Fig. 3 andSupporting Information S1). The secondN-methylation yielding either DMT or bufotenin was more efficient than the first. For example, k_cat/K_m values of 5.4 M⁻¹s⁻¹ and 56.2 M⁻¹s⁻¹ were obtained using tryptamine andN-methyltryptamine substrates, respectively (Table S1). The maximum velocity of RmNMT was 1134-fold greater than reported V_max of HsINMT, and K_m values were substantially lower than those reported for mammalian INMTs (11,12,13,37). Furthermore, the k_cat of RmNMT using tryptamine substrate was 1711-fold greater than the inferred k_cat for HsINMT (Table S1). Observing these dramatic differences prompted us to conduct our own, head-to-head comparison of recombinant RmNMT and the ‘best-performing’ mammalian INMT known to date. Of 10 human and rabbit INMT isoforms reviewed by Dean (34), the ‘reference’ or ‘lung’ rabbit INMT sequence (12) exhibited the lowest K_m with tryptamine substrate (270 μM) and was thus chosen for recombinant expression.P. cyanescens norbaeocystin methyltransferase (PcPsiM), which operates on indolethylamines within the psilocybin biosynthetic pathway (Fig. 1), was not expected to accept substrates lacking a 4-O-phosphoryl group and was therefore included as a negative control. The assays showed 18-, 27-, and 846-fold higher conversion of tryptamine,N-methyltryptamine, andN-methylserotonin, respectively, for RmNMT compared with OcINMT (Table 1). Failed OcINMT conversion of serotonin likely reflected a very low inherent activity falling below the detection limit of our assay, which was performed under linear product-forming conditions enabling quantitative comparison. Previously, OcINMT was shown to accept serotonin at 11% the value observed for tryptamine substrate (11).

Turnover numbers (k_cat) and catalytic efficiencies (k_cat/K_m) of ‘average’ enzymes are estimated to be between 10¹ s⁻¹ and 10⁵ s⁻¹M⁻¹, respectively (53). The k_cat and k_cat/K_m of recombinant RmNMT are several orders of magnitude lower than this average (10⁻⁴ s⁻¹ and 10¹ s⁻¹M⁻¹, respectively;Table S1) revealing the enzyme to be sluggish in the broader context. However, a sluggish character is typical in the context of small-molecule methyltransferases (39) and more generally among secondary or specialized metabolic enzymes, which are on average ∼30-fold slower than enzymes of central metabolism (53). Specialized metabolism inEphedra sinica yielding pseudoephedrine and related phenylalkylamines hinges on an NMT with turnover rates in the range of 10^-5 to 10⁻⁶ s⁻¹ (54), whereas human adrenaline production, an arguably more central or primary metabolic route, relies on a functionally comparable phenylalkylamine NMT exhibiting a relatively higher turnover rate in the order of ∼10⁻² s⁻¹ (55). Psychedelic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin are products of nonessential, secondary biochemistry, thus denoting RmNMT and HsINMT as enzymes of specialized metabolism. Yet, even among sluggish enzymes of this class, kinetic analyses show that mammalian INMTs are exceptionally slow (11,12,37) thereby rendering RmNMT as a superior biocatalyst strictly in comparison with INMTs using the same substrates.

RmNMT promiscuity facilitates production of new-to-nature indolethylamines

The apparent efficiency and promiscuity of RmNMT enabledin vivo turnover of a wide variety of products, several of which were subsequently purified for receptor-binding assays. However, substrates with 2,7-substitution patterns were either not accepted or showed poor conversion. More work is required to determine whether RmNMT accepts more complex indole-based ring structures (e.g., β-carboline, ergoline). The substrate displaying the highest turnover, percent conversion and overall product titer was 6-trifluoromethoxytryptamine, yielding the new-to-nature compound 6-trifluoromethoxy-DMT, demonstrating utility of RmNMT to generate novel, purifiable, and testable compounds. Future work can target the deployment of RmNMT in complex, multigene biosystems to achieve greater diversity from simpler feedstocks. Larger cultures facilitated product purification to 95% (w/w) using a relatively simple protocol suitable for the isolation of compounds exhibiting neutral character under basic (pH 10–12) conditions. Importantly, the method is better adapted to DMT derivatives compared with zwitterionic compounds, such as psilocybin, whose 4-O-phosphoryl group would preclude purification using the described procedure.

While efficiency and lack of product inhibition are desirable features in an indolethylamine NMT destined for deployment in bioproduction systems, catalyst performance is not expected to remain consistent across substrates and reaction conditions. To illustrate, while RmNMT converted most substrates to tertiary amines, assays with 5-butyltryptamine and 4-hydroxytryptamine yielded substantial levels of secondary amines (35% and 28% respectively, of total product). Incomplete conversion suggests that under specific conditions and with certain substrates, RmNMT may in fact exhibit some degree of product inhibition. Incomplete conversion represents a practical problem in terms of product isolation, as the similar chemical character ofN- andN,N-dimethyl products precludes facile purification procedures such as that outlined inFig. S11. Nonetheless, RmNMT was able to produce new-to-nature molecules beyond those found in toad, animals, fungi, and plants. Moreover, at the time of manuscript preparation, five products could not be found through a SciFinderⁿ exact structure search (https://scifinder-n.cas.org) and had yet to be assigned Chemical Abstract Service identifiers, suggesting these molecules have yet to be described (Fig. 3).

Reduced affinity at 5-HT_1A receptor for tertiary amines

Serotonergic receptor assays were selected for the preliminary,in vitro pharmacological screening of generated indolethylamines. Two subtypes of this receptor class, 5-HT_1A and 5-HT_2A, are commonly used to assess stress responses. Agonism at the 5-HT_1A receptor is an important aspect of anxiolytic/antidepressant drugs, such as buspirone and tandospirone (56), and atypical antidepressants, such as vortioxetine (57), although how exactly this feature contributes to overall efficacy remains unclear. The 5-HT_2A receptor is associated with hallucinations imparted by indolic-type drugs and has emerged as a target for the treatment of psychiatric pathologies (25). Results clearly showed enhanced 5-HT_1A receptor binding by primary amines compared to tertiary amines (Table 2). Direct 5-HT_2A receptor-binding assays yielded more nuanced results and did not support this general trend. Early work using pig brain cortex (58) or bovine hippocampus (59) membranes and [³H]8-OH-DPAT ligand suggested higher affinity at the 5-HT_1A receptor for serotonin compared with bufotenin. A recent report revealed higher-affinity 5-HT_1A receptor binding of 4-hydroxytryptamine (also known as norpsilocin) compared to psilocin (21) in agreement with this study. Practical implications of enhancedin vitro affinity are unclear, asin vivo factors may disproportionately affect the ability of primary amines to reach and act on their serotonin targets. For example, it is uncertain how easily primary amines pass through the blood brain barrier (BBB) compared to their tertiary counterparts and whether catabolism occurs more quickly for primary amines. Unlike its tertiary amine counterpart psilocin, 4-hydroxytryptamine does not induce head-twitch response in mice despite arguably sufficient affinity at the 5-HT_2A receptor (21) suggesting either (or both) an inability to permeate the BBB or that the compound undergoes rapid catabolism. On the other hand, effects of 4-hydroxytryptamine on mouse body temperature and locomotor activity were blocked by pretreatment with a 5-HT_1A-antagonist, implying that this primary amine can reach and modulate the 5-HT_1A receptor to some extent.

N-methylation serves as a protective group except for 6-substituted derivatives

Data presented herein show that mostN,N-dimethylated indolethylamines persist longer than their primary amine counterparts in HLM (Fig. 4), suggesting that degradation occurs more rapidly for primary amines. An important catabolic enzyme for many indolethylamines is MAO-A, which resides throughout the human body including the brain, particularly in catecholaminergic neurons (60). Early work with rat liver mitochondria suggested that while tryptamine andN-methyltryptamine were substrates of MAO-A, activity with DMT could not be detected (61) implying an inability of this enzyme to efficiently degrade tertiary amines. Similarly, recent evidence revealed that the tertiary amine sumatriptan was a poor substrate for MAO-A compared to its secondary and primary amine counterparts and thatN-demethylation of sumatriptan by hepatic CYPs may occur prior to MAO-A oxidation (33). Long-term kinetics for radiolabeled DMT and tryptamine injected into rabbits showed that whereas both compounds rapidly reached brain tissue in approximately equal abundance, the residence time for DMT was long (7 days) compared with tryptamine, which disappeared within minutes (62). While use of HLM is expected to yield information regarding liver catabolism and perhaps hint at general enzymatic lability, more sophisticated techniques (63) are needed to investigate the catabolism taking place in the brain for those drugs able to cross the BBB.

Notably,N,N-dimethylation did not offer much—or any—protection of 6-substituted indolethylamines. Like their primary amine counterparts, both 6-fluoro-DMT and 6-fluoromethoxy-DMT rapidly degraded upon exposure to liver microsomes. It is possible that 6-substituted indolethylamines are good substrates for MAO-A or other catabolic enzymes, regardless ofN-methylation status. This hypothesis could be tested by surveying the indolethylamine substrate acceptance profiles of MAO-A and other prospective catalysts. Interestingly, whereas 5-fluoro-DMT is hallucinogenic (41), 6-fluoro-DMT is a purported nonhallucinogen (64). It is possible that compound stability may be an important factor in determining the degree and/or duration of hallucinatory response.

While toads are notorious for their invasive behavior and problematic toxicity, their ability to biosynthesize an enormous diversity of compounds, ranging from hallucinogenic alkaloids to anticancer steroids to pesticidal peptides, justifies further research. RmNMT represents the first alkaloid biosynthetic enzyme isolated from toad and proved to be a superior catalyst compared with other known enzymes. While RmNMT did not accept all substrates, the enzyme was effective and promiscuous allowing production of new-to-nature, previously unreported derivatives. Subsequent purification and screening experiments revealed important observations on candidate stability and receptor activity. Further mining of toad genomics resources, and resources derived from indolethylamine-accumulating plant species, is expected to yield additional tools for deployment in biocatalytic platforms.

Transcriptome mining, candidate gene identification, and phylogenetic analysis

An annotated transcriptome ofRhinella marina was obtained from GigaScience Database (gigadb.org/dataset/100483) (48). A total of 33 CDS assigned annotations of INMT or NNMT were selected and aligned with the functionally established NMT enzymes HsINMT (NP_006765), OcINMT (NP_001075512), and HsNNMT (NP_001358974) to isolate the seven full-length candidates. The sevenR. marina full-length candidates are available in GenBank under the following accessions: Rm35569 (RmNMT),OQ557631; Rm43762,OQ557630; Rm29827,OQ557633; Rm30900,OQ557632; Rm25608,OQ557634; Rm8745,OQ557635; Rm54673,OQ557629. Subsequent identifications ofBufo bufo Bb82723 (XP_040282723),N. parkeri Np27127 (XP_01427127), andE. coqui Ec64034 (KAG9464034) were conducted by querying NCBI (blast.ncbi.nlm.nih.gov) withR. marina sequence Rm35569. Protein sequence alignments were generated using the Geneious Alignment feature of Geneious Prime (v. 2022.0.1), which in turn were used to construct phylogenetic trees using the Geneious Tree Builder feature, employing the Neighbor-Joining method and 100 replicates to calculate Bootstrap values. Trees were visualized using iTOL v. 6.6 (https://itol.embl.de) (65).

Chemicals

Most indolethylamine derivatives were from Combi-Blocks (combi-blocks.com) except forN-methyltryptamine hydrochloride from Cedarlane (cedarlanelabs.com); bufotenin, DMT, and 5-methoxy-DMT from Toronto Research Chemicals (trc-canada.com); 5-methoxytryptamine, 2-methyltryptamine, and serotonin hydrochloride from Oakwood Chemical (oakwoodchemical.com); 6-O-trifluoromethoxytryptamine hydrochloride from AstaTech (astatechinc.com); 7-fluorotryptamine hydrochloride from 1PlusChem (1pchem.com); and 5-hydroxy-N-methyltryptamine oxalate from Sigma-Aldrich (sigmaaldrich.com).S-(5′-Adenosyl)-l-methionine dihydrochloride was from Sigma-Aldrich. All other reagents were from Sigma-Aldrich and BioShop Canada (bioshopcanada.com).

Plasmid and strain construction

All candidate CDS were codon-optimized at Twist Bioscience (twistbioscience.com) for expression in yeast and subcloned into theNdeI andXhoI sites of pET28a(+), resulting in a His₆-tag fused to the proteinN-terminus. Yeast codon-optimization was performed (i) to facilitate alternative potential expression inS. cerevisiae and (ii) to avoid overabundant protein accumulation and inclusion body formation inE. coli. TheE. coli strain ArcticExpress (DE3) (Agilent Technologies) was used as the host forin vivo analysis ofN-methylation activity, production of protein (RmNMT, OcINMT, PcPsiM) for purification andin vitro work, and the large-scale preparative biosynthesis ofN,N-dimethylated indolethylamine derivatives.

In vivo biotransformation assay

A standardin vivo assay for NMT activity was performed as follows. Constructs of pET28a(+) containing candidate sequences (or the empty vector) were individually transformed intoE. coli ArcticExpress (DE3) competent cells. Fresh colonies were inoculated in 3 ml of TB medium supplemented with 50 μg/ml kanamycin at 30 °C for 16 h with shaking at 250 rpm. Seed cultures of 2 ml were used to inoculate 20 ml of TB medium with 50 μg/ml kanamycin. Cultures were grown at 37 °C for approximately 5 h to an A₆₀₀ of 2.5 to 3.0. Cultures were cooled to 30 °C and supplemented with IPTG (0.2 mM) and either tryptamine, serotonin, or an alternative indolethylamine substrate (80 mg/L). Following 24 h of shaking at 250 rpm, cells and broth were separated by centrifugation at 5000g for 10 min, and both fractions were stored at −80 °C. Broth was later thawed, mixed with an equal volume of acetonitrile, and centrifuged at 13,000g for 20 min. Ten microliters of supernatant were subjected to analysis forN-methylated products using an LC-equipped LTQ-Orbitrap-XL (full-scan 50–600 m/z) or to collision-induced dissociation (CID) analyses to confirm product identity, using an Agilent triple quadrupole LC-MS/MS. Conversely, frozen cell pellets were later thawed and resuspended in assay buffer (50 mM sodium phosphate, pH 7.0, 300 mM NaCl and 10% (v/v) glycerol) using 1 ml buffer per 2 ml of original culture. Following sonication, crude lysate was centrifuged at 10,000g for 25 min to remove insoluble matter. Fifty microliters of the supernatant were mixed with 10 μl of 6X loading buffer containing 375 mM Tris–HCl, 10% SDS (w/v), 50% glycerol (v/v), 0.03% bromophenol blue (w/v), and 9% β-mercaptoethanol (v/v) and boiled at 95 °C for 5 min. After cooling on ice for 10 min, an aliquot of 10 μl of the boiled sample was applied to SDS-PAGE followed by immunoblot analysis using anti-His₆ antibody visualized with a SuperSignal West Pico PLUS Chemiluminescent Substrate kit (thermofisher.com) according to manufacturer’s instructions.

Protein purification, standardin vitro assay, and enzyme kinetics

Constructs of pET28a(+) containing candidate sequences were individually transformed intoE. coli ArcticExpress (DE3) competent cells. Fresh colonies were inoculated in 50 ml of LB medium supplemented with 50 μg/ml kanamycin. The seed culture was grown at 30 °C for 16 h with shaking at 250 rpm and used to inoculate 500 ml of TB medium. The culture was grown at 37 °C to A₆₀₀ of ∼2.0, cooled to 16 °C, and supplemented with IPTG to a final concentration of 0.2 mM. Following 24 h of incubation, cells were harvested by centrifugation at 7000g for 10 min and stored at −80 °C. Cell pellets were resuspended and sonicated in a buffer containing 50 mM sodium phosphate, pH 7.0, 300 mM NaCl, and 10% (v/v) glycerol. Crude lysate was centrifuged at 10,000g for 25 min to remove insoluble debris, and the supernatant was loaded onto TALON cobalt-affinity resin (takarabio.com) for protein purification as recommended by the manufacturer. Briefly, the resin was washed with two 10-mL volumes of protein extraction buffer, followed by two additional 10-mL buffer volumes supplemented with 5 mM imidazole. Purified proteins were eluted stepwise using 4 ml of buffer supplemented with 150 mM imidazole. Eluted protein fractions were passed through PD-10 desalting columns (cytivalifesciences.com) to remove imidazole. Protein concentration was determined by the Quick Start Bradford Protein Assay (bio-rad.com) using bovine serum albumin as a standard. Protein purity was assessed by SDS-PAGE and immunoblot analysis. A standardin vitro enzyme assay consisted of a 100 μl reaction containing 20 mM Tris–HCl, pH 7.0, 20 μg of purified enzyme, 100 μM indolethylamine-derivative substrate, and 500 μM SAM. The assay proceeded for 1 h at 30 °C, followed by quenching with 200 μl acetonitrile and centrifugation at 21,000g at 4 °C for 30 min. Ten microliters of supernatant were subjected to analysis forN-methylated products by an LC-equipped LTQ-Orbitrap-XL (full-scan 50–600 m/z) or to CID analyses. For enzyme kinetics studies, thein vitro assay was adapted as follows: assay volumes consisted of 50 μl reactions, with identical components except (i) indolethylamine substrate concentrations were varied (5–400 μM), (ii) SAM concentrations were varied (100 μM–2 mM), and (iii) only 0.5 μg of purified enzyme was used. Assays were incubated at 30 °C for 10 min, followed by quenching with an equal volume of acetonitrile. Product identification and quantification was performed by LC-MS/MS. Saturation curves and kinetic parameters were determined based on the Michaelis–Menten equation using GraphPad PRISM software (Version 9.2.0;www.graphpad.com).

LC-MS analysis

High-resolution LC-HESI-LTQ-Orbitrap-XL (Thermo Fisher Scientific (thermofisher.com)) mass spectrometry coupled to an UltiMate 3000 HPLC (Thermo Fisher Scientific) equipped with a Poroshell 120 SB-C18 column (Agilent Technologies) was used. The HPLC portion of instrumentation was carried out as follows: 10 μl were injected at a flow rate of 0.5 ml min⁻¹ and a gradient of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile, 0.1% formic acid): 0 to 100% solvent B over 5 min, 100% solvent B for 1 min, 100 to 0% solvent B over 0.1 min, 100% solvent B for 1.9 min. The total run time was 8 min with data collected throughout the run. For the MS portion of instrumentation, positive-ion mode was used to run heated ESI (HESI) source and interface conditions with the following parameters: vaporizer temperature, 400 °C; source voltage, 3 kV; sheath gas, 50 au, auxiliary gas, 20 au; sweep gas, 2 au; capillary temperature, 380 °C; capillary voltage, 8 V; tube lens, 50 V. A single scan event was performed, with high-resolution Fourier-transform mass spectroscopy from 50 to 700 m/z with an ion injection time of 500 ms and a scan time of approximately 1.0 s. External and internal calibration ensured <2 ppm error, although a window of 5 ppm was used during extracted ion chromatograph data collection. Product identification and quantification was achieved through use of (i) quantitation curves for authentic standards and (ii) comparison of retention times (R_t), exactm/z (±5 ppm), and CID spectra compared with those of authentic standards. Quantitation curve analyses were used to determine linear ranges for all products. In cases where an authentic standard was unavailable, quantification was estimated using a standard curve for an available, closely related indolethylamine. Saturation curves and kinetic parameters were determined using the Michaelis–Menten equation using GraphPad PRISM software (Version 9.2.0;www.graphpad.com). CID spectra were determined to confirm product identities on an Agilent Technologies 1200 LC and 6410 triple quadrupole MS/MS (agilent.com) equipped with a Poroshell 120 SB-C18 column, and HPLC conditions were identical to those described for LC-HESI-LTQ-Orbitrap-XL analysis. Five microliters of sample or authentic standard (50 μM) were injected. MS conditions were as follows: electrospray ionization (ESI) capillary voltage 4 kV, fragmentor voltage 100 V, source temperature 350 °C, nebulizer pressure 50 psi, gas flow 10 L/min; precursor ion was selected in quadropole 1 and collision energies of 5 or 10 eV was applied in quadrupole 2. The resulting fragments were resolved by quadrupole 3, scanning from 50 to 10 m/z units greater than the precursorm/z.

Scaled fermentation for product purification

pET28a(+) containing RmNMT was transformed into competentE. coli ArcticExpress (DE3) cells. Fresh colonies were inoculated into 60 ml of TB medium supplemented with 50 μg/ml kanamycin for overnight culturing at 30 °C with shaking at 250 rpm. The seed culture (60 ml) was inoculated into 660 ml of TB medium supplemented with 50 μg/ml kanamycin and 80 mg/L appropriate indolethylamine substrate. This volume was separated into two one-L baffled flasks each containing 330 ml of culture medium to maintain sufficient aeration. Cultures were grown at 37 °C for ∼5 h to an A₆₀₀ of 2.5, cooled to 30 °C, and supplemented with IPTG (0.2 mM) and one primary indolethylamine at a final concentration of 80 mg/L. After 24 h incubation at 30 °C and 250 rpm, the culture was harvested and subjected to centrifugation at 7000g for 10 min. The supernatant was stored at 4 °C until further processing.

Compound purification and structural assignments

For the isolation of bufotenin, the culture medium was thawed, and concentrated ammonium hydroxide was added to pH 10. The culture was extracted with ethyl acetate (4 × 300 ml). The organic layer was combined and dried over sodium sulfate, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [from 2→5% (v/v) methanol in dichloromethane and 1.5% (w/v) ammonium hydroxide]. The compound was further purified by dissolving in 0.1 M HCl and extracted with dichloromethane, and the aqueous solution was basified to pH ∼9 with 0.1 M ammonium hydroxide solution, followed by extraction with dichloromethane to yield the pure compound (5 mg) after evaporation. Structural assignments:¹H NMR (400 MHz, CD₃OD): δ = 2.38 (s, 6H), 2.67 (m, 2H), 2.89 (m, 2H), 6.67 (dd,J = 8.7, 2.4 Hz, 1H), 6.92 (d,J = 2.4 Hz, 1H), 7.01 (s, 1H), 7.17 (d,J = 8.6, 0.5 Hz, 1H);¹³C NMR (100 MHz, CD₃OD): δ = 22.8, 43.9, 59.9, 102.0, 110.9, 111.2, 111.3, 119.0, 122.7, 127.9, 149.7; high-resolution mass spectroscopy (HRMS) with ESIm/z: calculated [M+H]⁺ for C₁₂H₁₆N₂O, 205.1335; measured [M+H]⁺, 205.1332.

For isolation of 5-fluoro-DMT, the culture medium was thawed, and 10 M sodium hydroxide was added to pH ∼12. The solution was extracted with ethyl acetate (3 × 300 ml). The organic layer was combined and dried over sodium sulfate, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [1→3% (v/v) methanol in dichloromethane with 1.5% (w/v) ammonium hydroxide]. The compound was further purified by dissolving in 0.1 M HCl and extracted with dichloromethane, and the aqueous solution was basified to pH ∼12 with 10 M sodium hydroxide. This was followed by extraction with dichloromethane to yield the pure compound (22 mg) after evaporation. Structural assignments:¹H NMR (400 MHz, CD₃OD): δ = 2.36 (s, 6H), 2.65 (m, 2H), 2.91 (m, 2H), 6.86 (ddd,J = 9.2, 9.2, 2.5 Hz, 1H), 7.13 (s, 1H), 7.20 (dd,J = 9.8, 2.5 Hz, 1H), 7.29 (dd,J = 8.8, 4.5 Hz, 1H);¹³C NMR (100 MHz, CD₃OD): δ = 22.8, 44.0, 59.8, 102.3 (d,J_{C, F} = 23.5 Hz), 108.9 (d,J_{C, F} = 26.4 Hz), 111.6 (d,J_{C, F} = 9.4 Hz), 112.4 (d,J_{C, F} = 4.8 Hz), 123.8, 127.5 (d,J_{C, F} = 9.5 Hz), 133.3, 157.4 (d,J_{C, F} = 231.7 Hz); HRMS (ESI)m/z: calculated [M+H]⁺ for C₁₂H₁₅FN₂; 207.1292; measured [M+H]⁺, 207.1291.

For isolation of 6-fluoro-DMT, the culture medium was thawed, and 10 M sodium hydroxide was added to pH ∼12. The culture was extracted with ethyl acetate (3 × 300 ml). The organic layer was combined and dried over sodium sulfate, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [1→3 % (v/v) methanol in dichloromethane with 1.5% (w/v) ammonium hydroxide]. The compound was further purified by dissolving in 0.1 M HCl and extracted with dichloromethane, and the aqueous solution was basified to pH ∼12 with 10 M sodium hydroxide, followed by extraction with dichloromethane to yield the pure compound (34 mg) after evaporation. Structural assignments:¹H NMR (400 MHz, CD₃OD): δ = 2.36 (s, 6H), 2.66 (m, 2H), 2.94 (m, 2H), 6.80 (ddd,J = 9.7, 8.7, 2.3 Hz, 1H), 7.04 (m, 2H), 7.49 (dd,J = 8.6, 5.4 Hz, 1H);¹³C NMR (100 MHz, CD₃OD): δ = 22.8, 44.0, 59.9, 96.7 (d,J_{C, F} = 25.8 Hz), 106.5 (d,J_{C, F} = 24.8 Hz), 112.4, 118.6 (d,J_{C, F} = 10.2 Hz), 112.1 (d,J_{C, F} = 3.5 Hz), 124.0, 136.7, 159.8 (d,J_{C, F} = 234.8 Hz); HRMS (ESI)m/z: calculated [M+H]⁺ for C₁₂H₁₅FN₂; 207.1292, measured [M+H]⁺, 207.1290.

For isolation of 2-methyl-DMT, the culture medium was thawed, and 10 M sodium hydroxide was added to pH ∼12. The culture was extracted with ethyl acetate (3 × 300 ml). The organic layer was combined and dried over sodium sulfate, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [1→ 3% (w/v) methanol in dichloromethane with 1.5% (w/v) ammonium hydroxide]. The compound was further purified by dissolving in 0.1 M HCl and extracted with dichloromethane, and the aqueous solution was basified to pH ∼12 with 10 M sodium hydroxide, followed by extraction with dichloromethane to afford the desired compound (24 mg) in pure form after evaporation. Structural assignments were as follows:¹H NMR (400 MHz, CD₃OD): δ = 2.37 (s, 6H), 2.38 (s, 3H), 2.54 (m, 2H), 2.89 (m, 2H), 6.98 (m, 2H), 7.23 (m, 1H), 7.42 (m, 1H);¹³C NMR (100 MHz, CD₃OD): δ = 9.9, 21.7, 43.9, 59.9, 107.5, 109.9, 116.8, 118.0, 119.9, 128.4, 131.3, 135.8; HRMS (ESI)m/z: calculated [M+H]⁺ for C₁₃H₁₈N₂ [M+H]⁺, 203.1543; measured [M+H]⁺, 207.1542.

For isolation of 6-trifluoromethoxy-DMT, the culture medium was thawed, and 10 M sodium hydroxide was added to pH ∼12. The culture was extracted with ethyl acetate (3 × 300 ml). The organic layer was combined and dried over sodium hydroxide, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [1→3 % (v/v) methanol in dichloromethane with 1.5% (w/v) sodium hydroxide]. The compound was further purified by dissolving in 0.1 M HCl and extracted with dichloromethane, and the aqueous solution was basified to pH ∼12 with 10 M sodium hydroxide, followed by extraction with dichloromethane to yield the pure compound (37.5 mg) after evaporation. Structural assignments were as follows:¹H NMR (400 MHz, CD₃OD): δ = 2.36 (s, 6H), 2.67 (m, 2H), 2.96 (m, 2H), 6.94 (m, 1H), 7.04 (m, 1H), 7.17 (br. s, 1H), 7.25 (m, 1H), 7.58 (d,J = 8.8 Hz, 1H);¹³C NMR (100 MHz, CD₃OD): δ = 22.7, 44.0, 59.9, 103.8, 112.0, 112.6, 118.6, 112.1, 123.5, 126.2, 136.2, 144.5; HRMS (ESI)m/z: calculated [M+H]⁺ for C₁₃H₁₅F₃N₂O, 273.1209; measured [M+H]⁺, 273.1206.

For 4-hydroxytryptamine, the culture medium was thawed, and concentrated ammonium hydroxide was added to pH ∼11. The culture was extracted with ethyl acetate (4 × 300 ml). The organic layer was combined and dried over sodium sulfate, followed by concentration under reduced pressure. The residue was purified by flash chromatography on silica gel [2→5 % (v/v) methanol in dichloromethane with 1.5% (w/v) ammonium hydroxide], to give the compound as a dark green solid (12 mg). Structural assignments were as follows:¹H NMR (400 MHz, CD₃OD): δ = 2.99 (m, 4H), 6.34 (dd,J = 7.4, 1.1, 1H), 6.84 (m, 3H);¹³C NMR (100 MHz, CD₃OD): δ = 29.0, 42.4, 102.6, 103.4, 112.3, 117.3, 120.8, 121.8, 139.3, 151.9; HRMS (ESI)m/z: calculated [M+H]⁺ for C₁₀H₁₂N₂O [M+H]⁺, 117.1022; measured [M+H]⁺, 117.1021.

Radioligand competition assays: 5-HT_1A receptor

Competition assays were performed as follows: scintillation proximity assay (SPA) beads (RPNQ0011), radiolabeled 8-hydroxy-DPAT [propyl-2,3-ring-1,2,3-³H] (labeled 7-(dipropylamino)-5,6,7,8-tetrahydronaphthalen-1-ol; NET929250UC), membranes containing 5HT_1A (6110501400UA), and isoplate-96 microplate (6005040) were from PerkinElmer (perkinelmer.com). Radioactive binding assays were carried out using SPA (66). For saturation-binding assays, mixtures of 10 μg of membrane containing HT_1A receptor was precoupled to 1 mg of SPA beads at room temperature in a tube rotator for 1 h in binding buffer [50 mM Tris–HCl pH 7.4, 10 mM magnesium sulfate, 0.5 mM EDTA, 3.7% (v/v) glycerol, 1 mM ascorbic acid, 10 μM pargyline HCl]. After precoupling, the beads and membrane were aliquoted in an isoplate-96 microplate with increasing amounts of 8-hydroxy-DPAT [propyl-2,3-ring-1,2,3-³H] (0.1525 nM to 5 nM) and incubated for 2 h at room temperature in the dark with shaking. After incubation, the samples were read on a MicroBeta 2 Microplate Counter (perkinelmer.com). Nonspecific binding was carried out in the presence of 100 μM of metergoline (M3668-500 MG, Sigma-Aldrich). Equilibrium binding constant for 8-hydroxy-DPAT (K_D) was determined from a saturation binding curve using one-site saturation-binding analysis from GraphPad PRISM software (Version 9.2.0). All test compounds were dissolved to 100 mM in dimethylsulfoxide, and dilutions were carried out in assay buffer. Competition binding assays were performed using 0.5 nM hot 8-hydroxy-DPAT and different concentrations of dimethylsulfoxide (up to 1%) or with unlabeled test compounds (3 nM to 1 mM), similar with the saturation binding assay. K_i values were calculated from the competition displacement data using the competitive binding analysis from GraphPad PRISM software.

Radioligand competition assays: 5-HT_2A receptor

Competition assays were performed as for 5-HT_1A assays with the following differences. SPA beads (RPNQ0010), [³H]ketanserin (NET1233025UC), and membranes containing 5-HT_2A (ES-313-M400UA) were from PerkinElmer. After precoupling, the beads and membrane were aliquoted in an isoplate-96 microplate with increasing amounts of [³H]ketanserin (0.1525 nM to 5 nM). Determination of nonspecific binding was carried out in the presence of 20 mM of spiperone (S7395-250 MG, Sigma-Aldrich). Equilibrium binding constant for ketanserin (K_d) was determined from saturation binding curves using the ‘one-site saturation binding analysis’ method in GraphPad PRISM software (Version 9.2.0). Competition binding assays were performed using fixed (1 nM) [³H]ketanserin and different concentrations of unlabeled test compounds (3 nM to 1 mM) similar to the saturation binding assay.

In vitro metabolic stability

HLMs were purchased from XenoTech (xenotech.com). In liver metabolism assays, candidate compounds (5 μM) were incubated with 400 μg/ml HLM in 50 mM potassium phosphate buffer, pH 7.4, containing 3 mM MgCl₂ and 1 mM EDTA at 37 °C in a total volume of 200 μl. Samples (50 μl) were drawn at the start of the assay and at 30 and 60 min and precipitated with a 1:1 volume of acetonitrile to quench the reaction before centrifugation at 10,000g for 20 min. Supernatants were analyzed for the presence of target compound using LC-LTQ-Orbitrap-XL MS analysis as described for other assays.

Author contributions

X. C., J. L., L. Y., F. M., L. C., and J. A. G. investigation; X. C., J. L., L. Y., F. M., L. C., J. A. G., D. J. P., and S. A. R. methodology; X. C., J. L., L. Y., F. M., L. C., J. A. G., D. J. P., and S. A. R. validation; J. M. H. data curation; J. M. H. writing–original draft; P. J. F. conceptualization; P. J. F. writing–review and editing; P. J. F. supervision; P. J. F. project administration; P. J. F. funding and additional information.

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Supplementary Materials

Data Availability Statement

All data is included in the manuscript and accompanying supporting information. Nucleotide sequence data have been deposited in Genbank under the following accession codes:OQ557631 (RmNMT),OQ557629 (Rm54673),OQ557630 (Rm43762),OQ557632 (Rm30900),OQ557633 (Rm29827),OQ557634 (Rm25608),OQ557635 (Rm8745),OQ557636 (BbNMT).