Precursor-directed Biosynthesis in Tabernaemontana catharinensis as a New Avenue for Alzheimerʼs Disease-modifying Agents

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• Plant Material
• T. catharinensis micropropagation
• PDB using 5-fluoro-tryptamine in T. catharinensis plantlets
• Feeding experiments using 2-13C-tryptophan, 1-13C-glyceraldehyde, and sodium pyruvate-3-13C in T. catharinensis plantlets
• Apparatus and operation conditions for PDB analysis
• Apparatus and operation conditions for feeding experiments analysis
• Molecular docking and therapeutic viability analysis
• Contributorsʼ Statement
• References

Abstract

Plants produce a high diversity of metabolites that can act as regulators of cholinergic dysfunction. Among plants, the potential of species of the genus Tabernaemontana to treat neurological disorders has been linked to iboga-type alkaloids that are biosynthesized by those species. In this context, precursor-directed biosynthesis approaches were carried out using T. catharinensis plantlets to achieve new-to-nature molecules as promising agents against Alzheimerʼs disease. Aerial parts of T. catharinensis, cultured in vitro, produced 7 unnatural alkaloids (5-fluoro-ibogamine, 5-fluoro-voachalotine, 5-fluoro-12-methoxy-Nb-methyl-voachalotine, 5-fluoro-isovoacangine, 5-fluoro-catharanthine, 5-fluoro-19-(S)-hydroxy-ibogamine, and 5-fluoro-coronaridine), while root extracts showed the presence of the same unnatural iboga-type alkaloids and 2 additional ones: 5-fluoro-voafinine and 5-fluoro-affinisine. Moreover, molecular docking approaches were carried out to evaluate the potential inhibition activity of T. catharinensis’ natural and unnatural alkaloids against AChE and BChE enzymes. Fluorinated iboga alkaloids (5-fluoro-catharanthine, 5-fluoro-voachalotine, 5-fluoro-affinisine, 5-fluoro-isovoacangine, 5-fluoro-corinaridine) were more active than natural ones and controls against AchE, while 5-fluoro-19-(S)-hydroxy-ibogamine, 5-fluoro-catharanthine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine showed better activity than natural ones and controls against BChE. Our findings showed that precursor-directed biosynthesis strategies generated “new-to-nature” alkaloids that are promising Alzheimerʼs disease drug candidates. Furthermore, the isotopic experiments also allowed us to elucidate the initial steps of the biosynthetic pathway for iboga-type alkaloids, which are derived from the MEP and shikimate pathways.

Introduction

Alzheimerʼs disease (AD) is a type of brain disease that causes dementia among older adults and is frequently accompanied by high levels of comorbidity in both psychiatric and nonpsychiatric patients. It is a progressive neurodegenerative disorder that slowly destroys the memory and other cognitive abilities [1]. Currently, around 40 to 50 million people are living with dementia, and by mid-century, the number of people with dementia is projected to reach 131.5 billion, with most of them living in middle and low income countries [2]. Among its well-described histopathological features, which include extracellular amyloid plaque and neurofibrillary tangle damages, AD also includes neuronal loss, extensive DNA damage, mitochondrial dysfunction, depletion of antioxidant system, and cholinergic impairment [3], [4], [5], [6], [7]. The loss of basal forebrain cholinergic neurons is, in particular, the base of the widely accepted cholinergic hypothesis, which is demonstrated by reduction in the number of cholinergic markers, such as choline acetyltransferase (ChAT), the neurotransmitter acetylcholine (ACh), and muscarinic and nicotinic ACh receptors. High levels of the acetylcholinesterase (AChE) enzyme acts as co-regulator of the cholinergic neurotransmission by hydrolyzing ACh [8], [9], [10]. All these changes together are highly correlated with the degree of dementia in AD.

The AChE was the first target to be investigated for the treatment of the pathology, and then 4 cholinergic inhibitors were internationally approved (tacrine, donepezil, rivastigmine, and galantamine). Interestingly, while all display AChE inhibitory activity, they have different action mechanisms. Recently, it was shown that the butyrylcholinesterase (BChE) enzyme also acts as a co-regulator of the cholinergic neurotransmission by hydrolyzing ACh [11], [12], [13]. In this context, several studies suggest that identification of nonselective molecules with capacity to inhibit both AChE and BChE enzymes could lead to better clinical outcomes.

Currently, a growing interest has emerged regarding the value and use of plants for the treatment and decrease of cognitive impairment in AD and other neurodegenerative diseases. Plants produce a high diversity of metabolites that can act as a regulator of the cholinergic dysfunction [14], [15], [16]. Among plants with therapeutic potential, the species of the genus Tabernaemontana (Apocynaceae) are widely used in traditional medicine due to several biological activities, including its neuropharmacological action [17]. The iboga-type alkaloids found in the genus Tabernaemontana have great potential as AChE inhibitors and can be significant compounds in the study of biochemical interactions involving cholinesterases [18]. Some of these alkaloids have been reported to show AChE inhibition and amyloid beta peptide (Aβ) aggregation inhibitory activities [19], [20], [21]. More than 100 iboga alkaloids have been reported since ibogaine, the first one, was elucidated [22]. An interesting example are the indole alkaloids coronaridine, voacangine, voacangine hydroxyindolenine, and voacristine, which exhibited an anticholinesterase action in the same concentration of the reference compounds, physostigmine and galantamine (0.01 mM) [23]. The iboga-type alkaloids ibogamine and voacangine are psychoactive and can be used in opioid addiction treatments [24].

T. catharinensis A. DC. is a species found in Argentina, Paraguay, Bolivia, and Brazil [25], and its crude extract and fractions, especially those with 12-methoxy-Nb-methyl-voachalotine, showed significant cholinesterase inhibition (IC₅₀ = between 2.1 and 2.5 µg/mL) [26]. In 2019, the FDAʼs Center for Drug Evaluation and Research approved 9 novel drugs for neurological purposes, which represent 19% of all drugs released, only behind anticancer drugs (11 new drugs; 23%) [27], showing the importance of new therapeutic alternatives in the area of neurology. Recently, Newman and Cragg (2020) reported that natural products and their derivatives have a significant role in the advance of drugs, since they represent 33% of all commercial drugs. Moreover, 30.5% of all synthetic pharmaceuticals have natural product core structures as an inspiration [28]. Due to the importance of natural product analogues, many strategies for modifying natural products have been explored in order to obtain “new-to-nature” molecules [29]. Recently, we have demonstrated that the biosynthesis of fluorinated and methylated analogues of oxindole alkaloids in Uncaria guianensis plantlets by precursor-directed biosynthesis (PDB) is a viable and efficient bioprocess, and this approach is unusual using plants [30]. The successful incorporation of unnatural precursors in the biosynthetic route of a given natural product can lead to the formation of an analogue that can be more biologically important than the natural product itself. An example is a fluorovinblastine analogue that exhibited remarkable antitumor activity compared with natural vinblastine [31]. In this context, the present work applied PDB strategies conducted with T. catharinensis plantlets to obtain unnatural alkaloid derivatives. Molecular docking approaches along with therapeutic viability analysis were carried out to evaluate the potential inhibition activity of the natural and unnatural alkaloids against AChE and BChE enzymes. Additionally, the biosynthetic pathway of iboga-type alkaloids was also evaluated by feeding strategy using ¹³C-precursor (2-¹³C-tryptophan, 1-¹³C-glyceraldehyde, and sodium pyruvate-3-¹³C).

Results and Discussion

Plantlets of T. catharinensis were transferred to sterile liquid media supplemented with the tryptamine analog (5-fluoro-tryptamine). It is important to highlight that all plants have grown healthy, without visible physiological change, which means that the unnatural precursor was not toxic for the plantlets. A methanolic extract from fresh shoots of T. catharinensis was prepared after 30 days of culture and analyzed by UPLC-DAD-MS. Aerial parts of T. catharinensis produced 7 unnatural alkaloids; 5-fluoro-ibogamine (m/z 298 [M + H]⁺), 5-fluoro-voachalotine (m/z 384 [M + H]⁺), 5-fluoro-12-methoxy-Nb-methyl-voachalotine (m/z 429 [M + H]⁺), 5-fluoro-isovoacangine (m/z 386 [M + H]⁺), 5-fluoro-catharanthine (m/z 354 [M + H]⁺), 5-fluoro-19-(S)-hydroxy-ibogamine (m/z 314 [M + H]⁺), and 5-fluoro-coronaridine (m/z 356 [M + H]⁺) were observed ([Fig. 1]; Fig. 1S–7S, Supporting Information). The extracted-ion chromatograms from roots showed the presence of the same unnatural iboga-type alkaloids that were found in aerial parts and 2 additional unnatural alkaloids: 5-fluoro-voafinine (m/z 330 [M + H]⁺) and 5-fluoro-affinisine (m/z 326 [M + H]⁺) ([Fig. 1]; Fig. 7S–8S, Supporting Information). Our results showed that PDB strategies using analogue precursor successfully produced unnatural alkaloids in T. catharinensis.

Additionally, molecular docking approaches along with therapeutic viability analysis were carried out to determine the potential inhibition activity of the natural and unnatural alkaloids against AChE and BChE enzymes. All 9 natural iboga-type alkaloids were selected because they were found in T. catharinensis, and then a fluorinated version of all was drawn ([Fig. 2]). [Table 1] shows molecular docking GOLD average scores between alkaloids and AChE, and fluorinated alkaloids 5-fluoro-catharanthine, 5-fluoro-voachalotine, 5-fluoro-affinisine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine showed higher scores than natural alkaloids and also higher than the galantamine (positive control). Regarding BChE/ligand complexes scores, all iboga-type alkaloids showed scores higher than the tacrine (positive control). Unnatural iboga alkaloids 5-fuoro-19-(S)-hydroxy-ibogamine, 5-fuoro-catharanthine, 5-fuoro-isovoacangine, and 5-fuoro-coronaridine also show scores higher than natural alkaloids. The boiled-egg graphic ([Fig. 3]) shows the evaluation of passive gastrointestinal absorption (HIA) and brain access (the blood-brain barrier). All of the alkaloids and their respective fluoro-alkaloids are located in the yellow region indicating a high probability of brain penetration. According to the computational model for predicting substrates or inhibitors of PGP substrate 4 of them (isovoacangine, 5-fluoro-isovoacangine, coronaridine, and 5-fluoro-coronaridine) are presented as a red dots. Therefore, they are considered as a PGP nonsubstrate (PGP-), and they were flagged for further analysis of therapeutic viability. All the others were predicted as PGP substrate (PGP+) (blue dots). Prediction of pharmacokinetic properties and compound toxicities of isovoacangine, 5-fluoro-isovoacangine, coronaridine, and 5-fluoro-coronaridine and the positive controls are shown in [Table 2]. In silico toxicity results prediction showed that the all the alkaloids had the same LD₅₀ values and the same toxicity profile. However, the 4 ligands showed a higher LD₅₀ value than galantamine and tacrine. Hydrogen bonds and hydrophobic interactions between alkaloids and positive controls with AChE and BChE receptors, as well as isovoacangine interaction with AChE via 1 H-bond at TYR332 and surrounded by 12 hydrophobic interactions, are shown in [Fig. 4]. One additional hydrophobic interaction was observed when 5-fluoro-isovoacangine interacted with AChE. Galantamine shows 2 H-bonds at SER200 and GLU199 and 11 hydrophobic interactions. Coronaridine complexed with BChE shows 1 H-bond at ASP68 and 9 hydrophobic interactions, and 5-fluoro-coronaridine complexed with BChE shows also 1 H-bond at ASP68 and 8 hydrophobic interactions. The tacrine control interacted with BChE via a single H-bond at HIS436 and 7 hydrophobic interactions. The number of H-bond and hydrophobic interactions observed for alkaloids show a profile similar to the positive control when in complex with the targets. The high GOLD scores obtained suggest a significant binding affinity between targets and receptors. Docking interaction of isovoacangine and 5-fluoro-isovoacangine at the binding site of AChE and coronaridine and 5-fluoro-coronaridine binding with BChE are showed in [Fig. 5] (top/bottom).

Additionally, isotopic labeling studies were carried out to elucidate the initial steps of iboga-type alkaloids biosynthesis. Firstly, plantlets were inoculated in liquid medium supplemented with 2-¹³C-tryptophan (10 mM) and sodium pyruvate-3-¹³C (10 mM). Then, in order to confirm the results, plantlets were inoculated in liquid medium supplemented with 1-¹³C – DL-glyceraldehyde (50 mM). After 4 wk of culture, a methanolic extract from fresh shoots was prepared and analyzed by UPLC-ESI-QTOF-MS (Fig. 9S–10S, Supporting Information). Data values based on the ratio between molecular mass (m/z) of compounds and their isotopes were either M + 1/or M + 2 and the smaller value of the ratio means ¹³C incorporation during biosynthetic process. The ¹³C enrichment patterns of vobasine, 19-(S)-hydroxy-ibogamine, voafinine, and voacristine from plant aerial parts after 2-¹³C-tryptophan incorporation indicated an increase of M + 1 isotope, once 1 ¹³C are included into iboga alkaloids. The same alkaloids showed an increase of M + 2 isotope after sodium pyruvate-3-¹³C and 1-¹³C – DL-glyceraldehyde and then 2 ¹³C are incorporated during metabolism. The iboga alkaloids voafinine and 12-methoxy-Nb-methyl-voachalotine from roots showed the same incorporation pattern ([Table 3]–[4]). The single exception was voafinine, which incorporated only 1 ¹³C from 1-¹³C – DL-glyceraldehyde because that molecule does not have the carboxymethoxy group. Additionally, main product ions of iboga alkaloids (vobasine, 19-(S)-hydroxy-ibogamine, voafinine, voacristine, and 12-methoxy-Nb-methyl-voachalotine) were confirmed by ESI-QTOF-MS/MS. All data allowed us to establish the structure of these iboga-type alkaloids in comparison with MS/MS from literature data [32], [33], [34], [35] ([Table 5]; Fig. 11S–16S, Supporting Information).

Table 5 Iboga-type alkaloids found in T. catharinensis extract by HRMS [UPLC-ESI(+)-QTOF-MS] and low-resolution mass spectrometry [UPLC-ESI(+)-MS/MS] after ¹³C incorporations.

Identification	Molecular Formula	Observed m/z	Calculated m/z	Error (ppm)	Fragmentation ESI(+)-MS/MS	Reference
Aerial parts
vobasine	C21H25N2O3	353.1862	353.1865	− 0.8	265; 221	[32]
19-(S)-hydroxy-ibogamine	C19H25N2O	297.1963	297.1967	− 1.3	279 (peak due to the loss of H2O); 211; 153	[33]
voafinine	C19H25N2O2	313.1915	313.1916	− 0.3	313; 295	[34]
voacristine	C22H29N2O4	385.2129	385.2127	0.5	384; 367(peak due to the loss of H2O); 335; 307	[35]
Roots
voafinine	C19H25N2O2	313.1915	313.1916	− 0.3	313; 295	[34]
12-methoxy-Nb-methyl-voachalotine	C24H31N2O4	411.2288	411.2284	1.0	381; 349; 199,9; 179,9	[33]

To elucidate steps of the iboga-type biosynthesis pathway, obtained data were compared to literature data ([Fig. 6]) [36], [37], [38], [39]. Our results showed that secologanin precursor is predominantly formed by the MEP pathway, since ¹³C-sodium pyruvate and ¹³C-glyceraldehyde were incorporated into the indole moiety. After incorporation of ¹³C-tryptophan, it was possible to confirm that the iboga-type alkaloid biosynthesis is derived from the shikimate pathway from T. catharinensis. In conclusion, our findings showed that PDB strategies using 5-fluoro-tryptamine precursor generate new-to-nature alkaloids with new promising potential AD drug candidates. Furthermore, fluorinated iboga alkaloids 5-fluoro-catharanthine, 5-fluoro-voachalotine, 5-fluoro-affinisine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine were more interactive than the natural ones and controls in inhibiting AchE activity, while 5-fluoro-19-(S)-hydroxy-ibogamine, 5-fluoro-catharanthine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine showed more enhanced activity than natural alkaloids and controls in inhibiting BChE. Moreover, the isotopic experiments also allowed us to establish the biosynthetic pathway for iboga-type alkaloids that are derived from the MEP and shikimate pathways.

Materials and Methods

Plant Material

T. catharinensis seeds were collected at Botanic Garden of Medicinal Plant Ordem e Progresso Jardinópolis, SP, Brazil by Dra. Ana Maria Soares Pereira and identified by Ana Carolina Devides Castello of Universidade Estadual de Campinas (UNICAMP), Instituto de Biologia, Departamento de Botânica, SP, Brazil. A voucher specimen from T. catharinensis was deposited in the Herbarium of Medicinal Plants at UNAERP (HPMU 3226), Ribeirão Preto, SP, Brazil. The collection of T. catharinensis specimens was previously authorized by the Brazilian Council for the Administration and Management of Genetic Patrimony (CGEN) of the Brazilian Ministry of the Environment (MMA) via the National Council for Scientific and Technological Development (CGEN/MMA Process number: A44DED2).

T. catharinensis micropropagation

Disinfected explants (by 1% (w/v) cercobin and captan solutions) were added to MS basal medium containing 3% (w/v) glucose, 0.3% phytagel (Sigma-aldrich), pH adjusted to 6.0 ± 0.05 and maintained at 25 ± 2 °C (55 – 60% relative humidity with a 16 h photoperiod of 40 µmol/m²/s intensity, provided by 85 W cool white GE fluorescent lamps). After 2 mo, a substantial number of micropropagated plants was transferred for WP medium containing 2% (w/v) glucose, 2.0 mg/L indole-3-butyric acid, 0.3% phytagel (Sigma-aldrich), pH adjusted to 6.00 ± 0.05 and micropropagated every 3 mo. The totally randomized experiment was conducted in triplicate with 3 repetitions (n = 5).

PDB using 5-fluoro-tryptamine in T. catharinensis plantlets

Precursor analogue 5-fluoro-tryptamine was solubilized in ultrapure H₂O (3 mM), filtered through a 0.2 µm sterile filter and added to WP medium, as described above, without phytagel. Then, T. catharinensis plantlets at 3 mo (n = 40 plantlets) were transferred to the WP medium, and another 40 plantlets were transferred to same medium except by the fluoro analogue and maintained at 25 °C ± 2 °C for 30 days (55 – 60% relative humidity with a photoperiod of 16 h light and 8 h dark). After incubation, the plantlets were separated into aerial parts and roots, of which each one was extracted separately with MeOH for 24 h in room temperature in order to obtain the crude extracts.

Feeding experiments using 2-¹³C-tryptophan, 1-¹³C-glyceraldehyde, and sodium pyruvate-3-¹³C in T. catharinensis plantlets

Having established that T. catharinensis could produce unnatural alkaloids, adult plantlets from 3 mo (n = 5 plantlets) were inoculated in liquid medium supplemented with 2-¹³C-tryptophan (10 mM), 1-¹³C – DL-glyceraldehyde (50 mM), and sodium pyruvate-3-¹³C (10 mM). Additionally, a control group of plantlets that did not receive ¹³C-precursors was also prepared. After 4 wk of culture, the plantlets were separated in aerial parts and roots, of which each one was extracted separately with MeOH. All the crude extract was filtered and analyzed by UPLC/MS-MS and UPLC/MS.

The statistical analysis was performed using multiple comparisons of mean values of analyte concentrations in the extracts, using the Scott Knott test at the 5% confidence level with the aid of Sisvar software [40].

Apparatus and operation conditions for PDB analysis

The analyses were performed in an UPLC ACQUITY UPLC H-Class system (Waters Corporation) coupled to the diode array detectors (DAD) and the Xevo TQ-S tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA), operating with an electrospray ionization source (ESI-MS) in positive analysis mode. A volume of 5 µL at a concentration of 100 µg/mL of the samples was injected into a Gemini 5 u C18 110A column (250 mm × 4.6 mm, 5 µ). The chromatographic condition used was (A: B; A – H₂O ultrapure + 0.1% formic acid; B-ACN + 0.1% formic acid); from 0 – 28 min starting at (95: 5) until (0: 100); 28 – 31 min (0: 100); and 31 – 35 min (95: 5). The flow of the mobile phase was 0.6 mL/min, and the compounds were monitored at λ = 313 and 280 nm. The operating parameters used in the Z-spray source were capillary voltage = 3.20 kV, cone voltage = 40 V, Z-spray source temperature = 150 °C, desolvation gas temperature = 350 °C, flow of the desolvation gas = 8000 mL/h. The mass to charge range used in the full-scan analysis mode was 100 to 600 Da.

Apparatus and operation conditions for feeding experiments analysis

UPLC and HRMS were performed on a Waters Acquity UPLC liquid chromatograph fitted to an Waters Xevo G2-X2 QTof spectrometer, operating with an ESI-MS in positive analysis mode. A volume of 1 µL of aerial part and 5 µL of roots at a concentration of 100 µg/mL of the samples was injected into an Acquity UPLC HSS T3 100A column (100 mm × 2.1 mm, 1.8 µm) coupled with Acquity UPLC HSS T3 vanguard pre-column. The chromatographic condition used was (A : B; A – H₂O ultrapure + 0.1% formic acid; B-ACN + 0.1% formic acid); from 0 – 8 min starting at (95: 5) until (0: 100); 8 – 13 min (0: 100); and 13 – 18 min (95: 5). The temperature of injector and column were 25 °C and 35 °C respectively. The flow of the mobile phase was 0.6 mL/min, and the compounds were monitored at λ = 313 and 280 nm. All analyses were made in triplicate and submitted to analysis of variance (ANOVA), using the statistical software SISVAR, and analyzed by the Scott-Knott [41] test at 5% probability.

UPLC and MS in low-resolution instrument were performed on a LC Shimadzu fitted to a Bruker Amazon SL spectrometer, operating with an ESI-MS in positive analysis mode and ion trap mass analyzer. A volume of 1 µL of aerial part and 5 µL of roots at a concentration of 2 mg/mL of the samples was injected into a Kinetex C18 100A column (150 × 2.1 mm, 5 µm). The chromatographic condition used was (A : B; A – H₂O ultrapure + 0.1% formic acid; B-ACN + 0.1% formic acid); from 0 – 30 min starting at (95: 5) until (0: 100); 30 – 35 min (0: 100); and 36 – 46 min (95: 5). The flow of the mobile phase was 1.0 mL/min, and the compounds were monitored at λ = 313 and 280 nm.

Molecular docking and therapeutic viability analysis

Molecular docking studies were performed on the ligands to determine potential interactions with 2 targets AChE and BChE for treating AD. The 3-dimensional structures of the targets were obtained from Protein Data Bank (AChE: PDB ID 4EY6; BChE: PDB ID 5DYW) [42]. Natural iboga-type alkaloids 19-(S)-hydroxy-ibogamine (1), voafinine (2), catharanthine (3), ibogamine (4), voachalotine (5), affinisine (6), 16-epi-affinine (7), isovoacangine (8), coronaridine (9), and unnatural iboga-type alkaloids 5-fluoro-19-(S)-hydroxyibogamine (1a), 5-fluoro-voafinine (2a), 5-fluoro-catharanthine (3a), 5-fluoro-ibogamine (4a), 5-fluoro-voachalotine (5a), 5-fluoro-affinisine (6a), 5-fluoro-16-epi-affinine (7a), 5-fluoro-isovoacangine (8a), 5-fluoro-coronaridine (9a) together with galantamine (CID 9651) and tacrine (CID 1935) were obtained from public repository and downloaded in mol file format. Commercial drugs galantamine and tacrine were used as positive controls to AChE and BChE, respectively. The GOLD (V 5.1) program was used in order to predict binding poses for protein/ligands complexes [43]. The CHEMPL scoring function was used during molecular docking. Pharmacokinetic properties prediction and compound toxicities of the ligands were performed by SwissADME platform [44] and Protx-II sever [45], respectively. Maestro program (Release 2019) was used to analyze the interface of the ligands with target binding site, and visual inspection of the protein/ligand complexes was done by using PyMol software (Version 2.0).

Contributorsʼ Statement

A. A. L. designed research; A. A. L., B. C., B. W. B., A. C. D. C., W. C.-O. and E. J. C. performed research; A. A. L., S. C. F., A. M. S. P. and S. G. analyzed data; A. A. L., B. C., W. C.-O. and S. G. wrote the manuscript and all authors reviewed the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors are grateful to the Biotechnology Unit (UNAERP). BM also thanks to CAPES for the award of scholarships (Grant #88882.365148/2019-01). The authors would like to thank Prof. Dr. Alberto J. Cavalheiro and Dr. Juliana Rodrigues (NuBBE – IQ/UNESP, Araraquara, SP, Brazil) for all MS analysis.

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• 18 Vieira IJC, Medeiros WLB, Monnerat CS, Souza JJ, Mathias L, Braz-Filho R, Epifanio RDA. Two fast screening methods (gc-ms and tlc-chei assay) for rapid evaluation of potential anticholinesterasic indole alkaloids in complex mixtures. An Acad Bras Ciênc 2008; 80: 419-426
  CrossrefPubMedSearch in Google Scholar
• 19 Athipornchai A, Ketpoo P, Saeeng R. Acetylcholinesterase inhibitor from Tabernaemontana pandacaqui flowers. Nat Prod Commun 2020; 15: 1-5
  CrossrefPubMedSearch in Google Scholar
• 20 Garcellano RC, Cort JR, Moinuddin SG, Franzblau SG, Ma R, Aguinaldo AM. An iboga alkaloid chemotaxonomic marker from endemic Tabernaemontana ternifolia with antitubercular activity. Nat Prod Res 2020; 34: 1175-1179
  CrossrefPubMedSearch in Google Scholar
• 21 Masondo N, Stafford G, Aremu A, Makunga N. Acetylcholinesterase inhibitors from Southern African plants: an overview of ethnobotanical, pharmacological potential and phytochemical research including and beyond Alzheimerʼs disease treatment. S Afr J Bot 2019; 120: 39-64
  CrossrefPubMedSearch in Google Scholar
• 22 Seong S, Lim H, Han S. Biosynthetically inspired transformation of iboga to monomeric post-iboga alkaloids. Cell 2018; 5: 353-363
  PubMedSearch in Google Scholar
• 23 Andrade MT, Lima JA, Pinto AC, Rezende CM, Carvalho MP, Epifanio RA. Indole alkaloids from Tabernaemontana australis (Müell. Arg) Miers that inhibit acetylcholinesterase enzyme. Bioorg Med Chem 2005; 13: 4092-4095
  CrossrefPubMedSearch in Google Scholar
• 24 Farrow SC, Kamileen MO, Caputi L, Bussey K, Mundy JEA, McAtee RC, Stephenson CRJ, OʼConnor SE. Biosynthesis of an anti-addiction agent from the iboga plant. J Am Chem Soc 2019; 141: 12979-12983
  Search in Google Scholar
• 25 Pereira PS, França SDC, Oliveira PVA, Breves CMS, Pereira SIV, Sampaio SV, Nomizo A, Dias DA. Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity. Quim Nova 2008; 31: 20-24
  CrossrefPubMedSearch in Google Scholar
• 26 Nicola C, Salvador M, Gower AE, Moura S, Echeverrigaray S. Chemical constituents antioxidant and anticholinesterasic activity of Tabernaemontana catharinensis . Sci World J 2013; 519858: 1-10
  PubMedSearch in Google Scholar
• 27 Mullard A. 2019, FDA drug approvals. Nat Rev Drug Discov 2020; 19: 79-84
  CrossrefPubMedSearch in Google Scholar
• 28 Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 0/9/2019. J Nat Prod 2020; 83: 770-803
  CrossrefPubMedSearch in Google Scholar
• 29 Runguphan W, OʼConnor SE. Metabolic reprogramming of periwinkle plant culture. Nat Chem Biol 2009; 5: 151-153
  CrossrefPubMedSearch in Google Scholar
• 30 Lopes AA, Chioca B, Musquiari B, Crevelin EJ, França SDC, Da Silva MFDGF, Pereira AMS. Unnatural spirocyclic oxindole alkaloids biosynthesis in Uncaria guianensis . Sci Rep 2019; 9: 1-8
  Search in Google Scholar
• 31 Sears JE, Boger DL. Total synthesis of vinblastine, related natural products, and key analogues and development of inspired methodology suitable for the systematic study of their structure-function properties. Acc Chem Res 2015; 48: 653-662
  CrossrefPubMedSearch in Google Scholar
• 32 Nicola C, Salvador M, Gower AE, Moura S, Echeverrigaray S. Chemical constituents antioxidant and anticholinesterasic activity of Tabernaemontana catharinensis . Sci World J 2013; 2013: 519858-519868
  CrossrefPubMedSearch in Google Scholar
• 33 Kam TS, Sim KM. Five new iboga alkaloids from Tabernaemontana corymbose . J Nat Prod 2002; 65: 669-672
  CrossrefPubMedSearch in Google Scholar
• 34 Kam TS, Pang HS, Lim TM. Biologically active indole and bisindole alkaloids from Tabernaemontana divaricate . Org Biomol Chem 2003; 1: 1292-1297
  CrossrefPubMedSearch in Google Scholar
• 35 Nakabayahi RM. MassBank of North America. Accessed October 15, 2020 at: https://mona.fiehnlab.ucdavis.edu/
• 36 Qu Y, Easson MEAM, Simionescu R, Hajicek J, Thamm AMK, Salim V, De Luca V. Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19e-geissoschizine. Proc Natl Acad Sci U S A 2018; 115: 3180-3185
  CrossrefPubMedSearch in Google Scholar
• 37 Caputi L, Franke J, Farrow SC, Chung K, Payne RME, Nguyen T, Dang TT, Carqueijeiro IST, Koudounas K, Bernonville TD, Ameyaw B, Jones DM, Vieira IJC, Courdavault V, OʼConnor SE. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 2018; 6394: 1235-1239
  CrossrefPubMedSearch in Google Scholar
• 38 Farrow SC, Kamileen MO, Meades J, Ameyaw B, Xiao XY, OʼConnor SE. Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga . J Biol Chem 2018; 293: 13821-13833
  CrossrefPubMedSearch in Google Scholar
• 39 Farrow SC, Kamileen MO, Caputi L, Bussey K, Mundy JEA, Mcatee RC, Stephenson CRJ, OʼConnor SE. Biosynthesis of an anti-addiction agent from the iboga plant. J Am Chem Soc 2019; 33: 12979-12983
  CrossrefPubMedSearch in Google Scholar
• 40 Ferreira DF. Sisvar: a computer statistical system. Ciênc agrotec 2011; 35: 1039-1042
  CrossrefPubMedSearch in Google Scholar
• 41 Scott AJ, Knott M. A cluster analysis method for grouping means in the analysis of variance. Biometrics 1974; 30: 507-512
  CrossrefPubMedSearch in Google Scholar
• 42 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissing H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acids Res 2000; 28: 235-242
  CrossrefPubMedSearch in Google Scholar
• 43 Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Bio 1997; 267: 727-748
  CrossrefPubMedSearch in Google Scholar
• 44 Banerjee P, Eckert AO, Schrey AK, Preissner R. Protox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res 2018; 46: 257-263
  CrossrefPubMedSearch in Google Scholar
• 45 Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep 2017; 7: 1-13
  Search in Google Scholar

Correspondence

Publication History

Received: 02 September 2020

Accepted after revision: 19 November 2020

Article published online:
15 December 2020

© 2020. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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  CrossrefPubMedSearch in Google Scholar
• 21 Masondo N, Stafford G, Aremu A, Makunga N. Acetylcholinesterase inhibitors from Southern African plants: an overview of ethnobotanical, pharmacological potential and phytochemical research including and beyond Alzheimerʼs disease treatment. S Afr J Bot 2019; 120: 39-64
  CrossrefPubMedSearch in Google Scholar
• 22 Seong S, Lim H, Han S. Biosynthetically inspired transformation of iboga to monomeric post-iboga alkaloids. Cell 2018; 5: 353-363
  PubMedSearch in Google Scholar
• 23 Andrade MT, Lima JA, Pinto AC, Rezende CM, Carvalho MP, Epifanio RA. Indole alkaloids from Tabernaemontana australis (Müell. Arg) Miers that inhibit acetylcholinesterase enzyme. Bioorg Med Chem 2005; 13: 4092-4095
  CrossrefPubMedSearch in Google Scholar
• 24 Farrow SC, Kamileen MO, Caputi L, Bussey K, Mundy JEA, McAtee RC, Stephenson CRJ, OʼConnor SE. Biosynthesis of an anti-addiction agent from the iboga plant. J Am Chem Soc 2019; 141: 12979-12983
  Search in Google Scholar
• 25 Pereira PS, França SDC, Oliveira PVA, Breves CMS, Pereira SIV, Sampaio SV, Nomizo A, Dias DA. Chemical constituents from Tabernaemontana catharinensis root bark: a brief NMR review of indole alkaloids and in vitro cytotoxicity. Quim Nova 2008; 31: 20-24
  CrossrefPubMedSearch in Google Scholar
• 26 Nicola C, Salvador M, Gower AE, Moura S, Echeverrigaray S. Chemical constituents antioxidant and anticholinesterasic activity of Tabernaemontana catharinensis . Sci World J 2013; 519858: 1-10
  PubMedSearch in Google Scholar
• 27 Mullard A. 2019, FDA drug approvals. Nat Rev Drug Discov 2020; 19: 79-84
  CrossrefPubMedSearch in Google Scholar
• 28 Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 0/9/2019. J Nat Prod 2020; 83: 770-803
  CrossrefPubMedSearch in Google Scholar
• 29 Runguphan W, OʼConnor SE. Metabolic reprogramming of periwinkle plant culture. Nat Chem Biol 2009; 5: 151-153
  CrossrefPubMedSearch in Google Scholar
• 30 Lopes AA, Chioca B, Musquiari B, Crevelin EJ, França SDC, Da Silva MFDGF, Pereira AMS. Unnatural spirocyclic oxindole alkaloids biosynthesis in Uncaria guianensis . Sci Rep 2019; 9: 1-8
  Search in Google Scholar
• 31 Sears JE, Boger DL. Total synthesis of vinblastine, related natural products, and key analogues and development of inspired methodology suitable for the systematic study of their structure-function properties. Acc Chem Res 2015; 48: 653-662
  CrossrefPubMedSearch in Google Scholar
• 32 Nicola C, Salvador M, Gower AE, Moura S, Echeverrigaray S. Chemical constituents antioxidant and anticholinesterasic activity of Tabernaemontana catharinensis . Sci World J 2013; 2013: 519858-519868
  CrossrefPubMedSearch in Google Scholar
• 33 Kam TS, Sim KM. Five new iboga alkaloids from Tabernaemontana corymbose . J Nat Prod 2002; 65: 669-672
  CrossrefPubMedSearch in Google Scholar
• 34 Kam TS, Pang HS, Lim TM. Biologically active indole and bisindole alkaloids from Tabernaemontana divaricate . Org Biomol Chem 2003; 1: 1292-1297
  CrossrefPubMedSearch in Google Scholar
• 35 Nakabayahi RM. MassBank of North America. Accessed October 15, 2020 at: https://mona.fiehnlab.ucdavis.edu/
• 36 Qu Y, Easson MEAM, Simionescu R, Hajicek J, Thamm AMK, Salim V, De Luca V. Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19e-geissoschizine. Proc Natl Acad Sci U S A 2018; 115: 3180-3185
  CrossrefPubMedSearch in Google Scholar
• 37 Caputi L, Franke J, Farrow SC, Chung K, Payne RME, Nguyen T, Dang TT, Carqueijeiro IST, Koudounas K, Bernonville TD, Ameyaw B, Jones DM, Vieira IJC, Courdavault V, OʼConnor SE. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 2018; 6394: 1235-1239
  CrossrefPubMedSearch in Google Scholar
• 38 Farrow SC, Kamileen MO, Meades J, Ameyaw B, Xiao XY, OʼConnor SE. Cytochrome P450 and O-methyltransferase catalyze the final steps in the biosynthesis of the anti-addictive alkaloid ibogaine from Tabernanthe iboga . J Biol Chem 2018; 293: 13821-13833
  CrossrefPubMedSearch in Google Scholar
• 39 Farrow SC, Kamileen MO, Caputi L, Bussey K, Mundy JEA, Mcatee RC, Stephenson CRJ, OʼConnor SE. Biosynthesis of an anti-addiction agent from the iboga plant. J Am Chem Soc 2019; 33: 12979-12983
  CrossrefPubMedSearch in Google Scholar
• 40 Ferreira DF. Sisvar: a computer statistical system. Ciênc agrotec 2011; 35: 1039-1042
  CrossrefPubMedSearch in Google Scholar
• 41 Scott AJ, Knott M. A cluster analysis method for grouping means in the analysis of variance. Biometrics 1974; 30: 507-512
  CrossrefPubMedSearch in Google Scholar
• 42 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissing H, Shindyalov IN, Bourne PE. The protein data bank. Nucleic Acids Res 2000; 28: 235-242
  CrossrefPubMedSearch in Google Scholar
• 43 Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Bio 1997; 267: 727-748
  CrossrefPubMedSearch in Google Scholar
• 44 Banerjee P, Eckert AO, Schrey AK, Preissner R. Protox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res 2018; 46: 257-263
  CrossrefPubMedSearch in Google Scholar
• 45 Daina A, Michielin O, Zoete V. SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep 2017; 7: 1-13
  Search in Google Scholar

• Supplementary Material