Characterization of an α 4/7-Conotoxin LvIF from Conus lividus That Selectively Blocks α3β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptor (nAChR), a member of pentameric ligand-gated ion channel transmembrane protein composed of five subunits, is widely distributed in the central and peripheral nervous system. The nAChRs are associated with various neurological diseases, including schizophrenia, Alzheimer’s disease, Parkinson’s disease, epilepsy and neuralgia. Receptors containing the α3 subunit are associated with analgesia, generating our interest in their role in pharmacological studies. In this study, α-conotoxin (α-CTx) LvIF was identified as a 16 amino acid peptide using a genomic DNA clone of Conus lividus (C. lividus). The mature LvIF with natural structure was synthesized by a two-step oxidation method. The blocking potency of α-CTx lvIF on nAChR was detected by a two-electrode voltage clamp. Our results showed that α-CTx LvIF was highly potent against rα3β2 and rα6/α3β2β3 nAChR subtypes, The half-maximal inhibitory concentration (IC₅₀) values of α-CTx LvIF against rα3β2 and rα6/α3β2β3 nAChRs expressed in Xenopus oocytes were 8.9 nM and 14.4 nM, respectively. Furthermore, α-CTx LvIF exhibited no obvious inhibition on other nAChR subtypes. Meanwhile, we also conducted a competitive binding experiment between α-CTxs MII and LvIF, which showed that α-CTxs LvIF and MII bind with rα3β2 nAChR at the partial overlapping domain. These results indicate that the α-CTx LvIF has high potential as a new candidate tool for the studying of rα3β2 nAChR related neurophysiology and pharmacology.     

Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)₃(β2)₂ nAChR was deduced using the information from the cryo-electron structure of (α4)₃(β2)₂ nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)₃(β2)₂ nAChR.     

4-(Phenylsulfanyl) Butan-2-One Attenuates the Inflammatory Response Induced by Amyloid-β Oligomers in Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is a progressive eye disease that causes irreversible impairment of central vision, and effective treatment is not yet available. Extracellular accumulation of amyloid-beta (Aβ) in drusen that lie under the retinal pigment epithelium (RPE) has been reported as one of the early signs of AMD and was found in more than 60% of Alzheimer’s disease (AD) patients. Extracellular deposition of Aβ can induce the expression of inflammatory cytokines such as IL-1β, TNF-α, COX-2, and iNOS in RPE cells. Thus, finding a compound that can effectively reduce the inflammatory response may help the treatment of AMD. In this research, we investigated the anti-inflammatory effect of the coral-derived compound 4-(phenylsulfanyl) butan-2-one (4-PSB-2) on Aβ_1-42 oligomer (oAβ_1-42) added to the human adult retinal pigment epithelial cell line (ARPE-19). Our results demonstrated that 4-PSB-2 can decrease the elevated expressions of TNF-α, COX-2, and iNOS via NF-κB signaling in ARPE-19 cells treated with oAβ_1-42 without causing any cytotoxicity or notable side effects. This study suggests that 4-PSB-2 is a promising drug candidate for attenuation of AMD.     

Anti-Alzheimer’s Molecules Derived from Marine Life: Understanding Molecular Mechanisms and Therapeutic Potential

Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the most common cause of dementia. It has been confirmed that the pathological processes that intervene in AD development are linked with oxidative damage to neurons, neuroinflammation, tau phosphorylation, amyloid beta (Aβ) aggregation, glutamate excitotoxicity, and cholinergic deficit. Still, there is no available therapy that can cure AD. Available therapies only manage some of the AD symptoms at the early stages of AD. Various studies have revealed that bioactive compounds derived from marine organisms and plants can exert neuroprotective activities with fewer adverse events, as compared with synthetic drugs. Furthermore, marine organisms have been identified as a source of novel compounds with therapeutic potential. Thus, there is a growing interest regarding bioactive compounds derived from marine sources that have anti-AD potentials. Various marine drugs including bryostatin-1, homotaurine, anabaseine and its derivative, rifampicins, anhydroexfoliamycin, undecylprodigioisin, gracilins, 13-desmethyl spirolide-C, and dictyostatin displayed excellent bioavailability and efficacy against AD. Most of these marine drugs were found to be well-tolerated in AD patients, along with no significant drug-associated adverse events. In this review, we focus on the drugs derived from marine life that can be useful in AD treatment and also summarize the therapeutic agents that are currently used to treat AD.     

Marine-Derived Compounds with Anti-Alzheimer’s Disease Activities

Alzheimer’s disease (AD) is an irreversible and progressive brain disorder that slowly destroys memory and thinking skills, and, eventually, the ability to perform simple tasks. As the aging population continues to increase exponentially, AD has become a big concern for society. Therefore, neuroprotective compounds are in the spotlight, as a means to tackle this problem. On the other hand, since it is believed—in many cultures—that marine organisms in an individual diet cannot only improve brain functioning, but also slow down its dysfunction, many researchers have focused on identifying neuroprotective compounds from marine resources. The fact that the marine environment is a rich source of structurally unique and biologically and pharmacologically active compounds, with unprecedented mechanisms of action, marine macroorganisms, such as tunicates, corals, sponges, algae, as well as microorganisms, such as marine-derived bacteria, actinomycetes, and fungi, have been the target sources of these compounds. Therefore, this literature review summarizes and categorizes various classes of marine-derived compounds that are able to inhibit key enzymes involved in AD, including acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), β-secretase (BACE-1), and different kinases, together with the related pathways involved in the pathogenesis of AD. The compounds discussed herein are emerging as promising anti-AD activities for further in-depth in vitro and in vivo investigations, to gain more insight of their mechanisms of action and for the development of potential anti-AD drug leads.     

Exploitation of Marine Molecules to Manage Alzheimer’s Disease

Neurodegenerative diseases are sociosanitary challenges of today, as a result of increased average life expectancy, with Alzheimer’s disease being one of the most prevalent. This pathology is characterized by brain impairment linked to a neurodegenerative process culminating in cognitive decline and behavioral disorders. Though the etiology of this pathology is still unknown, it is usually associated with the appearance of senile plaques and neurofibrillary tangles. The most used prophylaxis relies on anticholinesterase drugs and NMDA receptor antagonists, whose main action is to relieve symptoms and not to treat or prevent the disease. Currently, the scientific community is gathering efforts to disclose new natural compounds effective against Alzheimer’s disease and other neurodegenerative pathologies. Marine natural products have been shown to be promising candidates, and some have been proven to exert a high neuroprotection effect, constituting a large reservoir of potential drugs and nutraceutical agents. The present article attempts to describe the processes of extraction and isolation of bioactive compounds derived from sponges, algae, marine bacteria, invertebrates, crustaceans, and tunicates as drug candidates against AD, with a focus on the success of pharmacological activity in the process of finding new and effective drug compounds.     

Alzheimer’s Disease and Toxins Produced by Marine Dinoflagellates: An Issue to Explore

This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in β-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer’s disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce β-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.     

Fucoxanthin,a Marine Carotenoid,Reverses Scopolamine-Induced Cognitive Impairments in Mice and Inhibits Acetylcholinesterase in Vitro

Fucoxanthin, a natural carotenoid abundant in edible brown seaweeds, has been shown to possess anti-cancer, anti-oxidant, anti-obesity and anti-diabetic effects. In this study, we report for the first time that fucoxanthin effectively protects against scopolamine-induced cognitive impairments in mice. In addition, fucoxanthin significantly reversed the scopolamine-induced increase of acetylcholinesterase (AChE) activity and decreased both choline acetyltransferase activity and brain-derived neurotrophic factor (BDNF) expression. Using an in vitro AChE activity assay, we discovered that fucoxanthin directly inhibits AChE with an IC₅₀ value of 81.2 μM. Molecular docking analysis suggests that fucoxanthin likely interacts with the peripheral anionic site within AChE, which is in accordance with enzymatic activity results showing that fucoxanthin inhibits AChE in a non-competitive manner. Based on our current findings, we anticipate that fucoxanthin might exhibit great therapeutic efficacy for the treatment of Alzheimer’s disease by acting on multiple targets, including inhibiting AChE and increasing BDNF expression.     

Benefits under the Sea: The Role of Marine Compounds in Neurodegenerative Disorders

Marine habitats offer a rich reservoir of new bioactive compounds with great pharmaceutical potential; the variety of these molecules is unique, and its production is favored by the chemical and physical conditions of the sea. It is known that marine organisms can synthesize bioactive molecules to survive from atypical environmental conditions, such as oxidative stress, photodynamic damage, and extreme temperature. Recent evidence proposed a beneficial role of these compounds for human health. In particular, xanthines, bryostatin, and 11-dehydrosinulariolide displayed encouraging neuroprotective effects in neurodegenerative disorders. This review will focus on the most promising marine drugs’ neuroprotective potential for neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. We will describe these marine compounds’ potential as adjuvant therapies for neurodegenerative diseases, based on their antioxidant, anti-inflammatory, and anti-apoptotic properties.     

A Novel α4/7-Conotoxin QuIA Selectively Inhibits α3β2 and α6/α3β4 Nicotinic Acetylcholine Receptor Subtypes with High Efficacy

α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC₅₀ was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.     

α-Conotoxin TxID and [S9K]TxID, α3β4 nAChR Antagonists,Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice

Tobacco smoking has become a prominent health problem faced around the world. The α3β4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3β4 nAChR antagonist, which has weak inhibition activity of α6/α3β4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3β4 nAChR (IC₅₀ = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3β4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3β4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3β4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.     

Cysteine [2,4] Disulfide Bond as a New Modifiable Site of α-Conotoxin TxIB

α-Conotoxin TxIB, a selective antagonist of α6/α3β2β3 nicotinic acetylcholine receptor, could be a potential therapeutic agent for addiction and Parkinson’s disease. As a peptide with a complex pharmacophoric conformation, it is important and difficult to find a modifiable site which can be modified effectively and efficiently without activity loss. In this study, three xylene scaffolds were individually reacted with one pair of the cysteine residues ([1,3] or [2,4]), and iodine oxidation was used to form a disulfide bond between the other pair. Overall, six analogs were synthesized with moderate isolated yields from 55% to 65%, which is four times higher than the traditional two-step oxidation with orthogonal protection on cysteines. The cysteine [2,4] modified analogs, with higher stability in human serum than native TxIB, showed obvious inhibitory effect and selectivity on α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs), which was 100 times more than the cysteine [1,3] modified ones. This result demonstrated that the cysteine [2,4] disulfide bond is a new modifiable site of TxIB, and further modification can be a simple and feasible strategy for the exploitation and utilization of α-Conotoxin TxIB in drug discovery.     

Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu²⁺ and Zn²⁺, whereas Fe²⁺ displayed an intensification of enzymatic activity. The K_m and V_max of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.     

Talaromarins A–F: Six New Isocoumarins from Mangrove-Derived Fungus Talaromyces flavus TGGP35

Six new isocoumarin derivative talaromarins A-F (1–6), along with 17 known analogues (7–23), were isolated from the mangrove-derived fungus Talaromyces flavus (Eurotiales: Trichocomaceae) TGGP35. Their structures were identified by detailed IR, UV, 1D/2D NMR and HR-ESI-MS spectra. The absolute configurations of new compounds were determined by the modified Mosher’s method and a comparison of their CD spectra with dihydroisocoumarins described in the literature. The antioxidant, antibacterial, anti-phytopathogenic and inhibitory activity against α-glucosidase of all the isolated compounds were tested. Compounds 6–11, 17–19 and 21–22 showed similar or better antioxidant activity than the IC₅₀ values ranging from 0.009 to 0.27 mM, compared with the positive control trolox (IC₅₀ = 0.29 mM). Compounds 10, 18, 21 and 23 exhibited strong inhibitory activities against α-glucosidase with IC₅₀ values ranging from 0.10 to 0.62 mM, while the positive control acarbose had an IC₅₀ value of 0.5 mM. All compounds showed no antibacterial or anti-phytopathogenic activity at the concentrations of 50 μg/mL and 1 mg/mL, respectively. These results indicated that isocoumarins will be useful to developing antioxidants and as diabetes control agents.     

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

Exposure to β-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D₃BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%–32%), implying that D₃BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.     

Structural-Based Optimizations of the Marine-Originated Meridianin C as Glucose Uptake Agents by Inhibiting GSK-3β

Glycogen synthase kinase 3β (GSK-3β) is a widely investigated molecular target for numerous diseases, and inhibition of GSK-3β activity has become an attractive approach for the treatment of diabetes. Meridianin C, an indole-based natural product isolated from marine Aplidium meridianum, has been reported as a potent GSK-3β inhibitor. In the present study, applying the structural-based optimization strategy, the pyrimidine group of meridianin C was modified by introducing different substituents based on the 2-aminopyrimidines-substituted pyrazolo pyridazine scaffold. Among them, compounds B29 and B30 showed a much higher glucose uptake than meridianin C (<5%) and the positive compound 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8, 16%), with no significant toxicity against HepG2 cells at the same time. Furthermore, they displayed good GSK-3β inhibitory activities (IC₅₀ = 5.85; 24.4 μM). These results suggest that these meridianin C analogues represent novel lead compounds with therapeutic potential for diabetes.     

Dieckol Ameliorates Aβ Production via PI3K/Akt/GSK-3β Regulated APP Processing in SweAPP N2a Cell

The proteolytic processing of amyloid precursor protein (APP) by β-secretase (BACE1) and γ-secretase releases amyloid-β peptide (Aβ), which deposits in amyloid plaques and contributes to the initial causative events of Alzheimer’s disease (AD). In the present study, the regulatory mechanism of APP processing of three phlorotannins was elucidated in Swedish mutant APP overexpressed N2a (SweAPP N2a) cells. Among the tested compounds, dieckol exhibited the highest inhibitory effect on both intra- and extracellular Aβ accumulation. In addition, dieckol regulated the APP processing enzymes, such as α-secretase (ADAM10), β-secretase, and γ-secretase, presenilin-1 (PS1), and their proteolytic products, sAPPα and sAPPβ, implying that the compound acts on both the amyloidogenic and non-amyloidogenic pathways. In addition, dieckol increased the phosphorylation of protein kinase B (Akt) at Ser473 and GSK-3β at Ser9, suggesting dieckol induced the activation of Akt, which phosphorylated GSK-3β. The specific phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 triggered GSK-3β activation and Aβ expression. In addition, co-treatment with LY294002 noticeably blocked the effect of dieckol on Aβ production, demonstrating that dieckol promoted the PI3K/Akt signaling pathway, which in turn inactivated GSK-3β, resulting in the reduction in Aβ levels.     

Bioactive Compounds from Macroalgae in the New Millennium: Implications for Neurodegenerative Diseases

Marine environment has proven to be a rich source of structurally diverse and complex compounds exhibiting numerous interesting biological effects. Macroalgae are currently being explored as novel and sustainable sources of bioactive compounds for both pharmaceutical and nutraceutical applications. Given the increasing prevalence of different forms of dementia, researchers have been focusing their attention on the discovery and development of new compounds from macroalgae for potential application in neuroprotection. Neuroprotection involves multiple and complex mechanisms, which are deeply related. Therefore, compounds exerting neuroprotective effects through different pathways could present viable approaches in the management of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. In fact, several studies had already provided promising insights into the neuroprotective effects of a series of compounds isolated from different macroalgae species. This review will focus on compounds from macroalgae that exhibit neuroprotective effects and their potential application to treat and/or prevent neurodegenerative diseases.     

First Identification of 12β-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12β-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC)

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12β-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12β-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.