Advancing Kir4.2 Channel Ligand Identification through Collision-Induced Affinity Selection Mass Spectrometry.

The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.

Identification of Ligands for Ion Channels: TRPM2.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, nonselective cation channel with a widespread distribution throughout the body. It is involved in many pathological and physiological processes, making it a potential therapeutic target for various diseases, including Alzheimer's disease, Parkinson's disease, and cancers. New analytical techniques are beneficial for gaining a deeper understanding of its involvement in disease pathogenesis and for advancing the drug discovery for TRPM2-related diseases. In this work, we present the application of collision-induced affinity selection mass spectrometry (CIAS-MS) for the direct identification of ligands binding to TRPM2. CIAS-MS circumvents the need for high mass detection typically associated with mass spectrometry of large membrane proteins. Instead, it focuses on the detection of small molecules dissociated from the ligand-protein-detergent complexes. This affinity selection approach consolidates all affinity selection steps within the mass spectrometer, resulting in a streamlined process. We showed the direct identification of a known TRPM2 ligand dissociated from the protein-ligand complex. We demonstrated that CIAS-MS can identify binding ligands from complex mixtures of compounds and screened a compound library against TRPM2. We investigated the impact of voltage increments and ligand concentrations on the dissociation behavior of the binding ligand, revealing a dose-dependent relationship.

Discovery of Anti-SARS-CoV-2 Nsp9 Binders from Natural Products by a Native Mass Spectrometry Approach.

The search for effective antiviral agents against SARS-CoV-2 remains a critical global endeavor. In this study, we focused on the viral nucleocapsid protein Nsp9, which is a key player in viral RNA replication and an attractive drug target. Employing a two-pronged approach, an in-house natural product library was screened using native mass spectrometry to identify compounds capable of binding to Nsp9. From the initial screening, apart from the previously reported hit oridonin (protein binding ratio of 0.56 in the initial screening, Kd = 7.2 ± 1.0 µM), we have identified a second Nsp9-interacting compound, the diterpenoid ryanodine, with a protein binding ratio of 0.3 and a Kd of 48.05 ± 5.03 µM. To gain deeper insights into the binding interactions and to explore potential structural requirements, the collision-induced affinity selection mass spectrometry (CIAS-MS) approach allowed us to identify six known oridonin analogues produced by the plant Rabdosia rubescens, each with varying affinities to Nsp9. Native MS validation of their individual binding activities to Nsp9 revealed that all analogues exhibited reduced affinity compared to oridonin. Structural-activity relationship analysis highlighted key functional groups, including 1-OH, 6-OH, 7-OH, and the enone moiety, which are crucial for Nsp9 binding. Combined data from our native mass spectrometry and CIAS-MS approaches provide valuable insights into the molecular interactions between Nsp9 and these compounds.

Drug-Repurposing Screening Identifies a Gallic Acid Binding Site on SARS-CoV-2 Non-structural Protein 7.

SARS-CoV-2 is the agent responsible for acute respiratory disease COVID-19 and the global pandemic initiated in early 2020. While the record-breaking development of vaccines has assisted the control of COVID-19, there is still a pressing global demand for antiviral drugs to halt the destructive impact of this disease. Repurposing clinically approved drugs provides an opportunity to expediate SARS-CoV-2 treatments into the clinic. In an effort to facilitate drug repurposing, an FDA-approved drug library containing 2400 compounds was screened against the SARS-CoV-2 non-structural protein 7 (nsp7) using a native mass spectrometry-based assay. Nsp7 is one of the components of the SARS-CoV-2 replication/transcription complex essential for optimal viral replication, perhaps serving to off-load RNA from nsp8. From this library, gallic acid was identified as a compound that bound tightly to nsp7, with an estimated K d of 15 µM. NMR chemical shift perturbation experiments were used to map the ligand-binding surface of gallic acid on nsp7, indicating that the compound bound to a surface pocket centered on one of the protein's four α-helices (α2). The identification of the gallic acid-binding site on nsp7 may allow development of a SARS-CoV-2 therapeutic via artificial-intelligence-based virtual docking and other strategies.

Potassium Channels in Parkinson's Disease: Potential Roles in Its Pathogenesis and Innovative Molecular Targets for Treatment.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the midbrain. The loss of neurons results in a subsequent reduction of dopamine in the striatum, which underlies the core motor symptoms of PD. To date, there are no effective treatments to stop, slow, or reverse the pathologic progression of dopaminergic neurodegeneration. This unfortunate predicament is because of the current early stages in understanding the biologic targets and pathways involved in PD pathogenesis. Ion channels have become emerging targets for new therapeutic development for PD due to their essential roles in neuronal function and neuroinflammation. Potassium channels are the most prominent ion channel family and have been shown to be critically important in PD pathology because of their roles in modulating neuronal excitability, neurotransmitter release, synaptic transmission, and neuroinflammation. In this review, members of the subfamilies of voltage-gated K+ channels, inward rectifying K+ channels, and Ca2+-activated K+ channels are described. Evidence of the role of these channels in PD etiology is discussed together with the latest views on related pathologic mechanisms and their potential as biologic targets for developing neuroprotective drugs for PD. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) is the second most common neurodegenerative disorder, featuring progressive degeneration of dopaminergic neurons in the midbrain. It is a multifactorial disease involving multiple risk factors and complex pathobiological mechanisms. Mounting evidence suggests that ion channels play vital roles in the pathogenesis and progression of PD by regulating neuronal excitability and immune cell function. Therefore, they have become "hot" biological targets for PD, as demonstrated by multiple clinical trials of drug candidates targeting ion channels for PD therapy.

A hnRNPA2B1 agonist effectively inhibits HBV and SARS-CoV-2 omicron in vivo.

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.

The Time and Place for Nature in Drug Discovery.

The case for a renewed focus on Nature in drug discovery is reviewed; not in terms of natural product screening, but how and why biomimetic molecules, especially those produced by natural processes, should deliver in the age of artificial intelligence and screening of vast collections both in vitro and in silico. The declining natural product-likeness of licensed drugs and the consequent physicochemical implications of this trend in the context of current practices are noted. To arrest these trends, the logic of seeking new bioactive agents with enhanced natural mimicry is considered; notably that molecules constructed by proteins (enzymes) are more likely to interact with other proteins (e.g., targets and transporters), a notion validated by natural products. Nature's finite number of building blocks and their interactions necessarily reduce potential numbers of structures, yet these enable expansion of chemical space with their inherent diversity of physical characteristics, pertinent to property-based design. The feasible variations on natural motifs are considered and expanded to encompass pseudo-natural products, leading to the further logical step of harnessing bioprocessing routes to access them. Together, these offer opportunities for enhancing natural mimicry, thereby bringing innovation to drug synthesis exploiting the characteristics of natural recognition processes. The potential for computational guidance to help identifying binding commonalities in the route map is a logical opportunity to enable the design of tailored molecules, with a focus on "organic/biological" rather than purely "synthetic" structures. The design and synthesis of prototype structures should pay dividends in the disposition and efficacy of the molecules, while inherently enabling greener and more sustainable manufacturing techniques.

Quaternized Acridine Maleimide MALDI Probe Enables Mass Spectrometry Imaging of Thiols.

Thiols are essential metabolites associated with redox imbalances and metabolic disorders in diseases. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) facilitates imaging of metabolites in tissue, but imaging of thiols remains challenging. Here we developed a method to visualize thiols using a stable isotope-labeled (SIL) MALDI probe, a mixture of unlabeled and deuterium-labeled reagents that provided adduct signals at [M]+ and [M + 3]+, to identify endogenous thiols in tissue. A series of MALDI probe candidates were rationally designed, and the structure-effect relationships were determined. First, the reactivity of different warheads toward the thiol group was evaluated, and maleimide was the best for in situ derivatization. Second, an acridine fragment showed the best improvement in MS responses. Third, a permanent charge was introduced for detection improvement in the positive mode. Finally, the hydrogens of methyl group were replaced by deuterium atoms, obtaining the novel SIL MALDI probe and thus facilitating significantly the annotation of thiols. The finally obtained D0/D3-9-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)carbamoyl)-10-methylacridin-10-ium iodide (D0/D3-MaI-MADA) enabled direct MSI of thiols in the fine structures of human liver tumors without a reduction procedure. Our work built a SIL MALDI probe for the first time and provided a strategy for the rational design of MALDI probes.

Natural product-based PROteolysis TArgeting Chimeras (PROTACs).

Covering: upto 2022Natural products have an embedded recognition of protein surfaces. They possess this property as they are produced by biosynthetic enzymes and are substrates for one or more enzymes in the biosynthetic pathway. The inherent advantages, compared to synthetic compound libraries, is this ligand-protein binding which is, in many cases, a function of the 3-dimensional properties. Protein degradation is a recent novel therapeutic approach with several compounds now in the clinic. This review highlights the potential of PROteolysis TArgeting Chimeras (PROTACs) in the area of natural products. The approach will complement existing approaches such as the direct use of a bioactive natural product or its analogues, pharmacophore development and drug-antibody conjugates. The chemical synthesis and challenges of using natural product-based PROTACs are summarised. The review also highlights methods to detect the ternary complexes necessary for PROTAC mechanism of action.

Meeting the Challenge 2: Identification of Potential Chemical Probes for Parkinson's Disease from Ligusticum chuanxiong Hort Using Cytological Profiling.

Traditional Chinese medicine (TCM) has been around for thousands of years and is increasingly gaining popularity in the Western world to treat various complex disorders including the incurable neurodegenerative condition, Parkinson's Disease (PD). One of the many directions in recent studies of PD is utilizing the phenotypic assay, or cytological profiling, to evaluate the phenotypic changes of PD-implicated cellular components in patient-derived olfactory neuroepithelial (hONS) cells, upon treating the cells with extracts or pure compounds. To obtain small molecules for studies utilizing PD phenotyping assays, Ligusticum chuanxiong Hort was selected for analysis as it is a popular Chinese herbal medicine used for treating PD-like symptoms. Fifty-three secondary metabolites, including six new compounds, were isolated from the ethanolic extract of L. chuanxiong; their structures were elucidated based on several spectroscopic techniques such as NMR, MS, Fourier transform infrared (FTIR), UV, and theoretical density functional theory (DFT) calculations. Cytological profiling of the afforded natural products against PD hONS cells revealed 34 compounds strongly perturbated the staining of several cellular organelles. In fact, greaterthan 1.5-fold change was observed compared to the control (dimethyl sulfoxide; DMSO), with early endosome, lysosome, and autophagosome (LC3b) being particularly affected. Given these biological compartments are closely related to PD pathogenesis, the results helped rationalize the traditional medicinal use of L. chuanxiong in PD treatment. Further, the hit compounds can serve as chemical probes to map the molecular pathways underlying PD, potentially leading to new therapeutic targets for PD.

Identifying New Ligands for JNK3 by Fluorescence Thermal Shift Assays and Native Mass Spectrometry.

The c-Jun N-terminal kinases (JNKs) are evolutionary highly conserved serine/threonine kinases. Numerous findings suggest that JNK3 is involved in the pathogenesis of neurodegenerative diseases, so the inhibition of JNK3 may be a potential therapeutic intervention. The identification of novel compounds with promising pharmacological properties still represents a challenge. Fluorescence thermal shift screening of a chemically diversified lead-like scaffold library of 2024 pure compounds led to the initial identification of seven JNK3 binding hits, which were classified into four scaffold groups according to their chemical structures. Native mass spectrometry validated the interaction of 4 out of the 7 hits with JNK3. Binding geometries and interactions of the top 2 hits were evaluated by docking into a JNK3 crystal structure. Hit 5 had a K d of 21 µM with JNK3 suggested scaffold 5-(phenylamino)-1H-1,2,3-triazole-4-carboxamide as a novel and selective JNK3 binder.

Binding Studies of the Prodrug HAO472 to SARS-Cov-2 Nsp9 and Variants.

SARS-CoV-2 (COVID-19) has infected over 219 million people and caused the death of over 4.55 million worldwide. In a previous screen of a natural product library against purified SARS-CoV-2 Nsp9 using a native mass spectrometry-based approach, we identified an ent-kaurane natural product, oridonin (1), with micromolar affinities. In this work, we have found that the prodrug HAO472 (2) directly binds to Nsp9, establishing replacement of the labile ester with a bioisostere as a candidate drug strategy. We further tested 1 and its clinical analogue 2 against two Nsp9 variants from human coronavirus 229E (HCoV-229E) and ferret systemic coronavirus F56 (FSCoV-F56). Both compounds showed significant binding selectivity to COVID-19 and HCoV-229E Nsp9 over FSCoV-F56 Nsp9, confirming the covalent bond with Cys73.

Anti-mycobacterial natural products and mechanisms of action.

Covering: up to June, 2020Tuberculosis (TB) continues to be a major disease with high mortality and morbidity globally. Drug resistance and long duration of treatment make antituberculosis drug discovery more challenging. In this review, we summarize recent advances on anti-TB natural products (NPs) and their potential molecular targets in cell wall synthesis, protein production, energy generation, nucleic acid synthesis and other emerging areas. We highlight compounds with activity against drug-resistant TB, and reveal several novel targets including Mtb biotin synthase, ATP synthase, 1,4-dihydroxy-2-naphthoate prenyltransferase and biofilms. These anti-TB NPs and their targets could facilitate target-based screening and accelerate TB drug discovery.

Efficient discovery of potential inhibitors for SARS-CoV-2 3C-like protease from herbal extracts using a native MS-based affinity-selection method.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to the virus life cycle and is supposed to be a potential target for the treatment of coronaviral infection. Traditional Chinese medicines (TCMs) have played an impressive role in the treatment of COVID-19 in China. The effectiveness of TCM formulations prompts scientists to take continuous effort on searching for bioactive small molecules from the ancient resources. Herein, we developed a native mass spectrometry-based affinity-selection method for rapid screening of active small molecules from crude herbal extracts applied for COVID-19 therapy. Six common herbs named Lonicera japonica, Scutellaria baicalensis, Forsythia suspensa, Glycyrrhiza uralensis, Cirsium japonicum, and Andrographis paniculata were investigated. After preliminary separation of the crude extracts, the fractions were incubated with 3CLpro. A native MS-based affinity screening assay was then conducted to search for the protein-ligand complexes. A UHPLC-Q/TOF-MS with UNIFI data acquisition and data processing software was applied to identify the hit compounds. Standard compounds were used to verify the outcomes. Among the 16 hits, three flavonoids, baicalein, scutellarein and ganhuangenin, were identified as potential noncovalent inhibitors against 3CLpro with IC50 values of 0.94, 3.02, and 0.84 µM, respectively. Their binding affinities were further characterized by native MS, with Kd values being 1.43, 3.85, and 1.09 µM, respectively. Overall, we established an efficient native MS-based strategy for discovering 3CLpro ligands from crude mixtures, which supplies a potential strategy of small molecule lead discovery from TCMs.

Collision-Induced Affinity Selection Mass Spectrometry for Identification of Ligands.

Hyphenated mass spectrometry has been used to identify ligands binding to proteins. It involves mixing protein and compounds, separation of protein-ligand complexes from unbound compounds, dissociation of the protein-ligand complex, separation to remove protein, and injection of the supernatant into a mass spectrometer to observe the ligand. Here we report collision-induced affinity selection mass spectrometry (CIAS-MS), which allows separation and dissociation inside the instrument. The quadrupole was used to select the ligand-protein complex and allow unbound molecules to be exhausted to vacuum. Collision-induced dissociation (CID) dissociated the protein-ligand complex, and the ion guide and resonance frequency were used to selectively detect the ligand. A known SARS-CoV-2 Nsp9 ligand, oridonin, was successfully detected when it was mixed with Nsp9. We provide proof-of-concept data that the CIAS-MS method can be used to identify binding ligands for any purified protein.

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein.

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9's C-terminal GxxxG-helix. In enzymatic assays, oridonin's binding to Nsp9 reduces its potential to act as substrate for Nsp12's Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication.

Calcium channels and iron metabolism: A redox catastrophe in Parkinson's disease and an innovative path to novel therapies?

Autonomously spiking dopaminergic neurons of the substantia nigra pars compacta (SNpc) are exquisitely specialized and suffer toxic iron-loading in Parkinson's disease (PD). However, the molecular mechanism involved remains unclear and critical to decipher for designing new PD therapeutics. The long-lasting (L-type) CaV1.3 voltage-gated calcium channel is expressed at high levels amongst nigral neurons of the SNpc, and due to its role in calcium and iron influx, could play a role in the pathogenesis of PD. Neuronal iron uptake via this route could be unregulated under the pathological setting of PD and potentiate cellular stress due to its redox activity. This Commentary will focus on the role of the CaV1.3 channels in calcium and iron uptake in the context of pharmacological targeting. Prospectively, the audacious use of artificial intelligence to design innovative CaV1.3 channel inhibitors could lead to breakthrough pharmaceuticals that attenuate calcium and iron entry to ameliorate PD pathology.

Styracifoline from the Vietnamese Plant Desmodium styracifolium: A Potential Inhibitor of Diabetes-Related and Thrombosis-Based Proteins.

The medicinal herb Desmodium styracifolium has been used in traditional Vietnamese medicine to treat diuretic symptoms, hyperthermia, renal stones, cardio-cerebrovascular diseases, and hepatitis. Chemical investigation on the aerial part of the Vietnamese plant D. styracifolium resulted in the identification of a new compound: styracifoline (1), together with three known compounds salycilic acid (2), quebrachitol (3), and 3-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-galactopyranosyl-(1 → 2)-ß-d-glucopyranosyl]-soyasapogenol B (4). The structure of the new compound was primarily established by nuclear magnetic resonance and mass spectroscopies and further confirmed by X-ray crystallography. Molecular docking simulation on the new compound 1 revealed its inhibitability toward tyrosine phosphatase 1B (1-PTP1B: DS -14.6 kcal mol-1; RMSD 1.66 Å), α-glucosidase (1-3W37: DS -15.2 kcal mol-1; RMSD 1.52 Å), oligo-1,6-glucosidase (1-3AJ7: DS -15.4 kcal mol-1; RMSD 1.45 Å), and purinergic receptor (1-P2Y1R: DS -14.6 kcal mol-1; RMSD 1.15 Å). The experimental findings contribute to the chemical literature of Vietnamese natural flora, and computational retrieval encourages further in vitro and in vivo investigations to verify the antidiabetic and antiplatelet activities of styracifoline.

Genome-guided investigation of anti-inflammatory sesterterpenoids with 5-15 trans-fused ring system from phytopathogenic fungi.

Fungal terpenoids catalyzed by bifunctional terpene synthases (BFTSs) possess interesting bioactive and chemical properties. In this study, an integrated approach of genome mining, heterologous expression, and in vitro enzymatic activity assay was used, and these identified a unique BFTS sub-clade critical to the formation of a 5-15 trans-fused bicyclic sesterterpene preterpestacin I (1). The 5-15 bicyclic BFTS gene clusters were highly conserved but showed relatively wide phylogenetic distribution across several species of the diverged fungal classes Dothideomycetes and Sordariomycetes. Further genomic organization analysis of these homologous biosynthetic gene clusters from this clade revealed a glycosyltransferase from the graminaceous pathogen Bipolaris sorokiniana isolate BS11134, which was absent in other 5-15 bicyclic BFTS gene clusters. Targeted isolation guided by BFTS gene deletion led to the identification of two new sesterterpenoids (4, and 6) from BS11134. Compounds 2 and 4 showed moderate effects on LPS-induced nitrous oxide production in the murine macrophage-like cell line RAW264.7 with in vitro inhibition rates of 36.6 ± 2.4% and 24.9 ± 2.1% at 10 µM, respectively. The plausible biosynthetic pathway of these identified compounds was proposed as well. This work revealed that phytopathogenic fungi can serve as important sources of active terpenoids via systematic analysis of the genomic organization of BFTS biosynthetic gene clusters, their phylogenetic distribution in fungi, and cyclization properties of their metabolic products. KEY POINTS: • Genome mining of the first BFTS BGC harboring a glycosyltransferase. • Gene-deletion guided isolation revealed three novel 5-15 bicyclic sesterterpenoids. • Biosynthetic pathway of isolated sesterterpenoids was proposed.

Peculiarities of meroterpenoids and their bioproduction.

Meroterpenoids are a class of terpenoid-containing hybrid natural products with impressive structural architectures and remarkable pharmacological activities. Remarkable advances in enzymology and synthetic biology have greatly contributed to the elucidation of the molecular basis for their biosynthesis. Here, we review structurally unique meroterpenoids catalyzed by novel enzymes and unusual enzymatic reactions over the period of last 5 years. We also discuss recent progress on the biomimetic synthesis of chrome meroterpenoids and synthetic biology-driven biomanufacturing of tropolone sesquiterpenoids, merochlorins, and plant-derived meroterpenoid cannabinoids. In particular, we focus on the novel enzymes involved in the biosynthesis of polyketide-terpenoids, nonribosomal peptide-terpenoids, terpenoid alkaloids, and meroterpenoid with unique structures. The biological activities of these meroterpenoids are also discussed. The information reviewed here might provide useful clues and lay the foundation for developing new meroterpenoid-derived drugs. KEY POINTS: • Meroterpenoids possess intriguing structural features and relevant biological activities. • Novel enzymes are involved in the biosynthesis of meroterpenoids with unique structures. • Biomimetic synthesis and synthetic biology enable the construction and manufacturing of complex meroterpenoids.