Natural Β-carboline Alkaloid Research Articles

Studies on the total syntheses of β‑carboline alkaloids orthoscuticellines A and B

Orthoscuticellines A and B are newly isolated natural β-carboline alkaloids from the moss animal Orthoscuticella ventricosa. Herein, we report the first targeted total synthesis of orthoscuticelline B and an analogous synthetic method for the preparation of dihydro derivate of orthoscuticelline A. The new synthetic approach is based on commercially available and inexpensive reagents leading to a practical synthesis of the target molecules. The reaction sequence consisting of a T3P®-catalyzed amide formation followed by a Bischler–Napieralski cyclisation and a DDQ-assisted dehydrogenation step ensures a practical access to orthoscuticelline B in three steps with 58% overall yield.

Activation of endoplasmic reticulum stress by harmine suppresses the growth of esophageal squamous cell carcinoma.

The worldwide overall 5-year survival rate of esophageal squamous cell carcinoma (ESCC) patients is less than 20%, and novel therapeutic strategies for these patients are urgently needed. Harmine is a natural β-carboline alkaloid, which received great interest in cancer research because of its biological and anti-tumor activities. The aim of this study is to examine the effects of harmine on ESCC and its mechanism. We investigated the effects of harmine on proliferation, cell cycle, apoptosis, and tumor growth in vivo. RNA sequencing (RNA-seq), real-time PCR, and western blotting were used to detect the mechanism. Harmine inhibited ESCC cell growth in vitro and tumor growth in vivo. Differentially expressed genes in harmine-treated ESCC cells were mainly involved in protein processing in the endoplasmic reticulum (ER). Real-time PCR and western blotting confirmed harmine-induced cellular ER stress. CRISPR-Cas9 knockout of C/EBP homologous protein (CHOP) abolished harmine-induced expression of death receptor 5 and apoptosis. Harmine also induced the expression of CHOP-mediated sestrin-2, which in turn contributes to autophagosome formation via suppressing the AMP-activated protein kinase-protein kinase B-mammalian target of rapamycin signaling pathway. In conclusion, our results demonstrate that harmine inhibits the growth of ESCC through its regulation of ER stress, suggesting that it is a promising candidate for ESCC treatment.

Structural modelling and in silico pharmacology of β-carboline alkaloids as potent 5-HT1A receptor antagonists and reuptake inhibitors

Serotonin (5-HT) antagonists and reuptake inhibitors (SARIs) are atypical antidepressants for managing major depressive disorder. They are oftentimes applied as adjuvants for ameliorating aftereffects of SSRI antidepressants including insomnia and sexual dysfunction. The few available candidates of this class including lorpiprazole and trazodone also display some daunting side effects, making a continuous search for improved alternatives essential. Natural β-carboline alkaloids (NβCs) are interestingly renowned with broad pharmacological spectrum against several neuropsychiatric disorders including depression. However, their potentials as SARIs remain underexplored. In this study, 982 NβCs retrieved from the Ambinter-Greenpharma (Amb) database were virtually screened for potent SARI alternatives using computational and biocheminformatics approaches: homology modelling of 5-HT1A receptor, Glide HTVS, SP and XP molecular docking, molecular dynamics (MD) simulation, ADMET and mutagenicity predictions. The homology receptor was validated as a good representative of human 5HT1A receptor using the RCSB structure validation and quality protocols. From the virtual screening against the 5-HT1A receptor, Amb ligands, Amb18709727 and Amb37857532 showed higher binding affinities by XP scores of −8.725 and −7.976 kcal/mol, and MMGBSA of −87.972 and −107.585 kcal/mol respectively compared to lorpiprazole, a reference SARI with XP score and MMGBSA of −6.512 and −62.788 kcal/mol respectively. They maintained ideal contacts with pharmacologically essential amino acid residues implicated in SARI mechanisms and expressed higher stability and compactness than lorpiprazole throughout the trajectories of 100 ns MD simulation. They also displayed interesting ADME, druggability, low toxicity and mutagenicity profiles, ideal for CNS drug prospects, thus, recommended as putative SARI candidates for further study.

Harmine Alleviated Sepsis-Induced Cardiac Dysfunction by Modulating Macrophage Polarization via the STAT/MAPK/NF-κB Pathway.

Sepsis is a dysregulated systemic inflammatory response that often leads to cardiac dysfunction, which is termed sepsis-induced cardiomyopathy (SIC). Harmine, a natural β-carboline alkaloid compound, has been shown to exert pharmacological effects on several diseases. Here, we investigated whether harmine protected against SIC development and the underlying mechanisms. In vitro, the expression of the M1 phenotype markers iNOS and COX-2 was increased in RAW 264.7 cells stimulated with lipopolysaccharide (LPS), but this effect was reversed by the harmine intervention. Furthermore, LPS-induced increases in the levels of inflammatory cytokines, including IL-1β, IL-6, TNF-α, iNOS, COX-2, PGE2 and TXB2, generated by macrophages were suppressed when the cells were pretreated with harmine. Meanwhile, our findings showed that harmine administration effectively attenuated inflammation and apoptosis in H9c2 cells in the proinflammatory environment produced by macrophages, as evidenced by reductions in NLRP3 and cleaved caspase 3 levels and the p-NF-κB/NF-κB ratio. The western blot results indicated that the mechanisms underlying harmine-mediated inhibition of M1 polarization might be associated with suppression of STAT1/3, NF-κB and MAPK activation. Furthermore, an LPS injection induced cardiac dysfunction and decreased the survival rate of mice, which were alleviated by harmine treatment, and the relevant mechanism was possibly attributed to a drug-induced attenuation of the inflammatory and apoptotic processes in cardiomyocytes. Collectively, these results implied that harmine treatment protected against SIC by suppressing M1 phenotypic polarization and inflammation in macrophages.

Discovery and preliminary mechanism of 1-carbamoyl β-carbolines as new antifungal candidates.

Natural β-carboline alkaloids are ideal models for the discovery of pharmaceutically important entities. Various 1-substituted β-carbolines were synthesized from commercially inexpensive tryptophan and demonstrated significant invitro antifungal activity against G.graminis. Significantly, compound 4m (EC50=0.45μM) with carboxamide at 1-position displayed the best efficacy and nearly 20 folds enhancement in antifungal potential compared to Silthiopham (EC50=8.95μM). Moreover, compounds 6, 7, and 4i exhibited excellent invitro antifungal activities and invivo protective and curative activities against B.cinerea and F.graminearum. Preliminary mechanism studies revealed that compound 4m caused reactive oxygen species accumulation, cell membrane destruction, and deregulation of histone acetylation. These findings indicated that 1-carbamoyl β-carboline can be selected as a promising model for the discovery of novel and broad-spectrum fungicide candidates.

Design, synthesis and biological evaluation of harmine derivatives as potent GSK-3β/DYRK1A dual inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss and behavioral disturbances, ultimately leading to death. Glycogen synthase kinase 3β (GSK-3β) and dual-specificity tyrosine phosphorylation regulated kinase1A (DYRK1A) have gained a lot of attention for its role in tau pathology. To search for potential dual GSK-3β/DYRK1A inhibitors, we focused on harmine, a natural β-carboline alkaloid, which has been extensively studied for its various biological effects on the prevention of AD. In this study, a new series of harmine derivatives were designed, synthesized and evaluated as dual GSK-3β/DYRK1A inhibitors for their multiple biological activities. The invitro results indicated that most of them displayed promising activity against GSK-3β and DYRK1A. Among them, compound ZDWX-25 showed potent inhibitory effects on GSK-3β and DYRK1A with IC50 values of 71 and 103nM, respectively. Molecular modelling and kinetic studies verified that ZDWX-25 could interact with the ATP binding pocket of GSK-3β and DYRK1A. Western blot analysis revealed that ZDWX-25 inhibited hyperphosphorylation of tau protein in okadaic acid (OKA)-induced SH-SY5Y cells. In addition, ZDWX-25 showed good blood-brain barrier penetrability invitro. More importantly, ZDWX-25 could ameliorate the impaired learning and memory in APP/PS1/Tau transgenic mice. These results indicated that the harmine-based compounds could be served as promising dual-targeted candidates for AD.

β-Carboline as a Privileged Scaffold for Multitarget Strategies in Alzheimer's Disease Therapy.

The natural β-carboline alkaloids display similarities with neurotransmitters that can be favorably exploited to design bioactive and bioavailable drugs for Alzheimer's disease (AD) therapy. Several AD targets are currently and intensively being investigated, divided in different hypotheses: mainly the cholinergic, the amyloid β (Aβ), and the Tau hypotheses. To date, only symptomatic treatments are available involving acetylcholinesterase and NMDA inhibitors. On the basis of plethoric single-target structure-activity relationship studies, the β-carboline scaffold was identified as a powerful tool for fostering activity and molecular interactions with a wide range of AD-related targets. This knowledge can undoubtedly be used to design multitarget-directed ligands, a highly relevant strategy preferred in the context of multifactorial pathology with intricate etiology such as AD. In this review, we first individually discuss the AD targets of the β-carbolines, and then we focus on the multitarget strategies dedicated to the deliberate design of new efficient scaffolds.

Harmine, a natural β-carboline alkaloid, ameliorates apoptosis by decreasing the expression of caspase-3 in the kidney of diabetic male Wistar rats

BackgroundNumerous clinical studies have suggested that apoptosis in the kidney cells are associated with the diabetes. This study aimed to discover the anti-apoptotic activities of Peganum harmala seed extract and harmine in diabetic kidney disease. Materials and methodsIn this research, 64 male Wistar rats were divided into 8 groups (n = 8): diabetic (D), diabetic with vehicle (DV), diabetic with harmine (DH), diabetic with seed extract (DS), control (C), control with the vehicle (V), control with harmine (H), and control with seed extract (S). Diabetes was induced by a single dose of streptozotocin (STZ) (65 mg/kg). The extraction of the seed material was performed by methanol solvent. Analysis of seed extract was achieved by Fourier-transform infrared spectroscopy and high performance liquid chromatography. Harmine and the seed extract were used in a single dose of 6.5 and 150 mg/kg, respectively (daily by oral gavage for 28 days). Blood serum creatinine, urea, and albumin were examined and histological staining of kidney was analyzed. The percentages of apoptotic kidney cells were determined by flow cytometry and caspase-3 expression was performed by real-time polymerase chain reaction. ResultsThe mean of fasting blood urea level was significantly lower in DH (p < 0.001) and DS rats (p < 0.05), as compared to the D rats. A significant increase of albumin was observed in S and H rats compared to the C group of rats (p < 0.001). The seed extract has been alleviated damages to the kidney histology in D rats. Harmine and the seed extract caused a statistically significant reduction of caspase-3 in the renal diabetic cells compared to D group (p < 0.001). ConclusionOur findings suggest that active β-carboline of harmine in the P. harmala seed extract exerts its anti-apoptotic effects by decreasing caspase-3 in diabetic kidney disease.

Complexation with β-cyclodextrin enhances apoptosis-mediated cytotoxic effect of harman in chemoresistant BRAF-mutated melanoma cells

Harman, a natural β-carboline alkaloid, has recently gained considerable interest due to its anticancer properties. However, its physicochemical characteristics and poor oral bioavailability have been limiting factors for its pharmaceutical development. In this paper, we described the complexation of harman (HAR) with β-cyclodextrin (βCD) as a promising alternative to improve its solubility and consequently its cytotoxic effect in chemoresistant melanoma cells (A2058 cell line). Inclusion complexes (βCD-HAR) were prepared using a simple method and then characterized by FTIR, NMR and SEM techniques. Through in silico studies, the mechanism of complexation of HAR with βCD was elucidated in detail. Both HAR and βCD-HAR promoted cytotoxicity, apoptosis, cell cycle arrest and inhibition of cell migration in melanoma cells. Interestingly, complexation of HAR with βCD enhanced its pro-apoptotic effect by increasing of caspase-3 activity (p < 0.05), probably due to an improvement in HAR solubility. In addition, HAR and βCD-HAR sensitized A2058 cells to vemurafenib, dacarbazine and 5FU treatments, potentializing their cytotoxic activity. These findings suggest that complexation of HAR with natural polymers such as βCD can be useful to improve its bioavailability and antimelanoma activity.

Simplification of Natural β-Carboline Alkaloids to Obtain Indole Derivatives as Potent Fungicides against Rice Sheath Blight.

The increasing resistance of rice sheath blight caused by Rhizoctonia solani highlights the need for highly effective and environmentally benign agents. Natural β-carboline alkaloids were simplified to obtain a series of indole derivatives, and their fungicidal activity and preliminary mode of action against R. solani were also evaluated. The initial hit indole (7) displayed significant fungicidal activity with an EC50 value of 25.56 μg/mL, and was selected for further optimization. Importantly, compound 55, the most active compound, had an EC50 value of 0.62 μg/mL, and approximately 300-fold more potent than validamycin A (EC50 = 183.00 μg/mL). In vivo bioassay also demonstrated that compound 55 showed better fungicidal activities than validamycin A. Moreover, the mechanism studies revealed that compound 55 not only caused remarkable morphological and structural alterations of R. solani hyphae, but also induced the loss of mitochondrial membrane potential and interfered with DNA synthesis. Therefore, compound 55 showed superior fungicidal activity against R. solani, and the elucidated mode of action supported the potential application of compound 55 against rice sheath blight.

Combined transcriptomic and proteomic analysis of harmine on Spodoptera frugiperda Sf9 cells to reveal the potential resistance mechanism

The fall armyworm, Spodoptera frugiperda, is an important invasive pest and exhibits resistance to many insecticides. Harmine, a remarkable, natural β-carboline alkaloid, exhibits a variety of bioactivities and induces programmed cell death in Sf9 cells. In the present study, transcriptomic and proteomic analyses were combined to investigate the effects of harmine on Sf9 cells. A sublethal dose, 0.05 mM, was selected and the transcriptomic analysis revealed 2463 upregulated and 689 downregulated genes after harmine treatment. The most frequently enriched pathways of differentially expressed genes (DEGs) were mainly involved in drug and xenobiotic metabolism. The proteomics analysis revealed 36 upregulated and 77 downregulated proteins, and the results showed a nonlinear relationship with mRNA expression. All the genes related to detoxification and resistance in the transcriptome and DEGs were identified and annotated. Complete open reading frames of 27 cytochrome P450s (CYPs), 27 glutathione S-transferases (GSTs), 11 carboxylesterases (CarEs), 10 UDP-glucuronosyltransferases (UGTs) and 29 heat shock proteins (HSPs) were assembled and verified using qRT-PCR. Harmine exhibited a completely different detoxification mechanism from normal pesticides. The Sigma and Delta class GSTs and UGTs might play important roles, rather than CYP6 and CYP9 clans, CarEs or HSPs. Biological significanceHarmine, a natural β-carboline alkaloid, inhibits the cell proliferation and induces programmed cell death of Sf9 cells, which derived from Spodoptera frugiperda, an important world invasive pest. In the article, the combined transcriptomic and proteomic analysis is used to explore the potential solution for its resistance management. These results supporting that harmine can be applied as a novel adjuvant or pesticide. In addition, the systematically identified resistance-related genes in fall armyworm provide the foundation for potential resistance monitoring and management.

First total synthesis of β-carboline alkaloid trigonostemine G and its derivatives

The β-carboline core is the base structure of several biologically active natural and unnatural compounds. Herein, we report the first total synthesis of trigonostemine G, which is a newly isolated natural β-carboline alkaloid from the twigs of Trigonostemon filipes. Synthesis of two structurally close derivatives of trigonostemine G is also reported. Key step of the syntheses involves a nucleophilic addition of 5-{[tert-butyl(dimethyl) silyl]oxy}-1H-indole to 1-formyl-β-carboline building blocks.

Evolution from Natural β-Carboline Alkaloids to Obtain 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene Derivatives as Potent Fungicidal Agents against Rhizoctonia solani.

Rice sheath blight, caused by Rhizoctonia solani, is a globally important rice disease and the increasing resistance of this pathogen highlights the need for new active compounds against rice sheath blight. In this study, natural β-carboline alkaloids were optimized to obtain a series of 1,2,4,9-tetrahydro-3-thia-9-aza-fluorene derivatives and evaluated for their fungicidal activity and mode of action against R. solani. Of these compounds, 18 exhibited significant in vitro fungicidal activity against R. solani, with an EC50 value of 2.35 μg/mL, and was more active than validamycin A. In vivo bioassay also demonstrated that 18 displayed superior protective and curative activities as compared to validamycin A. Mechanistically, 18 not only induced the loss of mitochondrial membrane potential and accumulation of reactive oxygen species, but also interfered with DNA synthesis. Therefore, compound 18 displayed pronounced in vitro and in vivo fungicidal activity against R. solani and could be used as a potential candidate for the control of rice sheath blight.

Cytotoxic and Apoptotic Activity of the Novel Harmine Derivative ZC-14 in Sf9 Cells.

Harmine, one of the natural β-carboline alkaloids extracted from Peganum harmala L., exhibits broad spectrum but limited insecticidal ability against many pests. So there is an urgent need to synthesize novel derivatives with high efficiency. In the present study, a new synthetic compound, [1-(2-naphthyl)-3-(2-thioxo-1,3,4-oxadiazol-5-yl) β-carboline] (ZC-14), showed a strong proliferation inhibition effect against the Spodoptera frugiperda Sf9 cell line in a dose-dependent manner. Simultaneously, apoptosis induced by 7.5 μg/mL ZC-14 was confirmed with physiological and biochemical evidence, including typical apoptosis characteristics with shrinkage, apoptotic bodies, nuclear condensation/fragmentation, a clear DNA ladder, and a series of apoptotic rates. In addition, mitochondria were confirmed to be involved in apoptosis induced by ZC-14 accompanied with the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from mitochondria into the cytosol and increased expression of cleaved-caspase-3. However, harmine could not induce apoptosis at the same concentration. In summary, these data indicated that compound ZC-14 has a higher cytotoxicity than harmine against Sf9 cells. Besides, it exhibited an anti-proliferative effect in Sf9 cells via inducing apoptosis in which the mitochondrial apoptotic pathway plays a crucial role.

Harmine enhances GABAergic transmission onto basoamygdala projection neurons in mice

Emerging evidence indicates that loss of inhibitory tone in amygdala with its subsequent overactivation contributes to the development of multiple mental disorders such as anxiety disorders and post-traumatic stress disorder (PTSD). Harmine is a member of natural β-carboline alkaloids which can readily cross the blood brain barrier and displays significant antidepressant and anxiolytic effects in rodents. However, the underlying neurobiological mechanisms are largely unknown. Here, by using whole-cell patch clamp recordings in in vitro amygdala slices, we examined the effect of harmine on glutamatergic and GABAergic transmission onto basal amygdala (BA) projection neurons (PNs). Our results showed that harmine affected neither the amplitude nor the frequency of spontaneous and miniature excitatory postsynaptic currents (sEPSCs/mEPSCs) of PNs. By contrast, it markedly increased both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). For mIPSCs, only an increase of their frequency but not amplitude was observed following harmine perfusion, suggesting that harmine might act through presynaptic mechanism. In parallel, a reduction of paired-pulse ratio of evoked IPSCs emerged in the presence of harmine. Furthermore, the intrinsic excitability of PNs was dramatically decreased upon harmine treatment. Together, our study suggests that harmine selectively potentiates the inhibitory but not excitatory transmission onto BA PNs, which may contribute to its antidepressant and anxiolytic influence.

Harmine produces antidepressant-like effects via restoration of astrocytic functions

Depression is a world-wide disease with no effective therapeutic methods. Increasing evidence indicates that astrocytic pathology contributes to the formation of depression. In this study, we investigated the effects of harmine, a natural β-carboline alkaloid and potent hallucinogen, known to modulate astrocytic glutamate transporters, on chronic unpredictable stress (CUS)-induced depressive-like behaviors and astrocytic dysfunctions. Results showed that harmine treatment (10, 20mg/kg) protected the mice against the CUS-induced increases in the immobile time in the tail suspension test (TST) and forced swimming test (FST), and also reversed the reduction in sucrose intake in the sucrose preference experiment. Harmine treatment (20mg/kg) prevented the reductions in brain-derived neurotrophic factor (BDNF) protein levels and hippocampal neurogenesis induced by CUS. In addition, harmine treatment (20mg/kg) increased the protein expression levels of glutamate transporter 1 (GLT-1) and prevented the CUS-induced decreases in glial fibrillary acidic protein (GFAP) protein expressions in the prefrontal cortex and hippocampus, suggesting that restoration of astrocytic functions may be a potential mechanism underlying the antidepressant-like effects of harmine. This opinion was proved by the results that administration of mice with l-Alpha-Aminoadipic Acid (L-AAA), a gliotoxin specific for astrocytes, attenuated the antidepressant-like effects of harmine, and prevented the improvement effects of harmine on BDNF protein levels and hippocampal neurogenesis. These results provide further evidence to confirm that astrocytic dysfunction contributes critically to the development of depression and that harmine exerts antidepressant-like effects likely through restoration of astrocytic functions.

Effects of the Natural β-Carboline Alkaloid Harmine, a Main Constituent of Ayahuasca, in Memory and in the Hippocampus: A Systematic Literature Review of Preclinical Studies

ABSTRACTHarmine is a natural β-carboline alkaloid found in several botanical species, such as the Banisteriopsis caapi vine used in the preparation of the hallucinogenic beverage ayahuasca and the seeds of Syrian rue (Peganum harmala). Preclinical studies suggest that harmine may have neuroprotective and cognitive-enhancing effects, and retrospective/observational investigations of the mental health of long-term ayahuasca users suggest that prolonged use of this harmine-rich hallucinogen is associated with better neuropsychological functioning. Thus, in order to better investigate these possibilities, we performed a systematic literature review of preclinical studies analyzing the effects of harmine on hippocampal neurons and in memory-related behavioral tasks in animal models. We found two studies involving hippocampal cell cultures and nine studies using animal models. Harmine administration was associated with neuroprotective effects such as reduced excitotoxicity, inflammation, and oxidative stress, and increased brain-derived neurotrophic factor (BDNF) levels. Harmine also improved memory/learning in several animal models. These effects seem be mediated by monoamine oxidase or acetylcholinesterase inhibition, upregulation of glutamate transporters, decreases in reactive oxygen species, increases in neurotrophic factors, and anti-inflammatory effects. The neuroprotective and cognitive-enhancing effects of harmine should be further investigated in both preclinical and human studies.

Ene-hydrazide from enol triflate for the regioselective Fischer indole synthesis.

Ene-hydrazide prepared from enol triflate undergoes a Fischer indolization reaction to give the corresponding indole with complete regioselectivity. The starting enol triflate is readily accessed in regiochemically defined form from the ketone precursor via various well-established methods. This new protocol was successfully applied to the synthesis of desbromoarborescidine A, a natural β-carboline alkaloid, difficult to prepare with conventional Fischer indole synthesis.

DsDNA, ssDNA, G-quadruplex DNA, and nucleosomal DNA electrochemical screening using canthin-6-one alkaloid-modified electrodes

Microparticulate films of a canthin-6-one alkaloid (L), a natural β-carboline alkaloid presenting a characteristic naphtyridone motif, on glassy carbon electrodes yield different, separate voltammetric signals for dsDNA, ssDNA, G-quadruplex DNA, different degrees of DNA methylation and the biomimetic nucleosomal DNA with detection limit of 10−5M. This multiple-signal electrochemical behavior is in contrast with conventional use of DNA intercalators, only discriminating between different DNA forms by variations in the intensity of a unique signal. Complementary photochemical and scanning electrochemical microscopy (SECM) data suggest that the differences in the voltammetric response of the different DNA forms derive from the intercalating properties of the canthin-6-one.

Fatty Acid Modulated Human Serum Albumin Binding of the β-Carboline Alkaloids Norharmane and Harmane

Harmane and norharmane are representative members of the large group of natural β-carboline alkaloids featured with diverse pharmacological activities. In blood, these agents are transported by human serum albumin (HSA) which has a profound impact on the pharmacokinetic and pharmacodynamic properties of many therapeutic drugs and xenobiotics. By combination of various spectroscopic methods, the present contribution is aimed to elucidate how nonesterified fatty acids (FAs), the primary endogenous ligands of HSA, affect the binding properties of harmane and norharmane. Analysis of induced circular dichroism (CD) and fluorescence spectroscopic data indicates the inclusion of the neutral form of both molecules into the binding pocket of subdomain IIIA, which hosts two FA binding sites, too. The induced CD and UV absorption spectra of harmane and norharmane exhibit peculiar changes upon addition of FAs, suggesting the formation of ternary complexes in which the lipid ligands significantly alter the binding mode of the alkaloids via cooperative allosteric mechanism. To our knowledge, it is the first instance of the demonstration of drug-FA cobinding at site IIIA. In line with these results, molecular docking calculations showed two distinct binding positions of norharmane within subdomain IIIA. The profound increase in the affinity constants of β-carbolines estimated in the presence of FAs predicts that the unbound, pharmacologically active serum fraction of these compounds strongly depends on the actual lipid binding profile of HSA.