Isoflavone Derivatives Research Articles

Synthesis and evaluation of benzoylbenzofurans and isoflavone derivatives as sirtuin 1 inhibitors with antiproliferative effects on cancer cells

Isoflavone derivatives were prepared from benzoylbenzofuran precursors. The synthesized compounds were analyzed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as high-resolution mass spectrometry (HRMS) to confirm their structures. The benzoylbenzofuran and isoflavone analogues were evaluated for inhibition of sirtuin 1 (SIRT1) and cell proliferation in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Several isoflavone and benzoylbenzofuran derivatives exhibited potent antiproliferative effects against the MDA-MB-231 cancer cell line. Most of the isoflavone derivatives attenuated SIRT1 activity to below 50%. The most active compounds were the isoflavone quinones 38, 39, and 40, at IC50 values of 5.58 ± 0.373, 1.62 ± 0.0720, and 7.24 ± 0.823 μM, respectively. Importantly, the most active compound, 6-methoxy-4′,6′-dimethylisoflavone-2′,5′-quinone (39) displayed SIRT1 inhibitory activity comparable to that of the reference compound, suramin. The in silico docking simulations in the active site of SIRT1 further substantiated the experimental results and explored the binding orientations of potent compounds in the active site of the target.

Placovinane: 1″β-Ethoxy-6,4′-dimethoxy-3″,3″-dimethyl-1″,2″-dihydropyranoisoflavone, a New Isoflavone Derivative

Isoflavonoids possess a 3-phenylchroman skeleton and are the biologically active secondary metabolites of various plants that are used for different health promoting and restoring effects through a variety of mechanisms. Chromatographic separation of the n-hexane extract from the stems of Placolobium vietnamense led to the isolation of a new isoflavone derivative, placovinane (1), together with four known compounds (2–5). The structures of isolated compounds were identified from their spectroscopic data and by comparison with the literature. All isolated compounds were evaluated for their α-glucosidase inhibition. They all exhibited potent α-glucosidase inhibition with IC50 values ranging from 11.0 to 87.3 µM, which was significantly less than the positive control acarbose (IC50 179 µM). The cytotoxicity of 1 was evaluated against KB, Hep G2, and MCF7 cell lines, and displayed weak cytotoxicity toward KB and Hep G2 cell lines, with the IC50 values of 89.6 and 93.8 μM, respectively.

New Benzil and Isoflavone Derivatives with Cytotoxic and NO Production Inhibitory Activities from Placolobium vietnamense.

The phytochemical investigation of Placolobium vietnamense stems led to the isolation of a new isoflavone derivative (1) and three new benzil derivatives (2–4), together with four known pyranoisoflavones (5–8). The structures of all isolated compounds were determined on the basis of extensive spectroscopic analyses, including NMR and HRMS spectral data, as well as comparison of their spectroscopic data with those reported in the literature. The cytotoxicity of all isolated compounds was assessed against the human liver hepatocellular carcinoma (Hep G2) cell line, and compound 1 displayed the most significant cytotoxicity with an IC50 value of 8.0 μM. Furthermore, all isolated compounds were also tested for their inhibitory activity against NO production in RAW 264.7 macrophages. Of these, compound 1 exhibited the strongest inhibitory efficacy against the LPS-induced NO production with the IC50 value of 13.7 μM.

Role of Natural and Synthetic Flavonoids as Potential Aromatase Inhibitors in Breast Cancer: Structure-Activity Relationship Perspective.

World Health Organization categorized breast cancer as one of the leading cancer types in females worldwide, and its treatment remains challenging. Accumulated evidence suggested the role of estrogen and its metabolites in pre- and post-menopausal women. Upregulation of estrogen-dependent aromatase is significantly involved in the pathogenesis of breast cancer. Several aromatase inhibitors, such as exemestane, formestane, and letrozole, are being used clinically, owing to their estrogen suppression role. Apart from these drugs, several other molecules, such as natural and synthetic flavonoids, have been reported widely for a similar biological activity. However, some reasonable modifications are required for these structures to achieve desired efficacy and to alleviate toxicity. Designing a novel aromatase inhibitor will be possible if we can establish a rational correlation between the chemistry and biological features of the existing molecules. The benzopyranone- ring system, present in the flavonoid molecules, has been reported as a pharmacophore due to its inhibitory activity on aromatase, which helps repress breast cancer progression. This essential feature has been utilized to modify several natural flavonoids into 5 and 7 hydroxy/methoxy flavone, 4-imidazolyl/triazolyl flavone, 5,4'- diamino flavone, 7,8- benzo-4-imidazolyl flavone, α-naphthoflavone, and 2-azole/thiazolyl isoflavone derivatives. These scaffolds have been considered in this review for meticulous study in aspects of the structure-activity relationship for aromatase inhibitory activity, and it would likely pave the way for designing a potential lead candidate in the future.

Changes in Isoflavone Profile from Soybean Seeds during Cheonggukjang Fermentation Based on High-Resolution UPLC-DAD-QToF/MS: New Succinylated and Phosphorylated Conjugates.

In this study, thirty-eight isoflavone derivatives were comprehensively identified and quantified from the raw, steamed and fermented seeds of four selected soybean cultivars based on UPLC-DAD-QToF/MS results with reference to the previously reported LC-MS library and flavonoid database, and summarized by acylated group including glucosides (Glu), malonyl-glucosides (Mal-Glu), acetyl-glucosides (Ac-Glu), succinyl-glucosides (Suc-Glu) and phosphorylated conjugates (Phos) in addition to aglycones. Among them, Suc-Glu and Phos derivatives were newly generated due to fermentation by B. subtilis AFY-2 (cheonggukjang). In particular, Phos were characterized for the first time in fermented soy products using Bacillus species. From a proposed roadmap on isoflavone-based biotransformation, predominant Mal-Glu (77.5–84.2%, raw) decreased rapidly by decarboxylation and deesterification into Ac-Glu and Glu (3.5–8.1% and 50.0–72.2%) during steaming, respectively. As fermentation continued, the increased Glu were mainly succinylated and phosphorylated as well as gradually hydrolyzed into their corresponding aglycones. Thus, Suc-Glu and Phos (17.3–22.4% and 1.5–5.4%, 36 h) determined depending on cultivar type and incubation time, and can be considered as important biomarkers generated during cheonggukjang fermentation. Additionally, the changes of isoflavone profile can be used as a fundamental report in applied microbial science as well as bioavailability research from fermented soy foods.

In Vitro Evaluation of the Antioxidant Activity and Chemopreventive Potential in Human Breast Cancer Cell Lines of the Standardized Extract Obtained from the Aerial Parts of Zigzag Clover (Trifolium medium L.).

The aboveground parts of Trifolium medium L. (zigzag clover), a little-known representative of the family Fabaceae, collected during flowering in a wild stand (Sławin-Szerokie district, Lublin, Poland), were used in this study. Our previous investigations confirmed the higher content of phytoestrogenic isoflavones (especially biochanin A and formononetin derivatives) in T. medium compared to the closely related medicinal plant T. pratense (red clover) and the involvement of these compounds in anti-osteoporotic effects in ovariectomized female rats. The current study focused on evaluating other antibiodegenerative (antioxidant, chemopreventive, and cytostatic) effects for the lyophilisate (TML) obtained from wild zigzag clover. For this purpose, efficient ultrasound-assisted extraction (UAE) was employed, followed by vacuum drying and phytochemical standardization using a newly developed reversed-phase high-performance liquid chromatography (RP-LC) coupled with a PDA detection. Malonylglycosides of biochanin A and formononetin were the predominant compounds and were found to contribute more than 54% to the total isoflavone content determined in the standardized extract of zigzag clover. The antioxidant potential of TML was examined in vitro using the Folin–Ciocalteu and cupric ion-reducing (CUPRAC) methods in addition to the free radical (DPPH• and ABTS•+) scavenging assays. The cytotoxic effects of TML, formononetin, and ononin were evaluated on MCF-7 (estrogen-dependent) and MDA-MB-231 (estrogen-independent) human breast cancer cell lines using the MTT assay. The important role of malonyl isoflavone derivatives has been indicated both in chemoprevention and potential cytotoxic effects of TML against certain types of breast cancer.

7-Methoxyisoflavone ameliorates atopic dermatitis symptoms by regulating multiple signaling pathways and reducing chemokine production

7-Met, a derivative of soybean isoflavone, is a natural flavonoid compound that has been reported to have multiple signaling pathways regulation effects. This study investigated the therapeutic effects of 7-Met on mice with atopic dermatitis induced by fluorescein isothiocyanate (FITC), or oxazolone (OXZ). 7-Met ameliorated FITC or OXZ-induced atopic dermatitis symptoms by decreasing ear thickness, spleen index, mast cell activation, neutrophil infiltration and serum IgE levels in female BALB/c mice. In FITC-induced atopic dermatitis mice, 7-Met reduced Th1 cytokines production and regulated Th1/Th2 balance by downregulating the secretion of thymic stromal lymphopoietin (TSLP) via inactivation of the NF-κB pathway. In OXZ-induced atopic dermatitis, 7-Met functioned through the reduction of Th17 cytokine production. Our study showed that 7-Methoxyisoflavone alleviated atopic dermatitis by regulating multiple signaling pathways and downregulating chemokine production.

Regioselective One-Pot Synthesis of Hydroxy-(S)-Equols Using Isoflavonoid Reductases and Monooxygenases and Evaluation of the Hydroxyequol Derivatives as Selective Estrogen Receptor Modulators and Antioxidants.

Several regiospecific enantiomers of hydroxy-(S)-equol (HE) were enzymatically synthesized from daidzein and genistein using consecutive reduction (four daidzein-to-equol–converting reductases) and oxidation (4-hydroxyphenylacetate 3-monooxygenase, HpaBC). Despite the natural occurrence of several HEs, most of them had not been studied owing to the lack of their preparation methods. Herein, the one-pot synthesis pathway of 6-hydroxyequol (6HE) was developed using HpaBC (EcHpaB) from Escherichia coli and (S)-equol-producing E. coli, previously developed by our group. Based on docking analysis of the substrate or products, a potential active site and several key residues for substrate binding were predicted to interpret the (S)-equol hydroxylation regioselectivity of EcHpaB. Through investigating mutations on the key residues, the T292A variant was verified to display specific mono-ortho-hydroxylation activity at C6 without further 3′-hydroxylation. In the consecutive oxidoreductive bioconversion using T292A, 0.95 mM 6HE could be synthesized from 1 mM daidzein, while 5HE and 3′HE were also prepared from genistein and 3′-hydroxydaidzein (3′HD or 3′-ODI), respectively. In the following efficacy tests, 3′HE and 6HE showed about 30∼200-fold higher EC50 than (S)-equol in both ERα and ERβ, and they did not have significant SERM efficacy except 6HE showing 10% lower β/α ratio response than that of 17β-estradiol. In DPPH radical scavenging assay, 3′HE showed the highest antioxidative activity among the examined isoflavone derivatives: more than 40% higher than the well-known 3′HD. In conclusion, we demonstrated that HEs could be produced efficiently and regioselectively through the one-pot bioconversion platform and evaluated estrogenic and antioxidative activities of each HE regio-isomer for the first time.

Lupalbigenin Inhibiting NF-κB Translocation Associated with Anti-Inflammatory Responses in Lipopolysaccharide Stimulated RAW 264.7 Macrophages

Background: The dried stem of Derris scandens Benth. is well known as an Asian medicinal plant and is used for a variety of ailments. It is claimed to used for the relief of muscle aches and pain and revealed the presence of compounds that appear to modify inflammatory processes. Chemical analysis of D. scandens have revealed the presence of numerous isoflavone derivatives. Lupalbigenin, prenylated isoflavone is a key component of D. scandens stem ethanolic extract. Its anti-inflammatory activity in cell culture had never previously been reported which is why this study was performed. Objective: To investigate the inflammatory activity through molecular signaling pathways of lupalbigenin from D. scandens aqueous ethanol extract using LPS-induced cell changed in Raw 264.7 macrophages cells. Materials and Methods: Lupalbigenin was purified from aqueous extract of dried stem of D. scandens. Cell viability of lupalbigenin in Raw 264.7 cell was determined by MTT assay. Griess reagent has been used for nitric oxide determination. The protein expression of iNOS, cyclooxygenase-2 (COX-2), TNF-α, NF-κB and MAPK were performed by Western blotting. In addition, we also used immunofluorescent assay to examine the NF-κB translocation from nucleus to cytoplasm. Results: Lupalbigenin at 1.25 and 2.5 mM effectively inhibited the LPS-induced tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) as well as nuclear factor kappa B (NF-κB). In addition, lupalbigenin prevented LPSinduced inflammation by decreasing p38 and JNK expression in mitogen-activated protein kinase (MAPK) pathway. Conclusion: Lupalbigenin at low concentrations showed down-regulation of inflammatory gene and protein expressions as well as inhibiting NF-κB translocation. Lupalbigenin could be used as an anti-inflammatory agent. Keywords: Lupalbigenin; Anti-inflammatory activity; Nitric oxide (NO); NF-κB translocation

A Correlation Study on In Vitro Physiological Activities of Soybean Cultivars, 19 Individual Isoflavone Derivatives, and Genetic Characteristics.

The functionality of soybeans is an important factor in the selection and utilization of excellent soybean cultivars, and isoflavones are representative functional substances in soybeans, which exhibit effects on antioxidants, estrogen activity, and cancer, and prevent cardiovascular diseases. This study analyzed ABTS, DPPH, estrogen, ER (ER) alpha, UCP-1, and NO inhibition activities in 48 types of soybean cultivars, as well as the relationship with 19 isolated types of individual isoflavone derivatives. Statistical analysis was conducted to find individual isoflavone derivatives affecting physiological activities, revealing the high correlation of three types of derivatives: genistein 7-O-(6″-O-acetyl)glucoside (6″-O-acetylgenistin), genistein 7-O-(2″-O-apiosyl)glucoside, and glycitein. Based on these results, 15 types of soybean cultivars were selected (one control type, seven yellow types, six black types, and one green type), which have both high physiological activities and a high content of individual isoflavone derivatives. In addition, these high correlations were further verified through a genome-wide association study (GWAS) to determine the association between activities, substances, and genetic characteristics. This study comprehensively describes the relationship between the specific physiological activities of soybean resources, individual isoflavone derivative substances, and SNPs, which will be utilized for in-depth research, such as selection of excellent soybean resources with specific physiological activities.

The potential anti-amyloidogenic candidate, SPA1413, for Alzheimer's disease.

Recently, isoflavone derivatives have been shown to have neuroprotective effects against neurological disorders. For instance, genistein attenuated the neuroinflammation and amyloid-β accumulation in Alzheimer's disease animal models, suggesting the potential for use to prevent and treat Alzheimer's disease. Here, 50 compounds, including isoflavone derivatives, were constructed and screened for the inhibitory effects on amyloid-β42 fibrilization and oligomerization using the high-throughput screening formats of thioflavin T assay and multimer detection system, respectively. The potential neuroprotective effect of t3-(4-hydroxyphenyl)-2H-chromen-7-ol (SPA1413), also known as dehydroequol, idronoxil or phenoxodiol, was evaluated in cells and in 5xFAD (B6SJL) transgenic mouse, a model of Alzheimer's disease. SPA1413 had a potent inhibitory action on both amyloid-β fibrilization and oligomerization. In the cellular assay, SPA1413 prevented amyloid-β-induced cytotoxicity and reduced neuroinflammation. Remarkably, the oral administration of SPA1413 ameliorated cognitive impairment, decreased amyloid-β plaques and activated microglia in the brain of 5xFAD (B6SJL) transgenic mouse. Our results strongly support the repurposing of SPA1413, which has already received fast-track status from the US Food and Drug Administration (FDA) for cancer treatment, for the treatment of Alzheimer's disease due to its potent anti-amyloidogenic and anti-neuroinflammatory actions.

Effects of isoflavone derivatives on the production of inflammatory cytokines by synovial cells.

The present study investigated the effects of isoflavone derivatives (daidzein, genistein and glycitein) on the production of inflammatory cytokines (IL-6 and IL-8) by IL-1β-stimulated synovial cells. Synovial MH7A cells were stimulated with IL-1β in the absence or presence of isoflavone derivatives, and IL-6 and IL-8 production was measured by ELISA. The results of the present study indicated that daidzein significantly inhibited the production of IL-6, but not IL-8. Conversely, neither genistein nor glycitein exerted any inhibitory effects on the production of IL-6 or IL-8 by IL-1β-stimulated synovial cells. To elucidate the molecular mechanisms underlying the daidzein-mediated inhibition of IL-6 production, the present study examined the effects of daidzein on the phosphorylation (activation) of NF-κB p65, ERK1/2 and p38 MAPK. Daidzein significantly inhibited the phosphorylation of NF-κB p65 and ERK1/2, but not p38 MAPK in IL-1β-stimulated MH7A cells. The present study revealed that among the isoflavone derivatives examined (daidzein, genistein and glycitein), daidzein inhibited the production of IL-6, but not IL-8, by IL-1β-stimulated synovial MH7A cells via the suppression of NF-κB p65 and ERK1/2 activation. Collectively, these results suggested that daidzein may have potential as a therapeutic agent for the treatment of arthritic disorders through its anti-inflammatory effects via the inhibition of IL-6 production.

Effects of an equol-containing supplement on advanced glycation end products, visceral fat and climacteric symptoms in postmenopausal women: A randomized controlled trial.

Equol, an isoflavone derivative whose chemical structure is similar to estrogen, is considered a potentially effective agent for relieving climacteric symptoms, for the prevention of lifestyle-related diseases, and for aging care in postmenopausal women. We investigated the effect of an equol-containing supplement on metabolism and aging and climacteric symptoms with respect to internally produced equol in postmenopausal women. A single-center, randomized controlled trial (registration number: UMIN000030975) on 57 postmenopausal Japanese women (mean age: 56±5.37 years) was conducted. Twenty-seven women received the equol supplement, while the remaining received control. Metabolic and aging-related biomarkers were compared before and after the 3-month intervention. Climacteric symptoms were assessed every month using a validated self-administered questionnaire in Japanese postmenopausal women. Three months post-intervention, the treatment group showed significant improvement in climacteric symptoms compared to the control group (81% vs. 53%, respectively, p = 0.045). We did not observe any beneficial effect on metabolic and aging-related biomarkers in the intervention group. However, in certain populations, significant improvement in skin autofluorescence, which is a measurement of AGE skin products, and visceral fat area was observed, especially among equol producers. Women receiving equol supplementation showed improved climacteric symptoms. This study offered a new hypothesis that there may be a synergy between supplemented equol and endogenously produced equol to improve skin aging and visceral fat in certain populations.

Identification of novel Atg3-Atg8 inhibitors using virtual screening for autophagy modulation

A collection of 9050 natural products, their derivatives, and mimetics, was virtually screened against the human Atg3-Atg8 (Atg - autophagy) binding scaffold. By blocking this interaction, the lipidation of Atg8 does not occur and the formation of autophagosomes is inhibited. Forty-three (43) potential ligands were tested using enhanced Green Fluorescent Protein (eGFP) tagged LC3, the human ortholog of Atg8, in MCF7 breast cancer cells. Three hits showed single digit µM IC50 values with AT110, an isoflavone derivative, being the best at 1.2 ± 0.6 µM. Molecular modelling against Atg8 in conjunction with structural activity relationship (SAR) strongly supports the binding to this target. Testing in a panel of cancer cell lines showed little cytotoxic effect as compared to chloroquine. However, same concentration of AT110 was shown to be toxic to young zebrafish embryos. This can be explained in terms of the autophagy process being very active in the zebrafish embryos rendering them susceptible to AT110 whereas in the cancer cells tested the autophagy is not usually active. Nevertheless, AT110 blocks autophagy flux in the zebrafish confirming that the ligand is modulating autophagy. A small molecule non-cytotoxic autophagy inhibitor would open the door for adjunct therapies to bolster many established anticancer drugs, reducing their efficacious concentration thus limiting undesirable site effects. In addition, since many cancer types rely on the autophagy mechanism to survive a therapeutic regime, recurrence can potentially be reduced. The discovery of AT110 is an important step in establishing such an adjunct therapy.

Puerarin ameliorates depression-like behaviors of with chronic unpredictable mild stress mice by remodeling their gut microbiota

BackgroundPuerarin is an isoflavone derivative isolated from the traditional Chinese medicine Pueraria Lobelia, which has proven to relieve depression-like behavior. However, its underlying antidepressant mechanisms have been poorly characterized. Herein, we explored whether Puerarin's antidepressant effect is associated with changes in the gut microbiota (GM). MethodsThe model of depression in mice featuring chronic unpredictable mild stress (CUMS) was eastablished, and its antidepressant effect was estimated by sugar water preference and forced swimming test. Genomic DNA extracted from fecal samples was employed to sequence the 16S rRNA gene for gut microbiota identification. ResultsPuerarin (100 mg/kg) treatment was found to alleviate the CUMS-induced depression-like behaviors. Furthermore, chronic stress led to pathological microbial flora, which was principally marked by the increased abundance of pathogenic bacteria (Proteobacteria, Flexispira, Desulfovibrio) and the decreased abundance of beneficial bacteria (Firmicutes, Bacillales, Lactobacillus). Intriguingly, puerarin treatment reversed these changes. LimitationsThe specific role and anti-depression mechanism of characteristic gut microflora were not confirmed. ConclusionPuerarin can remedy stress-induced disruptions of normal gut microflora. It is suggested that the antidepressant mechanism of puerarin may closely interact with restoring beneficial microflora.

Enhanced treatment for cerebral ischemia-reperfusion injury of puerarin loading liposomes through neutrophils-mediated targeted delivery

The recovery of blood circulation following cerebral infarction-associated thrombolysis brings new damage to the brain, which is cerebral ischemia-reperfusion injury (CIRI). Inflammation is the main pathological mechanism of CIRI. The inflammatory response triggered by cerebral ischemia-reperfusion provides neutrophils with a special opportunity to facilitate drug delivery to the site of ischemic penumbra through the chemotaxis of inflammatory factors by neutrophils. Puerarin is an isoflavone derivative with a significant neuroprotective effect in vitro. But the blood-brain barrier (BBB) impedes its therapeutic efficacy on CIRI within the brain. Inspired by the pathological process, we have used neutrophils as carriers to enhance the BBB penetration of liposomes loaded with puerarin and improve the concentration of puerarin in the brain parenchyma in this study. These results showed that puerarin-containing liposomes were released in response to inflammatory conditions associated with brain injury to enhance the neuroprotection effect at the ischemic penumbra.

Natural Isoflavones and Semisynthetic Derivatives as Pancreatic Lipase Inhibitors.

Obesity, now widespread all over the world, is frequently associated with some chronic diseases. Thus, there is a growing interest in the prevention and treatment of obesity. To date, the only antiobesity drug is orlistat, a natural product-derived pancreatic lipase (PL) inhibitor with some undesired side effects. In the last decades, many natural compounds or derivatives have been evaluated as potential PL inhibitors, and natural polyphenols are among the most promising for possible exploitation as antiobesity agents. However, few studies have been devoted to isoflavones. In this work, we report a study on the PL inhibitory properties of a small library of semisynthetic isoflavone derivatives together with the natural leads daidzein (1), genistein (2), and formononetin (3). In vitro lipase inhibition assay showed that 2 is the most promising PL inhibitor. Among synthetic isoflavones, the hydroxylated and brominated derivatives were more potent than their natural leads. Detailed studies through fluorescence measurements and kinetics of lipase inhibition showed that 2 and the bromoderivatives 10 and 11 have the greatest affinity for PL. Docking studies corroborated these findings highlighting the interactions between isoflavones and the enzyme, confirming that hydroxylation and bromination are useful modifications.

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human CaV3.3 Channel.

Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, CaV3.1, CaV3.2, and CaV3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human CaV3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the CaV3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the CaV3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the CaV3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of in vitro and in silico techniques, we have identified interacting determinants relevant to the CaV3.3/genistein complex formation and propose that genistein directly blocks the function of the human CaV3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rational development of improved and novel low-voltage activated T-type calcium channel inhibitors.

Molecular docking analysis of an isoflavone derivative with the protein phosphatase 1 from Leishmania donovani.

Leishmaniasis is one of the most neglected diseases with high morbidity and mortality rate. Severe side effects with existing drug and lack of proper vaccine encouraged us to design alternative models to combat the disease. We showed that PP1 of Leishmania donovani mediates immunomodulation in host macrophages needed for parasite survival. Therefore, it is of interest to report the molecular docking analysis of 512 isoflavone derivatives with the phosphatase 1 protein from Leishmania donovani to highlight compound 362 (5-hydroxy-5-{9-[2-methoxy-2-(2-methylfuran-3-yl) ethyl]-1H, 3H, 4H, 10bH-pyrano[4,3-c]chromen-3-yl}pentanoic acid) having good binding features and acceptable ADMET properties for further consideration.