Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM) of Piperine.

In this article, we introduce a proof-of-concept strategy, Computational Predictive and Electrochemical Detection of Metabolites (CP-EDM), to expedite the discovery of drug metabolites. The use of a bioactive natural product, piperine, that has a well-curated metabolite profile but an unpredictable computational metabolism (Biotransformer v3.0) was selected. We developed an electrochemical reaction to oxidize piperine into a range of metabolites, which were detected by LC-MS. A series of chemically plausible metabolites were predicted based on ion fragmentation patterns. These metabolites were docked into the active site of CYP3A4 using Autodock4.2. From the clustered low-energy profile of piperine in the active site, it can be inferred that the most likely metabolic position of piperine (based on intermolecular distances to the Fe-oxo active site) is the benzo[d][1,3]dioxole motif. The metabolic profile was confirmed by comparison with the literature, and the electrochemical reaction delivered plausible metabolites, vide infra, thus, demonstrating the power of the hyphenated technique of tandem electrochemical detection and computational evaluation of binding poses. Taken together, we outline a novel approach where diverse data sources are combined to predict and confirm a metabolic outcome for a bioactive structure.

Dual Drug Repurposing: The Example of Saracatinib.

Saracatinib (AZD0530) is a dual Src/Abl inhibitor initially developed by AstraZeneca for cancer treatment; however, data from 2006 to 2024 reveal that this drug has been tested not only for cancer treatment, but also for the treatment of other diseases. Despite the promising pre-clinical results and the tolerability shown in phase I trials, where a maximum tolerated dose of 175 mg was defined, phase II clinical data demonstrated a low therapeutic action against several cancers and an elevated rate of adverse effects. Recently, pre-clinical research aimed at reducing the toxic effects and enhancing the therapeutic performance of saracatinib using nanoparticles and different pharmacological combinations has shown promising results. Concomitantly, saracatinib was repurposed to treat Alzheimer's disease, targeting Fyn. It showed great clinical results and required a lower daily dose than that defined for cancer treatment, 125 mg and 175 mg, respectively. In addition to Alzheimer's disease, this Src inhibitor has also been studied in relation to other health conditions such as pulmonary and liver fibrosis and even for analgesic and anti-allergic functions. Although saracatinib is still not approved by the Food and Drug Administration (FDA), the large number of alternative uses for saracatinib and the elevated number of pre-clinical and clinical trials performed suggest the huge potential of this drug for the treatment of different kinds of diseases.

Investigation of the formation mechanism of the pepper starch-piperine complex.

In this study, a new carrier for loading piperine was prepared using pepper starch, and its interaction mechanism was investigated. The porous pepper starch-piperine complex (PPS-PIP) showed higher loading efficiency (76.15 %) compared to the porous corn starch-piperine complex (PCS-PIP (52.34 %)). This may be ascribed to the hemispherical shell structure of porous pepper starch (PPS) compared to the porous structure of porous corn starch (PCS) based on the SEM result. PPS-PIP had smaller particle size (10.53 µm), higher relative crystallinity (38.95 %), and better thermal stability (87.45 °C) than PCS-PIP (17.37 µm, 32.17 %, 74.35 °C). Fourier transform infrared spectroscopy (FTIR) results implied that piperine not only forms a complex with amylose but may also be physically present in porous starch. This was demonstrated by the short-range order and X-ray type. Molecular dynamics simulations confirmed that hydrogen bonding is the primary interaction between amylose and piperine. Besides the formation of the amylose-piperine complex, some of the piperine is also present in physical form.

Formulation and evaluation of dissolving microneedle for transdermal delivery of piperine: the effect of polymers concentration.

This research aims to develop the formulation of Dissolving Microneedle Piperine (DMNs PIP) and evaluate the effect of polymer concentration on characterisation and permeation testing results in ex vivo. DMNs PIP were prepared from varying concentrations of piperine (PIP) (10, 15, and 20% w/w) and polymers of polyvinyl alcohol (PVA): Polyvinyl pyrrolidone (30:60 and 60:25), respectively. Then the morphological evaluation of the formula was carried out, followed by mechanical strength testing. Furthermore, the density, LOD, and weight percentage of piperine in the dried microneedle were calculated and the determination of volume, needle weight and piperine weight and analysed. Ex vivo testing, X-Ray Diffraction, FTIR and hemolysis tests were carried out. PIP with PVA and PVP (F1) polymers produced DMN with mechanical strength (8.35 ± 0.11%) and good penetration ability. In vitro tests showed that the F1 polymer mixture gave good penetration (95.02 ± 1.42 µg/cm2), significantly higher than the F2, F3, F4, and F5 polymer mixtures. The DMNs PIP characterisation results through XRD analysis showed a distinctive peak in the 20-30 region, indicating the presence of crystals. The FTIR study showed that the characteristics of piperine found in DMNs PIP indicated that piperine did not undergo interactions with polymers. The results of the ex vivo study through DMNs PIP hemolytic testing showed no hemolysis occurred, with the hemolysis index below the 5% threshold reported in the literature. These findings indicate that DMNs PIP is non-toxic and safe to use as alternative for treating inflammation.

Extraction, Characterization, and Evaluation of the Cytotoxic Activity of Piperine in Its Isolated form and in Combination with Chemotherapeutics against Gastric Cancer.

Gastric cancer is one of the most frequent types of neoplasms worldwide, usually presenting as aggressive and difficult-to-manage tumors. The search for new structures with anticancer potential encompasses a vast research field in which natural products arise as promising alternatives. In this scenario, piperine, an alkaloid of the Piper species, has received attention due to its biological activity, including anticancer attributes. The present work proposes three heating-independent, reliable, low-cost, and selective methods for obtaining piperine from Piper nigrum L. (Black pepper). Electronic (SEM) and optical microscopies, X-ray diffraction, nuclear magnetic resonance spectroscopies (13C and 1H NMR), and optical spectroscopies (UV-Vis, photoluminescence, and FTIR) confirm the obtention of piperine crystals. The MTT assay reveals that the piperine samples exhibit good cytotoxic activity against primary and metastasis models of gastric cancer cell lines from the Brazilian Amazon. The samples showed selective cytotoxicity on the evaluated models, revealing higher effectiveness in cells bearing a higher degree of aggressiveness. Moreover, the investigated piperine crystals demonstrated the ability to act as a good cytotoxicity enhancer when combined with traditional chemotherapeutics (5-FU and GEM), allowing the drugs to achieve the same cytotoxic effect in cells employing lower concentrations. These results establish piperine as a promising molecule for therapy investigations in aggressive gastric cancer, both in its isolated form or as a bioenhancer.

A mechanistic updated overview on Cepharanthine as potential anticancer agent.

The antitumor effects of traditional drugs have received increasing attention and active antitumor components extracted from traditional drugs have shown good efficacy with minimal adverse events. Cepharanthine(CEP for short) is an active component derived from the Stephania plants of Menispermaceae, which can regulate multiple signaling pathways alone or in combination with other therapeutic drugs to inhibit tumor cell proliferation, induce apoptosis, regulate autophagy, and inhibit angiogenesis, thereby inhibiting tumor progression. Therefore, we retrieved studies concerning CEP's antitumor effects in recent years and summarized the antitumor mechanism and targets, in order to gain new insights and establish a theoretical basis for further development and application of CEP.

Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

Semi-Preparative Separation, Absolute Configuration, Stereochemical Stability and Effects on Human Neuronal Cells of MDPV Enantiomers.

Synthetic cathinones, such as 3,4-methylenedioxypyrovalerone (MDPV), are widely abused due to their psychostimulant effects. As they are chiral molecules, studies of their stereochemical stability (racemization can occur in certain temperatures and acidic/basic environments) and of their biological and/or toxicity effects (enantiomers might display different properties) are of great relevance. In this study, the liquid chromatography (LC) semi-preparative enantioresolution of MDPV was optimized to collect both enantiomers with high recovery rates and enantiomeric ratio (e.r.) values. The absolute configuration of the MDPV enantiomers was determined by electronic circular dichroism (ECD) with the aid of theoretical calculations. The first eluted enantiomer was identified as S-(-)-MDPV and the second eluted enantiomer was identified as R-(+)-MDPV. A racemization study was performed by LC-UV, showing enantiomers' stability up to 48 h at room temperature and 24 h at 37 °C. Racemization was only affected by higher temperatures. The potential enantioselectivity of MDPV in cytotoxicity and in the expression of neuroplasticity-involved proteins-brain-derived neurotrophic factor (BDNF) and cyclin-dependent kinase 5 (Cdk5)-was also evaluated using SH-SY5Y neuroblastoma cells. No enantioselectivity was observed.

Impact of cholesterol and Lumacaftor on the folding of CFTR helical hairpins.

Cystic fibrosis (CF) is caused by mutations in the gene that codes for the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Recent advances in CF treatment have included use of small-molecule drugs known as modulators, such as Lumacaftor (VX-809), but their detailed mechanism of action and interplay with the surrounding lipid membranes, including cholesterol, remain largely unknown. To examine these phenomena and guide future modulator development, we prepared a set of wild type (WT) and mutant helical hairpin constructs consisting of CFTR transmembrane (TM) segments 3 and 4 and the intervening extracellular loop (termed TM3/4 hairpins) that represent minimal membrane protein tertiary folding units. These hairpin variants, including CF-phenotypic loop mutants E217G and Q220R, and membrane-buried mutant V232D, were reconstituted into large unilamellar phosphatidylcholine (POPC) vesicles, and into corresponding vesicles containing 70 mol% POPC +30 mol% cholesterol, and studied by single-molecule FRET and circular dichroism experiments. We found that the presence of 30 mol% cholesterol induced an increase in helicity of all TM3/4 hairpins, suggesting an increase in bilayer cross-section and hence an increase in the depth of membrane insertion compared to pure POPC vesicles. Importantly, when we added the corrector VX-809, regardless of the presence or absence of cholesterol, all mutants displayed folding and helicity largely indistinguishable from the WT hairpin. Fluorescence spectroscopy measurements suggest that the corrector alters lipid packing and water accessibility. We propose a model whereby VX-809 shields the protein from the lipid environment in a mutant-independent manner such that the WT scaffold prevails. Such 'normalization' to WT conformation is consistent with the action of VX-809 as a protein-folding chaperone.

Overview of Piperine: Bioactivities, Total Synthesis, Structural Modification, and Structure-Activity Relationships.

Natural products are an invaluable source for the discovery of drug and pesticide candidates. Piperine, a simple and pungent alkaloid, is isolated from several plants of Piperaceae. Piperine and its derivatives displayed a wide range of biological properties, such as antitumor activity, anti-inflammatory activity, antioxidant activity, neuroprotective activity, insecticidal activity, etc. In recent years, lots of works focused on the biological activities, mechanisms of action, total synthesis, and structural modifications of piperine and its derivatives have been conducted. To the best of our knowledge, however, few review articles related to the biological activities, mechanisms of action, total synthesis, and structural modifications of piperine and its derivatives have been reported to date. Therefore, this review summarizes the research advances (from 2014 to 2020) of piperine and its derivatives regarding bioactivity, mechanisms of action, total synthesis, and structural modifications. Meanwhile, the structure-activity relationships of piperine and its derivatives are also discussed.

The potential role of Piper guineense (black pepper) in managing geriatric brain aging: a review.

Brain aging is one of the unavoidable aspects of geriatric life. As one ages, changes such as the shrinking of certain parts (particularly the frontal cortex, which is vital to learning and other complex mental activities) of the brain may occur. Consequently, communications between neurons are less effective, and blood flow to the brain could also decrease. Efforts made at the biological level for repair become inadequate, leading to the accumulation of ß-amyloid peptide in the brain faster than its probable degradation mechanism, resulting in cognitive malfunction. Subsequent clinical usage of drugs in battling related brain-aging ailments has been associated with several undesirable side effects. However, recent research has investigated the potential use of natural compounds from food in combating such occurrences. This review provides information about the use of Piper guineense (black pepper) as a possible agent in managing brain aging because of its implications for practical brain function. P. guineense contains an alkaloid (piperine) reported to be an antioxidant, anti-depressant, and central nervous system stimulant. This alkaloid and other related compounds are neuroprotective agents that reduce lipid oxidation and inhibit tangles in the brain tissues.

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils.

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two ß-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

A Computational-Experimental Investigation of the Molecular Mechanism of Interleukin-6-Piperine Interaction.

Herein, we elucidate the biophysical aspects of the interaction of an important protein, Interleukin-6 (IL6), which is involved in cytokine storm syndrome, with a natural product with anti-inflammatory activity, piperine. Despite the role of piperine in the inhibition of the transcriptional protein NF-κB pathway responsible for activation of IL6 gene expression, there are no studies to the best of our knowledge regarding the characterisation of the molecular interaction of the IL6-piperine complex. In this context, the characterisation was performed with spectroscopic experiments aided by molecular modelling. Fluorescence spectroscopy alongside van't Hoff analyses showed that the complexation event is a spontaneous process driven by non-specific interactions. Circular dichroism aided by molecular dynamics revealed that piperine caused local α-helix reduction. Molecular docking and molecular dynamics disclosed the microenvironment of interaction as non-polar amino acid residues. Although piperine has three available hydrogen bond acceptors, only one hydrogen-bond was formed during our simulation experiments, reinforcing the major role of non-specific interactions that we observed experimentally. Root mean square deviation (RMSD) and hydrodynamic radii revealed that the IL6-piperine complex was stable during 800 ns of simulation. Taken together, these results can support ongoing IL6 drug discovery efforts.

A critical review on the extraction and pharmacotherapeutic activity of piperine.

Black pepper (Piper nigrum L.) is a climbing perennial plant in the Piperaceae family. Pepper has been known since antiquity for its use both as a medicine and a spice. It is particularly valued for its pungency attributed to its principal constituent - piperine. This review summarizes the information on the biological source of piperine, its extraction and isolation strategies, physicochemical properties, and pharmacological activity - analgesic, immunomodulatory, anti-depressive, anti-diarrheal, hepatoprotective, etc. The effect of piperine on biotransformation of co-administered drugs is also presented in this review, along with the mechanisms involved in its bioavailability-enhancing effect. Its important medicinal uses, including anti-hepatotoxic, anti-diarrheal, anti-depressive, analgesic, and immunomodulatory effects, besides many other traditional uses, are compiled. Based on an exhaustive review of literature, it may be concluded that piperine is a very promising alkaloid found in members of the Piperaceae family.

Neochrysosporazines: Precursor-Directed Biosynthesis Defines a Marine-Derived Fungal Natural Product P-Glycoprotein Inhibitory Pharmacophore.

Upregulation of ATP binding cassette (ABC) transporter efflux pumps (i.e. P-glycoprotein, P-gp) can impart multidrug resistance, rendering many chemotherapeutics ineffective and seriously limiting treatment regimes. While ABC transporters remain an attractive target for therapeutic intervention, the development of clinically useful small-molecule inhibitors has proved challenging. In this report, we describe the structure-activity relationship (SAR) analysis of a newly discovered P-gp inhibitory pharmacophore, phenylpropanoid piperazine chrysosporazines, produced by co-isolated marine-derived fungi. In the absence of any total syntheses, we apply an innovative precursor-directed biosynthesis strategy that successfully repurposed fungal biosynthetic output, allowing us to isolate, characterize, and identify the structurally diverse neochrysosporazines A-Q. SAR analysis utilizing all known (and new) neochrysosporazines, chrysosporazines, and azachrysosporazines, plus semi-synthetic analogues, established the key structure requirements for the P-gp inhibitory pharmacophore, and, in addition, identified non-essential sites that allow for the design of affinity and other conjugated probes.

Mechanism of CFTR correction by type I folding correctors.

Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.

(+)-Sesamin, a sesame lignan, is a potent inhibitor of gut bacterial tryptophan indole-lyase that is a key enzyme in chronic kidney disease pathogenesis.

The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with Ki values of 7 µM and 14 µM, respectively. These Ki values were smaller than that of 2-aza-l-tyrosine (143 µM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD.

Rapid estimation of piperine in black pepper: Exploration of Raman spectroscopy.

INTRODUCTION: The major chemical marker of black pepper (Piper nigrum L) is piperine and its estimation is extremely important for quality assessment of black pepper. The methods for piperine quantification, to date, are laboratory based and use high end instruments like chromatographs, which require tedious sample processing and cause sample destruction. OBJECTIVES: In this article, we present a simple, rapid and green analytical method based on Raman spectroscopy for the quantitative assessment of piperine. MATERIAL AND METHODS: To assess the potential of the technique, we report the complete vibrational characterisation of the piperine with density functional theory (DFT) calculations. RESULTS: The theoretical peaks were obtained at 1097 cm-1 , 1388 cm-1 , 1528 cm-1 , 1578 cm-1 , and at 1627 cm-1 , and this result was verified in a Raman spectrometer followed by a preliminary experiment. Twenty black pepper samples were analysed using high-performance liquid chromatography (HPLC) and used as reference data for Raman analysis. The Raman shift spectra were analysed using partial least squares (PLS) and good prediction accuracy with correlation coefficient of prediction (Rp2 ) = 0.93, root mean square error of prediction (RMSEP) = 0.13 and residual prediction deviation (RPD) = 3.9 obtained. CONCLUSIONS: The results demonstrate the efficacy of the Raman technique for the estimation of piperine in the dry fruit of Piper nigrum.

Natural inspired piperine-based ureas and amides as novel antitumor agents towards breast cancer.

In this work, the natural piperine moiety was utilised to develop two sets of piperine-based amides (5a-i) and ureas (8a-y) as potential anticancer agents. The anticancer action was assessed against triple negative breast cancer (TNBC) MDA-MB-231, ovarian A2780CP and hepatocellular HepG2 cancer cell lines. In particular, 8q stood out as the most potent anti-proliferative analogue against TNBC MDA-MB-231 cells with IC50 equals 18.7 µM, which is better than that of piperine (IC50 = 47.8 µM) and 5-FU (IC50 = 38.5 µM). Furthermore, 8q was investigated for its possible mechanism of action in MDA-MB-231 cells via Annexin V-FITC apoptosis assay and cell cycle analysis. Moreover, an in-silico analysis has proposed VEGFR-2 as a probable enzymatic target for piperine-based derivatives, and then has explored the binding interactions within VEGFR-2 active site (PDB:4ASD). Finally, an in vitro VEGFR-2 inhibition assay was performed to validate the in silico findings, where 8q showed good VEGFR-2 inhibitory activity with IC50 = 231 nM.

Piperine analogs arrest c-myc gene leading to downregulation of transcription for targeting cancer.

G-quadruplex (G4) structures are considered a promising therapeutic target in cancer. Since Ayurveda, Piperine has been known for its medicinal properties. Piperine shows anticancer properties by stabilizing the G4 motif present upstream of the c-myc gene. This gene belongs to a group of proto-oncogenes, and its aberrant transcription drives tumorigenesis. The transcriptional regulation of the c-myc gene is an interesting approach for anticancer drug design. The present study employed a chemical similarity approach to identify Piperine similar compounds and analyzed their interaction with cancer-associated G-quadruplex motifs. Among all Piperine analogs, PIP-2 exhibited strong selectivity, specificity, and affinity towards c-myc G4 DNA as elaborated through biophysical studies such as fluorescence emission, isothermal calorimetry, and circular dichroism. Moreover, our biophysical observations are supported by molecular dynamics analysis and cellular-based studies. Our study showed that PIP-2 showed higher toxicity against the A549 lung cancer cell line but lower toxicity towards normal HEK 293 cells, indicating increased efficacy of the drug at the cellular level. Biological evaluation assays such as TFP reporter assay, quantitative real-time PCR (qRT- PCR), and western blotting suggest that the Piperine analog-2 (PIP-2) stabilizes the G-quadruplex motif located at the promoter site of c-myc oncogene and downregulates its expression. In conclusion, Piperine analog PIP-2 may be used as anticancer therapeutics as it affects the c-myc oncogene expression via G-quadruplex mediated mechanism.