DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing.

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

Post-functionalization of sulfur quantum dots and their aggregation-dependent antibacterial activity.

Sulfur quantum dots (SQDs) have emerged as an intriguing class of luminescent nanomaterial due to their exceptional physiochemical and optoelectronic properties. However, their biomedical application is still in its infancy due to the limited scope of their surface functionalization. Herein, we explored the surface functionalization of SQDs through different thiol ligands with tuneable functionality and tested their antibacterial efficacy. Notably, very high antibacterial activity of functionalized SQDs (10-25 ng ml-1) was noted, which is 105 times higher compared to that of nonfunctionalized SQDs. Moreover, a rare phenomenon of the reverse trend of antibacterial activity through surface modification was observed, with increasing surface hydrophobicity of various nanomaterials as the antibacterial activity increased. However, we also noted that as the surface hydrophobicity increased, the SQDs tended to exhibit a propensity for aggregation, which consequently decreased their antibacterial efficacy. This identical pattern was also evident in in vivo assessments. Overall, this study illuminates the importance of surface modifications of SQDs and the role of surface hydrophobicity in the development of antibacterial agents.

DNA-Mediated Control of Protein Function in Semi-Synthetic Systems.

The development of strategies for controlling protein function in a precise and predictable manner has the potential to revolutionize catalysis, diagnostics, and medicine. In this regard, the use of DNA has emerged as a powerful approach for modulating protein activity. The programmable nature of DNA allows for constructing sophisticated architectures wherein proteins can be placed with control over position, orientation, and stoichiometry. This ability is especially useful considering that the properties of proteins can be influenced by their local environment or their proximity to other functional molecules. Here, we chronicle the different strategies that have been developed to interface DNA with proteins in semi-synthetic systems. We further delineate the unique applications unlocked by the unprecedented level of structural control that DNA affords. We end by outlining outstanding challenges in the area and discuss future research directions towards potential solutions.

Graphene oxide as a dual template for induced helicity of peptides.

Artificial template-mediated fabrication of secondary structures within peptides always attracts great interest in biological systems due to several biomimetic interactions. In all earlier studies, a uniform template containing molecules/nanomaterials was used to target only one type of peptide at a time, which extensively limits the diversity in the generation of artificial protein surface/binding sites. This limitation can be overcome by the incorporation of more than one binding template (heterogeneity) in a single system, for example, Janus nanomaterials, which are challenging and difficult to synthesize. In this context, graphene oxide (GO) is considered an artificial binding site (template). It contains two distinctive binding zones, i.e., surface and edge, which can induce the secondary structure of peptides based on complementary interactions. To establish our concept, we have implemented a hybrid sequence i.e., i, i + 4, i + 7 and i + 11 pattern peptides, which defines a more linear surface, suitable for recognition by the two-dimensional GO. Depending on the amino acid residue at the specific locations, we observed substantial enhancement of peptide helicity either at the surface or at the edges of GO from the random coil. However, non-interacting peptides remain as a random coil. We have established this by circular dichroism study at various conditions, as well as atomic force microscopy and optical imaging study. Furthermore, we have also established our observations using molecular dynamics (MD) simulations. This study reveals that the synthesized GO-peptides composite with different secondary structures and recognition residues can mimic biological systems.

Surface engineered amphiphilic carbon dots: solvatochromic behavior and applicability as a molecular probe.

Carbon dots (C-dots) have attracted great attention in the fields of nanotechnology and bioengineering owing to their unique and tunable optical properties with excellent photoluminescence characteristics. Herein, we have engineered amphiphilic C-dots (AC-dots) using positional isomers of diamino benzene with citric acid under mild microwave irradiation to minimize any background reactions. The optical properties changed from excitation-dependent to excitation-independent depending on the isomer used. This unique optical property of the AC-dots was studied in the presence of various solvents and we extensively inspected the AC-dot-solvent interactions. The intensity of the emission wavelength varied with solvent polarity and showed a linear relationship. Furthermore, we extended this property to investigate the molecular environment in biomolecular systems such as proteins. Interestingly, we found that, in the presence of various proteins, the emission intensity was enhanced, quenched or remained unchanged depending on the nature of the protein surface. The mode of interaction between AC-dots and protein was determined using temperature-dependent fluorescence spectroscopy. This study could provide vital information about the surfaces of proteins and the potential application of C-Dots as a fluorescent probe to detect biological molecules and environments.

One-pot bottom-up synthesis of a 2D graphene derivative: application in biomolecular recognition and nanozyme activity.

The synthesis of two-dimensional (2D) nanosheets such as graphene and its derivatives through a bottom-up approach has many advantages such as growth control and functionalization, but it is always challenging to get the desired material. Herein, we have reported the synthesis of water soluble 2D-nanosheets through a bottom-up approach from 2,4,6-tribromo-3-hydroxybenzoic acid via a self-coupling pathway and characterized them using several techniques. AFM and TEM analyses reveal that the synthesized material has a layered structure with a thickness of ∼1.2 nm. Also, the prepared nanosheets are amorphous in nature with high negative charge (-38 ± 2.5 mV). The flexible nature of 2D-nanosheets and their functionality can be used in many related applications. Therefore, we have utilized the synthesized 2D-nanosheets in biomolecular recognition studies. It was found that the enzymatic activity of α-chymotrypsin can be controlled reversibly in the presence of the synthesized 2D-nanosheets. The kinetic study revealed that the nanosheet surface selectively binds to the active sites of the enzyme through a competitive pathway. Furthermore, we explored the nanozyme activity of the material in a peroxidase-like activity assay of two bio-active molecules: Nicotinamide Adenine Dinucleotide Phosphate (NADH) and dopamine. The results suggest that the prepared material efficiently catalyzed the oxidation of NADH to biological cofactor NAD+ and dopamine to aminochrome in the presence of H2O2. These synthesized graphene-like 2D-nanosheets with functional groups can be further tuned with other functionalities, which can open a new window for other related applications.

In Situ Synthesis of Amino Acid Functionalized Carbon Dots with Tunable Properties and Their Biological Applications.

Emissive carbon dots (C-Dots) are known for displaying versatile properties, which have been widely utilized in many applications such as bioimaging, light-emitting devices, and photocatalysis. Further functionalization can tune their physical and chemical properties, which play the key role in many biologically and chemically relevant applications. Amino acids provide an attractive means for introducing functionality with a structural diversity. In this work, water-soluble emissive C-Dots were synthesized from pyrolysis of citric acid in the presence of various amino acids under hydrothermal conditions. We established that the diverse functionality-bearing polyamides and the polyester backbone of C-Dots are the origin of optical properties with a high quantum yield. The amino acid based C-Dots were systematically characterized using various analytical methods to confirm the core structure as well as the functionality. The results show that, depending on functionalization, the quantum yield can be varied and enhanced up to 62% and surface charge and hydrophobicity can be tuned. Most importantly, we observed a correlation between the quantum yield and properties of the side chain residue of amino acids such as hydrophobicity index and volume. Furthermore, it was found that the synthesized, diversely functionalized C-Dots exhibit no cellular toxicity and can be used for cell imaging. Due to variable surface functionality, we have also applied these C-Dots for array-based protein sensing. Overall, based on this reported method, we can easily tune the optical as well as surface properties of C-Dots, which will be suitable for many future applications.

Graphene Oxide as a Carbocatalyst for a Diels-Alder Reaction in an Aqueous Medium.

The Diels-Alder (DA) reaction, a [4+2] cycloaddition reaction, is highly important in synthetic organic chemistry and is frequently used in the synthesis of natural products containing six-membered rings. Herein, we report an efficient protocol for the DA reaction between 9-hydroxymethylanthracene and N-substituted maleimides using two-dimensional graphene oxide (GO) as a heterogeneous carbocatalyst in an aqueous medium at room temperature. High yields, a wide substrate scope, low temperature, excellent functional group tolerance, atom economy, and water as a green solvent are noteworthy features of this protocol. The heterogeneous GO catalyst can be easily recovered and used multiple times without any significant loss in catalytic activity.

Safety assessment of selected medicinal food plants used in Ayurveda through CYP450 enzyme inhibition study.

BACKGROUND: Andrographis paniculata, Bacopa monnieri and Centella asiatica are mentioned in Ayurveda for the management of neurodegenerative disorders. These plants and their phytomolecules, such as andrographolide, bacoside A and asiaticoside, were studied for their inhibition potential on pooled CYP450 as well as human CYP3A4, CYP2D6, CYP2C9 and CYP1A2 by CYP-CO complex assay and fluorogenic assay respectively followed by IC50 determination. Quantification of bioactive compounds present in the extracts was done by RP-HPLC. Heavy metal content in the selected medicinal plants was determined by atomic absorption spectroscopy. RESULT: CYP-CO complex assay indicated significantly less inhibition potential than standard inhibitor (P < 0.05 and above). A. paniculata showed highest inhibitory activity against CYP3A4 and CYP2D6 (IC50 = 63.06 ± 1.35 µg mL-1 ; 88.80 ± 3.32 µg mL-1 ), whereas C. asiatica and B. monnieri showed least inhibitory activity against CYP1A2 (IC50 = 288.83 ± 1.61 µg mL-1 ) and CYP2C9 (184.68 ± 3.79 µg mL-1 ), respectively. In all cases the extract showed higher inhibition than the single bioactive compounds. The heavy metals content in the plant extracts were within the permissible limits. CONCLUSION: The findings suggested that selected food plants and bioactive compounds contributed negligible interaction potential with CYP isozymes and may not possess any harmful effect with regard to their therapeutic application. © 2016 Society of Chemical Industry.

Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress. AIM OF THE STUDY: Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay. MATERIALS AND METHODS: Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes. RESULTS: Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (P<0.001) IC50 value against CYP1A2 (IC50-13.80±1.96µg/mL), 2C19 (IC50-14.343±2.28µg/mL), 2D6 (IC50-0.897±0.28µg/mL) and 3A4 (IC50-32.057±2.51µg/mL) compared to positive controls such as furafylline, tranylcypromine, quinidine and ketoconazole respectively. Cocktail of herbal formulation and cardio protective, antihypertensive, anti-diabetic drugs showed significantly (P<0.001and P<0.01) less or negligible HDI. CONCLUSION: Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated HDI.

Metabolism-mediated interaction potential of standardized extract of Tinospora cordifolia through rat and human liver microsomes.

OBJECTIVE: Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. MATERIALS AND METHODS: Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC50 values were determined. RESULTS: The content of tinosporaside was found to be 1.64% (w/w) in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC50 (µg/ml) value was 136.45 (CYP3A4), 144.37 (CYP2D6), 127.55 (CYP2C9), and 141.82 (CYP1A2). Tinosporaside and extract showed higher IC50 (µg/ml) value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. CONCLUSIONS: Plant extract showed significantly higher IC50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics.

Effect of Coleus forskohlii and its major constituents on cytochrome P450 induction.

Coleus forskohlii Briq. has been used traditionally for the treatment of several ailments since antiquity in Ayurveda. In the present study, an approach has been made to evaluate the effect of C. forskohlii and its major constituents on cytochrome P450 (CYP3A, CYP2B, and CYP2C) mRNA expression in rat hepatocytes. To gain better understanding of the herb-drug interaction potential of the chemical constituents present in C. forskohlii, the extract was subjected to column chromatography followed by standardization with respect to forskolin, 1-deoxyforskolin, and 1,9-dideoxyforskolin using reversed-phase high-performance liquid chromatography (RP-HPLC). Hepatocytes were treated with extracts, fractions, and phytoconstituents, followed by extraction and purification of total mRNA. Study of mRNA expression was carried out through reverse transcription polymerase chain reaction, followed by agarose gel electrophoresis. Results revealed that the test substances did not show any significant mRNA expression compared to the control against CYP3A, CYP2B, and CYP2C. Positive controls such as dexamethasone and rifampin showed significantly high (p < 0.001) induction potential compared to the control. It can be concluded that C. forskohlii and its major constituents may not be involved in CYP450 induction-based drug interaction.

Pueraria tuberosa: a review on its phytochemical and therapeutic potential.

Pueraria tuberosa (Willd.) DC is a perennial herb commonly known as 'vidarikanda', distributed throughout south east Asia. The plant's tuber is widely used in ethanomedicine as well as in traditional systems of medicine, particularly in ayurveda. It has been used in various ayurvedic formulations as restorative tonic, antiaging, spermatogenic and immune booster and has been recommended for the treatment of cardiovascular diseases, hepatosplenomegaly, fertility disorders, menopausal syndrome, sexual debility and spermatorrhoea. Numerous bioactive phytochemicals, mostly isoflavonoids such as puerarin, genistein, daidzein, tuberosin and so on have been identified in the tuber. In vivo and in vitro studies have provided the support against traditional demands of the tuber as spermatogenic, immune booster, aphrodisiac, anti-inflammatory, cardiotonic and brain tonic. However, further studies are required to define the active phytochemical compositions and to validate its clinical utilisation in the herbal formulations for human uses. This review provides an overview of traditional applications, current knowledge on the phytochemistry, pharmacology and toxicology of P. tuberosa. This review also provides plausible hypotheses about how various isoflavones particularly puerarin, genistein and daidzein, individually or collectively, may be responsible for the therapeutic potential against a wide range of ailments.

Botanicals as medicinal food and their effects on drug metabolizing enzymes.

Botanicals fall under different regulations in different countries and are mostly consumed without the consultation of the healthcare professional. Over the last decade, utilization of herbal therapies has been extensively documented. The findings indicate the possibility of potential herb-drug interactions due to the concomitant administration of herbal extracts and prescription/over-the-counter drugs. Simultaneously, with the increasing public awareness and search for safer herbal remedies, the study on herbal-drug interactions has gained momentum through the study of drug metabolizing enzymes. Cytochrome P450 (CYP) inhibition or induction is probably the most common mechanism for the pharmacokinetic interactions of herbs and drugs. Any inhibition of CYP enzymes by herbal extracts may result in enhanced plasma and tissue concentration of drugs, leading to toxicity, while induction results in reduced drug concentration leading to decreased drug efficacy and treatment failure. Considering the rapidly growing herbal markets, these types of clinical interactions remain under-reported and unclear. With the increasing consumption of herbal extracts along with prescription medicines, the safety of herbs has become a concern. This article reviews the potential for drug interactions by herbal extracts through drug metabolizing enzymes.

RP-HPLC-DAD for simultaneous estimation of mahanine and mahanimbine in Murraya koenigii.

Murraya koenigii leaves (Rutaceae) are widely used as food condiments in various food preparations in India. They possess a wide range of biological activities including antioxidant, antibacterial, anticancer, hypoglycemic and hypolipidemic activity. A rapid reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed for quantitative estimation of mahanine and mahanimbine, two major bioactive alkaloids in this plant. The amounts of mahanine and mahanimbine were detected as 9.56 ± 1.04 and 4.32 ± 0.81% w/w in the extract, with the retention times of 6.26 ± 0.66 and 10.40 ± 0.95 minutes. The limits of detection and quantification were estimated to be 29.30 and 81.12 µg/mL and 1.67 and 6.31 µg/mL, respectively. This specific and precise validated method can be useful for the routine analysis and quantitative determination of mahanine and mahanimbine in this therapeutically potent medicinal plant.

Exploring the possible metabolism mediated interaction of Glycyrrhiza glabra extract with CYP3A4 and CYP2D6.

The rhizome of Glycyrrhiza glabra L. (licorice) is used very widely in Indian and Chinese traditional medicine, and it is a popular flavor ingredient of drinks, sweets and candies. Its medicinal uses include treating bronchitis, dry cough, respiratory infections, liver disorders and diabetes. Glycyrrhizin is normally considered to be its biologically active marker, so a rapid RP-HPLC method was developed for the quantitative estimation of glycyrrhizin in the extract. The effect of the standardized extract and its marker on drug metabolizing enzymes was evaluated through CYP3A4 and CYP2D6 inhibition assays to evaluate the safety through its drug interaction potential. The inhibition of CYP3A4 and CYP2D6 isozymes was analysed by the fluorescent product formation method. In the CYP450-CO assay, the interaction potential of the standardized extract and pooled microsomes (percentage inhibition 23.23 ± 1.84%), was found to be less than the standard inhibitor. In the fluorimetric assay, G. glabra extracts showed higher IC(50) values than their positive inhibitors, ketoconazole and quinidine for CYP3A4 and CYP2D6, respectively. Furthermore, the interaction potential of the plant extract was greater than the pure compound. The results demonstrate that G. glabra and its principle bioactive compound, glycyrrhizin, when co-administered with conventional medicines showed only a weak interaction potential with drug metabolizing enzymes.

Cytochrome P450 inhibitory potential of Triphala--a Rasayana from Ayurveda.

ETHNOPHARMACOLOGICAL RELEVANCE: 'Triphala' is one of the age-old, most commonly used polyherbal preparation from Ayurveda as Rasayana drug. AIM OF THE STUDY: This study was aimed at evaluating the effect of 'Triphala' on drug modulating enzymes to assess its safety through its potential to interact with co-administered drugs. MATERIALS AND METHODS: The cytochrome P450 inhibitory effect of 'triphala' formulation was investigated on rat liver microsomes using CYP450-CO complex assay and on individual isoform such as CYP3A4 and 2D6 using fluorescence screening. RP-HPLC method was developed to standardize 'triphala' and its individual components using gallic acid as analytical marker compound. RESULTS: RP-HPLC analysis demonstrated the presence of gallic acid (4.30±2.09 mg/g) in the formulation. The formulation showed 23% inhibition of the rat liver microsomes through CYP450-CO complex assay which is comparatively less when compared with the individual components. Further, the effect of standardized formulation dissolved in ethanol showed CYP3A4 and CYP2D6 inhibitory activity at the IC(50) values of 119.65±1.91 µg/ml and 105.03±0.98 µg/ml respectively. Gallic acid was also found to inhibit both the isoforms at the IC(50) values of 87.24±1.11 µg/ml and 92.03±0.38 µg/ml respectively. CONCLUSIONS: Various concentrations of the formulation and its individual components showed significantly less inhibitory activity (p<0.001) on individual isoforms when compared with the positive control. Assessment on the in vitro effect of 'triphala' on drug modulating enzymes has important implications for predicting the likelihood of herb-drug interactions if these are administered concomitantly.

Metabolism mediated interaction of α-asarone and Acorus calamus with CYP3A4 and CYP2D6.

The present study was aimed to investigate the possible interaction of the standardized extract of Acorus calamus (AC) with Cytochrome P450 enzyme, quantitative determination of the α-asarone in the AC rhizome was performed by RP-HPLC method. In vitro interaction of the plant extract was evaluated by CYP450-carbon monoxide complex (CYP450-CO) assay. Effect on individual isoforms such as CYP3A4 and CYP2D6 isozymes were analyzed through fluorescence product formation and respective IC(50) values were determined. CYP450-CO assay showed moderate interaction potential. Extract showed higher IC(50) values (46.84±1.83-32.99±2.21 µg/ml) comparing to the standard inhibitors and lower IC(50) value than α-asarone (65.16±2.37-42.15±2.45 µg/ml).

Quantification of glycyrrhizin in Glycyrrhiza glabra extract by validated HPTLC densitometry.

Glycyrrhiza glabra Linn (Family-Fabaceae) is active as an anti-allergic, anti-inflammatory, spasmolytic, mild laxative, antistress, antidepressive, antiulcer, liver protective, estrogenic, emmenagogue, and antidiabetic substance, and is widely used in the Indian system of medicine. The major bioactive constituent is glycyrrhizin. A simple HPTLC method has been developed to control the quality of raw as well as finished glycyrrhiza using glycyrrhizin as the bioactive marker. The solvent system was optimized to chloroform-methanol-water (65 + 36 + 7.5, v/v/v). Extract and standard were dissolved in 70% methanol and applied on a precoated TLC plate. After development, the plate was scanned at 254 nm to create a chromatogram, then the quantity of glycyrrhizin was determined in the extract. The method was validated in terms of specificity, linearity, precision, LOD, and LOQ. Linearity range was found to be 0.96-4.80 microg per spot. The linearity relationship was described by the equation: Y = 612.706 + 1.091X (with r = 0.99904 and SD = 2.52%), where Y is the area under curve and X is the amount of glycyrrhizin (ng). The amount of glycyrrhizin found in the extract was 9.1%. Thus, the method provides a rapid and cost-effective quality measure for Glycyrrhiza glabra hydroalcoholic extract.