Synthesis and characterization of new thiosemicarbazones,as potent urease inhibitors: In vitro and in silico studies

A new series of N-substituted thiosemicarbazones (3a-u) bearing 2-naphthyl and dihydrobenzofuranyl scaffolds were synthesized in good to excellent yields (78–95%). The synthesized compounds were characterized by advanced spectroscopic techniques, such as FTIR, ¹HNMR, ¹³CNMR and ESI-MS and evaluated as urease inhibitors. The structure of compound 3m was unambiguously confirmed by single crystal X-ray analysis. All compounds showed remarkable activities against urease enzyme with IC₅₀ values in range of 1.4–36.1 µM. The majority of the synthesized compounds showed higher activity than the standard compound thiourea. Molecular docking was performed to study the mode of interaction of these compounds and their structure-activity relationship. These studies revealed that the compounds bind at the active site and interacts with the nickel atom present in the binding site. The molecular docking demonstrated excellent co-relations with the experimental findings.     

Tyrosinase inhibition by some flavonoids: Inhibitory activity,mechanism by in vitro and in silico studies

Flavonoids are main polyphenolic groups widely distributed to fruits, vegetables and beverages we consumed daily. They exhibit many biological effects. We tested tyrosinase inhibitor potential of structurally related (1–9) flavonoids and found that all the tested materials possessed tyrosinase inhibitory effect compared to the positive control, kojic acid. 2 exhibited the strongest tyrosinase inhibitory effect with an IC₅₀ value of 40.94 ± 0.78 µM in a competitive manner. According to kinetic analysis 1, 4 and 7 were found to be competitive inhibitors, 3, 5, and 6 noncompetitive inhibitors of tyrosinase. According to the docking studies, A and C ring of the flavonoid structure, hydroxyl substituent at the 7th position, and hydroxyl substituents at para or para and meta position of ring B play key role for competitive inhibition of the enzyme.     

Importance of oligomerisation on Pseudomonas aeruginosaLectin-II binding affinity. In silico and in vitro mutagenesis

The effect of terminal GLY114* deletion on the binding affinity of the PA-IIL lectin toward l-fucose was investigated. Both experimental (isothermal titration calorimetry) and computational (molecular dynamics simulations) methods have shown that the deletion mutation decreases the L-fucose affinity. It implies that the PA-IIL saccharide binding affinity is influenced by the dimerization of the lectin. A detailed analysis of computational data confirms the key role of electrostatic interactions in the PA-IIL/saccharide binding.     

In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE

The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel's hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC₅₀ 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC₅₀ value of 126 μM, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.     

In vitro and in silico elucidation of antidiabetic and anti-inflammatory activities of bioactive compounds from Momordica charantia L.

Bitter melon (Momordica charantia) has been used to manage diabetes and related conditions in various parts of the world. In the present study, ten compounds were isolated from acetone and methanol extracts of bitter melon. The chemical structures of compounds were unambiguously elucidated by 1D, 2D NMR, and high-resolution mass spectra. Identified compounds 1–7 exhibited significant inhibition of α-amylase and moderate inhibition of α-glucosidase activities. Momordicoside G and gentisic acid 5-O-β-d-xyloside showed the highest inhibition of α-amylase (70.5%), and α-glucosidase (56.4%), respectively. Furthermore, molecular docking studies of isolated compounds 1–7 were able to bind to the active sites of both enzymes. Additionally, the isolated compounds 1–7 significantly attenuated lipopolysaccharide (LPS)-induced inflammation, downregulating the expression of pro-inflammatory markers NF-κB, INOS, IL-6, IL-1β, TNF-α, and Cox-2 in murine macrophage RAW 264.7 cells. One phenolic derivative, gentisic acid 5-O-β-d-xyloside, was isolated and identified for the first time from bitter melon, and significantly suppressed the expression of Cox-2 and IL-6 compared to the LPS-treated group. α-Amylase and α-glucosidase are targets of anti-diabetes drugs, our findings suggest that compounds purified from bitter melon may have potential to use as functional food ingredients for the prevention of type 2 diabetes and related inflammatory conditions.     

In vitro and in silico evaluation of new thiazole compounds as monoamine oxidase inhibitors

New twenty compounds bearing thiazole ring (3a-3t) were designed and synthesized as monoamine oxidase (MAO) inhibitors. The fluorometric enzyme inhibition assay was used to determine the biological effects of synthesized compounds. Most of them showed remarkable inhibitory activity against both MAO-A and MAO-B. By comparing their IC₅₀ values, it can be seen that active derivatives displayed generally selectivity on MAO-B enzyme. Compounds 3j and 3t, which bear dihydroxy moiety at the 3rd and 4th position of phenyl ring, were the most active derivatives in the series against both isoenzymes. Compounds 3j and 3t showed significant inhibition profile on MAO-A with the IC₅₀ values of 0.134 ± 0.004 µM and 0.123 ± 0.005 µM, respectively, while they performed selectivity against MAO-B with the IC₅₀ values of 0.027 ± 0.001 µM and 0.025 ± 0.001 µM, respectively. Also, docking studies about these compounds were carried out to evaluate their binding modes on the active regions of MAO-A and MAO-B.     

The immunomodulation potential of the synthetic derivatives of benzothiazoles: Implications in immune system disorders through in vitro and in silico studies

Benzothiazole and its natural or synthetic derivatives have been used as precursors for several pharmacological agents for neuroprotective, anti-bacterial, and anti-allergic activities. The objecctive of the present study was to evaluate effects of benzothiazole analogs (compounds 1–26) for their immunomodulatory activities. Eight compounds (2, 4, 5, 8–10, 12, and 18) showed potent inhibitory activity on PHA-activated peripheral blood mononuclear cells (PBMCs) with IC₅₀ ranging from 3.7 to 11.9 μM compared to that of the standard drug, prednisolone <1.5 μM. Some compounds (2, 4, 8, and 18) were also found to have potent inhibitory activities on the production of IL-2 on PHA/PMA-stimulated PBMCs with IC₅₀ values ranging between <4.0 and 12.8 μM. The binding interaction of these compounds was performed through silico molecular docking. Compounds 2, 8, 9, and 10 significantly suppressed oxidative burst ROS production in phagocytes with IC₅₀ values between <4.0 and 15.2 μM. The lipopolysaccharide (LPS)-induced nitrites in murine macrophages cell line J774 were found to be inhibited by compounds 4, 8, 9, and 18 at a concentration of 25 μg/mL by 56%, 91%, 58%, and 78%, respectively. Furthermore, compounds 5, 8, 12, and 18 showed significant (P < 0.05) suppressive activity on Th-2 cytokine, interleukin 4 (IL-4) with an IC₅₀ range of <4.0 to 40.3 μM. Interestingly compound 4 has shown a selective inhibitory activity on IL-2 and T cell proliferation (naïve T cell proliferation stage) rather than on IL-4 cytokine, while compound 12 displayed an interference with T-cell proliferation and IL-4 generation. Moreover compound 8 and 18 exert non-selective inhibition on both IL-2 and IL-4 cytokines, indicating a better interference with stage leading to humoral immune response and hence possible application in autoimmune diseases.     

Marine-derived sea urchin compounds as potential anti-cancer drug candidate against colorectal cancer: In silico and in vitro studies

Sea urchin-derived compounds are potential candidates for the development of effective drugs for the treatment of cancer diseases. In this study, 19 compounds derived from sea urchin (Diadema savignyi) were used to treat colorectal cancer using the HCT116 cell line. However, molecular docking, ADME (absorption, distribution, metabolism, and excretion), toxicity, molecular dynamic (MD) simulation, and molecular mechanics generalized Born surface area (MM-GBSA) were used to confirm the ligand–protein interaction. Interactions of Importin-11 receptor with sea urchin compounds reveal that four compounds have higher binding affinities (ranging from -8.6 to -7.1 kcal/mol). In vitro testing revealed that the CID 6432458 compound was effective (docking score of −8.6 kcal/mol) against the HCT116 cell line. The cytotoxicity of HCT116 has been documented, with an IC50 value of 6.885 ± 4. MTT assay, apoptosis analysis, and cell cycle assay were utilized to examine cell death in colorectal cancer. In the MTT experiment, 15 µM and 20 µM dosages were associated with 77% cell death; however, flow cytometry analysis using the IC50 value revealed that the selected chemical induced greater apoptosis in the HCT116 cell line (58.5%). The gene expression data revealed that the apoptotic gene BAX is expressed at a higher level than the BCL-2 gene. The IPO11 gene was downregulated during treatment. In the experiment involving the cell cycle, the S phase for the 30  µM dose showed 75.1% apoptosis, which was greater than the other concentrations used alone. These in silico and in vitro analysis will not only provide new information about Importin-11 receptor and insight into colorectal cancer but will also facilitate the development of natural compounds in a significant and worthwhile manner.     

Strong binding active constituents of phytochemical to BMPR1A promote bone regeneration: In vitro,in silico docking,and in vivo studies

Two of the most problematic orthopedic and neurosurgeon visits are associated with spine and craniofacial fractures. Therefore, more attention needs to be paid to finding a medicine to repair these fractures. Amongst the most mysterious herbs, Aloe vera stands out. In the present study, the ameliorating function of A. vera on osteogenesis was studied in vitro and in vivo. Osteoblast-like cells were exposed to A. vera, followed by analysis of cell viability, lactate dehydrogenase release, and intracellular reactive oxygen species (ROS) production. The results showed an enhanced cell biocompatibility in a dose-dependent manner due to attenuated intracellular ROS production. Furthermore, a docking study indicated that the strong affinity of A. vera constituents to type I bone morphogenic protein receptor (BMPR1A) without the involvement of the BMPR1A chain B. The induction of osteogenesis prompts extracellular calcium deposition by osteoblasts, which affirms successful in vitro bone regeneration. However, injection of A. vera in rats with critical size calvarial defects induced Runx2, alkaline phosphatase (ALP), OCN, and BMP2 genes overexpression, which led to the formation of victorious bone with enhanced bone density and ALP activity. It is worthy to note that Aloin has the highest affinity to BMPR1A, whereas there are no reports regarding the impact of Aloenin, Aloesin, and γ-sitosterol on osteogenesis. Furthermore, some of them have antitumor potency, and it might be proposed that they are considered as a bone substitute in the osteotomy site of osteosarcoma with the aim of bone recovery and suppression of osteosarcoma. The whole consequences of this investigation manifests the plausibility of using A. vera as an antioxidant and osteoconductive substitute.     

Synthesis of new ibuprofen derivatives with their in silico and in vitro cyclooxygenase-2 inhibitions

Cyclooxygenase-2 (COX-2) is one of the important targets for treatment of inflammation related diseases. In the literature, most of drug candidates are first synthesized and then their COX-2 inhibitory activities are tested by in vitro and in vivo experiments. However, synthesis of dozens of drug analogues without any interpretations on their inhibitory activity can result in loss of time and chemicals. Therefore, synthetic drug designs with molecular modeling are of importance to synthesize selective drug candidates against inflammatory diseases. The synthesis of the novel ibuprofen derivatives through their in silico and in vitro COX-2 inhibitory activities were investigated in the present study. Starting from ibuprofen, ibuprofen amide and ibuprofen acyl hydrazone derivatives were synthesized. According to the results of the in silico molecular docking and in vitro enzyme inhibition studies, the synthesized novel ibuprofen derivatives have selective COX-2 inhibition, and molecule 3a and 3c were showed higher inhibition compared to ibuprofen. In conclusion, the newly synthesized ibuprofen derivatives can be used in model in vivo studies.     

Combined in vitro and in silico approach to evaluate the inhibitory potential of an underutilized allium vegetable and its pharmacologically active compounds on multidrug resistant Candida species

Candida infections and related mortality have become a challenge to global health. Nontoxic and natural bioactive compounds from plants are regarded as promising candidates to inhibit these multidrug resistant strains. In the present study, in vitro assays and in silico molecular docking approach was combined to evaluate the inhibitory effect of crude extracts from Allium ampeloprasum and its variety A. porrum on Candida pathogens. Phytochemical screening revealed the presence of phenolic acids and flavonoids in higher quantity. Spectral studies of the extracts support the presence of phenols, flavonoids and organosulfur compounds. Aqueous extract of A. ampeloprasum showed a total antioxidant capacity of 68 ± 1.7 mg AAE/ g and an IC₅₀ value of 0.88 ± 2.1 mg/ml was obtained for DPPH radicals scavenging assay. C. albicans were highly susceptible (19.9 ± 1.1 mm) when treated with aqueous A. ampeloprasum extract. Minimum inhibitory concentrations were within the range of 19–40 μg/ml and the results were significant (p ≤ 0.05). In silico molecular docking studies demonstrated that bioactive phytocompounds of A. ampeloprasum and A. porrum efficiently interacted with the active site of Secreted aspartyl proteinase 2 enzyme that is responsible for the virulence of pathogenic yeasts. Rosmarinic acid and Myricetin exhibited low binding energies and higher number of hydrogen bond interactions with the protein target. Thus the study concludes that A. ampeloprasum and A. porrum that remain as underutilized vegetables in the Allium genus are potential anti-candida agents and their pharmacologically active compounds must be considered as competent candidates for drug discovery.     

Epitope specificity of two anti‐morphine monoclonal antibodies: In vitro and in silico studies

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine‐containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three‐dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti‐morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure‐based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti‐cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.     

Benzylidine indane-1,3-diones: As novel urease inhibitors; synthesis,in vitro,and in silico studies

Current study deals with the evaluation of indane-1,3-dione based compounds as new class of urease inhibitors. For that purpose, benzylidine indane-1,3-diones (1–30) were synthesized and fully characterized by different spectroscopic techniques including EI-MS, HREI-MS, ¹H, and ¹³C NMR. All synthetic molecules 1–30 were evaluated for urease inhibitory activity and showed good to moderate inhibitory potential within the range of (IC₅₀ = 11.60 ± 0.3–257.05 ± 0.7 µM) as compared to the standard acetohydroxamic acid (IC₅₀ = 27.0 ± 0.5 µM). Compound 1 (IC₅₀ = 11.60 ± 0.3 µM) was found to be most potent inhibitor amongst all derivatives. The key binding interactions of most active compounds within the enzyme pocket were evaluated through in silico studies.     

In silico binding analysis and SAR elucidations of newly designed benzopyrazine analogs as potent inhibitors of thymidine phosphorylase

Thymidine phosphorylase (TP) is up regulated in wide variety of solid tumors and therefore presents a remarkable target for drug discovery in cancer. A novel class of extremely potent TPase inhibitors based on benzopyrazine (1–28) has been developed and evaluated against thymidine phosphorylase enzyme. Out of these twenty-eight analogs eleven (11) compounds 1, 4, 14, 15, 16, 17, 18, 19, 20, 24 and 28 showed potent thymidine phosphorylase inhibitory potentials with IC₅₀ values ranged between 3.20 ± 0.30 and 37.60 ± 1.15 μM when compared with the standard 7-Deazaxanthine (IC₅₀ = 38.68 ± 4.42 μM). Structure-activity relationship was established and molecular docking studies were performed to determine the binding interactions of these newly synthesized compounds. Current studies have revealed that these compounds established stronger hydrogen bonding networks with active site residues as compare to the standard compound 7DX.     

Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies

Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and ¹H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM) while other four compounds exhibited good to moderate inhibition with IC₅₀ values between 29.45 ± 1.1 μM and 69.53 ± 0.9 μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.     

Cucurbitane-type compounds from Momordica charantia: Isolation,in vitro antidiabetic,anti-inflammatory activities and in silico modeling approaches

Momordica charantia L., commonly known as bitter melon, belongs to the Cucurbitaceae family. Various in vitro and in vivo studies have indicated that extracts of bitter melons have anti-diabetic properties. However, very little is known about the specific purified compounds responsible for these antidiabetic properties. In the present study, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al, charantal, charantoside XI, and 25ξ-isopropenylchole-5, 6-ene-3-O-d-glucopyranoside were isolated from bitter melon fruit. The structures of the purified compounds were elucidated by HR-ESIMS, 1D, and 2D NMR experiments. All compounds exhibited significant inhibition of α-amylase and α-glucosidase comparable to acarbose. Molecular docking studies demonstrated that purified compounds were able to bind to the active sites of proteins. Additionally, the purified compounds showed significant anti-inflammatory activity, downregulating the expression of NF-κB, iNOS, IL-6, IL-1β, TNF-α, and Cox-2 in lipopolysaccharide-activated macrophage RAW 264.7 cells. Our findings suggest that the purified compounds have potential anti-diabetic and anti-inflammatory activities and therefore hold promise for the development of plant-based management for diabetic and inflammatory conditions.     

Functional characterization of hypothetical proteins of Mycobacterium tuberculosis with possible esterase/lipase signature: a cumulative in silico and in vitro approach

The functional aspect of several mycobacterium proteins annotated as hypothetical are yet to be discovered. In the present investigation, in silico approaches were used to predict the biological function of some of the unknown Mtb proteins, which were further validated by wet lab experiments. After screening thousands of Mtb proteins, functionally unknown hypothetical proteins Rv0421c, Rv0519c, Rv0774c, Rv1191, Rv1592c, and Rv3591c were chosen on the basis of their importance in Mtb life cycle. All these proteins posses the α/β-hydrolase topological fold, characteristic of lipases/esterases, with serine, aspartate, and histidine as the putative members of the catalytic triad. The catalytic serine is located in pentapeptide motif “GXSXG” and oxyanion residue is in dipeptide motif HG. To further support our observation, molecular docking was performed with conventional synthetic lipolytic substrates (pNP-esterss) and specific lipase/esterase inhibitors (tetrahydrolipstatin and phenylmethanesulfonyl fluoride (PMSF)). Significant docking score and strong interaction of substrates/inhibitors with these proteins revealed that these could be possible lipases/esterases. To validate the in silico studies, these genes were cloned from Mtb genome and the proteins were over-expressed in pQE-30/Escherichia coli M15 system. The expressed proteins were purified to homogeneity and enzymatic activity was determined using pNP esters as substrate. The enzyme activity of recombinant proteins was inhibited by tetrahydrolipstatin and PMSF pre-treatment. Outcome of the present investigation provided a basic platform to analyze and characterize unknown hypothetical proteins.     

In vitro and in silico studies on cell adhesion protein peroxinectin from Fenneropenaeus indicus and screening of heme blockers against activity

In invertebrates, the prophenoloxidase (proPO) pathway is involved in the phenol‐like antioxidant production against invading pathogens. Overproduction of melanin and phenolic substances leads to the disruption of hemocytes (own host cells); therefore, there is a prerequisite to regulate the antioxidant production, which is performed by the proteases and proPO‐associated cell adhesion protein peroxinectin (PX). PX is a macromolecular structure consisting of protein involved in the proPO pathway, which is a potential target in the regulatory mechanism in crustaceans. In the proPO cascade, pattern recognition proteins initiate the proPO cascade by the consequent reaction, and PX is involved in the key step in the regulatory mechanism of phenoloxidase enzyme synthesis. In the present study, Indian white shrimp Fenneropenaeus indicus PX (Fein‐PX) gene sequence was used. Upregulation of Fein‐PX was determined using immunostimulants β‐glucan (agonists) and examined its expression by quantitative RT‐PCR. To find the downregulation or negative regulation of Fein‐PX, inhibitors were screened, and its 3D model provides molecular insights into the rationale inhibitor design for developing an effective molecule against Fein‐PX. Copyright © 2015 John Wiley & Sons, Ltd.