Assay for protein by dye binding

A proposed assay for protein [Bradford, M. M. (1976) Anal. Biochem. 72, 248–254] by binding of Coomassie brilliant blue G-250 has been evaluated. Some proteins give standard curves similar to those reported by Bradford, but others deviate widely; caution is urged in application of the procedure to general assay for protein concentration.     

Protein determination of cells immobilized in cross-linked synthetic gels

Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.     

Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-acyltransferase-1 inhibitor

We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared from 1 using a combination reagent of hydrogen peroxide and sodium tungstate with either phosphoric acid or trifluoroethanol as the solvent to control the regioselectivity. Upon exposure of 4b to tert-butyl hypochlorite at −78 °C, the monosulfoxidation afforded synthetic intermediate of M3 in excellent yield. The efficient synthesis of M4 was established. The in vitro metabolic study exhibited a high clearance value (720 μL/min/mg protein) of 1 using human liver microsomes. We orally administered a single dose of 10 mg/kg of 1 to monkeys because the in vitro metabolic patterns are quite similar. Fortunately, the drug concentration of 1 was much higher than those of M1, M2, M3 and M4.     

Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface

We have discovered a new molecule naphthyridine–azaquinolone hybrid (Npt–Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt–Azq, the melting temperature (T_m) of 5′-d(CTA ACG GAA TG)-3′/3′-d(GAT TGA CTT AC)-5′ containing a single G-A mismatch increased by 15.4°C, whereas fully matched duplex increased its T_m only by 2.2°C. Npt–Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt–Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was ~6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 µM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt–Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.     

Improved binding affinities of pyrrolidine derivatives as Mcl-1 inhibitors by modifying amino acid side chains

As an important member of anti-apoptotic Bcl-2 protein, myeloid cell leukemia sequence 1 (Mcl-1) protein is an attractive target for cancer therapy. In this study, a new series of pyrrolidine derivatives as Mcl-1 inhibitors were developed by mainly modifying the amino acid side chain of compound 1. Among them, compound 18 (K_i = 0.077 μM) exhibited better potent inhibitory activities towards Mcl-1 protein compared to positive control Gossypol (K_i = 0.18 μM). In addition, compound 40 possessed good antiproliferative activities against PC-3 cells (K_i = 8.45 μM), which was the same as positive control Gossypol (K_i = 7.54 μM).     

Quantitative determination of the protein content of citrus leaf extracts: A comparative study

Three spectrophotometric methods for the quantitative determination of proteins were compared taking as a reference the method of Kjeldahl. The abnormally high protein values obtained when these methods were applied to soluble extracts were decreased by using protective and precipitating agents. The method of M. M. Bradford (Anal. Biochem. 72, 248–254 (1976)) gave values identical to the method of Kjeldahl when ethylenediaminetetracetic acid, 2-mercaptoethanol, and polyvinylpolypyrrolidone were added to the extraction medium and the soluble proteins were precipitated with trichloroacetic acid prior to protein determination.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein

The Coomassie brilliant blue G assay for proteins described by Bradford (1976) (Anal. Biochem.72, 248) was reexamined. It was found that the extinction coefficient of the dye-protein complex solution remained constant over the protein concentration range of 0.8 to 10 μg/ml of solution. This unchanging extinction coefficient, $\text{A}{}_{595}\text{=}\text{0.60 ± 0.01}\text{10}\text{μ}\text{g}$ of protein/ml of solution, enhances both the sensitivity and versatility of the assay. Selection of a volume of dye-reagent (0.5 to 5.0 ml) which dilutes the protein sample to a final concentration of 0.8 to 10 μg/ml permits the application of Beer's Law for accurate determinations of ≤0.5 to 50 μg of protein. A combination of Bradford's study and the present one indicates that most common laboratory reagents and chemicals exert little or no influence on the A₅₉₅ of the dye-reagent.     

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC₅₀s of (2.4–52.5?µM), and α-glucosidase with IC₅₀ values of (1.7–72.7?µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC₅₀?=?2.4?µM for 3, 2.8?µM for 7) and α-glucosidase (IC₅₀?=?4.8?µM for 3, 1.7?µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K_i^app?=?2.4?µM; k₅?=?0.05001?µM^?1?S^?1 and k₆?=?0.02076?µM^?1?S^?1.     

Stereoisomeric guaiacylglycerol-β-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells

Two 8-O-4′-type neolignan epimers erythro-guaiacylglycerol-β-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-β-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC₅₀ = 45.56 μM) and 2b (IC₅₀ = 39.02 μM) had more cytotoxic effect than its enantiomers 1a (IC₅₀ = 82.66 μM) and 2a (IC₅₀ = 67.97 μM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 μM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-β-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.     

Eudesmanolide sesquiterpenes and protein tyrosine phosphatase 1B inhibitory ent-kaurene diterpenes from aerial parts of Indonesian Wedelia prostata

Seven eudesmanolide sesquiterpenes (1–7) and two ent-kaurene diterpenes (8 and 9) including two new (9R)-eudesman-9,12-olides, named wedelolides I and J (1 and 2), were isolated from the aerial parts of Indonesian Wedelia prostata. The structures of 1 and 2 were assigned based on their spectroscopic data. Diterpenes 8 and 9 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC₅₀ values of 8.3 and 28 μM, respectively. Among sesquiterpenes 1–7, compound 4, wedelolide D, exhibited 32% inhibitory activity against PTP1B at 20 μM.     

Filamenting temperature-sensitive mutant Z inhibitors from Glycyrrhiza glabra and their inhibitory mode of action

FtsZ is an essential protein for bacterial cell division, and an attractive and underexploited novel antibacterial target protein. Screening of Indonesian plants revealed the inhibitory activity of the methanol extract of Glycyrrhiza glabra on the Bacillus subtilis FtsZ (BsFtsZ) GTPase, and further bioassay-guided fractionation of the active methanol extract led to the isolation of seven known polyketides (1–7). Among them, gancaonin I (1), glycyrin (3), and isolicoflavanol (5) exhibited anti-BsFtsZ GTPase activities, at levels comparable to that of the synthetic FtsZ inhibitor, Zantrin Z3. Enzymatic assays using a BsFtsZ Val307R mutant protein and in silico simulations suggested that 1, 3, and 5 bind to the cleft on BsFtsZ, as in the case of the previously reported uncompetitive FtsZ inhibitor, PC190723, and thereby display their significant anti-BsFtsZ inhibitory activities. Furthermore, 1 also showed significant inhibitory activity against B. subtilis, with a MIC value of 5 μM. The present study provides new insights into the naturally occurring B. subtilis growth inhibitors.     

Structure-based design of some isonicotinic acid hydrazide analogues as potential antitubercular agents

New pyridine derivatives were designed and synthesized as Isonicotinic acid hydrazide (INH) analogues. The synthesized compounds were evaluated for their antitubercular activity against Mycobacterium tuberculosis strain H₃₇R_v. Ten compounds (3c, 3e-g, 5a-c, 6h, 10 and 11b) showed promising antitubercular activity with MIC range 7.30 µM–19.39 µM. Compounds 3e, 3g, 5b and 11b were the most potent analogues, with MIC 7.30–8.74 µM. They were equipotent to the standard drug Ethambutol (MIC 7.64 µM) and more active than the standard drug Pyrazinamide (MIC 50.77 µM). They were further examined for cytotoxicity in human embryonic kidney (HEK) cell line at the concentration of 50 µg/mL using MTT assay. Results declared that most compounds showed acceptable safety margin. Molecular Docking studies into 2-trans-enoyl-acyl carrier protein reductase, called InhA have been conducted for compounds 3e, 3g, 5b and 11b using Molecular Operating Enviroment software (MOE 2016.0802), where reasonable binding interactions have been identified and effective overall docking scores have been recorded. Their drug-likeness has been assessed using Molinspiration and Osiris software.     

Synthesis,biological evaluations and computational studies of N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors

In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline substituents at 2nd position were synthesized and screed for biofilm inhibitory, antifungal and antibacterial activity. The result of biofilm inhibition of C. albicans suggested that compounds 5j (IC₅₀ value?=?51.2?μM) and 5a (IC₅₀ value?=?66.2?μM) possess promising antibiofilm inhibition when compared with the standard antifungal drug fluconazole (IC₅₀?=?40.0?μM). Two compounds 5a (MIC?=?94.2?μg/mL) and 5f (MIC?=?98.8?μg/mL) also exhibited good antifungal activity comparable to standard drug fluconazole (MIC?=?50.0?μg/mL). The antibacterial screening against four strains of bacteria viz. E. coli, P. aeruginosa, B. subtilis, and S. aureus suggested their potential antibacterial activity and especially all the compounds except 5g were found more active than the standard drug ciprofloxacin against B. subtilis. To further gain insights into the possible mechanism of these compounds in biofilm inhibition through the agglutinin like protein (Als), molecular docking and molecular dynamics simulation studies were carried out. Molecular modeling studies suggested the clear role in inhibition of this protein and the resulting biofilm inhibitory activity.     

Discovery of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups as potential PTP1B inhibitors

Two series of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, IIIv was found to have the best in vitro inhibition activity against PTP1B (IC₅₀?=?0.67?±?0.09?µM) and the best selectivity (9-fold) between PTP1B and T-cell protein tyrosine phosphatase (TCPTP). Molecular docking studies demonstrated that compounds IIIm, IIIv and IVg could occupy simultaneously at both the catalytic site and the adjacent pTyr binding site. These results provide novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein

This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change (∼80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC₅₀ values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.     

Development of benzo[d]oxazol-2(3H)-ones derivatives as novel inhibitors of Mycobacterium tuberculosis InhA

A series of twenty seven substituted 2-(2-oxobenzo[d]oxazol-3(2H)-yl)acetamide derivatives were designed based on our earlier reported Mycobacterium tuberculosis (MTB) enoyl-acyl carrier protein reductase (InhA) lead. Compounds were evaluated for MTB InhA inhibition study, in vitro activity against drug-sensitive and -resistant MTB strains, and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, 2-(6-nitro-2-oxobenzo[d]oxazol-3(2H)-yl)-N-(5-nitrothiazol-2-yl)acetamide (30) was found to be the most promising compound with IC₅₀ of 5.12 ± 0.44 μM against MTB InhA, inhibited drug sensitive MTB with MIC 17.11 μM and was non-cytotoxic at 100 μM. The interaction with protein and enhancement of protein stability in complex with compound 30 was further confirmed biophysically by differential scanning fluorimetry.     

Protein tyrosine phosphatase 1B inhibitory effects of depsidone and pseudodepsidone metabolites from the Antarctic lichen Stereocaulon alpinum

Seven phenolic lichen metabolites (1–7) have been isolated from a methanol extract of the Antarctic lichen Stereocaulon alpinum by various chromatographic methods. The structures of these compounds were determined mainly by analysis of NMR spectroscopic data. A depsidone-type compound, lobaric acid (1) and two pseudodepsidone-type compounds, 2 and 3, exhibited potent inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) with IC₅₀ values of 0.87 μM, 6.86 μM, and 2.48 μM, respectively. Kinetic analyses of PTP1B inhibition by compounds 1 and 2 suggested that these compounds inhibited PTP1B activity in a non-competitive manner.     

Design,synthesis and biological evaluation of N,N-3-phenyl-3-benzylaminopropanamide derivatives as novel cholesteryl ester transfer protein inhibitor

A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP (cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al., 2014). Based on L10 (IC₅₀ 8.06 μM), compound HL6 (IC₅₀ 10.7 μM) was discovered following systematic structure variation and biological tests. Further optimization of the structure–activity relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay. Among them, HL16 (IC₅₀ 0.69 μM) was a highly potent CETP inhibitor in vitro. In addition, HL16 exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied the CETP binding site and formed interactions with the key amino acid residues.