Mevashuntin, a novel metabolite produced by inhibition of the mevalonate pathway in Streptomyces prunicolor

Inhibition of the mevalonate pathway by an HMG-CoA reductase inhibitor, mevalotin, in Streptomyces prunicolor possessing both mevalonate and MEP pathways resulted in the production of a new metabolite mevashuntin that consisted of conjugated thiazolone and pyranonaphthoquinone moieties.     

Fluorescence detection techniques for protein kinase assay

Traditional methods for protein kinase (PK) assay are mainly based on use of ³²P-labeled adenosine triphosphate (ATP); applications of such methods are, however, hampered by radioactive waste and short half-life of ³²P-labeled ATP. Therefore non-radioactive methods, such as fluorescence detection techniques are good alternative. In this review, we describe the principles of four fluorescence techniques (fluorescence intensity endpoint measurement, fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and fluorescence lifetime imaging) and provide an overview of applications of these fluorescence detection techniques in protein kinase assay, underlining their relative advantages and limitations. Research trends in this field are also highlighted. Figure Schematic representation of kinase assay based on direct fluorescence polarization measurements. The fluorescent peptide, on phosphorylation by kinase, binds to a phosphospecific antibody, which leads to a high FP value     

Structure‐Based Design and Synthesis of the First Weak Non‐Phosphate Inhibitors for IspF,an Enzyme in the Non‐Mevalonate Pathway of Isoprenoid Biosynthesis

In this paper, we describe the structure‐based design, synthesis, and biological evaluation of cytosine derivatives and analogues that inhibit IspF, an enzyme in the non‐mevalonate pathway of isoprenoid biosynthesis. This pathway is responsible for the biosynthesis of the C₅ precursors to isoprenoids, isopentenyl diphosphate (IPP, 1 ) and dimethylallyl diphosphate (DMAPP, 2 ; Scheme 1). The non‐mevalonate pathway is the sole source for 1 and 2 in the protozoan Plasmodium parasites. Since mammals exclusively utilize the alternative mevalonate pathway, the enzymes of the non‐mevalonate pathway have been identified as attractive new drug targets in the fight against malaria. Based on computer modeling (cf. Figs. 2 and 3), new cytosine derivatives and analogues (Fig. 1) were selected as potential drug‐like inhibitors of IspF protein, and synthesized (Schemes 2–5). Determination of the enzyme activity by ¹³C‐NMR spectroscopy in the presence of the new ligands showed inhibitory activities for some of the prepared cytosine and pyridine‐2,5‐diamine derivatives in the upper micromolar range (IC₅₀ values; Table). The data suggest that it is possible to inhibit IspF protein without binding to the polar diphosphate binding site and the side chain of Asp56′, which interacts with the ribose moiety of the substrate and substrate analogues. Furthermore, a new spacious sub‐pocket was discovered which accommodates aromatic spacers between cytosine derivatives or analogues (binding to ‘Pocket III’) and rings that occupy the flexible hydrophobic region of ‘Pocket II’. The proposed binding mode remains to be further validated by X‐ray crystallography.     

Difference spectrophotometric assay of benzaldehyde in benzyl alcohol

A simple and rapid procedure is described for the selective determination of benzaldehyde in benzyl alcohol intended for use in the manufacture of parenteral dosage form. The assay is based upon the measurement of the difference absorbance between two equimolar solutions of benzaldehyde in buffer solution pH 5.75, one of which also contains sodium bisulphite. The difference absorbance which has a maximum at 248 nm is due to the different spectral characteristics of benzaldehyde and its adduct form with sodium bisulphite is proportional to concentration of benzaldehyde. The accuracy, precision, sensitivity and specificity of the procedure are discussed. Application of the assay is described for different batches of benzyl alcohol. The results are compared with the official (BP) GLC method.     

A real-time,fluorescence-based assay for Rho-associated protein kinase activity

Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z’-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC₅₀ values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.     

Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

A spectrophotometric assay for quantification of artemisinin

A sensitive and rapid spectrophotometric method for determination of artemisinin concentration is described. The method is based on the measurement of a reaction product of the drug in strong alkali solution. The interaction produces a homogenous electronic transition band from 250 to 330 nm with maximum transition at around 291 nm. The absorption curve shows Gaussian distribution with identical half bandwidth, thus providing information for formation of a possible mono-type reaction product. The 291 nm absorption intensity increases with increasing concentration of artemisinin and obeys Beer's law in the range of 0.44-172 nmol (ml⁻¹). The optimum reaction conditions and other analytical parameters were evaluated including its recovery from human plasma and erythrocyte samples.     

Interference study of the Pu(III) spectrophotometric assay

The study was undertaken to determine the immunity of the Pu(III) spectrophotometric assay to diverse species. We acquired spectra of a set of solutions containing 10 mM Pu(III), reduced with ascorbic acid-aminoguanidine in 2M HCl, and of a similar set of solutions containing a spike of test species. The initial molar ratio of plutonium to species was 101, and after interference it was increased to 100.1, etc., until no interference occurred. The species examined were element numbers 1, 9, 11–13, 17, 19, 22–31, 35, 42, 44–46, 48, 50, 53, 57, 58, 60, 62, 73, 74, 76, 77, 79, 83, 90, 92, 93, 95 and nitrate, phosphate, sulfate, and oxalic acid.     

Simpler spectrophotometric assay of paracetamol in tablets and urine samples

A very fast, economical and simpler direct spectrophotometric method was investigated for paracetamol (PC) determination in aqueous medium without using any chemical reagents. The method is based on the photo-absorption of the analyte at 243 nm after dissolution in water. The change in structure of PC after addition of water was studied by comparing the corresponding FTIR spectra. Optimization studies were conducted by using a 5 microg ml(-1) standard solution of the analyte. Various parameters studied include, time for stability and measurement of spectra, effect of HCl, NaOH, CH(3)COOH and NH(3) for change in absorbance and shift in spectra, interference by some analgesic drugs and some polar solvents and temperature effect. After optimization, Beer's law was obeyed in the range of 0.3-20 microg ml(-1) PC solution with a correlation coefficient of 0.9999 and detection limit of 0.1 microg ml(-1). The newly developed method was successfully applied for PC determination in some locally available tablets and urine samples. The proposed method is very useful for quick analysis of various types of solid and liquid samples containing PC.     

Estimation of background interference in prompt-gamma neutron activation using MCNP

Prompt-gamma neutron activation (PGNA) is used to measure total-bodynitrogen and hydrogen in humans. Background interference in the gamma spectraarises from both subject and shielding. A Monte Carlo simulation program (MCNP4B2)was used to examine the neutron and gamma signals in the PGNA system ( ²⁴¹AmBe source). N and H peak regions were assessed in the presenceand absence of calibration phantoms. The simulations suggested extracorporealH peak contributions of up to 30%, depending on subject body habitus. MostN background could be attributed to detector pileup events. The MCNP resultsallowed us to improve shielding design and develop background correction algorithmsto improve measurement precision.     

A spectrophotometric assay for biotinbinding sites of immobilized avidin

A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB₁. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 μmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

A novel photoelectrochemical biosensor for protein kinase activity assay based on phosphorylated graphite-like carbon nitride

Protein kinases are general and significant regulators in the cell signaling pathway, and it is still greatly desired to achieve simple and quick kinase detection. Herein, we develop a simple and sensitive photoelectrochemical strategy for the detection of protein kinase activity based on the bond between phosphorylated peptide and phosphorylated graphite-like carbon nitride (P-g-C₃N₄) conjugates triggered by Zr⁴⁺ ion coordination. Under optimal conditions, the increased photocurrent is proportional to the protein kinase A (PKA) concentration ranging from 0.05 to 50 U/mL with a detection limit of 0.077 U/mL. Moreover, this photoelectrochemical assay can be also applied to quantitative analysis of kinase inhibition. The results indicated that the IC₅₀ value (inhibitor concentration producing 50% inhibitor) for ellagic acid was 9.1 μM. Moreover, the developed method is further applied to detect PKA activity in real samples, which contains serum from healthy person and gastric cancer patients and breast tissue from healthy person and breast cancer patients. Therefore, the established protocol provides a new and simple tool for assay of kinase activity and its inhibitors with low cost and high sensitivity.     

紫外分光光度法表征Lipozyme TL IM脂肪酶转酯化活性

建立了一种新的有机相脂肪酶转酯化活性测定方法. 以正己烷为溶剂,脂肪酶催化棕榈酸对硝基苯酯和正丁醇的转酯化反应为模型反应,通过测定反应液中310 nm下吸光值的变化计算反应转化率. 以气相色谱法对新建的紫外分光光度法进行验证,分别采用这两种方法测定了七种商品化脂肪酶的转酯化活性,两种方法所得实验结果基本一致. 利用紫外分光光度检测法考察了Lipozyme TL IM脂肪酶催化转酯化的时间进程及合成活性与酶量的关系,并对Lipozyme TL IM催化转酯化的性质（最适溶剂、酰基受体特异性、醇耐受性、最优反应温度和热力学稳定性）进行了表征.     

A CE‐based assay for human protein kinase CK2 activity measurement and inhibitor screening

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid at the C‐terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non‐phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6‐methyl‐1,3,8‐trihydroxyanthraquinone and 4,5,6,7‐tetrabromobenzotriazole resulted in IC₅₀ values of 1.33 and 0.27 μM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.     

Highly sensitive electrogenerated chemiluminescence biosensor in profiling protein kinase activity and inhibition using a multifunctional nanoprobe

We presented a novel electrogenerated chemiluminescence (ECL) biosensor for monitoring the activity and inhibition of protein kinases based on signal amplification using enzyme-functionalized Au NPs nanoprobe. In this design, the biotin-DNA labeled glucose oxidase/Au NPs (GOx/Au NPs/DNA-biotin) nanoprobes, prepared by conjugating Au NPs with biotin-DNA and GOx, were bound to the biotinylated anti-phosphoserine labeled phosphorylated peptide modified electrode surface through a biotin−avidin interaction. The GOx assembled on the nanoprobe can catalyze glucose to generate H₂O₂ in the presence of O₂ while the ECL reaction occurred in the luminol ECL biosensor. At a higher concentration of kinase, there are more nanoprobes on the electrode, which gives a higher amount of GOx at the electrode interface and thus higher electrocatalytic efficiency to the luminol ECL reaction. Therefore, the activity of protein kinases can be monitored by ECL with high sensitivity. Protein kinase A (PKA), an important enzyme in regulation of glycogen, sugar, and lipid metabolism in the human body, was used as a model to confirm the present proof-of-concept strategy. The as-proposed biosensor presents high sensitivity, low detection limit of 0.013 U mL⁻¹, wide linear range (from 0.02 to 40 U mL⁻¹), and excellent stability. Moreover, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent ECL signal, the half-maximal inhibition value IC₅₀ of ellagic acid, a typical PKA inhibitor, was estimated, which is in agreement with those obtained using the conventional kinase assay. The simple and sensitive biosensor is promising in developing a high-through assay of in vitro kinase activity and inhibitor screening for clinic diagnostic and drug development.     

Rapid ultraviolet spectrophotometric assay of benzoyl metronidazole in an oral suspension.

A procedure is described for the rapid determination of benzoyl metronidazole in an oral suspension which is based on measurement of the change in absorbance at 276 nm during alkaline hydrolysis of the compound in 2 mol dm-3 NaOH. The change in absorbance follows a linear relationship with concentration in the range 3-18 micrograms ml-1. Results of the determination of benzoyl metronidazole in an oral suspension and of the recovery experiments performed on this formulation, and also on various individual excipients and other additives, confirmed the applicability of the proposed method to complex formulations.