Fluorous-based peptide microarrays for protease screening

As ever more protease sequences are uncovered through genome sequencing projects, efficient parallel methods to discover the potential substrates of these proteases becomes crucial. Herein we describe the first use of fluorous-based microarrays to probe peptide sequences and begin to define the scope and limitations of fluorous microarray technologies for the screening of proteases. Comparison of a series of serine proteases showed that their ability to cleave peptide substrates in solution was maintained upon immobilization of these substrates onto fluorous-coated glass slides. The fluorous surface did not serve to significantly inactivate the enzymes. However, addition of hydrophilic components to the peptide sequences could induce lower rates of substrate cleavage with enzymes such as chymotrypsin with affinities to hydrophobic moieties. This work represents the first step to creating robust protease screening platforms using noncovalent microarray interface that can easily incorporate a range of compounds on the same slide.     

Electrophoretic assay for penicillinase: substrate specificity screening by parallel CE with an active pixel sensor

We report application of a new UV imaging detector incorporating an active pixel sensor in an electrophoretic enzyme assay for penicillinase (beta-lactamase) with multiple substrates. The method based on electrophoretically mediated microanalysis was developed on a standard CE system with a single-point diode array detector and 200 nm UV wavelength, then transferred to a parallel capillary setup with the UV imaging detector for screening of penicillinase substrate specificity. One capillary is used for the assay and the other for reference, with an enzyme solution plug introduced into the first at the same time as a water plug into the second capillary. A mixture of antibiotics and markers is subsequently introduced as a sample plug to both capillaries, and driven through the enzyme (or water) plug by application of voltage. Most individual reactant and product peaks were separated and compounds amenable to beta-lactam hydrolysis could readily be identified and the extent of the reaction quantified within a single electrophoretic run.     

Microbial sensor for screening mutagens

Preliminary screening of mutagens has been achieved using microbial sensors composed of immobilized microorganisms and oxygen electrodes. This microbial sensor system can be applied to the detection of various chemical mutagens.     

Peptide microarrays for the determination of protease substrate specificity

A method is described for the preparation of substrate microarrays that allow for the rapid determination of protease substrate specificity. Peptidyl coumarin substrates, synthesized on solid support using standard techniques, are printed onto glass slides using DNA microarraying equipment. The linkage from the peptide to the slide is formed through a chemoselective reaction, resulting in an array of uniformly displayed fluorogenic substrates. The arrays can be treated with proteases to yield substrate specificity profiles. Standard instrumentation for visualization of microarrays can be used to obtain comparisons of the specificity constants for all of the prepared substrates. The utility of these arrays is demonstrated by the selective cleavage of preferred substrates with trypsin, thrombin, and granzyme B, and by assessing the extended substrate specificity of thrombin using a microarray of 361 different peptidyl coumarin substrates.     

A surface-based mass spectrometry method for screening glycosidase specificity in environmental samples

A new surface-based MALDI-Tof-MS glycosyl hydrolase assay has been developed in which lipid-tagged oligosaccharides, representing defined fragments of major plant cell wall polysaccharides, are immobilized via hydrophobic interactions on an alkylthiol functionalised gold sample plate and employed in the functional screening of several purified enzymes, environmental samples and saliva.     

A disposable paper-based electrochemical sensor with an addressable electrode array for cancer screening

A novel addressable electrode array based on paper was assembled on the crossing points of the row/column electrodes to form a 4 × 6 sensor array by a facile home-made device-holder, one paper layer contained the sensing sites, the other paper layer the printed counter electrode and reference electrode.     

Enhancing specificity and sensitivity of pharmacophore-based virtual screening by incorporating chemical and shape features--a case study of HIV protease inhibitors

Virtual screening (VS), if applied appropriately, could significantly shorten the hit identification and hit-to-lead processes in drug discovery. Recently, the version of VS that is based upon similarity to a pharmacophore has received increased attention. This is due to two major factors: first, the public availability of the ZINC1 conformational database has provided a large selection pool with high-quality and purchasable small molecules; second, new technology has enabled a more accurate and flexible definition of pharmacophore models coupled with an efficient search speed. The major goal of this study was to achieve improved specificity and sensitivity of pharmacophore-based VS by optimizing the variables used to generate conformations of small molecules and those used to construct pharmacophore models from known inhibitors or from inhibitor-protein complex structures. By using human immunodeficiency virus protease and its inhibitors (PIs) as a case study, the impact of the key variables, including the selection of chemical features, involvement of excluded volumes (EV), the tolerance radius of excluded volumes, energy windows, and the maximum number of conformers in conformation generation, was explored. Protein flexibility was simulated by adjusting the sizes of EV. Our best pharmacophore model, combining both chemical features and excluded volumes, was able to correctly identify 60 out of 75 structurally diverse known PIs, while misclassifying only 5 out of 75 similar compounds that are not inhibitors. To evaluate the specificity of the model, 1193 oral drugs on the market were screened, and 25 original hits were identified, including 5 out of 6 known PI drugs.     

Virtual screening for SARS-CoV protease based on KZ7088 pharmacophore points

Pharmacophore modeling can provide valuable insight into ligand-receptor interactions. It can also be used in 3D (dimensional) database searching for potentially finding biologically active compounds and providing new research ideas and directions for drug-discovery projects. To stimulate the structure-based drug design against SARS (severe acute respiratory syndrome), a pharmacophore search was conducted over 3.6 millions of compounds based on the atomic coordinates of the complex obtained by docking KZ7088 (a derivative of AG7088) to SARS CoV M(pro) (coronavirus main proteinase), as reportedly recently (Chou, K. C.; Wei, D. Q.; Zhong, W. Z. Biochem. Biophys. Res. Commun. 2003, 308, 148-151). It has been found that, of the 3.6 millions of compounds screened, 0.07% are with the score satisfying five of the six pharmacophore points. Moreover, each of the hit compounds has been evaluated for druggability according to 13 metrics based on physical, chemical, and structural properties. Of the 0.07% compounds thus retrieved, 17% have a perfect score of 1.0; while 23% with one druggable rule violation, 13% two violations, and 47% more than two violations. If the criterion for druggability is set at a maximum allowance of two rule violations, we obtain that only about 0.03% of the compounds screened are worthy of further tests by experiments. These findings will significantly narrow down the search scope for potential compounds, saving substantial time and money. Finally, the featured templates derived from the current study will also be very useful for guiding the design and synthesis of effective drugs for SARS therapy.     

Chromogenic radical based optical sensor membrane for screening of antioxidant activity

Solid-state optical sensor membranes based on immobilised chromogenic radicals for the assessment of antioxidant activity have been studied. Two stable lipophilic chromogenic radicals, DPPH (2,2-diphenyl-1-picrylhydrazyl radical) and galvinoxyl radical, GV, (2,6-di-tert-butyl-α-(3,5-di-tert-butyl-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy radical), were immobilised in plasticised PVC films and screened for suitability as indicators of antioxidative activity. The spectrophotometric characterisation of the polymer films containing immobilised free radicals was performed, and the response of the immobilised free radicals toward standard antioxidants was studied. It has been demonstrated that the immobilised radicals retain their reactivity towards antioxidants and the results suggest that the reactivity of immobilised radicals is comparable to standard solution-based DPPH assays. Polymer films containing immobilised DPPH radical respond to standard antioxidants in aqueous solutions by changing colour irreversibly from purple (absorption maximum at 520 nm) to yellow. The initial slopes of the response curves to the phenolic antioxidant gallic acid, obtained in the 1-50 mM concentration range, gave a linear calibration plot in a 1 min exposure cuvette test. The polymer films were used to screen antioxidative activity of beverage and food samples known to contain antioxidants, such as black and green tea, coffee, red wine, fruit juice, olive oil and sunflower oil. It has been demonstrated that a rapid and simple qualitative screening test of untreated samples is possible using a test strip based on immobilised DPPH radical.     

A model for biological specificity

The phenomenon of biological specificity is described, and a history of discoveries related to the phenomenon is presented. Aspects of biological specificity described include the mechanism of the immune system, chemotherapy, enzyme-substrate specificity, neurotransmitters, autoassembly of viruses, autoassembly of subcellular organelles, differentiation, and cellular recognition. A model for biological specificity involving both steric and electrostatic complementarity is presented and the role of structured water and hydrophobic forces is also discussed.     

Pharmacophore model-aided virtual screening combined with comparative molecular docking and molecular dynamics for identification of marine natural products as SARS-CoV-2 papain-like protease inhibitors

Targeting SARS-CoV-2 papain-like protease using inhibitors is a suitable approach for inhibition of virus replication and dysregulation of host anti-viral immunity. Engaging all five binding sites far from the catalytic site of PLpro is essential for developing a potent inhibitor. We developed and validated a structure-based pharmacophore model with 9 features of a potent PLpro inhibitor. The pharmacophore model-aided virtual screening of the comprehensive marine natural product database predicted 66 initial hits. This hit library was downsized by filtration through a molecular weight filter of ≤ 500 g/mol. The 50 resultant hits were screened by comparative molecular docking using AutoDock and AutoDock Vina. Comparative molecular docking enables benchmarking docking and relieves the disparities in the search and scoring functions of docking engines. Both docking engines retrieved 3 same compounds at different positions in the top 1 % rank, hence consensus scoring was applied, through which CMNPD28766, aspergillipeptide F emerged as the best PLpro inhibitor. Aspergillipeptide F topped the 50-hit library with a pharmacophore-fit score of 75.916. Favorable binding interactions were predicted between aspergillipeptide F and PLpro similar to the native ligand XR8-24. Aspergillipeptide F was able to engage all the 5 binding sites including the newly discovered BL2 groove, site V. Molecular dynamics for quantification of Cα-atom movements of PLpro after ligand binding indicated that it exhibits highly correlated domain movements contributing to the low free energy of binding and a stable conformation. Thus, aspergillipeptide F is a promising candidate for pharmaceutical and clinical development as a potent SARS-CoV-2 PLpro inhibitor.     

Cell-active dual specificity phosphatase inhibitors identified by high-content screening

Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC(50) of <10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.     

Physicochemical properties and substrate specificity of protease C from dormant cotton seeds

The substrate specificity of a proteolytic enzyme — protease C — isolated from cotton seeds has been studied. The activity of protease C is suppressed completely under the action of diisopropyl phosphorofluoridate. Protein inhibitors — duck ovomucoid, soybean inhibitor, and also TPCK — suppressed the activity of protease C to different degrees. On the basis of results obtained in the hydrolysis of the cottonseed reserve proteins, 7S and 11S globulins, and the B chain of insulin, protease C has been assigned to the serine group of endopeptidases. The optimum conditions — pH, time, and temperature — at which protease C exhibits its maximum activity has been determined.Institute of Chemistry of Plant Substances, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 744–746, September–October, 1988.     

A sensor for hydrogen sulphide

A sensor for hydrogen sulphide is described. It is based on the catalytic effect of the gas on the iodine-azide reaction. An iodine-azide solution is exuded from a sintered-glass ball (10mm diameter) at a flow-rate of 3.30 ml min whilst its potential is monitored by two platinum electrodes. one inside and the other outside the ball. Sample is carried in a gas at a flow-rate of 350 ml min . Carbon disulphide, methyl mercaptan or sulphur dioxide in amounts up to a hundred times that of the hydrogen sulphide do not interfere. The detection limit is 5 ng and the determination limit is 8 ng, with a relative standard deviation of 10%.     

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.