Surface Plasmon Resonance Assay for Identification of Small Molecules Capable of Inhibiting Aß Aggregation.

Toxic aggregates of amyloid-beta (Aß) have importance in the pathology of Alzheimer's disease, and inhibition of aggregate formation is considered to be a promising strategy for drug development. Here, we report a simple and rapid surface plasmon resonance (SPR) assay method that can identify potential Aß aggregation inhibitors. Our assay is based on the SPR shifting of the Aß-gold nanoparticle (Aß-GNP) aggregates by size under the influence of an Aß aggregation inhibitor. This user-friendly assay features a short assay time with a low reagent consumption that can be easily adapted as a high-throughput screen. We demonstrated that an effective Aß aggregation inhibitor induces the blue-shifted SPR peaks of the Aß-GNP aggregates by hindering the formation of long fibrillar aggregates. Moreover, the blue shifting was correlated to the efficacy and concentrations of an Aß aggregation inhibitor. Overall, our findings suggest that our simple SPR assay can be a powerful tool to screen small molecules targeting Aß aggregation.

Dopamine Receptor D1 Agonism and Antagonism Using a Field-Effect Transistor Assay.

The field-effect transistor (FET) has been used in the development of diagnostic tools for several decades, leading to high-performance biosensors. Therefore, the FET platform can provide the foundation for the next generation of analytical methods. A major role of G-protein-coupled receptors (GPCRs) is in the transfer of external signals into the cell and promoting human body functions; thus, their principle application is in the screening of new drugs. The research community uses efficient systems to screen potential GPCR drugs; nevertheless, the need to develop GPCR-conjugated analytical devices remains for next-generation new drug screening. In this study, we proposed an approach for studying receptor agonism and antagonism by combining the roles of FETs and GPCRs in a dopamine receptor D1 (DRD1)-conjugated FET system, which is a suitable substitute for conventional cell-based receptor assays. DRD1 was reconstituted and purified to mimic native binding pockets that have highly discriminative interactions with DRD1 agonists/antagonists. The real-time responses from the DRD1-nanohybrid FET were highly sensitive and selective for dopamine agonists/antagonists, and their maximal response levels were clearly different depending on their DRD1 affinities. Moreover, the equilibrium constants (K) were estimated by fitting the response levels. Each K value indicates the variation in the affinity between DRD1 and the agonists/antagonists; a greater K value corresponds to a stronger DRD1 affinity in agonism, whereas a lower K value in antagonism indicates a stronger dopamine-blocking effect.

Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation.

Ultrasensitive quantitative LC-MS/MS of an inhibitor of apoptosis protein's antagonist in plasma using protein target affinity extraction.

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.

Microarray-based screening of heat shock protein inhibitors.

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 µM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family.

Simultaneous and confirmative detection of multi-residues of ß2-agonists and ß-blockers in urine using LC-MS/MS/MS coupled with ß-receptor molecular imprinted polymer SPE clean-up.

A liquid chromatography-linear ion-trap spectrometry (LC-MS³) method using ß-receptor molecular-imprinted polymer (MIP) solid-phase extraction (SPE) as clean-up was developed to determine simultaneously and confirmatively residues of 25 ß2-agonists and 21 ß-blockers in urine samples. Urine samples were subjected to enzymatic hydrolysis by ß-glucoronidase/arylsulphatase, and then extracted with perchloric acid. Sample clean-up was performed using ß-receptor MIP SPE. A Supelco Ascentis® express Rp-Amide column was used to separate the analytes, and MS³ detection used an electrospray ionisation source in positive-ion mode. Recovery studies were carried out using blank urine samples fortified with the 46 analytes at the levels of 0.5, 1.0 and 2.0 µg l⁻¹. Recoveries were obtained ranging from 60.1% to 109.9% with relative standard deviations (RSDs, n = 7) from 0.5% to 19.4%. The limits of detection (LODs) and limits of quantitation (LOQs) of the 46 analytes in urine were 0.02-0.18 and 0.05-0.60 µg l⁻¹, respectively. As a result of the selective clean-up by MIP SPE and MS³ detection of the target drugs, the sensitivity and accuracy of the present method was high enough for monitoring ß2-agonist and ß-blocker residues in urine samples. Satisfactory results were obtained in the process of the determination of positive urine samples.

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2.

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677-23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2.

Interaction of cepharanthine with immobilized heat shock protein 90α (Hsp90α) and screening of Hsp90α inhibitors.

Heat shock protein 90α (Hsp90α) immobilized on aminopropyl silica gels was prepared via the N- or C-terminal, which was termed Hsp90α-NT or Hsp90α-CT, respectively. Binding interactions of biscoclaurine alkaloids (cepharanthine (CEP), berbamine (BBM), isotetrandrine (ITD), and cycleanine (CCN)) with Hsp90α were examined using the Hsp90α-NT or -CT columns by frontal and zonal chromatography studies. The dissociation constants of CEP, BBM, ITD, and CCN to Hsp90α-NT were estimated to be 5.3, 18.6, 46.3, and 159 µM, respectively, by frontal chromatography techniques. Similar results were obtained with the Hsp90α-CT column. These data suggest that these biscoclaurine alkaloids interact with the middle domain of Hsp90α. This was confirmed by demonstrating that CEP competed with endothelial nitric oxide synthase at the middle domain of Hsp90α, where it was shown to have a dissociation constant of 15 nM. Furthermore, the Hsp90α-NT column was applied for preliminary screening of natural Hsp90α inhibitors by zonal chromatography studies.

A metal-based inhibitor of tumor necrosis factor-α.

Staying in the pocket: A cyclometalated iridium(III) biquinoline complex targets the protein-protein interface (see picture; C yellow, N blue, Ir dark green) of the tumor necrosis factor-α (TNF-α) trimer. Molecular-modeling studies confirm the nature of this interaction. Both enantiomers of the iridium complex display comparable in vitro potency to the strongest small-molecule inhibitor of TNF-α.

Screening of an α-amylase inhibitor peptide by photolinker-peptide array.

Peptide arrays in which peptides were immobilized on cellulose membranes through photolinkers were synthesized. The peptides were subsequently detached from the arrays by ultraviolet (UV) photolysis for 3 h, and were used to search for functional peptides that inhibit the activity of α-amylase derived from human pancreatic juice. Amino acid replacement with high-molecular-size amino acids, Arg (R), Phe (F), Trp (W), or Tyr (Y), for the first and seventh residues of amylase inhibitor peptide, GHWYYRCW, as previous reported, led to enhancement of the inhibitory effect of the peptide on α-amylase. In particular, one of the resulting peptides, RHWYYRYW, showed a stronger inhibitory effect than acarbose (which is used as a hypoglycemic agent) or inhibitor peptide GHWYYRCW.

Immobilizing CC chemokine receptor 4's N-terminal extracellular tail on a capillary to study its potential ligands by capillary electrophoresis.

ML40 is the equivalent peptide derived from the N terminal of CCC4 (CC chemokine receptor 4), which plays a pivotal role in allergic inflammation. A new capillary electrophoresis method was developed to study the interactions between ML40 and its potential ligands in which ML40 was immobilized on the inner wall of capillary as the stationary phase based on the covalent linking technique. The interaction between S009, a known CCR4 antagonist, and the immobilized ML40 was studied to validate the bioactivity of ML40. The electropherogram of S009 showed that the peak height was reduced and the peak width was broadened in the ML40 immobilized capillary. Otherwise, 25 computer-aided design and drafting compounds were screened out using this method. Four compounds' peak widths were broadened and their peak heights were reduced, as with S009. Meanwhile, nonlinear chromatography was used to calculate the constants for the ligand-receptor complex formation. Furthermore, the tertiary amine compounds belonging to the chiral tertiary amines of the type NRR'R″, which are optically inactive resulting from rapid pyramide inversion, were chiral separated by our protein immobilization method for the first time. In general, the methodology presented would be applicable to study compound-ML40 interactions as a reliable and robust screening method for CCR4 antagonist discovery.

Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC.

Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics and the discovery process to find new ones. In this study, we reconstituted and characterized by surface plasmon resonance the assembly of AcrAB-TolC, the archetypal multidrug efflux pump from Escherichia coli. We report that the periplasmic AcrA and the outer membrane channel TolC assemble high-affinity complexes with AcrB transporter independently from each other. Antibiotic novobiocin and MC-207,110 inhibitor bind to the immobilized AcrB but do not affect interactions between components of the complex. In contrast, DARPin inhibits interactions between AcrA and AcrB. Mutational opening of TolC channel decreases stability of interactions and promotes disassembly of the complex. The conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC and could be targeted for the development of new inhibitors.

SPR biosensor as a tool for screening prion protein binders as potential antiprion leads.

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative disorders affecting animals and humans. No effective treatments are currently available for the diseases, vCJD in particular. It is believed that the formation of protease-resistant insoluble prion protein (PrP(Sc)), which is the main component of amyloidal deposits, from the cellular prion protein (PrP(C)), is essential for the progression of the disease. Therefore, both PrP(Sc) and PrP(C) are currently being used as potential drug targets.This protocol details an optimised experimental protocol to conduct an affinity screening of compound libraries by the immobilisation of PrP(C) using an SPR-based instrument, Biacore 3000.

The first Hes1 dimer inhibitors from natural products.

In the present study, we developed a high-throughput screening system for small molecule-inhibitors of the basic helix-loop-helix (bHLH) transcriptional repressor factor Hes1. Successful dimerization of Hes1 immobilized on a microplate and fluorophore (Cy3)-labelled Hes1 was confirmed. Using this system, several natural products were identified as the first Hes1 dimer inhibitors. Of these, two compounds which were isolated from myxomycetes (true slime molds) inhibited Hes1 from N box-dependent suppression of the gene expression in C3H10T1/2 cells.

Interaction of bupropion with muscle-type nicotinic acetylcholine receptors in different conformational states.

To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion (a) inhibits epibatidine-induced Ca(2+) influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with approximately 100-fold higher potency compared to that in the open state, (c) increases the desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits binding of [(3)H]TCP and [(3)H]imipramine to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/alpha-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively, our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process.