The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease.

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson’s and Alzheimer’s diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson’s disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson’s disease and related conditions.

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (ka) measured at 25 °C ranged from a low of 2.42×10(4) M(-1) s(-1) to the highest value of 8.3×10(5) M(-1) s(-1). Rate constants of dissociation (kd) ranged from 1.09×10(-4) s(-1) (corresponding to a residence time of close to three hours), to the fastest of 0.028 s(-1). Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aβ generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aβ generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aβ in the CSF of healthy human volunteers.

Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of alpha-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition in vivo. One such compound significantly decreased phosphorylation of alpha-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.

Surface Plasmon Resonance (SPR) is rarely used as a primary High-throughput Screening (HTS) tool in fragment-based approaches.With SPR instruments becoming increasingly high-throughput it is now possible to use SPR as a primary tool for fragment finding.SPR becomes, therefore, a valuable tool in the screening of difficult targets such as the ubiquitin E3 ligase Parkin.As a prerequisite for the screen, a large number of SPR tests were performed to characterize and validate the active form of Parkin.A set of compounds was designed and used to define optimal SPR assay conditions for this fragment screen.Using these conditions, more than 5000 pre-selected fragments from our in-house library were screened for binding to Parkin.Additionally, all fragments were simultaneously screened for binding to two off target proteins to exclude promiscuous binding compounds.A low hit rate was observed that is in line with hit rates usually obtained by other HTS screening assays.All hits were further tested in dose responses on the target protein by SPR for confirmation before channeling the hits into Nuclear Magnetic Resonance (NMR) and other hit-confirmation assays.

The structure-activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.

Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.

Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak μM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.

The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1′ region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2′ substituent of chromane–HEA(s) with polar substituents provided improvements in the compound’s in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.

Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive and devastating neurodegenerative disease resulting from injury and death of upper and lower motor neurons. Symptoms initially include muscle weakness and twitching and subsequently progress to muscle atrophy, complete loss of limb use, respiratory difficulties and ultimately death. Multiple biological mechanisms have been implicated in ALS and a complex etiology has been described. As a result, drug discovery researchers have few validated targets to pursue and patients have few therapeutic options.Areas covered: Identification of new drug targets in ALS can be facilitated by a detailed understanding of the processes and genes that contribute to pathogenesis. Accordingly, this review summarizes current hypotheses regarding underlying mechanisms for motor neuron susceptibility in ALS. An overview of emerging and tractable drug targets that could result in therapeutic breakthroughs is provided.Expert opinion: Despite the immense progress that has been made in understanding ALS over the last decade, riluzole remains the only approved drug to treat ALS. Combining structure-guided drug design applied to validated and pharmaceutically tractable targets with disease-relevant phenotypic screens will allow for the identification of novel drug targets and potentially breakthrough therapeutics for ALS.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z′ values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization–based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.

Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

BioanalysisVol. 4, No. 6 EditorialFree AccessCombining label-free technologies: discovery in strengthRobin Barbour & Michael P BovaRobin Barbour* Author for correspondenceDirector of Antibody Technologies, Neotope Biosciences, a division of Elan Pharmaceuticals Inc., 650 Gateway Blvd, San Francisco, CA, USA. & Michael P BovaDirector of Target Advancement, Elan Pharmaceuticals Inc., CA, USAPublished Online:28 Mar 2012https://doi.org/10.4155/bio.12.45AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: biolayer interferometrybiomolecular screeningdifferential scanning calorimetrydrug discoveryisothermal calorimetrylabel-free technologyplate-based optical biosensorsquartz crystal microbalancesurface plasmon resonanceSince the early 1990s when Biacore entered the market with the introduction of surface plasmon resonance (SPR) for use in elucidating interactions of proteins, label-free technologies have gained acceptance into all stages of drug discovery. The myriad of technologies now available to researchers allows for label-free methods to impact drug development from early discovery to entry into the clinic and beyond. However, each of these methods comes with its own advantages and disadvantages. While many of these methods can be used at several stages of drug discovery and development, researchers may find any individual technology lacking either the throughput, sensitivity, or the ease of use that they may require. In addition, each method requires that care be used in the design and analysis of the experiments to ensure quality data are obtained. However, by combining several technologies one can leverage the strengths of the individual technology while avoiding the pitfalls.In early discovery, the incorporation of label-free methods requires high throughput as the number of individual entities to be screened can be in the tens to hundreds of thousands. One can consider the addition of plate-based technologies, such as the SRU Bind™ (Woburn, MA, USA) or Corning Epic® (Lowell, MA). These optically based instruments illuminate a plate containing an optical grating with white light. The optical grating acts as a cut-off filter that only allows specific wavelengths of light to be refracted back to the reader [1]. The shift in wavelength is proportional to the mass bound to the sensor. Refractive index changes affect the readings, so the instruments are sensitive to DMSO concentration and to the intrinsic refractive differences of some small-molecule compounds. The Corning Epic has internal calibration to help compensate for these differences, while the SRU Bind does not. Both instruments are available in 96- and 384-well formats, while the SRU Bind additionally allows for use in a 1536-well format. Either of the instruments can be incorporated into high-throughput screening (HTS) with great success for screening both biochemical and cell-based assays.In small-molecule discovery, the plate-based instruments are quite useful in hit triage of a large number of positives, elimination of ill-behaved compounds, identification of probable 1:1 binders and demonstration of saturation binding. However, there are issues that limit their usefulness for a true HTS campaign. These plate-based technologies all require large quantities of highly active protein and the instruments may not have the needed sensitivity if the target protein is large (>75 kD). Due to these issues, at the early stages of discovery a more conventional labeled biochemical method may be required to meet the assay needs. In addition, due to mass transport limitations of passive diffusion of compound to the sensor and sensor configuration, compounds may not be accurately rank ordered and false negatives may arise, particularly with low solubility, low affinity compounds [2].The use of this technology in cell-based label-free assays, in the opinion of this author, are where the true strengths of these instruments lie. Their utility in identifying antagonists and agonists to G protein-coupled receptors, ion channels or integrins in cell-based assays has been demonstrated by many researchers, and their adoption into HTS for this market is becoming common. The signals in these assays are robust and are not significantly affected by DMSO mismatches. Since the assay shows actual changes in cell signaling it may be more biologically relevant than binding to a membrane preparation or a recombinant protein [3,4].At the completion of a HTS screen, the number of hits will typically number in the range of several hundred to several thousand, and at this point label-free methods such as SPR or biolayer interferometry can be utilized. These methods provide more information than the plate-based instruments, and are especially useful in confirmation, early characterization, and prioritization of hits from other screening methods. These technologies allow for ranking based on association and/or dissociation constants, confirm 1:1 binding of a compound to its target protein or allow for epitope binning of antibodies. The three most commonly used instruments at this stage of drug development are the Biacore instruments (4000, A100 and T200), ForteBio QK or Red and the Biorad ProteOn.The ForteBio QK and Red series of instruments are based on biolayer interferometry, and unlike both SPR and the optical sensors in the plate readers where readings are proportional to mass, these instruments measure shifts in an interference pattern of light caused by the thickness of the layer of molecules bound to the sensor [5]. Since shifts of the interference pattern only occur by binding or dissociation to or from the sensor, changes in refractive index due to DMSO mismatches or the intrinsic properties of a compound, do not cause a significant change in signal. The ForteBio Red has been making in-roads into the small molecule screening market, with several companies publishing on its utility for hit confirmation. It is available in a 384-well plate format and simultaneously runs sixteen samples, which allows higher daily throughput than the Biacore [6]. Currently, only biotinylation of the target protein, and attachment to high capacity strepavidin sensors are available to the researcher for immobilizing target proteins to the sensor. Unlike Biacore, there is limited literature available on the types of and sizes of targets where successful small molecule screening campaigns were run.The speed and ease of use of the ForteBio QKe and Red, however, can be invaluable in the antibody discovery environment. The ForteBio is very useful in quickly ranking antibody hits by off-rates, allowing researchers to rapidly identify antibodies they wish to further characterize. As the antibodies are optimized through humanization or maturation, many versions can be quickly screened and characterized for ka and kd. The ForteBio has a clear advantage due to speed and ease of use in the quantitation of antibody concentration in supernatant, which facilitates selection of clones for manufacturing, and monitoring production runs of antibody [7]. In antibody characterization, one can epitope bin all antibodies or choose antibody pairs for sandwich assay development [8].There are limitations to the ForteBio technology for antibody development. It is recommended that off-rate ranking be done with antigen immobilized on the sensor which does not allow for an accurate affinity determination due to avidity issues. If true kinetics are desired, the anti-mouse Fc or anti-human Fc sensors must be used and the addition of multiple layers can diminish sensitivity. Since the ForteBio technology is based on Bio-Layer Interferometry, which relies on the thickness of the layer bound to the sensor and not the mass bound, we have found obtaining true kinetics on antibodies to unstructured proteins to be difficult. We have also found that occasionally the sensorgrams are not intuitive since binding of two proteins of the same weight and activity can have differing maximum binding levels, which can be disconcerting to those familiar with SPR-based instruments. Determining off-rates of very high affinity antibodies can also be troublesome due to re-binding and evaporation in the well with the required long dissociation times, and assay modifications must be made in order to determine an accurate off-rate. However, even with these caveats, with a well-designed experiment ka, kd and KD values are often similar to those obtained in mass-based instruments [9].The ProteOn XPR36 is also an SPR-based instrument with the advantage of allowing the researcher to interrogate 6×6 interactions per experiment. The instrument has been mainly used in antibody development where it offers similar benefits of speed in off-rate ranking and epitope binning as the ForteBio [10]. There are application notes for use in the small molecule market, but it has not been widely used in this application.The Biacore 4000 is based on SPR. Prism-based surface plasmon resonance technology has been successfully used in the Biacore platform technology to characterize small molecule and antibody interactions with proteins since the early 1990s. SPR occurs when polarized light under conditions of total internal reflection strikes a gold layer at the interface between media of different refractive indices. The refractive indices at the interface between the biosensor surface and a solution flowing over the biosensor changes as molecules bind to the sensor surface. A change in angle of reflection occurs when molecules bind or dissociate from the surface and is proportional to the mass of bound material and is recorded in a Biacore sensorgram in real time [11].The Biacore 4000 allows the throughput needed to look at several thousand small molecule compounds per experiment in a fairly short time frame of several days to a week. It is very sensitive, and its numerous coupling chemistries allow the user versatility, both with the size of the target protein and the method of coupling used to obtain high levels of active protein on the sensor. Biacore instrumentation in small molecule and fragment screening has long been the gold standard [12].The Biacore 4000 is also widely used in high-throughput antibody screens where accurate ka, kd and KD can be obtained using cell culture supernatants early in the screening process [13]. In antibody development, however, the throughput of even the 4000 may not be sufficient to quickly prioritize antibody hits, and valuable time may be wasted on maintaining low value clones while waiting for needed information.New technologies have been introduced using acoustic wave microbalances, which may allow real time label-free kinetics of protein interactions with whole cells. Both the Attana Sensor Technologies (Cell 200) and Saw Instruments (SAM 5) have released application notes describing their instruments utility in this research area. This may give valuable information at early-stage development of antibodies to cell surface antigens allowing for rich biologically relevant binding information not obtainable using recombinant proteins or cell membranes attached to a sensor surface [14,15].In later stages of development of potential therapeutic candidates, several low-throughput technologies can make a significant impact. Isothermal calorimetry allows thermodynamic properties of compound or antibody binding to be investigated, and this information can be instructive in SAR or affinity maturation for finding more potent therapeutics. It is the only instrument that directly follows the enthalpy of binding. Large changes in ΔH can indicate that the binding characteristic of your compound have changed and this can be instructive in molecular design [16].Differential Scanning Calorimetry measures the enthalpy of protein unfolding due to heat denaturatation. It is mainly used in biotherapeutic development, where it is used to assess whether engineering changes to the protein affect stability, to compare stability of several possible candidates and assess biocomparability when engineering process changes are made. This information allows for the advancement of the most rugged therapeutic into the clinic [17].Lastly, all of these methods require knowledge of proper analysis of label-free data in order to reach proper conclusions. After review of the literature where label-free technology was used, Rich and Myszka concluded each year that a number of experiments using label-free technology in published papers were improperly designed or interpreted [18]. Researchers incorporating label-free technology need to have a working knowledge of the limitations of each of the technologies to insure the integrity of their data, and should consider enlisting the help of the application engineers when incorporating a new technology to ensure proper design of experiments and mathematical interpretation data.In summary, researchers now have a wide range of technologies available to meet their assay criteria. Decisions can be made as to which technology should be applied at each of the phases of drug development – from hit identification, to clinical characterization. Researchers should evaluate which stages of development they wish to incorporate label-free technology based on the throughput, sensitivity and amount of information needed. All of these factors will influence which instruments fit best into the development flow. While many instruments can be used throughout development, incorporating only one technology may fall short in at least one desired characteristic, but with a combination of technologies one can improve the odds of success.Financial & competing interests disclosureThe authors are employees of Neotope Biosciences, a division of Elan Pharmaceuticals, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1 Cunningham BT, Li P, Schulz S et al. Label-free assays on the BIND system. J. Biomol. Screen.9(6),481–490 (2004).Crossref, Medline, CAS, Google Scholar2 Barbour R. Combining label free assay platforms to support both large and small molecule drug discovery. Presented at: Screening Summit-World Pharmaceutical Congress. Philadelphia, PA, USA, 8 June 2011.Google Scholar3 Bova MP, Nguyen L, Wallace W et al. A label-free approach to identify inhibitors of α4β7-mediated cell adhesion to MadCAM. J. Biomol. Screen.16(5),536–544 (2011).Crossref, Medline, CAS, Google Scholar4 McGuinness RP, Proctor JM, Gallant DL et al. Enhanced selectivity screening of GPCR ligands using a label-free cell based assay technology. Comb. Chem. High Throughput Screen.12(8),812–823 (2009).Crossref, Medline, CAS, Google Scholar5 Concepcion J, Witte K, Wartchow C et al. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb. Chem. High Throughput Screen.12(8),791–800 (2009).Crossref, Medline, CAS, Google Scholar6 Wartchow CA, Podlaski F, Li S et al. Biosensor-based small molecule fragment screening with biolayer interferometry. J. Comput. Aided Mol. Des.25(7),669–676 (2011).Crossref, Medline, CAS, Google Scholar7 Do T, Ho F, Heidecker B, Witte K, Chang L, Lerner L. A rapid method for determining dynamic binding capacity of resins for the purification of proteins. Protein Exp. Purif.60(2),147–150 (2008).Crossref, Medline, CAS, Google Scholar8 Abdiche YN, Malashock DS, Pinkerton A, Pons J. Exploring blocking assays using Octet, ProteOn, and Biacore biosensors. Anal. Biochem.386(2),172–180 (2009).Crossref, Medline, CAS, Google Scholar9 Abdiche Y, Malashock D, Pinkerton A, Pons J. Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Anal. Biochem.377(2),209–217 (2008).Crossref, Medline, CAS, Google Scholar10 Abdiche YN, Lindquist KC, Pinkerton A, Pons J, Rajpal A. Expanding the ProteOn XPR36 biosensor into a 36-ligand array expedites protein interaction analysis. Anal. Biochem.411(1),139–151 (2011).Crossref, Medline, CAS, Google Scholar11 Jason-Moller L, Murphy M, Bruno J. Overview of Biacore systems and their applications. Curr. Protoc. Protein. Sci.19,19.13 (2006).Google Scholar12 Löfås S. Optimizing the hit-to-lead process using SPR analysis. Assay. Drug. Dev. Technol.2(4),407–415 (2004).Crossref, Medline, Google Scholar13 Säfsten P, Klakamp SL, Drake AW, Karlsson R, Myszka DG. Screening antibody-antigen interactions in parallel using Biacore A100. Anal. Biochem.353(2),181–190 (2006).Crossref, Medline, Google Scholar14 Stumbaum M, Burkhard C, Gronewold T et al. Binding kinetics of therapeutic antibodies to native cell surgace proteins on living cells. Presented at: Miptec. Basel, Switzerland, 22 September 2011.Google Scholar15 Pei Z, Elovsson K, Käck C, Kaiser L, Aastrup T. Label free cell based biosensors: bridging the gap between biosensors and cell based assays. Presented at: Next Generation Pharma Summit. Scottsdale, AZ, USA, 26 May 2011.Google Scholar16 Chaires JB. Calorimetry and thermodynamics in drug design. Annu. Rev. Biophys.37,135–151 (2008).Crossref, Medline, CAS, Google Scholar17 Ahrer K, Buchacher A, Iberer G, Jungbauer A. Thermodynamic stability and formation of aggregates of human immunoglobulin G characterised by differential scanning calorimetry and dynamic light scattering. J. Biochem. Biophys. Methods66(1–3),73–86 (2006).Crossref, Medline, CAS, Google Scholar18 Rich RL, Myszka DG. Survey of the 2009 commercial optical biosensor literature. J. Mol. Recognit.24(6),892–914 (2011).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByNiloticin inhibits osteoclastogenesis by blocking RANKL–RANK interaction and suppressing the AKT, MAPK, and NF-κB signaling pathwaysBiomedicine & Pharmacotherapy, Vol. 149Selectivity of mTOR-Phosphatidic Acid Interactions Is Driven by Acyl Chain Structure and Cholesterol30 December 2021 | Cells, Vol. 11, No. 1Exopolysaccharide from Streptococcus thermophilus as stabilizer in fermented dairy: Binding kinetics and interactions with casein of milkInternational Journal of Biological Macromolecules, Vol. 140Interactions between β-cyclodextrin and tea catechins, and potential anti-osteoclastogenesis activity of the (−)-epigallocatechin-3-gallate–β-cyclodextrin complex1 January 2019 | RSC Advances, Vol. 9, No. 48Covalent affixation of histidine-tagged proteins tethered onto Ni-nitrilotriacetic acid sensors for enhanced surface plasmon resonance detection of small molecule drugs and kinetic studies of antibody/antigen interactions1 January 2019 | The Analyst, Vol. 144, No. 2Biophysical Analysis of the Protein-Small Molecule Interactions to Develop Small Molecule Drug DiscoveryYAKUGAKU ZASSHI, Vol. 138, No. 8Part-of-the-sites binding and reactivity in the homooligomeric enzymes – facts and artifactsArchives of Biochemistry and Biophysics, Vol. 642A label-free screening approach targeted protease-activated receptor 1 based on dynamic mass redistribution in living cells1 January 2017 | RSC Advances, Vol. 7, No. 68Kinetic analysis of a high-affinity antibody/antigen interaction performed by planar waveguide fluorescence immunosensor1 January 2016 | RSC Advances, Vol. 6, No. 17Recognization of receptors on bone marrow-derived dendritic cells bound with Pholiota nameko polysaccharidesInternational Journal of Biological Macromolecules, Vol. 72Solid-Phase Biological Assays for Drug DiscoveryAnnual Review of Analytical Chemistry, Vol. 7, No. 1Application of the log-normal model for long term high affinity antibody/antigen interactions using Bio-Layer Interferometry6 December 2013 | Journal of Mathematical Chemistry, Vol. 52, No. 2Application of Bio-Layer Interferometry for the analysis of protein/liposome interactionsJournal of Pharmaceutical and Biomedical Analysis, Vol. 72Label-free methods of reporting biomolecular interactions by optical biosensorsThe Analyst, Vol. 138, No. 13 Vol. 4, No. 6 Follow us on social media for the latest updates Metrics History Published online 28 March 2012 Published in print March 2012 Information© Future Science LtdKeywordsbiolayer interferometrybiomolecular screeningdifferential scanning calorimetrydrug discoveryisothermal calorimetrylabel-free technologyplate-based optical biosensorsquartz crystal microbalancesurface plasmon resonanceFinancial & competing interests disclosureThe authors are employees of Neotope Biosciences, a division of Elan Pharmaceuticals, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

p75(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75(NTR) is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75(NTR), no p75(NTR) signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75(NTR) modulators and studying p75(NTR) function. Stimulation of p75(NTR)-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75(NTR) mutant could not substitute for wild-type p75(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75(NTR) as well as further characterizing downstream signaling events.

Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4β7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg(2+) was greater than Ca(2+) at promoting cell adhesion. This homogeneous and label-free format exhibited low variability with a calculated Z' of 0.6. In addition to measuring α4β7-mediated 8866 cell adhesion to MadCAM, the authors also demonstrate that this platform can measure adhesion of Jurkat cells expressing α4β1 to the vascular cell adhesion molecule. Thus, the SRU BIND platform is widely applicable to measuring cell adhesion events mediated by other integrins binding to their receptors in an assay format that is amenable to high-throughput screening.

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aβ(x-40) levels in FVB mice via a single 100 mpk IP dose are highlighted.

Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease, and non-selective inhibitors are protective in rodent disease models. Because of their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far, and it is still unknown whether selective LRRK2, inhibition can attenuate mutant LRRK2-dependent toxicity inhuman neurons. Here, we employ a chemoproteomics strategy to identify potent, selective, and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.

The structure–activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate β-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aβx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.