Free‐solution interaction assay of carbonic anhydrase to its inhibitors using back‐scattering interferometry

Morcos, Ereny F.; Kussrow, Amanda; Enders, Carolyn S.; Bornhop, Darryl J.

doi:10.1002/elps.201000389

Cited by 15 publications

(14 citation statements)

References 17 publications

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“…The probe acts through a turn-on fluorescence mechanism whereby HS – reduces the nonfluorescent azide in solution to give the corresponding fluorescent amine (Da-NH 2 ). Although Da-NH 2 is a known CA inhibitor, 32 we used the initial rates of Da-N 3 reduction by NTA1 to compare the rates of H 2 S production in the presence and absence of CA.…”

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confidence: 99%

Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

et al. 2016

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Carbonyl sulfide (COS) is a gas that may play important roles in mammalian and bacterial biology, but its study is limited by a lack of suitable donor molecules. We report here the use of N-thiocarboxyanhydrides (NTAs) as COS donors that release the gas in a sustained manner under biologically relevant conditions with innocuous peptide byproducts. Carbonic anhydrase converts COS into H2S, allowing NTAs to serve as either COS or H2S donors, depending on the availability of the enzyme. Analysis of the pseudo-first-order H2S release rate under biologically relevant conditions revealed a release half-life of 75 min for the small molecule NTA under investigation. A polynorbornene bearing pendant NTAs made by ring-opening metathesis polymerization was also synthesized to generate a polymeric COS/H2S donor. A half-life of 280 min was measured for the polymeric donor. Endothelial cell proliferation studies revealed an enhanced rate of proliferation for cells treated with the NTA over untreated controls.

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confidence: 99%

Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

et al. 2016

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“…BSI can be used to measure binding interactions in free solution without the need for surface immobilization, specialized reagents, or fluorescent probes, thus providing a clear advantage over techniques such as isothermal titration calorimetry (ITC),11 surface plasmon resonance (SPR),12 and fluorometric assays 13. Furthermore, cosolvents, such as DMSO1b and acetonitrile,1c can be used without assay interference.…”

Section: Methodsmentioning

confidence: 99%

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

et al. 2012

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A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. Keywords detection method; interferometry; acetylcholinesterase; high-throughput screening; enzyme assayIn recent years, the emphasis of drug discovery and optimization has shifted from traditional methods to rapid microfluidic screening to minimize cost and maximize output. Backscattering interferometry (BSI),[1] a technique which quantifies refractive index (RI) changes arising from intermolecular binding interactions, is a novel biosensing platform that may be useful in drug discovery. Acetylcholinesterase (AChE), a widely studied serine hydrolase that plays pivotal roles in Alzheimer's disease (AD), [2] inflammatory processes, [3] and nerve-agent poisoning, [4] is an interesting system with which to study the use of BSI for the rapid detection of drug candidates. The search for potent AChE inhibitors (AChEI) has been driven largely by the need for an effective AD treatment, and is based on the longstanding cholinergic hypothesis [2] and the more recent amyloid hypothesis. [5] It is wellestablished that AChE accelerates amyloid-beta (Ab) peptide deposition, a process which may be mediated by an interaction between plaque precursors and the peripheral anionic site (PAS) of the enzyme.[6] Recent drug discovery efforts have focused on the design of AChEIs that are able to interact with the PAS to target both cholinergic and noncholinergic AD pathologies. [7] Seminal work in this field has led to the discovery of dual-binding inhibitors, such as bisgalantamine [8] and pseudo-irreversible carbamate inhibitors. [9] Given the large number of studies on several diverse classes of AChEIs, this system is a widely accepted benchmark for the development and testing of novel screening techniques.Traditional methods used to detect interactions with AChE focus on the indirect measurement of substrate procedures. [10] To explore the utility of BSI to detect AChEIs, several known and novel inhibitors with diverse potencies and inhibitory mechanisms have been screened. Herein, we show that BSI can: 1) quantify AChE-inhibitor interactions in the We first wanted to probe the utility of BSI to screen AChEIs using a set of known and novel AChEIs (Scheme 1). Edrophonium, a competitive inhibitor, propidium, a noncompetitive inhibitor, and the mixed inhibitors, 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) and galantamine, were the standards used to validate BSI as a method for the detection of AChEIs. Two separate and distinct saturation curves were observed for inhibitors 4 and 6 (Figure 2 and Supporting Information, Figure S7, respectively). For ligand 4, the dual-binding curve is shown in the graph inset of while the fit of the second bindin...

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“…Insensitivity of a biosensor to refractive index changes is of considerable interest for drug screening because most drug candidates are stored as DMSO (dimethylsulfoxide) solutions (refractive index n ≈ 1.5). This constitutes a real challenge for refractometric sensors, especially in assays where a reference channel cannot be used [54]. For this reason, we also investigate the refractive index change susceptibility of unreferenced refractometric biosensors.…”

Section: Experimental Noise Rejection Performance Of a Spatial Lock-imentioning

confidence: 99%

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part II. Experimental Demonstration

Frutiger

Gatterdam

Blickenstorfer

et al. 2020

Sensors

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Label-free optical biosensors are an invaluable tool for molecular interaction analysis. Over the past 30 years, refractometric biosensors and, in particular, surface plasmon resonance have matured to the de facto standard of this field despite a significant cross reactivity to environmental and experimental noise sources. In this paper, we demonstrate that sensors that apply the spatial affinity lock-in principle (part I) and perform readout by diffraction overcome the drawbacks of established refractometric biosensors. We show this with a direct comparison of the cover refractive index jump sensitivity as well as the surface mass resolution of an unstabilized diffractometric biosensor with a state-of-the-art Biacore 8k. A combined refractometric diffractometric biosensor demonstrates that a refractometric sensor requires a much higher measurement precision than the diffractometric to achieve the same resolution. In a conceptual and quantitative discussion, we elucidate the physical reasons behind and define the figure of merit of diffractometric biosensors. Because low-precision unstabilized diffractometric devices achieve the same resolution as bulky stabilized refractometric sensors, we believe that label-free optical sensors might soon move beyond the drug discovery lab as miniaturized, mass-produced environmental/medical sensors. In fact, combined with the right surface chemistry and recognition element, they might even bring the senses of smell/taste to our smart devices.

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Free‐solution interaction assay of carbonic anhydrase to its inhibitors using back‐scattering interferometry

Cited by 15 publications

References 17 publications

Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part II. Experimental Demonstration

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Free‐solution interaction assay of carbonic anhydrase to its inhibitors using back‐scattering interferometry

Cited by 15 publications

References 17 publications

Therapeutic Delivery of H2S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Therapeutic Delivery of H2S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Back‐Scattering Interferometry: An Ultrasensitive Method for the Unperturbed Detection of Acetylcholinesterase–Inhibitor Interactions

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part II. Experimental Demonstration

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Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts

Therapeutic Delivery of H₂S via COS: Small Molecule and Polymeric Donors with Benign Byproducts