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IntroductionFragment-based drug design (FBDD) has become an increasingly popular platform for the identification and design of lead candidates in drug discovery programs. The detection and characterization of chemical fragment binding events is facilitated by sensitive biophysical technologies capable of detecting low affinity interactions of low molecular weight compounds. Over the last decade, approaches such as nuclear magnetic resonance (NMR), X-ray crystallography, differential scanning fluorimetry (DSF), and surface plasmon resonance (SPR) have become core technologies in many pharma and biotech settings for the identification of these low-affinity fragment compounds. In particular, SPR-based biosensors have sufficient sensitivity and throughput to provide complete fragment screens on libraries of several thousand compounds in just a few weeks per target. This poster describes how the Pioneer FE SPR system can improve on the efficiency of the fragment screening and characterization process to support FBDD.
Process Improvement with diSPR®Fragment screening is typically hindered by a host of technical challenges including large numbers of samples, low molecular weight analytes (< 300 Da) and weak affinity interactions (KD: 10 μM to 10 mM). Low molecular weight, weak affinity, and solubility limitations make it impractical to test for binding using sample concentrations above the KD and frequently results in making decisions based on ambiguous (small square-shaped) SPR data. To effectively analyze this limited fragment binding information, a process workflow has been defined for conventional SPR as shown in Figure 1.
FIguRE 5: An illustration of NeXtStep Injection. Rapid dispersion of two analyte components form a sigmoidal concentration gradient injected over the biosensor.
FIguRE 6: NeXtStep competition assay design shown by relative component concentration. Competitor concentration remains constant and fragment concentration ranges from 0–1. The illustration shows that at the beginning of the NeXtStep Injection, the competitor is at top concentration and stays constant while the fragment concentration goes from low to high concentration. This method is accomplished by merging the competitor sample as component B and the fragment plus competitor sample as component A. A reference injection of competitor only is also run to perform double-referencing of the data and show only the signal from fragment binding.
FIguRE 7: Examples of three common competition mechanisms binned by NeXtStep Injections. Fragments from the primary set of 24 were binned into three common competition mechanisms: non-competitive, competitive, and un-competitive. Identification of the competition mechanism is simple using NeXtStep Injections and characterization of un- and non-competitive fragments is possible from a single injection.
FIguRE 4: OneStep primary fragment screen example. OneStep data for fragments (color differentiated) and control (blue curves) are processed, hits are selected, and binding data are analyzed for kinetics and/or affinity in a single workflow.
The conventional SPR fragment screening process can be streamlined by integrating dynamic injection SPR (diSPR) available only with the Pioneer platform. At the core of diSPR are two unique injection methodologies: OneStep® Injections and NeXtStep™ Injections that can be used to advance binding characterization in multiple application areas throughout the drug discovery process. The two techniques can be used in tandem to fully characterize the kinetics parameter and binding specificity of screened drug molecules. A OneStep Injection is a Taylor dispersion injection of analyte producing a continuous concentration gradient that provides reliable affinity measurements in a single injection and is suitable for rapid screening. It offers improvements in screening time and provides higher information content that allows for confident characterization of hits in the primary screen. The Pioneer FE system’s NeXtStep Injection technology has the unique ability to determine full kinetics and affinity of a fragment in the presence of a competitor molecule. using the NeXtStep Injection’s rapid dispersion injection (similar to OneStep Injections), fragment and competitor are dispersed and injected over the biosensor surface to determine the fragment’s competition mechanism. Figure 2 shows the diSPR optimized fragment screening workflow.
Fragment Screening by OneStep and NeXtStep Injection TechniquesBy increasing sample throughput and data content through the generation of sample gradients, OneStep Injections can dramatically improve upstream SPR-based fragment screening. The technique is designed to streamline binding analysis by testing a full concentration series in a single injection. Figure 3 illustrates the dynamic gradient formation process which is performed at the time of analyte injection with a OneStep Injection.
SPR-based competition assays are often used to determine a molecule’s competition mechanism to either confirm binding to an active site or find un-competitive fragments. The Pioneer FE system’s NeXtStep Injection technology has the unique ability to determine full kinetics and affinity in the presence of a competitor molecule. using a rapid dispersion injection, two sample components are dispersed and injected over a biosensor surface. Like OneStep Injections, NeXtStep Injections test a concentration gradient of a fragment and by adding a competitor molecule to both the fragment and buffer components, it enables full kinetic analysis in the presence of the competitor.
Fragment screening process improvement with Pioneer FE and dynamic injection SPR Eric L. Reese, PhD, SensiQ Technologies, Inc.; Aaron Martin* and David O. Apiyo, PhD, Application Managers, Pall ForteBio LLC Pall ForteBio, 47661 Fremont Boulevard, Fremont, CA 94538 www.fortebio.com
© 2017, Pall Corporation. Pall, , ForteBio, OneStep and NeXtStep are trademarks of Pall Corporation. ® indicates a trademark registered in the uSA and ™ indicates a common law trademark.
* Author for correspondence: [email protected]
FIguRE 3: Illustration of OneStep Injection Taylor dispersion gradient formation. Sample (pink) is injected and dispersed through a buffer-filled capillary (blue) to produce a continuous concentration gradient of analyte that passes through the biosensor flow cell. Resolution of analyte concentration versus time enables a highly accurate analysis of kinetic and affinity constants for fragment hits.
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This not only saves sample preparation time and materials, but also reduces the cost of human error by avoiding the need for the preparation of multiple sample dilutions. OneStep also represents a significant improvement in how SPR biosensor assays are performed by streamlining the efficiency and accuracy of the workflow process. Secondary screening can be completely avoided as fragment candidates selection and characterization (KD and ligand efficiency) is optimized during primary screening.
Key Advantages of the OneStep Technique: • Allows users to make decisions early on by obtaining reliable affinity (KD) and
kinetics (ka, kd) data directly from the primary screen.
• Reduces time to result, is fully automated, and requires minimal assay development to arrive at the correct fragment candidate(s).
• Provides reproducible identification of fragment actives via integrated, cutting-edge hit selection software.
• Provides high throughput fragment actives analysis: up to 768 samples in 24 hours with the Pioneer FE system.
OneStep Injections eliminate the need for multiple sample preparations by generating a continuous concentration gradient using the sample and the running buffer. Therefore, primary screening data are ready for KD analysis, without the need for laborious secondary screening and extra sample preparation steps.
To demonstrate the NeXtStep competition technique on the Pioneer FE system, carbonic anhydrase II (CAII) was immobilized on a COOHV sensor chip via amine coupling and the Pioneer FE system was primed in HEPES-buffered saline, pH 7.4, with 0.005% Tween-20 (HBST). 24 fragment hits were previously identified from the Maybridge Ro3 1,000 compound library using the Pioneer FE system and its integrated hit selection. These fragments were prepared in two sample solutions, one in buffer and another in buffer with 100 µM furosemide. NeXtStep Injections (50 µL/min) were performed on each sample by dispersing the buffer solutions with buffer and the furosemide solutions with furosemide (100 µM). Control injections of buffer and furosemide were also performed for double referencing. Data analysis was performed using Qdat Analysis software (Pall ForteBio) and results were compared to determine the fragment competition mechanism in the presence and absence of furosemide (active site competitor).
ConclusionThe detection and characterization of fragment binding events is dependent upon ultrasensitive technologies that are able to detect interactions of weak affinity and low molecular weight compounds. As demonstrated throughout this poster, in comparison to other SPR-based biosensor technologies, the Pioneer FE system has the extraordinary advantage of both increasing the rate at which fragment screening is accomplished and increasing information content of the data. Content-rich data can be derived from a primary screen with OneStep Injections, enabling the rapid selection of active fragment hits without the need for secondary screening. NeXtStep competition assays can be performed to determine fragment competition mechanisms and characterize un- and non-competitive fragments. The result is a streamlined and optimized fragment screening process with the integration of two diSPR methods demonstrating that the Pioneer FE system is an important tool in the fragment-based drug discovery pipeline.
FIguRE 1: Conventional SPR-based fragment screening workflow: A pilot screen and primary screen is performed to identify poor quality fragments and fragments which bind target. Secondary screening follows where samples have to be prepared at different concentrations and analyzed separately to allow for affinity characterization and fragment hit confirmation. Hits are then ranked and the data are provided to medicinal chemistry for hit expansion.
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FIguRE 2: Optimized workflow using diSPR: Hit selection and affinity characterization is accomplished in a single OneStep screen. A competition follow-up assay with a NeXtStep Injection determines if hits are competitive or non/un-competitive. The optimized process results in faster and better characterization of fragment hits with greater confidence in their mechanism of action and specificity.
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