To Affinity and Beyond: Tools to Optimize Assay Design and Evaluation in Fragment Based Lead Discovery and Characterization with SPR-based Biosensors.
SPR-based biosensors are becoming a powerful tool in lead generation due to the sensitivity required to identify and rank low molecular weight and low affinity binders. This approach offers novel ways of efficiently identifying true hits for hit to lead development, complementing more classical assay technologies used within fragment based lead discovery (FBLD). In this workshop, the basic strategy of the technology used, key applications in the identification of site specific binders and best practices/troubleshooting of FBLD via SPR will be discussed.
Compound Titrations Using OneStep(TM): A Practical Tool for Label-Free Screening
Implementation of Taylor Dispersion (OneStep) based SPR Experiments in Fragment Screening and Characterization.
The standard assay format for label-free screening has not changed since the inception of of the field over two decades ago. In the standard approach a series of binding response curves are recorded over a range of concentrations and used to plot an affinity isotherm thereby revealing the KD of the interaction. This titration approach can tolerate multisite binding assuming a sufficient density of equilibrium responses points are included . Despite the success of this approach there remain several limitations including the following: (a) Time consuming - at least six compound dilutions must be prepared and analysed. A higher point density is required to resolve complex binding where a high affinity site is masked by the presence of non-specific binding or a DMSO offset. (b) Systematic error - variability accumulates due to the preparation/processing of multiple separate compound dilutions over an extended period of time where baseline drift, loss of compound/target activity and non-specific binding can be variable. (c) Poor resolution - an isotherm defined by a small number of points provides less reliable estimates of KD . Higher resolution is particularly useful when the affinity is low and the maximum measured response is less than one third of the expected saturation response. We introduce a real-time compound titration (OneStep™) that provides an isotherm as a function of injection time that can be recorded in less than one minute and containing over a thousand binding response points. An in-line capillary is used to spontaneously generate a predictable smoothly changing compound titration that increases, or decreases. Two component titrations are readily produced providing a simple means of conducting screens in competitive binding mode. Using model interactions we demonstrate OneStep™ as a practical method that addresses the aforementioned limitations of the standard approach. In addition we provide a more accurate approach to estimation of Rmax when using the Rmax lock-in method.
Fragment Based De Novo Discovery Workshop Scaffold Replacement | Growing | Linking.
The workshop will cover hands-on examples of the fragment-based drug design tools in MOE. Topics will include scaffold replacement, fragments linking & growing, as well as combinatorial design in the pocket. This will include protein:ligand preparation, applying automatic 2D and 3D descriptors, pharmacophore constraints, ligand refinement in a (flexible) pocket and scoring. Tools for generating custom fragment databases will also be discussed. Please note that seats are limited so early registration is strongly recommended. Computers will be provided for the hands-on exercises. For any questions, please contact Raul Alvarez at ralvarez@chemcomp.com