Abstract
Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008).
Highlights
WHY USE IN VITRO PROFILING ASSAYS TO UNDERSTAND LEAD SELECTIVITY AND SPECIFICITY? The drug discovery and development process is long, and resourceintensive, often fraught with unforeseeable pitfalls, and historically challenged by low probability of success (Tollman et al, 2011)
Millipore offers a wide variety of profiling services that utilize biochemical assays for over 300 kinases and phosphatases and cellbased functional assays for over 150 G-protein coupled receptors (GPCRs) and over 50 ion channels that can be utilized to query compounds at any development stage for specificity and selectivity for primary targets, related family members, and for cardiac safety
The other is to profile hits employing a panel of enzyme and receptor binding assays, gather the respective profile of offtarget activities predicted for each compound, and assess their profile in vivo, in an animal model chosen to reveal ancillary pharmacological activities
Summary
OF IN VIVO SPECIFICITY AND SELECTIVITY ASSESSMENT There are a number of confounding aspects related to using such in vivo assessment approaches, and the data generated can be highly misleading without a through understanding gained from in vitro assays (e.g., Raehal et al, 2011). There is no bona fide substitute to definitively identify putative ion channel or transporter modulators other than by direct measurement of protein function This is especially true when evaluating novel chemical structures without previous pharmacological history, because many examples of false negatives and false positives have been noted when using binding protocols to profile such compound decks. BIRB0796, which was developed as an allosteric inhibitor of p38 MAP kinase (aka SAPK2A/SAPK2B; Pargellis et al, 2002) was profiled at a single concentration of 10 μM in a functional Safety and Liability Screening Panel consisting of 125 targets (76 GPCRs, 39 Kinases, two Phosphatases, and eight Cardiac Ion Channels). 10 μM BIRB0796 inhibited five GPCRs (Table 2) and two of seven Cardiac Ion Channels, hERG, and Kv1.5 (Figure 1), by
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