Abstract
Candidate drugs rationally designed in vitro often fail due to low efficacy in vivo caused by low tissue availability or because of unwanted side effects. To overcome the limitations of in vitro rational drug design, the binding of candidate drugs to their target needs to be evaluated in the cellular context. Here, we applied in-cell NMR to investigate the binding of a set of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. Some compounds were originally developed toward other targets and were later found to inhibit CAs. We observed strikingly different dose- and time-dependent binding, wherein some drugs exhibited a more complex behavior than others. Specifically, some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, therefore preventing the quantitative formation of a stable protein–ligand complex. Such observations could be correlated to the known off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design.
Highlights
Classical rational drug design approaches aim at maximizing the activity toward a specific target in vitro
Ideally, the binding to an intracellular target should be evaluated in vitro, in isolated conditions, and directly in the cellular context, where poor cell penetrance or the occurrence of off-target binding can negatively affect the activity of potential drugs toward their main target
We focused on a selection of sulfonamide compounds representative of different categories of drugs, including anti-inflammatory drugs originally designed to inhibit non-carbonic anhydrase (CA) targets, diuretics, and anticonvulsants, which exert their function through inhibition of multiple targets, including CAs, and an anticancer drug currently under clinical trials that was designed to inhibit the tumor-associated isoform CA IX
Summary
Classical rational drug design approaches aim at maximizing the activity toward a specific target in vitro. Drug efficacy in vivo can be affected by many factors, such as low tissue availability, binding to off-target biomolecules, or unwanted side effects. Ideally, the binding to an intracellular target should be evaluated in vitro, in isolated conditions, and directly in the cellular context, where poor cell penetrance or the occurrence of off-target binding can negatively affect the activity of potential drugs toward their main target. Cell-based activity assays provide an indirect measure of the effect of a drug, but may not discriminate the molecular origin of the cellular response. The interaction of a potential drug with the intracellular target should be monitored directly at atomic resolution. Among the existing atomic-resolution structural techniques, Nuclear Magnetic Resonance (NMR) spectroscopy stands out for its ability to investigate protein−ligand interactions in solution at physiological temperatures.[1−3] in-cell
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