Compound Aggregation in Drug Discovery: Implementing a Practical NMR Assay for Medicinal Chemists

Abstract

The pharmaceutical industry has recognized that many drug-like molecules can self-aggregate in aqueous media and have physicochemical properties that skew experimental results and decisions. Herein, we introduce the use of a simple NMR strategy for detecting the formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that (1)H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation and for determining the concentrations of single molecule, aggregate entities, and precipitate. The critical concentrations above which aggregation begins can be found and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.

Anion transport and binding properties of N N′-(phenylmethylene)dibenzamide based receptors

“…Typical NMR spectra of aggregators can show changes in the number of signals present, indicative of multiple aggregates species in solution. Additionally, changes in chemical shifts (d ppm) may be detected resulting from local environmental changes in the (33). Proton NMR spectra were obtained for receptors 1 -7 at a range of concentrations (1 -100 mM).…”

Non-stoichiometric inhibition in integrated lead finding – a literature review

“…Inversely, the efficacy of molecules that are potent bioactive inhibitors in their monomeric form can be significantly reduced upon aggregate formation, especially if this blocks their uptake into cells. [49] Another technique to detect aggregation of a compound is nuclear magnetic resonance (NMR), which either analyzes the signal from the compound itself [50] or uses the conformational distortion of the human La antigen as a surrogate readout in what is called ALARM NMR. [18,51] An indirect approach compares compound potency at different concentrations of enzyme [25,52]; bellshaped dose-response curves in cellular assays are another warning signal.…”

“…[89] The most likely reason is that the throughput of devices for dynamic [44] or static [45] light scattering is limited. NMR is also used primarily to characterize compounds synthesized during a medicinal chemistry program [50] or to confirm results by higher-throughput methods [29] rather than for screening hits at an early stage of the flowchart. Methods to test larger numbers of screening hits for redox activity, in particular the resazurin-to-resorufin conversion [67] and the phenol redhorseradish peroxidase assay, [66] are primarily applied for redox-sensitive targets.…”

NMR methods in fragment based drug discovery

“…By examining the concentration-dependent peak intensity and chemical shift, fragment aggregation is readily determined. 17 One of more practical strategies to conduct fragment quality control is measuring simple 1D proton spectra and comparing peak intensities of fragments with an internal standard. In our laboratory, we measure 1D proton spectra of 1 mM fragments mixed with 0.1 mM internal standard 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) in phosphate buffer.…”