Robust Strategy for Hit-to-Lead Discovery: NMR for SAR.

Establishing robust structure-activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an "undruggable" protein target, HRas.

Probing the free-state solution behavior of drugs and their tendencies to self-aggregate into nano-entities.

The free-state solution behaviors of drugs profoundly affect their properties. Therefore, it is critical to properly evaluate a drug's unique multiphase equilibrium when in an aqueous enviroment, which can comprise lone molecules, self-associating aggregate states and solid phases. To date, the full range of nano-entities that drugs can adopt has been a largely unexplored phenomenon. This protocol describes how to monitor the solution behavior of drugs, revealing the nano-entities formed as a result of self-associations. The procedure begins with a simple NMR 1H assay, and depending on the observations, subsequent NMR dilution, NMR T2-CPMG (spin-spin relaxation Carr-Purcell-Meiboom-Gill) and NMR detergent assays are used to distinguish between the existence of fast-tumbling lone drug molecules, small drug aggregates and slow-tumbling colloids. Three orthogonal techniques (dynamic light scattering, transmission electron microscopy and confocal laser scanning microscopy) are also described that can be used to further characterize any large colloids. The protocol can take a non-specialist between minutes to a few hours; thus, libraries of compounds can be evaluated within days.

Click-Functionalization of a Poly(Tetrazine-co-Fluorene)-Conjugated Polymer with a Series of trans-Cyclooctene Derivatives.

A soluble poly(tetrazine) polymer was prepared via Suzuki polycondensation of 3,6-bis(5-bromofuran-2-yl)-1,2,4,5-tetrazine and a fluorene diboronate derivative. It can undergo efficient and quantitative post-polymerization inverse-electron-demand Diels-Alder click reactions with a variety of trans-cyclooctene (TCO) derivatives. The resulting polymers were oxidized to convert dihydropyridazine rings into pyridazines. The absorption spectra of the product polymers, both before and after oxidation, showed hypsochromic shifts that correlated with steric hindrance of the appended side chains. They also exhibited a significantly enhanced fluorescence intensity relative to the original poly(tetrazine). While gel-permeation chromatography indicated that the product polymers exhibited longer retention times, NMR end-group analysis showed that the polymers retained relatively constant degrees of polymerization. Graft copolymers were easily prepared via reaction with TCO-functionalized poly(ethylene glycol) chains and a cross-linked foam was produced by reacting the poly(tetrazine) with a bis-TCO crosslinker.