Richard W. Dixon

Alchemical free energy methods for drug discovery: progress and challenges

Improved rational drug design methods are needed to lower the cost and increase the success rate of drug discovery and development. Alchemical binding free energy calculations, one potential tool for rational design, have progressed rapidly over the past decade, but still fall short of providing robust tools for pharmaceutical engineering. Recent studies, especially on model receptor systems, have clarified many of the challenges that must be overcome for robust predictions of binding affinity to be useful in rational design. In this review, inspired by a recent joint academic/industry meeting organized by the authors, we discuss these challenges and suggest a number of promising approaches for overcoming them.

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode.

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.

Identification of a selective thieno[2,3-c]pyridine inhibitor of COT kinase and TNF-α production

COT (Tpl2 in mice) is a serine/threonine MAP3 kinase that regulates production of TNF-alpha and other pro-inflammatory cytokines such as IL-1beta via the ERK/MAP kinase pathway. As TNF-alpha and IL-1beta are clinically validated targets for therapeutic intervention in rheumatoid arthritis (RA), blocking COT provides a potential avenue for amelioration of disease. Herein we describe identification of a cellular active selective small molecule inhibitor of COT kinase.

2,4-Diaminopyrimidine MK2 inhibitors. Part II: Structure-based inhibitor optimization

We describe structure-based optimization of a series of novel 2,4-diaminopyrimidine MK2 inhibitors. Co-crystal structures (see accompanying Letter) demonstrated a unique inhibitor binding mode. Resulting inhibitors had IC(50) values as low as 19nM and moderate selectivity against a kinase panel. Compounds 15, 31a, and 31b inhibit TNFalpha production in peripheral human monocytes.

Enabling structure-based drug design of Tyk2 through co-crystallization with a stabilizing aminoindazole inhibitor

BackgroundStructure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds.ResultsA comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5–2.6 Å resolution. This structure revealed initial “hot-spot” regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods.ConclusionsPractical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.