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

BACKGROUND: Structure-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. RESULTS: A 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. CONCLUSIONS: Practical 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.

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 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.

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.

Novel boron-containing, nonclassical antifolates: synthesis and preliminary biological and structural evaluation.

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.

Theoretical and experimental studies of biotin analogues that bind almost as tightly to streptavidin as biotin.

We have used a newly developed qualitative computational approach, PROFEC (Pictorial Representation of Free Energy Changes), to visualize the areas of the ligand biotin where modifications of its structure might lead to tighter binding to the protein streptavidin. The PROFEC analysis, which includes protein flexibility and ligand solvation/desolvation, led to the suggestion that the pro-9R hydrogen atom of biotin, which is in alpha-position to the CO(2)(-) group, might be changed to a larger group and lead to better binding with streptavidin and avidin. Free energy calculations supported this suggestion and predicted that the methyl analogue should bind approximately 3 kcal/mol more tightly to streptavidin, with this difference coming exclusively from the relative desolvation free energy of the ligand. The PROFEC analysis further suggested little or no improvement for changing the pro-9S hydrogen atom to a methyl group, and great reduction in changing the ureido N-H groups to N-CH(3). Stimulated by these results, we synthesized 9R-methylbiotin and 9S-methylbiotin, and their binding free energies and enthalpies were measured for interaction with streptavidin and avidin, respectively. In contrast to the calculated results, experiments found both 9-methylbiotin isomers to bind more weakly to streptavidin than biotin. The calculated preference for the binding of the 9R- over the 9S-stereoisomer was observed. In addition, 9-methylbiotin is considerably less soluble in water than biotin, as predicted by the calculation, and the 9R isomer is, to our knowledge, thus far the tightest binding analogue of biotin to streptavidin. Subsequently, X-ray structures of the complexes between streptavidin and both 9R- and 9S-methylbiotin were determined, and the structures were consistent with those used in the free energy calculations. Thus, the reason for the discrepancy between the calculated and experimental binding free energy does not lie in unusual binding modes for the 9-methylbiotins.