Bruton tyrosine kinase (BTK) is a key enzyme in B-cell development whose improper regulation causes severe immunodeficiency diseases. Design of selective BTK therapeutics would benefit from improved, ...in-silico structural modeling of the kinase's solution ensemble. However, this remains challenging due to the immense computational cost of sampling events on biological timescales. In this work, we combine multi-millisecond molecular dynamics (MD) simulations with Markov state models (MSMs) to report on the thermodynamics, kinetics, and accessible states of BTK's kinase domain. Our conformational landscape links the active state to several inactive states, connected via a structurally diverse intermediate. Our calculations predict a kinome-wide conformational plasticity, and indicate the presence of several new potentially druggable BTK states. We further find that the population of these states and the kinetics of their inter-conversion are modulated by protonation of an aspartate residue, establishing the power of MD & MSMs in predicting effects of chemical perturbations.
Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically ...differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallography, kinetic, and computational studies, interaction of cyanamide 12 with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by 32. In relevant cell-based assays and in agreement with previous results from this group, 32 demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for 12. This work also gave key insights into a productive approach to decrease glutathione/glutathione S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.
Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant ...effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.
Tanaproget represents a potential first-in-class nonsteroidal PR agonist for contraception with improved safety and side effect profiles versus currently available steroidal oral contraceptives. ...Additional SAR, biological activity, and structural information from a tanaproget/hPR-LBD (hPR-LBD = human progesterone receptor ligand binding domain) cocrystal structure will also be presented.
A high-throughput screening campaign to discover small molecule leads for the treatment of bone disorders concluded with the discovery of a compound with a 2-aminopyrimidine template that targeted ...the Wnt β-catenin cellular messaging system. Hit-to-lead in vitro optimization for target activity and molecular properties led to the discovery of (1-(4-(naphthalen-2-yl)pyrimidin-2-yl)piperidin-4-yl)methanamine (5, WAY-262611). Compound 5 has excellent pharmacokinetic properties and showed a dose dependent increase in the trabecular bone formation rate in ovariectomized rats following oral administration.
The rational design and discovery of a new series of pyrrole2,3-
dazepino FXR agonists with aqueous solubility and improved pharmaceutical properties is reported.
Pyrrole2,3-
dazepines have been ...identified as potent agonists of the farnesoid X receptor (FXR). Based on the planar X-ray crystal structure of WAY-362450
1 in the ligand binding domain and molecular modeling studies, non-planar reduced compounds were designed which led to agonists that exhibit high aqueous solubility and retain moderate in vitro potency.
Anisole and fluoroanisoles display distinct conformational preferences, as evident from a survey of their crystal structures. In addition to altering the free ligand conformation, various degrees of ...fluorination have a strong impact on physicochemical and pharmacokinetic properties. Analysis of anisole and fluoroanisole matched molecular pairs in the Pfizer corporate database reveals interesting trends: 1) PhOCF3 increases log D by ~1 log unit over PhOCH3 compounds; 2) PhOCF3 shows lower passive permeability despite its higher lipophilicity; and 3) PhOCF3 does not appreciably improve metabolic stability over PhOCH3 . Emerging from the investigation, difluoroanisole (PhOCF2 H) strikes a better balance of properties with noticeable advantages of log D and transcellular permeability over PhOCF3 . Synthetic assessment illustrates that the routes to access difluoroanisoles are often more straightforward than those for trifluoroanisoles. Whereas replacing PhOCH3 with PhOCF3 is a common tactic to optimize ADME properties, our analysis suggests PhOCF2 H may be a more attractive alternative, and greater exploitation of this motif is recommended.
The energetics of rotation around single bonds (torsions) is a key determinant of the three-dimensional shape that druglike molecules adopt in solution, the solid state, and in different biological ...environments, which in turn defines their unique physical and pharmacological properties. Therefore, accurate characterization of torsion angle preference and energetics is essential for the success of computational drug discovery and design. Here, we analyze torsional strain in crystal structures of druglike molecules in Cambridge structure database (CSD) and bioactive ligand conformations in protein data bank (PDB), expressing the total strain energy as a sum of strain energy from constituent rotatable bonds. We utilized cloud computing to generate torsion scan profiles of a very large collection of chemically diverse neutral fragments at DFT(B3LYP)/6-31G*//6-31G** or DFT(B3LYP)/6-31+G*//6-31+G** (for sulfur-containing molecule). With the data generated from these ab initio calculations, we performed rigorous analysis of strain due to deviation of observed torsion angles relative to their ideal gas-phase geometries. Contrary to the previous studies based on molecular mechanics, we find that in the crystalline state, molecules generally adopt low-strain conformations, with median per-torsion strain energy in CSD and PDB under one-tenth and one-third of a kcal/mol, respectively. However, for a small fraction (<5%) of motifs, external effects such as steric hindrance and hydrogen bonds result in strain penalty exceeding 2.5 kcal/mol. We find that due to poor quality of PDB structures in general, bioactive structures tend to have higher torsional strain compared to small-molecule crystal conformations. However, in the absence of structural fitting artifacts in PDB structures, protein-induced strain in bioactive conformations is quantitatively similar to those due to the packing forces in small-molecule crystal structures. This analysis allows us to establish strain energy thresholds to help identify biologically relevant conformers in a given ensemble. The work presented here is the most comprehensive study to date that demonstrates the utility and feasibility of gas-phase quantum mechanics (QM) calculations to study conformational preference and energetics of drug-size molecules. Potential applications of this study in computational lead discovery and structure-based design are discussed.
Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As ...JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.
PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the ...comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1β production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.
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