Target the More Druggable Protein States in a Highly Dynamic Protein–Protein Interaction System

The proteins of the Bcl-2 family play key roles in the regulation of programmed cell death by controlling the integrity of the outer mitochondrial membrane and the initiation of the apoptosis process. We performed extensive molecular dynamics simulations to investigate the conformational flexibility of the Bcl-x_L protein in both the apo and holo (with Bad peptide and ABT-737) states. The accelerated molecular dynamics method implemented in Amber 14 was used to produce broader conformational sampling of 200 ns simulations. The pocket mining method based on the variational implicit-solvent model tracks the dynamic evolution of the ligand binding site with a druggability score characterizing the maximal affinity achievable by a drug-like molecule. Major movements were observed around the α3-helical domain and the loop region connecting the α1 and α2 helices, reshaping the ligand interaction in the BH3 binding groove. Starting with the apo crystal structure, which is recognized as “closed” and undruggable, the BH3 groove transitioned between the “open” and “closed” states during equilibrium simulation. Further analysis revealed a small percentage of the trajectory frames (∼10%) with a moderate degree of druggability that mimic the ligand-bound states. The ability to attain and detect by computer simulation the most suitable conformational states for ligand binding in advance of compound synthesis and crystal structure solution is of immense value to the application and success of structure-based drug design

ATP-Mediated Kinome Selectivity: The Missing Link in Understanding the Contribution of Individual JAK Kinase Isoforms to Cellular Signaling

Kinases constitute an important class of therapeutic targets being explored both by academia and the pharmaceutical industry. The major focus of this effort was directed toward the identification of ATP competitive inhibitors. Although it has long been recognized that the intracellular concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays, little thought has been devoted to incorporating this discrepancy into our understanding of translation from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed in the literature for this class of inhibitors. Herein, we demonstrate utilizing the four JAK family members that the difference in the ATP <i>K</i>_m of each individual kinase has a significant impact on the enzyme to cell inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or <i>K</i>_m ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been thought to have a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling, conducting similar analyses for other kinases by taking into account potency at high ATP rather than <i>K</i>_m ATP may provide crucial insights into a compound’s activity and selectivity in cellular contexts

Microfluidic-Enabled Intracellular Delivery of Membrane Impermeable Inhibitors to Study Target Engagement in Human Primary Cells

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure–activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1‑((2<i>S</i>,5<i>R</i>)‑5-((7<i>H</i>‑Pyrrolo[2,3‑<i>d</i>]pyrimidin-4-yl)­amino)-2-methylpiperidin-1-yl)­prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

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 <b>11</b> (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor <b>11</b> led to its evaluation in several human clinical studies

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1‑((2<i>S</i>,5<i>R</i>)‑5-((7<i>H</i>‑Pyrrolo[2,3‑<i>d</i>]pyrimidin-4-yl)­amino)-2-methylpiperidin-1-yl)­prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans

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 <b>11</b> (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor <b>11</b> led to its evaluation in several human clinical studies

Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition

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. <i>In vitro</i>, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and <i>in vivo</i> 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