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

Early Process Development of Two Vanin‑1 Inhibitors: Solid Form Challenges and Control of Ambident Reactivity

Discovery chemistry efforts within Pfizer identified a new vanin-1 inhibitor, (S)-1, bearing a chiral methyl substituent, which exhibited an excellent profile as a potential drug-candidate selection except for the propensity to exist as an amorphous solid. Based on an improved solid form proposition, the project team chose to prioritize 2, the corresponding des-methyl compound. Both compounds were scaled to supply toxicology studies in preclinical species, and kilograms of compound 2 were manufactured to support the preclinical development work. The development of our synthetic chemistry and solid form work on this program are described in the paper. Included are computational studies to rationalize both an expected TBD-mediated epimerization as well as the control of ambident reactivity of activated 2-chloro-pyrimidine-5-carboxylic acid

Identification of Morpholino‑2<i>H</i>‑pyrido[3,2‑<i>b</i>][1,4]­oxazin-3(4<i>H</i>)‑ones as Nonsteroidal Mineralocorticoid Antagonists

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit <b>9</b> that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead <b>10</b>. After further optimization, <i>cis</i> disubstituted morpholine <b>22</b> was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to <b>13</b>. While <b>22</b> had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na⁺/K⁺ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of <b>22</b> were adequate, suggesting that it could be suitable as a potential clinical asset

Identification of Morpholino‑2<i>H</i>‑pyrido[3,2‑<i>b</i>][1,4]­oxazin-3(4<i>H</i>)‑ones as Nonsteroidal Mineralocorticoid Antagonists

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit <b>9</b> that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead <b>10</b>. After further optimization, <i>cis</i> disubstituted morpholine <b>22</b> was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to <b>13</b>. While <b>22</b> had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na⁺/K⁺ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of <b>22</b> were adequate, suggesting that it could be suitable as a potential clinical asset

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