Anti-citrullinated protein antibodies (ACPA) could not be detected in mice with CIA.

<p>CIA was induced in DBA/1J mice. After 70–90 days, serum was harvested and anti-CarP and ACPA levels were determined by ELISA. Every symbol represents an individual mice and the line indicates the median. (<b>A</b>) IgG1 levels. (<b>B</b>) IgG2a levels. Statistical analysis was performed using a kruskall-wallis test followed by a Dunn's Multiple comparison test (* p<0.05).</p

Anti-CarP antibodies can be detected in mice.

<p>(<b>A</b>) Schematic picture of the carbamylation process. (<b>B</b>) DBA/1J mice were immunized with CII in CFA. The antibody binding to FCS and Carbamylated-FCS (Ca-FCS) was determined by ELISA. The OD value for Ca-FCS binding and FCS binding of each sample are connected with a line. Statistical analysis was performed using a Wilcoxon paired test (n = 29). (<b>C</b>) Sera from anti-CarP positive mice were pre-incubated with different concentrations of Ca-FCS and FCS. The Ig binding to FCS and Ca-FCS was determined by ELISA (n = 4). (<b>D</b>) C57Bl/6 mice were immunized with CII in CFA. Equal amounts of Ca-FCS and FCS were blotted on a membrane. The presence of antibodies reactive to the Ca-FCS or FCS on the blots was analyzed by incubating the blots with sera from the immunized mice. A representative example of 2 independent experiments is depicted.</p

Kinetics of the anti-CarP response during CIA.

<p>(<b>A</b>) CIA was induced in DBA/1J mice (n = 19). The mice were immunized at day 0 and boosted at day 21. Blood was harvested from the mice at the indicated time points and serum was stored at −80. The anti-CarP antibody levels were determined in all serum samples simultaneously by ELISA at the end of the experiment. The data shown are the pooled data from 2 independent experiments that showed a similar trend. (<b>B</b>) Anti-CarP IgG2a levels were determined by ELISA. Arbitrary units were calculated using a standard curve of pooled serum from mice with CIA. Every symbol represents one individual mouse and the line indicates the median. (<b>C</b>) Anti-CarP IgG2a antibody levels are plotted on the left Y axes and indicated by the dots with the solid line. The clinical score is plotted on the right Y-axis and indicated by the squares and the dashed line. The error bars indicate the SEM.</p

Mice with CIA harbor anti-CarP antibodies.

<p>CIA was induced in DBA/1J mice. After 70–90 days, serum of naïve non-immunized mice (HC; squares) and mice with CIA (dots) was harvested and anti-CarP levels were determined by ELISA. Arbitrary units were calculated using a standard curve of pooled serum from mice with CIA. The number indicates the number of mice per group. (<b>A</b>) Total Ig levels (<b>B</b>) Total Ig levels plotted against the clinical score (<b>C</b>) IgG1 levels (<b>D</b>) IgG1 levels plotted against the clinical score (<b>E</b>) IgG2a levels (<b>F</b>) IgG2a levels plotted against the clinical score. The data is pooled data from 4 independent experiments. Every symbol represents one mouse and the bar indicates the median. Statistical analysis was performed using a Mann-Whitney test (** p<0.01, *** p<0.001).</p

Anti-CarP can be detected in CFA immunized mice.

<p>DBA/1J were immunized with CII (CIA; (n = 19)) or PBS in CFA followed by a booster with CII or PBS in IFA (CFA; n = 9). Anti-CarP IgG2a levels were determined by ELISA. The data shown are the pooled data from 2 independent experiments that showed a similar trend. CFA +PBS immunized mice are depicted as circles and CIA mice are depicted as squares. The error bars indicate the SEM. In the right panel the area under curve is depicted. Every symbol represents one individual mouse and the line indicates the median. Statistical analysis was performed by comparing the area under the curve followed by a Mann-Whitney test (** p<0.01).</p

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

Identification of <i>N</i>‑{<i>cis</i>-3-[Methyl(7<i>H</i>‑pyrrolo[2,3‑<i>d</i>]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases

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 (<b>25</b>) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases

Discovery of Clinical Candidate 1‑{[(2<i>S</i>,3<i>S</i>,4<i>S</i>)‑3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin‑1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound <b>40</b>). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration