Demonstration of In Vitro to In Vivo Translation of a TYK2 Inhibitor That Shows Cross Species Potency Differences.

Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 → V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.

Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors.

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.

Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841).

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

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. 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-1ß 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.

Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a ß-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

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 Km 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 Km 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 Km ATP may provide crucial insights into a compound's activity and selectivity in cellular contexts.

The use of plasma aldosterone and urinary sodium to potassium ratio as translatable quantitative biomarkers of mineralocorticoid receptor antagonism.

BACKGROUND: Accumulating evidence supports the role of the mineralocorticoid receptor (MR) in the pathogenesis of diabetic nephropathy. These findings have generated renewed interest in novel MR antagonists with improved selectivity against other nuclear hormone receptors and a potentially reduced risk of hyperkalemia. Characterization of novel MR antagonists warrants establishing translatable biomarkers of activity at the MR receptor. We assessed the translatability of urinary sodium to potassium ratio (Na+/K+) and plasma aldosterone as biomarkers of MR antagonism using eplerenone (Inspra®), a commercially available MR antagonist. Further we utilized these biomarkers to demonstrate antagonism of MR by PF-03882845, a novel compound. METHODS: The effect of eplerenone and PF-03882845 on urinary Na+/K+ and plasma aldosterone were characterized in Sprague-Dawley rats and spontaneously hypertensive rats (SHR). Additionally, the effect of eplerenone on these biomarkers was determined in healthy volunteers. Drug exposure-response data were modeled to evaluate the translatability of these biomarkers from rats to humans. RESULTS: In Sprague-Dawley rats, eplerenone elicited a rapid effect on urinary Na+/K+ yielding an EC50 that was within 5-fold of the functional in vitro IC50. More importantly, the effect of eplerenone on urinary Na+/K+ in healthy volunteers yielded an EC50 that was within 2-fold of the EC50 generated in Sprague-Dawley rats. Similarly, the potency of PF-03882845 in elevating urinary Na+/K+ in Sprague-Dawley rats was within 3-fold of its in vitro functional potency. The effect of MR antagonism on urinary Na+/K+ was not sustained chronically; thus we studied the effect of the compounds on plasma aldosterone following chronic dosing in SHR. Modeling of drug exposure-response data for both eplerenone and PF-03882845 yielded EC50 values that were within 2-fold of that estimated from modeling of drug exposure with changes in urinary sodium and potassium excretion. Importantly, similar unbound concentrations of eplerenone in humans and SHR rats yielded the same magnitude of elevations in aldosterone, indicating a good translatability from rat to human. CONCLUSIONS: Urinary Na+/K+ and plasma aldosterone appear to be translatable biomarkers of MR antagonism following administration of single or multiple doses of compound, respectively. TRIAL REGISTRATION: For clinical study reference EE3-96-02-004, this study was completed in 1996 and falls out scope for disclosure requirements. Clinical study reference A6141115: http://clinicaltrials.gov, http://NIHclinicaltrails.gov; NCTID: NCT00990223.