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.
Type 2 diabetes (T2D) and its complications can have debilitating, sometimes fatal consequences for afflicted individuals. The disease can be difficult to control, and therapeutic strategies to ...prevent T2D-induced tissue and organ damage are needed. Here we describe the results of administering a potent and selective inhibitor of Protein Kinase C (PKC) family members PKCα and PKCβ, Cmpd 1, in the ZSF1 obese rat model of hyperphagia-induced, obesity-driven T2D. Although our initial intent was to evaluate the effect of PKCα/β inhibition on renal damage in this model setting, Cmpd 1 unexpectedly caused a marked reduction in the hyperphagic response of ZSF1 obese animals. This halted renal function decline but did so indirectly and indistinguishably from a pair feeding comparator group. However, above and beyond this food intake effect, Cmpd 1 lowered overall animal body weights, reduced liver vacuolation, and reduced inguinal adipose tissue (iWAT) mass, inflammation, and adipocyte size. Taken together, Cmpd 1 had strong effects on multiple disease parameters in this obesity-driven rodent model of T2D. Further evaluation for potential translation of PKCα/β inhibition to T2D and obesity in humans is warranted.
A diaryl ketone series was identified as vanin-1 inhibitors from a high-throughput screening campaign. While this novel scaffold provided valuable probe 2 that was used to build target confidence, ...concerns over the ketone moiety led to the replacement of this group. The successful replacement of this moiety was achieved with pyrimidine carboxamides derived from cyclic secondary amines that were extensively characterized using biophysical and crystallographic methods as competitive inhibitors of vanin-1. Through optimization of potency and physicochemical and ADME properties, and guided by co-crystal structures with vanin-1, 3 was identified with a suitable profile for advancement into preclinical development.
Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 ...cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.
Display omitted
•USP18 is the primary negative regulator of Type I interferon signaling•In contrast to mouse USP18, human USP18 possesses weak enzymatic activity•USP18 acts chiefly through its scaffold function•Targeting USP18 scaffold function leads to cancer cell killing
Immunity; Immune response; Cell biology; Cancer
BackgroundUSP18 is a key negative regulator of Type I interferon (IFN) signaling. USP18 cleaves ISG15, a ubiquitin-like modification, through its canonical catalytic function, and directly inhibits ...the Type I IFN receptor through its scaffold role. USP18 loss-of-function dramatically impacts autoimmune disease, viral susceptibility, and cancer cell survival. However, published studies have reached different conclusions as to whether catalytic or scaffold function is essential.MethodsThe ability of USP18 to deconjugate ISG15 was determined using purified protein against a reporter substrate or cell lysates. Scaffolding activity was quantified using STAT1 phosphorylation upon IFN stimulation. After characterizing the impact of specific point mutations on USP18 function, mutations that selectively impair catalytic (USP18 C64S) or scaffold function (USP18 I60N) were evaluated for their effects on cancer cell vulnerability. To assess USP18 mechanism-of-action across multiple cancer cell lineages, guide RNAs that introduce the C64S mutation or knock out USP18 were introduced into cancer cells, and cells were passaged for 2 weeks in the presence or absence of IFN. IFN-dependent changes in wild-type, knockout, or C64S USP18 allelic frequency were assessed by DNA sequencing. To determine the effect of USP18 in vivo, mouse Usp18 was knocked out in CT26 colorectal cancer cells, and cells were implanted subcutaneously in wild-type or immunocompromised (NSG) mice.ResultsDeletion of USP18 in a variety of human cancer cell lines of multiple lineages (blood, breast, colon, lung) rendered them vulnerable to IFN. Furthermore, Usp18 knockout in cancer cells prevented tumor formation in wild-type mice, and resulted in partial tumor growth inhibition in immunocompromised mice. Introducing the C64S mutation fully impaired catalytic activity, yet did not result in IFN sensitivity. Furthermore, preventing ISGylation by knocking out the E1 enzyme required for ISGylation, UBA7, did not rescue IFN sensitivity in USP18-deficient cells. By contrast, the I60N mutation led to partial scaffold impairment and partial cancer cell sensitivity. Finally, human USP18 exhibited minimal catalytic activity, in stark contrast to mouse USP18. Therefore, deISGylase activity of USP18 does not mediate IFN sensitivity in human cancer cells.ConclusionsLoss of USP18 creates a key vulnerability of human cancer cells to IFN. This is primarily due to loss of scaffold function, rather than loss of catalytic function (figure 1). Indeed, human USP18 does not appear to function as an enzyme under physiological conditions, unlike mouse USP18. These findings resolve the mechanistic basis for human USP18 function, paving the way to target USP18 for cancer treatment.Ethics ApprovalAll procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.Abstract 1079 Figure 1The mechanistic basis for human USP18 function and cancer cell vulnerability.
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.
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.
Herein, we report the discovery of novel, proline‐based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of ...1‐(4‐amino‐3‐fluoro‐phenyl)‐1H‐pyridin‐2‐one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.
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 K 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 K 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 K m ATP may provide crucial insights into a compound’s activity and selectivity in cellular contexts.
