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Letter

Selective Downregulation of JAK2 and JAK3 by an ATP-Competitive pan-JAK Inhibitor S. Denise Field, Jacob Arkin, Jing Li, and Lyn H. Jones ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00116 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

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ACS Chemical Biology

[PF-956980] JAK2 JAK3

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Selective Downregulation of JAK2 and JAK3 by an ATP-Competitive pan-JAK Inhibitor S. Denise Field,‡ Jacob Arkin,‡ Jing Li‡* and Lyn H. Jones‡* ‡

Medicine Design, Pfizer, 610 Main Street, Cambridge, MA, 02139, USA

PF-956980 has been used previously as a JAK3-selective chemical probe in numerous cell-based experiments. Here, we report that not only is PF-956980 a pan-JAK ATP-competitive inhibitor, but it also causes selective reduction of endogenous JAK2 and JAK3 protein levels in human primary immune cells (in a time-dependent manner), leaving the other JAK family members (JAK1 and TYK2) unchanged. We found that PF-956980 selectively downregulated JAK2 and JAK3 mRNA, corresponding to changes observed at the protein level. This work highlights therapeutic opportunities for the development of pharmacological inhibitors that also modulate the expression of their cognate binding proteins.

The cellular functions of proteins are often studied using genetic methods, such as knock-out (KO) or knock-down (KD) strategies that include the application of RNA interference (RNAi) or antisense oligonucleotides. Discrepancies between pharmacological modulation and KO/KD experiments are not uncommon because protein-protein interactions (and potentially important scaffolding functions) are eliminated following protein depletion, but may be retained with


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pharmacological modulation. As a result, the functional efficacy of a small molecule ligand does not always match genetic manipulation of the same target protein. Therefore, ligands that are able to downregulate binding proteins may possess a different therapeutic efficacy profile, but with the potential for improved delivery and safety as compared to oligonucleotide-based approaches. A number of technologies have emerged to enable target protein degradation by small molecule conjugates. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that contain an E3 ligase-binding moiety tethered to a ligand for the protein targeted for degradation.1 The resulting PROTAC-induced proximity causes ubiquitination and subsequent proteasomal degradation of the target. Ligands that are tagged with hydrophobic groups may also cause target protein degradation. Protein ligands conjugated to adamantyl groups are believed to mimic a partially denatured protein state, and can result in chaperonemediated proteasomal degradation.2-3 Tris tert-butyl carbamate (Boc3)-arginine hydrophobic conjugates can induce degradation by localizing target proteins to the 20S proteasome.4 Although these approaches hold significant promise, they currently face considerable challenges as therapeutic modalities. For example, PROTAC and hydrophobically-tagged conjugates possess a lower propensity for oral bioavailability (and brain penetration) due to their tendency towards higher molecular weight and lipophilicity.5-6 The rational design of small molecule degraders is also complicated by the need to satisfy complex bifunctional structureactivity relationships. However, some recent progress has been made to address these issues,7-9 including an approach that relies on the self-assembly of the individual precursor ligands in-cell using click chemistry.6


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Interestingly, there are some reported examples of ATP-site kinase inhibitors that induce degradation of their cognate binding proteins without the need for conjugation to an E3 ligase recruiting group. Inhibitors of EGFR,10 B-RAF,10 ErbB2,10-11 BTK,12 LRRK2,13-17 MELK,18 CHK119 and SK120 have been shown recently to trigger proteasome-dependent degradation of their respective kinase targets. Chemical probes may also transcriptionally regulate genes that correlate to binding proteins of the ligand under investigation, although such molecules often induce changes in the expression of multiple genes (e.g. bromodomain and histone deacetylase inhibitors)21-22 potentially resulting in the modulation of a myriad of phenotypic effects. Orallybioavailable pharmacological agents that selectively downregulate binding protein levels would thus be of considerable interest from the perspective of drug discovery. Janus kinases (JAKs) are intracellular tyrosine kinases that are activated by cytokine binding to cell surface receptors. Signal transducer and activator of transcription (STAT) proteins are subsequently phosphorylated and translocate to the nucleus to induce the transcription of target genes. Aberrant JAK-STAT signaling is involved in many immune-related disorders and cancers and small molecule JAK inhibitors have demonstrated therapeutic utility.23 Indeed, many selective inhibitors of the JAK family (JAK1, JAK2, JAK3 and TYK2) have been developed,24 although the possibility of such a ligand mediating JAK downregulation has not been investigated thus far. A previous study25 had shown that the promiscuous and very weak kinase inhibitor AG49026 caused a reduction in the protein levels of JAK1 and JAK2 in colorectal cancer cell lines, albeit at very high concentrations (100 and 150 µM). However, AG490 is a poor JAK chemical probe, despite its (inappropriate) widespread use as a JAK inhibitor.26 Similarly, the promiscuous kinase inhibitor staurosporine has been shown to regulate JAK gene


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expression.27 We were therefore interested to explore potential JAK downregulation by a selective ATP-competitive JAK inhibitor. PF-956980 (Figure 1a) was previously described as a selective JAK3 inhibitor, although the activity of the compound against JAK1, JAK2 and TYK2 was not reported.28 However, due to the reported kinase selectivity profile of PF-956980,28 we chose this compound as an appropriate chemical probe to explore potential JAK regulation. Surprisingly, we discovered that the original structure associated with “PF-956980” in the Chemical Abstracts Service (CAS) registry was incorrect. Following our request, CAS has subsequently updated the structure and registry number for the commercially available hydrate form of PF-956980 (CAS 1262750-62-8). Therefore, care should be taken if ordering this compound from a commercial vendor to ensure it is the correct material.

Figure 1. a) Structure of the pan-JAK inhibitor PF-956980. b) Broad kinome selectivity profile of PF-956980 (Invitrogen). The selectivity profile of PF-956980 was confirmed in a broader panel of 38 kinases (Figure 1b, S1 and Table S1) and a wide-ligand profile of 65 proteins at CEREP (Figure S2). JAK3 is the only protein inhibited to a significant extent at 1 µM, although MARK1, LCK and PDE4 appear to be targets of this compound at higher concentrations (Figures S1 and S2, Table S1). Due to the


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similarity of the ATP-sites of the JAK family of kinases (particularly when bound to kinase inhibitors),29-30 PF-956980 potency was determined against JAK1/2/3 and TYK2. PF-956980 was found to be a pan-JAK inhibitor, with modest selectivity over TYK2 within the family (Table 1). This is in itself an important result, since PF-956980 has been used several times as a selective JAK3 inhibitor in cell-based experiments.31-37 Table 1. JAK selectivity profile of PF-956980a Km ATP

1 mM ATP

JAK1 IC50/nM

7.5 (5.6-11.7; n = 7)

38 (23.9-49.0; n = 5)

JAK2 IC50/nM

7.1 (5.3-15.0; n = 6)

124 (92.1-156; n = 5)

JAK3 IC50/nM

2.8 (0.9-4.6; n = 6)

139 (89.2-176; n = 6)

TYK2 IC50/nM

128 (50.6-311; n = 7)

1632 (1118-2327; n = 5)

a

Geomean data for at least n = 5

Human peripheral blood mononuclear cells (PBMCs) are immune cells often used in immunological studies, including investigations into JAK-STAT signaling.38 For that reason, PBMCs were chosen as a physiologically-relevant cell-based system to explore the potential for small molecule-mediated changes in endogenous JAK protein levels. Following 4 hours incubation of PBMCs with PF-956980 there appeared to be no change in JAK levels by Western blot (Figures 2a, S3-S6). However, following 22 hours incubation, a significant reduction in JAK2 and JAK3 protein levels was observed, even at concentrations as low as 100 nM (Figures 2a, S3-S6). The specificity of this effect was also demonstrated against representative kinases not inhibited by PF-956980 (BTK, ERK2 and p38) – these kinases maintained their levels in PBMCs, even at high concentrations of the inhibitor (Figure 2b, S4-S6).


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Figure 2. a) PF-956980 selectively reduced the protein levels of JAK2 and JAK3 in PBMCs after 22 hours incubation (boxes) and at low concentrations. b) PF-956980 had no effect on the total protein levels of representative non-binding kinases BTK, ERK2 and p38 in PBMCs. c) JAK2 and JAK3 mRNA levels (determined by qPCR, relative to DMSO) were selectively reduced following treatment of PBMCs with PF-956980 for 22 hours. We next explored the mechanism of small molecule-induced reduction of JAK2 and JAK3 levels to ascertain if PF-956980 reduced protein synthesis or increased protein degradation. PBMCs were treated with the proteasome inhibitors MG132 or carfilzomib and PF-956980. Unfortunately, the proteasome inhibitors were found to reduce the levels of JAK2 and JAK3 in the absence of PF-956980 (Figure S7) and therefore this method could not be used to understand if protein reductions were mediated by proteasomal degradation. The rate of degradation of JAK2 and JAK3 did not appear to be significantly different with or without PF-956980 in the presence of the protein synthesis inhibitor cycloheximide, (Figure S8, S9), indicative of a mechanism that modulates protein synthesis. As a result, we measured changes in mRNA levels by qPCR which showed that PF-956980 caused a significant reduction in JAK2 and JAK3 gene expression, but had no effect on JAK1 and TYK2, corresponding to the observed changes in protein levels (Figure 2c and Supporting Information). The lack of an effect on JAK1 and TYK2


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is intriguing since the ATP-sites of both kinases were fully engaged by PF-956980 in PBMCs, particularly at the higher concentrations used in the study. These effects are either ‘onmechanism’ i.e. JAK-STAT signaling directly regulates JAK2 and JAK3 transcription (but not JAK1/TYK2), or they are the consequence of off-target pharmacology. Since certain JAK inhibitors are also known to inhibit the epigenetic reader domain of BRD4,39-41 and are thus likely to modulate the expression of many genes, we screened PF-956980 for BRD4 inhibition. However, PF-956980 was found to be essentially inactive in a fluorescence polarization BRD4 binding assay42 (4.8% inhibition at 32 µM). Broad proteomic and transcriptomic profiling of PF956980 will be required to further elucidate the off-target and network pharmacology43 of this molecule. Nevertheless, irrespective of any additional proteins that may be regulated by PF956980, it should be noted that the compound does not possess cytotoxicity in PBMCs (by trypan blue exclusion),44 the immortalized human liver epithelial (THLE) or liver carcinoma HepG2 cell-based assays (IC50 > 300 µM)45 suggesting further therapeutic potential for this modality. Regardless of the underlying mechanisms at play, our observations highlight a new aspect of kinase inhibitor selectivity i.e. differential transcriptional modulation. Such effects should be assessed during the course of a medicinal chemistry optimization program to ensure all relevant pharmacological and selectivity profiles are appropriately considered. The temporal components of JAK3 kinase activity were recently investigated using a JAK3 inhibitor.46 This study identified a second wave of IL-2 receptor signaling in CD4+ T cells that is highly sensitive to the inhibition of JAK3. Our work describes another interesting pharmacological feature of a selective JAK inhibitor in human immune cells that is also time-


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dependent. Future work in our group will explore the effects of cytokine activation on JAK downregulation caused by PF-956980. PF-956980 inhibited the delayed-type hypersensitivity swelling response in a mouse model of inflammation at a dose of 5 mg/Kg and an exposure of 30 ng/mL (88 nM).28 The reductions in JAK2/3 levels were therefore observed at concentrations in our study which are relevant to the anti-inflammatory effects of PF-956980 in vivo. We believe it is entirely possible that certain JAK inhibitors could derive a component of their clinical phenotypic effects by causing a reduction in target protein levels. Further work is warranted to study these effects in vivo and in different cell lines. In summary, a previously reported JAK3 chemical probe, PF-956980, was found to be a panJAK inhibitor that additionally downregulated JAK2 and JAK3. Important scaffolding functions of the JAK enzymes have been described,47 and as a result, inhibitors that also reduce protein levels may have additional functional effects. Consequently, small molecules that modulate both canonical (kinase-dependent) and non-canonical (kinase-independent) JAK signaling could possess enhanced therapeutic efficacy.48 For instance, JAK2 kinase inhibitors are of limited utility in the treatment of JAK2-dependent myeloproliferative neoplasms, but combinations with JAK2 protein downregulators (through HDAC49, Hsp9050 or BET bromodomain inhibition51) would be expected to deliver improved efficacy for this disease. This study demonstrates that a selective type I kinase inhibitor, which satisfies the Lipinski’s rule of 5,5 and does not affect cell viability, is able to selectively downregulate therapeutically important binding proteins in human immune cells. As described, certain kinase inhibitors have been reported to induce proteasome-mediated degradation of their cognate binding targets.10-20 Kinases may also regulate the expression of their own genes, as seen for BTK.52 Therefore, since


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multiple mechanisms exist for small molecule-mediated kinase regulation (that may be the result of on- or off-target effects) we recommend that all kinase inhibitors, including chemical probes, drugs, compounds in clinical development and allosteric modulators, are assessed for their ability to change target kinase levels in relevant cell types. More broadly, due to the simplicity of measuring cellular protein levels, these effects should be investigated for all small molecule modalities in order to drive a greater understanding of molecular pharmacology and to potentially unearth new therapeutic opportunities.53

ASSOCIATED CONTENT Supporting Information. Experimental procedures; selectivity profile of PF-956980 (Figures S1 and S2, and Table S1); additional Western blots showing the effects of PF-956980 on protein levels in PBMCs (Figures S3-S6); effects of MG132 or carfilzomib plus PF-956980 on JAK2 and JAK3 levels (Figure S7); effects of cycloheximide plus PF-956980 on JAK2 and JAK3 levels (Figure S8 and S9). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *Email: [email protected] and [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Notes LHJ and JL are full time employees and shareholders of Pfizer. SDF is a Postdoctoral Research Fellow at Pfizer. JA performed research included in this manuscript whilst on a co-op placement at Pfizer from Northeastern University. Research was conducted in accordance with all acceptable Pfizer policies including IRB/IEC approval.

ACKNOWLEDGMENT We thank H. Xu, E. Hett and M. Banker for advice and helpful discussions.

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Selective Downregulation of JAK2 and JAK3 by an ... - ACS Publications

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