Functional and Structural Characterization of Clinical-Stage Janus Kinase 2 Inhibitors Identifies Determinants for Drug Selectivity.

Janus kinase 2 (JAK2) plays a critical role in orchestrating hematopoiesis, and its deregulation leads to various blood disorders, most importantly myeloproliferative neoplasms (MPNs). Ruxolitinib, fedratinib, momelotinib, and pacritinib are FDA-/EMA-approved JAK inhibitors effective in relieving symptoms in MPN patients but show variable clinical profiles due to poor JAK selectivity. The development of next-generation JAK2 inhibitors is hampered by the lack of comparative functional analysis and knowledge of the molecular basis of their selectivity. Here, we provide mechanistic profiling of the four approved and six clinical-stage JAK2 inhibitors and connect selectivity data with high-resolution structural and thermodynamic analyses. All of the JAK inhibitors potently inhibited JAK2 activity. Inhibitors differed in their JAK isoform selectivity and potency for erythropoietin signaling, but their general cytokine inhibition signatures in blood cells were comparable. Structural data indicate that high potency and moderate JAK2 selectivity can be obtained by targeting the front pocket of the adenosine 5'-triphosphate-binding site.

Molecular basis of JAK2 activation in erythropoietin receptor and pathogenic JAK2 signaling.

Janus kinase 2 (JAK2) mediates type I/II cytokine receptor signaling, but JAK2 is also activated by somatic mutations that cause hematological malignancies by mechanisms that are still incompletely understood. Quantitative superresolution microscopy (qSMLM) showed that erythropoietin receptor (EpoR) exists as monomers and dimerizes upon Epo stimulation or through the predominant JAK2 pseudokinase domain mutations (V617F, K539L, and R683S). Crystallographic analysis complemented by kinase activity analysis and atomic-level simulations revealed distinct pseudokinase dimer interfaces and activation mechanisms for the mutants: JAK V617F activity is driven by dimerization, K539L involves both increased receptor dimerization and kinase activity, and R683S prevents autoinhibition and increases catalytic activity and drives JAK2 equilibrium toward activation state through a wild-type dimer interface. Artificial intelligence-guided modeling and simulations revealed that the pseudokinase mutations cause differences in the pathogenic full-length JAK2 dimers, particularly in the FERM-SH2 domains. A detailed molecular understanding of mutation-driven JAK2 hyperactivation may enable novel therapeutic approaches to selectively target pathogenic JAK2 signaling.

Differences in JAK Isoform Selectivity Among Different Types of JAK Inhibitors Evaluated for Rheumatic Diseases Through In Vitro Profiling.

OBJECTIVE: The selectivity of JAK inhibitors (Jakinibs) forms the basis for understanding their clinical characteristics; however, evaluation of selectivity is hampered by the lack of comprehensive head-to-head studies. Our objective was to profile in parallel Jakinibs indicated or evaluated for rheumatic diseases for their JAK and cytokine selectivity in vitro. METHODS: We analyzed 10 Jakinibs for JAK isoform selectivity by assaying their inhibition of JAK kinase activity, binding to kinase and pseudokinase domains, and inhibition of cytokine signaling using blood samples from healthy volunteers and using isolated peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis and from healthy donors. RESULTS: Pan-Jakinibs effectively suppressed kinase activity of 2 to 3 JAK family members, whereas isoform-targeted Jakinibs possessed varying degrees of selectivity for 1 or 2 JAK family members. In human leukocytes, Jakinibs predominantly inhibited the JAK1-dependent cytokines interleukin-2 (IL-2), IL-6, and interferons (IFNs). In PBMCs from patients with rheumatoid arthritis compared with healthy controls, inhibition of these cytokines was more pronounced, and some cell-type and STAT isoform differences were observed. Novel Jakinibs demonstrated high selectivity: the covalent Jakinib ritlecitinib showed 900- to 2,500-fold selectivity for JAK3 over other JAKs and specific suppression of IL-2-signaling, whereas the allosteric TYK2 inhibitor deucravacitinib inhibited IFNα signaling with high specificity. Interestingly, deucravacitinib targeted the regulatory pseudokinase domain and did not affect JAK in vitro kinase activity. CONCLUSION: Inhibition of JAK kinase activity did not directly translate into cellular inhibition of JAK/STAT signaling. Despite differences in JAK selectivity, the cytokine inhibition profiles of currently approved Jakinibs were highly similar, with preference for JAK1-mediated cytokines. Novel types of Jakinibs showed narrow cytokine inhibition profile specific for JAK3- or TYK2-mediated signaling.

The RNA-binding protein Snd1/Tudor-SN regulates hypoxia-responsive gene expression.

Snd1 is an evolutionarily conserved RNA-binding protein implicated in several regulatory processes in gene expression including activation of transcription, mRNA splicing, and microRNA decay. Here, we have investigated the outcome of Snd1 gene deletion in the mouse. The knockout mice are viable showing no gross abnormalities apart from decreased fertility, organ and body size, and decreased number of myeloid cells concomitant with decreased expression of granule protein genes. Deletion of Snd1 affected the expression of relatively small number of genes in spleen and liver. However, mRNA expression changes in the knockout mouse liver showed high similarity to expression profile in adaptation to hypoxia. MicroRNA expression in liver showed upregulation of the hypoxia-induced microRNAs miR-96 and -182. Similar to Snd1 deletion, mimics of miR-96/182 enhanced hypoxia-responsive reporter activity. To further elucidate the function of SND1, BioID biotin proximity ligation assay was performed in HEK-293T cells to identify interacting proteins. Over 50% of the identified interactors were RNA-binding proteins, including stress granule proteins. Taken together, our results show that in normal growth conditions, Snd1 is not a critical factor for mRNA transcription in the mouse, and describe a function for Snd1 in hypoxia adaptation through negatively regulating hypoxia-related miRNAs and hypoxia-induced transcription consistent with a role as stress response regulator.

Development of an enzyme-coupled activity assay for Janus kinase 2 inhibitor screening.

JAK2 transmits signals of several important cytokines, such as growth hormone and erythropoietin. The interest toward the therapeutic targeting of JAK2 was boosted in 2005, when the somatic JAK2 V617F mutation, responsible for the majority of myeloproliferative neoplasms (MPNs) was discovered. JAK2 inhibitors have been approved for MPN therapy and they are effective in alleviating symptoms and improving the quality of life of the patients, but they do not lead to molecular remission. This calls for the discovery of new compounds for JAK2-targeted therapeutic approaches. Here we describe the development of a fluorescence-based activity assay for the screening of versatile inhibitor types against JAK2. The assay was utilized to screen a diverse set of small molecule weight natural products and the assay performance was compared to that of differential scanning fluorimetry. We identified 37 hits and further analysis of the most potent hits revealed that most of them displayed non-ATP competitive binding modes. The hits were profiled against other JAK family members and showed distinctive selectivity profiles. The developed assay is consistent, simple and inexpensive to use, and can be utilized for inhibitor screening of diverse compound classes against all JAK family members.

Identification of Novel Small Molecule Ligands for JAK2 Pseudokinase Domain.

Hyperactive mutation V617F in the JAK2 regulatory pseudokinase domain (JH2) is prevalent in patients with myeloproliferative neoplasms. Here, we identified novel small molecules that target JH2 of JAK2 V617F and characterized binding via biochemical and structural approaches. Screening of 107,600 small molecules resulted in identification of 55 binders to the ATP-binding pocket of recombinant JAK2 JH2 V617F protein at a low hit rate of 0.05%, which indicates unique structural characteristics of the JAK2 JH2 ATP-binding pocket. Selected hits and structural analogs were further assessed for binding to JH2 and JH1 (kinase) domains of JAK family members (JAK1-3, TYK2) and for effects on MPN model cell viability. Crystal structures were determined with JAK2 JH2 wild-type and V617F. The JH2-selective binders were identified in diaminotriazole, diaminotriazine, and phenylpyrazolo-pyrimidone chemical entities, but they showed low-affinity, and no inhibition of MPN cells was detected, while compounds binding to both JAK2 JH1 and JH2 domains inhibited MPN cell viability. X-ray crystal structures of protein-ligand complexes indicated generally similar binding modes between the ligands and V617F or wild-type JAK2. Ligands of JAK2 JH2 V617F are applicable as probes in JAK-STAT research, and SAR optimization combined with structural insights may yield higher-affinity inhibitors with biological activity.

Inhibition of RNA Binding in SND1 Increases the Levels of miR-1-3p and Sensitizes Cancer Cells to Navitoclax.

SND1 is an RNA-binding protein overexpressed in large variety of cancers. SND1 has been proposed to enhance stress tolerance in cancer cells, but the molecular mechanisms are still poorly understood. We analyzed the expression of 372 miRNAs in the colon carcinoma cell line and show that SND1 silencing increases the expression levels of several tumor suppressor miRNAs. Furthermore, SND1 knockdown showed synergetic effects with cancer drugs through MEK-ERK and Bcl-2 family-related apoptotic pathways. To explore whether the SND1-mediated RNA binding/degradation is responsible for the observed effect, we developed a screening assay to identify small molecules that inhibit the RNA-binding function of SND1. The screen identified P2X purinoreceptor antagonists as the most potent inhibitors. Validation confirmed that the best hit, suramin, inhibits the RNA binding ability of SND1. The binding characteristics and mode of suramin to SND1 were characterized biophysically and by molecular docking that identified positively charged binding cavities in Staphylococcus nuclease domains. Importantly, suramin-mediated inhibition of RNA binding increased the expression of miR-1-3p, and enhanced sensitivity of cancer cells to Bcl-2 inhibitor navitoclax treatment. Taken together, we demonstrate as proof-of-concept a mechanism and an inhibitor compound for SND1 regulation of the survival of cancer cells through tumor suppressor miRNAs.

Janus Kinases in Leukemia.

Janus kinases (JAKs) transduce signals from dozens of extracellular cytokines and function as critical regulators of cell growth, differentiation, gene expression, and immune responses. Deregulation of JAK/STAT signaling is a central component in several human diseases including various types of leukemia and other malignancies and autoimmune diseases. Different types of leukemia harbor genomic aberrations in all four JAKs (JAK1, JAK2, JAK3, and TYK2), most of which are activating somatic mutations and less frequently translocations resulting in constitutively active JAK fusion proteins. JAKs have become important therapeutic targets and currently, six JAK inhibitors have been approved by the FDA for the treatment of both autoimmune diseases and hematological malignancies. However, the efficacy of the current drugs is not optimal and the full potential of JAK modulators in leukemia is yet to be harnessed. This review discusses the deregulation of JAK-STAT signaling that underlie the pathogenesis of leukemia, i.e., mutations and other mechanisms causing hyperactive cytokine signaling, as well as JAK inhibitors used in clinic and under clinical development.

Selective JAKinibs: Prospects in Inflammatory and Autoimmune Diseases.

Cytokines, many of which signal through the JAK-STAT (Janus kinase-Signal Transducers and Activators of Transcription) pathway, play a central role in the pathogenesis of inflammatory and autoimmune diseases. Currently three JAK inhibitors have been approved for clinical use in USA and/or Europe: tofacitinib for rheumatoid arthritis, psoriatic arthritis and ulcerative colitis, baricitinib for rheumatoid arthritis, and ruxolitinib for myeloproliferative neoplasms. The clinical JAK inhibitors target multiple JAKs at high potency and current research has focused on more selective JAK inhibitors, almost a dozen of which currently are being evaluated in clinical trials. In this narrative review, we summarize the status of the pan-JAK and selective JAK inhibitors approved or in clinical trials, and discuss the rationale for selective targeting of JAKs in inflammatory and autoimmune diseases.

Characterization of JAK1 Pseudokinase Domain in Cytokine Signaling.

The Janus kinase-signal transducer and activator of transcription protein (JAK-STAT) pathway mediates essential biological functions from immune responses to haematopoiesis. Deregulated JAK-STAT signaling causes myeloproliferative neoplasms, leukaemia, and lymphomas, as well as autoimmune diseases. Thereby JAKs have gained significant relevance as therapeutic targets. However, there is still a clinical need for better JAK inhibitors and novel strategies targeting regions outside the conserved kinase domain have gained interest. In-depth knowledge about the molecular details of JAK activation is required. For example, whether the function and regulation between receptors is conserved remains an open question. We used JAK-deficient cell-lines and structure-based mutagenesis to study the function of JAK1 and its pseudokinase domain (JH2) in cytokine signaling pathways that employ JAK1 with different JAK heterodimerization partner. In interleukin-2 (IL-2)-induced STAT5 activation JAK1 was dominant over JAK3 but in interferon-γ (IFNγ) and interferon-α (IFNα) signaling both JAK1 and heteromeric partner JAK2 or TYK2 were both indispensable for STAT1 activation. Moreover, IL-2 signaling was strictly dependent on both JAK1 JH1 and JH2 but in IFNγ signaling JAK1 JH2 rather than kinase activity was required for STAT1 activation. To investigate the regulatory function, we focused on two allosteric regions in JAK1 JH2, the ATP-binding pocket and the αC-helix. Mutating L633 at the αC reduced basal and cytokine induced activation of STAT in both JAK1 wild-type (WT) and constitutively activated mutant backgrounds. Moreover, biochemical characterization and comparison of JH2s let us depict differences in the JH2 ATP-binding and strengthen the hypothesis that de-stabilization of the domain disturbs the regulatory JH1-JH2 interaction. Collectively, our results bring mechanistic understanding about the function of JAK1 in different receptor complexes that likely have relevance for the design of specific JAK modulators.

Structural basis for DNA break recognition by ARTD2/PARP2.

Human ARTD2 (or PARP2) is an ADP-ribosyltransferase, which is catalytically activated by binding to damaged DNA. ARTD2 subsequently ADP-ribosylates itself and other proteins, initiating a cascade of events leading to DNA repair. In contrast to ARTD1, the founding member of the enzyme family, ARTD2 does not have specialized zinc-fingers for detecting DNA damage. The domain organization of ARTD2 includes disordered N-terminus, WGR and catalytic domains. However, the N-terminus of ARTD2 is not strictly required for the DNA dependent activity. While it is known that ARTD2 requires the WGR domain for efficient DNA binding and subsequent catalytic activation, the mechanism of DNA damage detection and subsequent catalytic activation are not completely understood. Here, we report crystal structures of ARTD2 WGR domain bound to double-strand break mimicking DNA oligonucleotides. Notably, the crystal structures revealed DNA binding mode of ARTD2 involving DNA end to end interaction. Structures demonstrate how ARTD2 recognizes nicked DNA, how it interacts with the 5'-phosphate group, and how it can mediate joining of DNA ends in vitro. Extensive mutagenesis of the ARTD2-DNA interface combined with activity, binding, and stoichiometry measurements demonstrate that the WGR domain is the key for DNA break detection.

Small-Molecule Screening Assay for Mono-ADP-Ribosyltransferases.

Mono-ADP-ribosyltransferases of the PARP/ARTD enzyme family are enzymes catalyzing the transfer of a single ADP-ribose unit to target proteins. The enzymes have various roles in vital cellular processes such as DNA repair and transcription, and many of the enzymes are linked to cancer-relevant functions. Thus inhibition of the enzymes is a potential way to discover and develop new drugs against cancer. Here we describe an activity-based screening assay for mono-ADP-ribosyltransferases. The assay utilizes the natural substrate of the enzymes, NAD+, and it is based on chemically converting the leftover substrate to a fluorophore and measuring its relative concentration after the enzymatic reaction. The assay is homogenous, robust, and cost-effective and, most importantly, applicable to mono-ADP-ribosyltransferases as well as poly-ADP-ribosyltransferases for screening of small-molecule inhibitors against the enzymes.

2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors.

Tankyrases (TNKSs) are enzymes specialized in catalyzing poly-ADP-ribosylation of target proteins. Several studies have validated TNKSs as anti-cancer drug targets due to their regulatory role in Wnt/ß-catenin pathway. Recently a lot of effort has been put into developing more potent and selective TNKS inhibitors and optimizing them towards anti-cancer agents. We noticed that some 2-phenylquinazolinones (2-PQs) reported as CDK9 inhibitors were similar to previously published TNKS inhibitors. In this study, we profiled this series of 2-PQs against TNKS and selected kinases that are involved in the Wnt/ß-catenin pathway. We found that they were much more potent TNKS inhibitors than they were CDK9/kinase inhibitors. We evaluated the compound selectivity to tankyrases over the ARTD enzyme family and solved co-crystal structures of the compounds with TNKS2. Comparative structure-based studies of the catalytic domain of TNKS2 with selected CDK9 inhibitors and docking studies of the inhibitors with two kinases (CDK9 and Akt) revealed important structural features, which could explain the selectivity of the compounds towards either tankyrases or kinases. We also discovered a compound, which was able to inhibit tankyrases, CDK9 and Akt kinases with equal µM potency.

Development of an Inhibitor Screening Assay for Mono-ADP-Ribosyl Hydrolyzing Macrodomains Using AlphaScreen Technology.

Protein mono-ADP-ribosylation is a posttranslational modification involved in the regulation of several cellular signaling pathways. Cellular ADP-ribosylation is regulated by ADP-ribose hydrolases via a hydrolysis of the protein-linked ADP-ribose. Most of the ADP-ribose hydrolases share a macrodomain fold. Macrodomains have been linked to several diseases, such as cancer, but their cellular roles are mostly unknown. Currently, there are no inhibitors available targeting the mono-ADP-ribose hydrolyzing macrodomains. We have developed a robust AlphaScreen assay for the screening of inhibitors against macrodomains having mono-ADP-ribose hydrolysis activity. We utilized this assay for validatory screening against human MacroD1 and identified five compounds inhibiting the macrodomain. Dose-response measurements and an orthogonal assay further validated four of these compounds as MacroD1 inhibitors. The developed assay is homogenous, easy to execute, and suitable for the screening of large compound libraries. The assay principle can also be adapted for other ADP-ribose hydrolyzing macrodomains, which can utilize a biotin-mono-ADP-ribosylated protein as a substrate.

Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite responsible for African trypanosomiasis or sleeping sickness. It contains a single PARP enzyme opposed to many higher eukaryotes, which have numerous PARPs. PARPs are responsible for a post-translational modification, ADP-ribosylation, regulating a multitude of cellular events. T. brucei PARP, like human PARPs-1-3, is activated by DNA binding and it potentially functions in DNA repair processes. Here we characterized activation requirements, structure and subcellular localization of T. brucei PARP. T. brucei PARP was found to be selectively activated by 5' phosphorylated and 3' phosphorylated DNA breaks. Importantly, the N-terminal region is responsible for high-affinity DNA-binding and required for DNA-dependent enzymatic activation. This module is also required for nuclear localization of the protein in response to oxidative stress. Solution structures of activating and non-activating PARP-DNA complexes were determined with small-angle X-ray scattering revealing distinct differences in their DNA-binding modes.

Highly Potent and Isoform Selective Dual Site Binding Tankyrase/Wnt Signaling Inhibitors That Increase Cellular Glucose Uptake and Have Antiproliferative Activity.

Compounds 13 and 14 were evaluated against 11 PARP isoforms to reveal that both 13 and 14 were more potent and isoform selective toward inhibiting tankyrases (TNKSs) than the "standard" inhibitor 1 (XAV939)5, i.e., IC50 = 100 pM vs TNKS2 and IC50 = 6.5 µM vs PARP1 for 14. In cellular assays, 13 and 14 inhibited Wnt-signaling, enhanced insulin-stimulated glucose uptake, and inhibited the proliferation of DLD-1 colorectal adenocarcinoma cells to a greater extent than 1.

Characterization of the DNA dependent activation of human ARTD2/PARP2.

Human ADP-ribosyltransferase 2 (ARTD2/PARP2) is an enzyme catalyzing a post-translational modification, ADP-ribosylation. It is one of the three DNA dependent ARTDs in the 17 member enzyme family. ADP-ribosylation catalyzed by ARTD2 is involved in the regulation of multiple cellular processes such as control of chromatin remodeling, transcription and DNA repair. Here we used a combination of biochemical and biophysical methods to elucidate the structure and function of ARTD2. The solution structures revealed the binding mode of the ARTD2 monomer and dimer to oligonucleotides that mimic damaged DNA. In the complex, DNA binds between the WGR domain and the catalytic fragment. The binding mode is supported by biophysical data that indicate all domains contribute to DNA binding. Also, our study showed that ARTD2 is preferentially activated by short 5'-phosphorylated DNA oligonucleotides. We demonstrate that the N-terminus functions as a high-affinity DNA-binding module, while the WGR domain contributes to DNA binding specificity and subsequent catalytic activation. Our data further suggest that ARTD2 would function in double strand break repair as a dimeric module, while in single strand break repair it would function as a monomer.

Small-Molecule Chemical Probe Rescues Cells from Mono-ADP-Ribosyltransferase ARTD10/PARP10-Induced Apoptosis and Sensitizes Cancer Cells to DNA Damage.

Members of the human diphtheria toxin-like ADP-ribosyltransferase (ARTD or PARP) family play important roles in regulating biological activities by mediating either a mono-ADP-ribosylation (MARylation) of a substrate or a poly-ADP-ribosylation (PARylation). ARTD10/PARP10 belongs to the MARylating ARTDs (mARTDs) subfamily, and plays important roles in biological processes that range from cellular signaling, DNA repair, and cell proliferation to immune response. Despite their biological and disease relevance, no selective inhibitors for mARTDs are available. Here we describe a small-molecule ARTD10 inhibitor, OUL35, a selective and potent inhibitor for this enzyme. We characterize its selectivity profile, model its binding, and demonstrate activity in HeLa cells where OUL35 rescued cells from ARTD10 induced cell death. Using OUL35 as a cell biology tool we show that ARTD10 inhibition sensitizes the cells to the hydroxyurea-induced genotoxic stress. Our study supports the proposed role of ARTD10 in DNA-damage repair and provides a tool compound for selective inhibition of ARTD10-mediated MARylation.

Structure-activity relationships of 2-arylquinazolin-4-ones as highly selective and potent inhibitors of the tankyrases.

Tankyrases (TNKSs), members of the PARP (Poly(ADP-ribose)polymerases) superfamily of enzymes, have gained interest as therapeutic drug targets, especially as they are involved in the regulation of Wnt signalling. A series of 2-arylquinazolin-4-ones with varying substituents at the 8-position was synthesised. An 8-methyl group (compared to 8-H, 8-OMe, 8-OH), together with a 4'-hydrophobic or electron-withdrawing group, provided the most potency and selectivity towards TNKSs. Co-crystal structures of selected compounds with TNKS-2 revealed that the protein around the 8-position is more hydrophobic in TNKS-2 compared to PARP-1/2, rationalising the selectivity. The NAD(+)-binding site contains a hydrophobic cavity which accommodates the 2-aryl group; in TNKS-2, this has a tunnel to the exterior but the cavity is closed in PARP-1. 8-Methyl-2-(4-trifluoromethylphenyl)quinazolin-4-one was identified as a potent and selective inhibitor of TNKSs and Wnt signalling. This compound and analogues could serve as molecular probes to study proliferative signalling and for development of inhibitors of TNKSs as drugs.