A Comparative Study of the Stability, Transport, and Structure-Activity Relationship of Round Scad Derived Peptides with Antineuroinflammatory Ability.

Our previous study identified round scad neuroprotective peptides with different characteristics. However, the intrinsic relationship between their structure and bioactivity, as well as their bioavailability, remains unclear. The aim of this study is to elucidate the bioavailability of these peptides and their structure-activity relationship against neuroinflammation. Results showed that the SR and WCP peptides were resistant to gastrointestinal digestion. Additionally, peptides SR, WCP, and WCPF could transport Caco-2 monolayers as intact peptides. The permeability coefficients (Papp) of SR, WCP, and WCPF in Caco-2 monolayer were (1.53 ± 0.01) × 10-5, (2.12 ± 0.01) × 10-5, and (8.86 ± 0.03) × 10-7 cm/s, respectively. Peptides SR, WCP, and WCPF, as promising inhibitors of JAK2 and STAT3, could attenuate the levels of pro-inflammatory cytokines and regulate the NFκB and JAK2/STAT3 signaling pathway in LPS-treated BV-2 cells. WCPF exerted the highest anti-inflammatory activity. Moreover, bioinformatics, molecular docking, and quantum chemistry studies indicated that the bioactivity of SR was attributed to Arg, whereas those of WCP and WCPF were attributed to Trp. This study supports the application of round-scad peptides and deepens the understanding of the structure-activity relationship of neuroprotective peptides.

Comparison of the crystal structures of the JAK1/2 inhibitor ruxolitinib and its hydrate and phosphate.

Ruxolitinib {RUX; systematic name: (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile, C17H18N6} is an orally bioavailable JAK1/2 inhibitor approved for treating intermediate- or high-risk myelofibrosis (MF) and high-risk polycythemia vera (PV). Recent patents claim that RUX can exist in many different forms, information for which is important for the clinical utilization of RUX, especially for the formulation and bioavailability of the drug. But there has been no detailed study on its forms so far. Herein crystals of RUX and its dihydrate (RUX-2H; C17H18N6·2H2O) and phosphate (RUX-P; systematic name: 4-{1-[(1R)-2-cyano-1-cyclopentylethyl]-1H-pyrazol-4-yl}-7H-pyrrolo[2,3-d]pyrimidin-3-ium dihydrogen phosphate, C17H19N6+·H2PO4-) were prepared successfully and their structures studied in detail for the first time. Our study shows that the three crystals of RUX differ in the orientation of the pyrimidine ring relative to the pyrazole ring of the RUX molecule, and in their hydrogen-bond interactions. The water molecules in RUX-2H and the dihydrogen phosphate anion in RUX-P enrich the hydrogen-bond networks in these forms. The expected proton transfer occurs in RUX phosphate and the protonated N atom is engaged in a charge-assisted hydrogen bond with the counter-anion. Hydrogen-bonding interactions dominate in the crystal packing of the three forms. The detailed conformations and packing of the three forms were compared through the calculation of both Hirshfeld surfaces and fingerprint plots.

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.

A computationally affordable approach for accurate prediction of the binding affinity of JAK2 inhibitors.

Janus kinase 2 (JAK2) inhibitors are potential anticancer drugs in the treatment of lymphoma, leukemia, thrombocytosis and particularly myeloproliferative diseases. However, the resemblance among JAK family members has challenged the identification of highly selective inhibitors for JAK2 to reduce undesired side effects. As a result, a robust search for promising JAK2 inhibitors using a computational approach that can effectively nominate new potential candidates to be further analyzed through laborious experimental operations has become necessary. In this study, the binding affinities of JAK2 inhibitors were rapidly and precisely estimated using the fast pulling of ligand (FPL) simulations combined with a modified linear interaction energy (LIE) method. The approach correlates with the experimental binding affinities of JAK2 inhibitors with a correlation coefficient of R = 0.82 and a root-mean-square error of 0.67 kcal•mol-1. The data reveal that the FPL/LIE method is highly approximate in anticipating the relative binding free energies of known JAK2 inhibitors with an affordable consumption of computational resources, and thus, it is very promising to be applied in in silico screening for new potential JAK2 inhibitors from a large number of molecules available.

Insights into the Potential Mechanisms of JAK2V617F Somatic Mutation Contributing Distinct Phenotypes in Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPN) are a group of blood cancers in which the bone marrow (BM) produces an overabundance of erythrocyte, white blood cells, or platelets. Philadelphia chromosome-negative MPN has three subtypes, including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The over proliferation of blood cells is often associated with somatic mutations, such as JAK2, CALR, and MPL. JAK2V617F is present in 95% of PV and 50-60% of ET and PMF. Based on current molecular dynamics simulations of full JAK2 and the crystal structure of individual domains, it suggests that JAK2 maintains basal activity through self-inhibition, whereas other domains and linkers directly/indirectly enhance this self-inhibited state. Nevertheless, the JAK2V617F mutation is not the only determinant of MPN phenotype, as many normal individuals carry the JAK2V617F mutation without a disease phenotype. Here we review the major MPN phenotypes, JAK-STAT pathways, and mechanisms of development based on structural biology, while also describing the impact of other contributing factors such as gene mutation allele burden, JAK-STAT-related signaling pathways, epigenetic modifications, immune responses, and lifestyle on different MPN phenotypes. The cross-linking of these elements constitutes a complex network of interactions and generates differences in individual and cellular contexts that determine the phenotypic development of MPN.

Structural and functional analysis of target recognition by the lymphocyte adaptor protein LNK.

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.

IL-6 regulates autophagy and chemotherapy resistance by promoting BECN1 phosphorylation.

Extracellular cytokines are enriched in the tumor microenvironment and regulate various important properties of cancers, including autophagy. However, the precise molecular mechanisms underlying the link between autophagy and extracellular cytokines remain to be elucidated. In the present study, we demonstrate that IL-6 activates autophagy through the IL-6/JAK2/BECN1 pathway and promotes chemotherapy resistance in colorectal cancer (CRC). Mechanistically, IL-6 triggers the interaction between JAK2 and BECN1, where JAK2 phosphorylates BECN1 at Y333. We demonstrate that BECN1 Y333 phosphorylation is crucial for BECN1 activation and IL-6-induced autophagy by regulating PI3KC3 complex formation. Furthermore, we investigate BECN1 Y333 phosphorylation as a predictive marker for poor CRC prognosis and chemotherapy resistance. Combination treatment with autophagy inhibitors or pharmacological agents targeting the IL-6/JAK2/BECN1 signaling pathway may represent a potential strategy for CRC cancer therapy.

Structural Insights into the Interaction of Heme with Protein Tyrosine Kinase JAK2*.

Janus kinase 2 (JAK2) is the most important signal-transducing tyrosine kinase in erythropoietic precursor cells. Its malfunction drives several myeloproliferative disorders. Heme is a small metal-ion-carrying molecule that is incorporated into hemoglobin in erythroid precursor cells to transport oxygen. In addition, heme is a signaling molecule and regulator of various biochemical processes. Here, we show that heme exposure leads to hyperphosphorylation of JAK2 in a myeloid cancer cell line. Two peptides identified in JAK2 are heme-regulatory motifs and show low-micromolar affinities for heme. These peptides map to the kinase domain of JAK2, which is essential for downstream signaling. We suggest these motifs to be the interaction sites of heme with JAK2, which drive the heme-induced hyperphosphorylation. The results presented herein could facilitate the development of heme-related pharmacological tools to combat myeloproliferative disorders.

Synthesis, In Silico Prediction and In Vitro Evaluation of Antitumor Activities of Novel Pyrido[2,3-d]pyrimidine, Xanthine and Lumazine Derivatives.

Ethyl 5-arylpyridopyrimidine-6-carboxylates 3a-d were prepared as a one pot three component reaction via the condensation of different aromatic aldehydes and ethyl acetoacetate with 6-amino-1-benzyluracil 1a under reflux condition in ethanol. Additionally, condensation of ethyl 2-(2-hydroxybenzylidene) acetoacetate with 6-amino-1-benzyluracil in DMF afforded 6-acetylpyridopyrimidine-7-one 3e; a facile, operationally, simple and efficient one-pot synthesis of 8-arylxanthines 6a-f is reported by refluxing 5,6-diaminouracil 4 with aromatic aldehydes in DMF. Moreover, 6-aryllumazines 7a-d was obtained via the reaction of 5,6-diaminouracil with the appropriate aromatic aldehydes in triethyl orthoformate under reflux condition. The synthesized compounds were characterized by spectral (1H-NMR, 13C-NMR, IR and mass spectra) and elemental analyses. The newly synthesized compounds were screened for their anticancer activity against lung cancer A549 cell line. Furthermore, a molecular-docking study was employed to determine the possible mode of action of the synthesized compounds against a group of proteins highly implicated in cancer progression, especially lung cancer. Docking results showed that compounds 3b, 6c, 6d, 6e, 7c and 7d were the best potential docked compounds against most of the tested proteins, especially CDK2, Jak2, and DHFR proteins. These results are in agreement with cytotoxicity results, which shed a light on the promising activity of these novel six heterocyclic derivatives for further investigation as potential chemotherapeutics.

The Natural Compound Notopterol Binds and Targets JAK2/3 to Ameliorate Inflammation and Arthritis.

The traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang has anti-rheumatism activity, and a mass spectrometry assay of patients' serum after administration of the herb revealed that notopterol is the most abundant component enriched. However, the functions of notopterol and its molecular target in rheumatoid arthritis (RA) treatment remain unknown. Here, we show in different RA mouse strains that both oral and intraperitoneal administration of notopterol result in significant therapeutic effects. Mechanistically, notopterol directly binds Janus kinase (JAK)2 and JAK3 kinase domains to inhibit JAK/signal transducers and activators of transcription (JAK-STAT) activation, leading to reduced production of inflammatory cytokines and chemokines. Critically, combination therapy using both notopterol and tumor necrosis factor (TNF) blocker results in enhanced therapeutic effects compared to using TNF blocker alone. We demonstrate that notopterol ameliorates RA pathology by targeting JAK-STAT signaling, raising the possibility that notopterol could be effective in treating other diseases characterized by aberrant JAK-STAT signaling pathway.

Fraxetin inhibits the growth of colon adenocarcinoma cells via the Janus kinase 2/signal transducer and activator of transcription 3 signalling pathway.

OBJECTIVE: Fraxetin, extracted from the bark of Fraxinus rhynchophylla, has been shown to exhibit antitumour and anti-inflammatory pharmacological properties. However, the mechanism underlying its anticancer activity towards colon adenocarcinoma (COAD) is not well understood. We aimed to determine the antitumour effect of fraxetin on COAD cell lines and elucidate its biochemical and molecular targets. METHODS: The cell lines HCT116 and DLD-1 were used to evaluate the in vitro antitumour efficacy of fraxetin. Cytotoxicity and viability were assessed by CCK-8 and plate colony formation assays. Flow cytometry was used to assess apoptosis and cell cycle progression in fraxetin-treated COAD cells. Western blot, RT-qPCR, molecular docking, immunohistochemical, and immunofluorescence analyses were used to gain insights into cellular and molecular mechanisms. Preclinical curative effects were evaluated in nude mouse xenograft models. RESULTS: Fraxetin significantly inhibited COAD cell proliferation in both dose- and time-dependent manners, specifically by inducing S-phase cell cycle arrest and triggering intrinsic apoptosis. Additionally, the level of p-JAK2 was decreased by fraxetin via the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway. Interestingly, in COAD cells, fraxetin directly targeted the Y1007 and Y1008 residues of JAK2 to suppress its auto- or transphosphorylation, leading to decreased activation of its downstream effector STAT3 and blocking its nuclear translocation. Finally, fraxetin exhibited good tumour growth suppression activity and low toxicity. CONCLUSIONS: Fraxetin inhibits the proliferation of COAD cells by regulating the JAK2/STAT3 signalling pathway, providing evidence that targeting JAK2 with fraxetin may offer a novel potential auxiliary therapy for COAD treatment.

Synthetic antiprotozoal thiazolide drug induced apoptosis in colorectal cancer cells: implications of IL-6/JAK2/STAT3 and p53/caspases-dependent signaling pathways based on molecular docking and in vitro study.

Colorectal cancer (CRC) is a global pressing healthcare priority. Dysregulation of the IL6/JAK2/STAT3 and p53/caspase downstreaming pathways are significantly involved in the progression of CRC, and mainly affecting apoptosis. Discovery of new anti-cancer agents is laborious, time consuming, and costly with obvious socioeconomic burden. In the present study, we are proposing new molecular insights on the anti-proliferative and apoptotic therapeutic effects of nitazoxanide (NTZ) on CRC. NTZ is FDA-approved thiazolide antiparasitic agent, which has excellent safety and pharmacokinetic profiles. The molecular docking study revealed that NTZ has better binding affinity and docking score against JAK2 and BCL2 proteins compared to 5-Fluorouracil, which is the standard drug for treatment of CRC. The current in vitro work on a human HCT116 cell line displayed that NTZ had lower IC50 value (11.20 µM) than 5-flurouracil (23.78 µM), and NTZ induced a statistically significant down-regulation of IL6/JAK2/STAT3. NTZ also modulated significantly the p53/caspases-dependent signaling pathways, leading to enhancement of apoptosis and an increase of DNA fragmentation. Moreover, NTZ regulated the Bcl-2 gene family and promoted the loss of mitochondrial function which was depicted by release of cytochrome c (Cyt c), and caspase activation in apoptotic HCT116 cells. Additionally, NTZ was able to reduce the expression of VEGF in CRC cell line, which needs future thorough molecular investigations. In conclusion, our findings provided a novel evidence that NTZ could be a dual potential IL6/JAK2/STAT3 signaling inhibitor and p53/caspases-dependent pathway activator in CRC cell line. These potentials support further exploratory molecular researches targeting the therapeutic roles of NTZ in CRC; individually and simultaneously with current approved chemotherapeutic regimens.

Selective Janus Kinase 2 (JAK2) Pseudokinase Ligands with a Diaminotriazole Core.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are essential components of the JAK-STAT signaling pathway. Associated aberrant signaling is responsible for many forms of cancer and disorders of the immune system. The present focus is on the discovery of molecules that may regulate the activity of JAK2 by selective binding to the JAK2 pseudokinase domain, JH2. Specifically, the Val617Phe mutation in JH2 stimulates the activity of the adjacent kinase domain (JH1) resulting in myeloproliferative disorders. Starting from a non-selective screening hit, we have achieved the goal of discovering molecules that preferentially bind to the ATP binding site in JH2 instead of JH1. We report the design and synthesis of the compounds and binding results for the JH1, JH2, and JH2 V617F domains, as well as five crystal structures for JH2 complexes. Testing with a selective and non-selective JH2 binder on the autophosphorylation of wild-type and V617F JAK2 is also contrasted.

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations.

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.

Construction of Quantitative Structure Activity Relationship (QSAR) Models to Predict Potency of Structurally Diversed Janus Kinase 2 Inhibitors.

Janus kinase 2 (JAK2) inhibitors represent a promising therapeutic class of anticancer agents against many myeloproliferative disorders. Bioactivity data on pIC 50 of 2229 JAK2 inhibitors were employed in the construction of quantitative structure-activity relationship (QSAR) models. The models were built from 100 data splits using decision tree (DT), support vector machine (SVM), deep neural network (DNN) and random forest (RF). The predictive power of RF models were assessed via 10-fold cross validation, which afforded excellent predictive performance with R 2 and RMSE of 0.74 ± 0.05 and 0.63 ± 0.05, respectively. Moreover, test set has excellent performance of R 2 (0.75 ± 0.03) and RMSE (0.62 ± 0.04). In addition, Y-scrambling was utilized to evaluate the possibility of chance correlation of the predictive model. A thorough analysis of the substructure fingerprint count was conducted to provide insights on the inhibitory properties of JAK2 inhibitors. Molecular cluster analysis revealed that pyrazine scaffolds have nanomolar potency against JAK2.

Discovery of Potent and Orally Effective Dual Janus Kinase 2/FLT3 Inhibitors for the Treatment of Acute Myelogenous Leukemia and Myeloproliferative Neoplasms.

Herein, we describe the design, synthesis, and structure-activity relationships of a series of unique 4-(1H-pyrazol-4-yl)-pyrimidin-2-amine derivatives that selectively inhibit Janus kinase 2 (JAK2) and FLT3 kinases. These screening cascades revealed that 18e was a preferred compound, with IC50 values of 0.7 and 4 nM for JAK2 and FLT3, respectively. Moreover, 18e was a potent JAK2 inhibitor with 37-fold and 56-fold selectivity over JAK1 and JAK3, respectively, and possessed an excellent selectivity profile over the other 100 representative kinases. In a series of cytokine-stimulated cell-based assays, 18e exhibited a higher JAK2 selectivity over other JAK isoforms. The oral administration of 60 mg/kg of 18e could significantly inhibit tumor growth, with a tumor growth inhibition rate of 93 and 85% in MV4-11 and SET-2 xenograft models, respectively. Additionally, 18e showed an excellent bioavailability (F = 58%), a suitable half-life time (T1/2 = 4.1 h), a satisfactory metabolic stability, and a weak CYP3A4 inhibitory activity, suggesting that 18e might be a potential drug candidate for JAK2-driven myeloproliferative neoplasms and FLT3-internal tandem duplication-driven acute myelogenous leukemia.

Roles of T875N somatic mutation in the activity, structural stability of JAK2 and the transformation of OCI-AML3 cells.

Activating mutations in JAK2 have been described in patients with various hematologic malignancies including acute myeloid leukemia (AML) and myeloproliferative neoplasms. However, mechanism of these mutations in JAK2's activity, structural stability and pathology of AML remains poorly understood. The JAK2 T875N somatic mutation has been detected in about 5.2% of AML patients. But the structural basis and mechanism of JAK2 T875N mutation in the pathology of AML is still unclear. Our results suggested that JAK2 T875N mutation disrupted the T875 and D873 interaction which destroyed the compact structure of JH1 domain, forced it into the active conformation, facilitated the entrance of substrate and thus led to JAK2 hyperactivation. Mutations (T875N, T875A, D873A and D873G) disrupted the T875 and D873 interaction enhanced JAK2's activity, decreased its structural stability and JH2 domain's activity which further enhanced JAK2's activity, while mutations (T875R, D873E, T875R/D873E) repaired this interaction displayed opposite results. Moreover, JAK2 T875N mutation enhanced the activity of JAK2-STAT5 pathway, promoted the proliferation and transformation of OCI-AML3 cells. This study provides clues in understanding structural basis of T875N mutation caused JAK2 hyperactivation and its roles in the pathology of AML.

3-Deoxy-2ß,16-dihydroxynagilactone E, a natural compound from Podocarpus nagi, preferentially inhibits JAK2/STAT3 signaling by allosterically interacting with the regulatory domain of JAK2 and induces apoptosis of cancer cells.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, especially the JAK2/STAT3 pathway, play vital roles in the development of many malignancies. Overactivation of STAT3 promotes cancer cell survival and proliferation. Therefore, the JAK2/STAT3-signaling pathway has been considered a promising target for cancer therapy. In this study, we identified a natural compound 3-deoxy-2ß,16-dihydroxynagilactone E (B6) from the traditional Chinese medicinal plant Podocarpus nagi as a potent inhibitor of STAT3 signaling. B6 preferentially inhibited the phosphorylation of STAT3 by interacting with and inactivating JAK2, the main upstream kinase of STAT3. B6 dose-dependently inhibited IL-6-induced STAT3 signaling with an IC50 of 0.2 µM. In contrast to other JAK2 inhibitors, B6 did not interact with the catalytic domain but instead with the FERM-SH2 domain of JAK2. This interaction was JAK-specific since B6 had little effect on other tyrosine kinases. Furthermore, B6 potently inhibited the growth and induced apoptosis of MDA-MB-231 and MDA-MB-468 breast cancer cells with overactivated STAT3. Taken together, our study uncovers a novel compound and a novel mechanism for the regulation of JAK2 and offers a new therapeutic approach for the treatment of cancers with overactivated JAK2/STAT3.

Disruption of R867 and Y613 interaction plays key roles in JAK2 R867Q mutation caused acute leukemia.

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations were important for the leukemogenesis of acute leukemia (AL). The JAK2 R867Q somatic mutation is detected in a subset of AL patients. However, roles of JAK2 R867Q mutation in the pathogenesis of AL remain unclear. In this study, homology modeling analysis showed that loss of interaction between R867 and Y613 disrupted the JAK2 JH1/JH2 domain's interactions was responsible for its activation. JAK2 R867Q and mutations (R867A and R867G) abolished this interaction caused JAK2 constitutive activation. While, mutations (R867K, Y613E, R867K/Y613E) repairing this interaction reduced JAK2 R867Q mutation's activity. Furthermore, our studies showed that abolished R867 and Y613 interaction disrupted JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, mutations (R867Q, R867A and R867G) disrupted this interaction enhanced the activity of JAK2-STAT5 pathway and the proliferation of Ba/F3 and MV4-11 cells. Further study showed that JAK2 R867Q mutation promoted the expression of proliferation marker and inhibited the differentiation marker of Ba/F3 and MV4-11 cells. Thus our studies provide clues in understanding the pathogenesis of JAK2 R867Q mutation in AL.

Discovery and Structural Characterization of ATP-Site Ligands for the Wild-Type and V617F Mutant JAK2 Pseudokinase Domain.

The oncogenic V617F mutation lies in the pseudokinase domain of JAK2, marking it as a potential target for development of compounds that might inhibit the pathogenic activity of the mutant protein. We used differential scanning fluorimetry to identify compounds that bind the JAK2 pseudokinase domain. Crystal structures of five candidate compounds with the wild-type domain reveal their modes of binding. Exploration of analogs of screening hit BI-D1870 led to the identification of compound 2, a 123 nM ligand for the pseudokinase domain. Interestingly, crystal structures of the V617F domain in complex with two unrelated compounds reveal a conformation that is characteristic of the wild-type domain, rather than that previously observed for the V617F mutant. These structures suggest that certain ATP-site ligands can modulate the V617F allosteric site, thereby providing a mechanistic rationale for targeting the pseudokinase domain and a structural foundation for development of more potent and pseudokinase-selective compounds.