ATP Site Research Articles

Sophoridine Inhibits Human Colorectal Cancer Progression via Targeting MAPKAPK2.

Radian Sophorae flavescentis is a traditional Chinese medicine commonly used to treat cancer in China. However, its active components and underlying mechanism remain ambiguous. In this study, we have screened the pharmacokinetic parameters of the main chemical constituents of Radian Sophorae flavescentis by Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform and have found that Sophoridine is one of the best antitumor active ingredients. We have found that MAPKAPK2 is a potential target for Sophoridine by the PharmMapper and KEGG databXase analysis. Moreover, we have found that Sophoridine selectively inactivates phospho-MAPKAPK2 (Thr222) and directly binds into the ATP site of MAPKAPK2 by molecular docking. Furthermore, we have found out a direct binding between MAPKAPK2 and Sophoridine by cellular thermal shift assay and drug affinity responsive targets stability assay. The inhibition effects are further confirmed by Western blot: Sophoridine significantly decreases phospho-MAPKAPK2 (Thr222) in a time-dependent manner, but there is no obvious change in its total expression in colorectal cancer cells. Clinical studies have shown that a higher level of MAPKAPK2 is associated with a poorer percent survival rate (prognosis). Furthermore, a higher level of MAPKAPK2 is positively associated with the enrichment of downregulation of apoptosis and autophagy by gene set enrichment analysis, as well as upregulation of proliferation and cell-cycle arrest. Taken together, our results suggest that the MAPKAPK2 plays a key role in Sophoridine-inhibited growth and invasion in colorectal cancers. IMPLICATIONS: These studies show that Sophoridine may be a promising therapeutic strategy that blocks tumorigenesis in colorectal cancers.

Critical c-Met-inhibitor interactions resolved from molecular dynamics simulations of different c-Met complexes

Overexpression or deregulation of c-Met enzyme has been reported to be associated with the formation and development of various types of human cancers, so an intense scientific work was devoted to design more potent inhibitors. We ran 17 molecular dynamics simulations of potent type I c-Met inhibitors to investigate the critical and stable interactions regarding the inherent flexibility of protein and ligand via dynamics ensembles. Protein-ligand interactions of hydrogen bond, pi-pi stacked and hydrophobic have been investigated throughout the equilibrium time to suggest critical pharmacophores. Also, the fluctuations of amino acid residues were monitored by root mean square fluctuation (RMSF) measure. MM-PBSA approach was also used to estimate the binding free energy of different complexes. The pi-pi stacked interaction of c-Met inhibitors with Tyr1230 or both Tyr1230 and Tyr1159 was shown critical for a good inhibitory activity and was defined as the most important interaction type in terms of binding energy. Almost in all codes, ligands showed van der Waals interactions with hydrophobic residues of ATP site. In addition, hydrogen bond interaction with Met1160 was found necessary for a ligand being a potent inhibitor of c-Met. RMSF analysis showed that Met1160 and Pro1158 which make the key hydrogen bond interactions and Tyr1230 that contributes in pi-pi stacked interactions with the ligands were quite fixed during ligand binding.

Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib's effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.

Lead identification and characterization of hTrkA type 2 inhibitors.

Virtual in silico structure-guided modeling, followed by in vitro biochemical screening of a subset of commercially purchasable compound collection resulted in the identification of several human tropomyosin receptor kinase A (hTrkA) inhibitors that bind the orthosteric ATP site and exhibit binding preference for the inactive kinase conformation. The type 2 binding mode with the DFG-out and αC-helix out hTrkA kinase domain conformation was confirmed from X-ray crystallographic solution of a representative inhibitor analog, 1b. Additional hTrkA and hTrkB (selectivity) assays in recombinant cells, neurite outgrowth inhibition using rat PC12 cells, early ADME profiling, and preliminary pharmacokinetic evaluation in rodents guided the lead inhibitor progression in the discovery screening funnel.

MARK4 protein can explore the active-like conformations in its non-phosphorylated state

Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules. In the current study, the non-phosphorylated conformation of the activation loop was modeled in a structure representing the enzymatically inactive form of this protein, and its dynamics were evaluated through a 100 ns initial all-atom simulation, which was prolonged by another 2 μs. Although the activation loop was folding on itself and was leaning toward ATP site in the initial modeled structure, soon after the initiating the simulation, this loop stretched away from the ATP binding site and stably settled in its new position for the rest of simulation time. A network of hydrogen bonds, mainly between the activation segment residues, αC-helix and the catalytic loop reinforced this conformation. Interestingly, several features of active kinase conformation such as formation of R-spine, Glu106-Lys88 salt-bridge, and DFG-In motif were observed during a considerable number of trajectory frames. However, they were not sustainably established during the simulation time, except for the DFG-In motif. Consequently, this study introduces a stable conformation of the non-phosphorylated form of MARK4 protein with a partially stretched activation loop conformation as well as partial formation of R-spine, closely resembling the active kinase.

Structural Analyses of Helicobacter Pylori FolC Conducting Glutamation in Folate Metabolism

FolC plays important roles in the folate metabolism of cells by attaching l-Glu to dihydropteroate (DHP) and folate, which are known activities of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS), respectively. Here, we determined the crystal structure of Helicobacter pylori FolC (HpFolC) at 1.95 Å resolution using the single-wavelength anomalous diffraction method. HpFolC has globular N- and C-terminal domains connected by a single loop, and a binding site for ATP is located between the two domains. Apo-HpFolC was crystallized in the presence of citrate in a crystallization solution, which was held in the ATP-binding site. Structural motifs such as the P-loop and Ω-loop of HpFolC for binding of ATP and two magnesium ions are well conserved in spite of the low overall sequence similarity to other FolC/FPGSs. The Ω-loop would also recognize a folate molecule, and the DHP-binding loop of HpFolC is expected to exhibit a unique recognition mode on DHP, compared with other FolCs. Because human FolC is known to only exhibit FPGS activity, the DHFS activity of bacterial FolC is an attractive target for the eradication of pathogenic bacteria. Consequently, our structural analyses of HpFolC provide a valuable foundation for a universal antibacterial strategy against H. pylori as well as other pathogenic bacteria.

Highly Selective Fluorescence Sensing and Imaging of ATP Using a Boronic Acid Groups-Bearing Polythiophene Derivate.

A boronic acid groups-bearing polythiophene derivate (L) was designed and synthesized for highly sensitive fluorescence detection of ATP based on a multisite-binding coupled with analyte-induced aggregation strategy. L has a polythiophene backbone as fluorophores and two functional side groups, i.e., quaternary ammonium group and boronic acid group, as multibinding sites for ATP. When various structural analogues such as ADP, AMP, and various inorganic phosphates were added into the aqueous solution of L, only ATP caused a remarkable fluorescence quenching of about 60-fold accompanied by obvious color changes of solution from yellow to purple. The detection limit is estimated to be 2 nM based on 3σ/slope. With the advantage of good water solubility, low toxicity, and highly selective response to ATP, L was successfully utilized as a probe to real-time assay activity of adenylate kinase (ADK) and map fluorescent imaging of ATP in living cells.

Effects of purine-scaffold inhibitors on HUVECs: Involvement of the purinergic pathway and interference with ATP. Implications for preventing the adverse effects of extracellular Grp94

BackgroundExtracellular Glucose-regulated protein94 (Grp94) is linked to pathological conditions disrupting the obligatory intracellular location of this Heat Shock Protein (HSP). In plasma, Grp94 is linked to IgG in complexes that drive adverse effects on vascular cells and are biomarker of gastro-intestinal cancer. By blocking ATP site in different HSPs, purine-scaffold inhibitors are used as promising anti-cancer compounds, but their effects on vasculature are not known. MethodsWe tested the capacity of two purine-scaffold inhibitors, PU-H71 and PU-WS13, to prevent the binding of Grp94 to IgG and to antagonize the effects of Grp94 and native Grp94-IgG complexes on HUVECs in different experimental conditions. ResultsPU-H71 and PU-WS13 blocked Grp94 and the formation of Grp94-IgG complexes in absence of cells. Instead, in presence of HUVECs rather than Grp94 PU-inhibitors targeted cells causing stimulation of Akt and VEGF pathways and displaying angiogenic-like effects similar to, although less intense than that provoked by Grp94 and Grp94-IgG complexes. Unlike Grp94 and Grp94-IgG complexes, PU-inhibitors also activated the purinergic pathway and increased the expression of the ATP receptor P2X7. Effects of PU-inhibitors on HUVECs were reversed by ATP and in presence of ATP PU-inhibitors were again able to block Grp94. ConclusionsPU-inhibitors can display direct effects on endothelial cells by targeting the ATP receptor P2X7. In absence of ATP, PU-inhibitors preferentially bind to cells rather than Grp94. ATP antagonizes the PU-inhibitor binding to cells thus restoring the capacity to block Grp94 and Grp94-IgG complex formation. Results have implications for enhancing the therapeutic efficacy of PU-inhibitors against circulating pathogenic Grp94.

ABCE1 Controls Ribosome Recycling by an Asymmetric Dynamic Conformational Equilibrium

SummaryThe twin-ATPase ABCE1 has a vital function in mRNA translation by recycling terminated or stalled ribosomes. As for other functionally distinct ATP-binding cassette (ABC) proteins, the mechanochemical coupling of ATP hydrolysis to conformational changes remains elusive. Here, we use an integrated biophysical approach allowing direct observation of conformational dynamics and ribosome association of ABCE1 at the single-molecule level. Our results from FRET experiments show that the current static two-state model of ABC proteins has to be expanded because the two ATP sites of ABCE1 are in dynamic equilibrium across three distinct conformational states: open, intermediate, and closed. The interaction of ABCE1 with ribosomes influences the conformational dynamics of both ATP sites asymmetrically and creates a complex network of conformational states. Our findings suggest a paradigm shift to redefine the understanding of the mechanochemical coupling in ABC proteins: from structure-based deterministic models to dynamic-based systems.

Structure-based identification of potent VEGFR-2 inhibitors from in vivo metabolites of a herbal ingredient.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is one of the regulatory elements of angiogenesis that is expressed highly in various diseases and is also essential for solid tumor growth. The present study was aimed at identifying potent inhibitors of VEGFR-2 by considering herbal secondary metabolites; as natural molecules are less toxic than synthetic derivatives. A structure-based virtual screening protocol consisting of molecular docking, MM-GBSA and ADME/T analysis was initially used to screen a library of in vivo metabolites of the herbal ingredient. Using a fixed cutoff value, four potent virtual hits were identified from molecular docking, ADME/T and binding affinity calculations, which were considered further for molecular dynamics (MD) simulation to broadly describe the binding mechanisms to VEGFR-2. The results suggested that these molecules have high affinity for the catalytic region of VEGFR-2, and form strong hydrophobic and polar interactions with the amino acids involved in the binding site of ATP and linker regions of the catalytic site. Subsequently, the stability of the docked complexes and binding mechanisms were evaluated by MD simulations, and the energy of binding was calculated through MM-PBSA analysis. The results uncovered two virtual hits, designated ZINC14762520 and ZINC36470466, as VEGFR-2 inhibitors, and suggested that they bind to kinase domainin an ATP-competitive manner. These virtual hits will offer a suitable starting point for the further design of their various analogs, allowing a rational search for more effective inhibitors in the future. Graphical abstract.

Defining a new nomenclature for the structures of active and inactive kinases

Targeting protein kinases is an important strategy for intervention in cancer. Inhibitors are directed at the active conformation or a variety of inactive conformations. While attempts have been made to classify these conformations, a structurally rigorous catalog of states has not been achieved. The kinase activation loop is crucial for catalysis and begins with the conserved DFGmotif. This motif is observed in two major classes of conformations, DFGin-a set of active and inactive conformations where the Phe residue is in contact with the C-helix of the N-terminal lobe-and DFGout-an inactive form where Phe occupies the ATP site exposing the C-helix pocket. We have developed a clustering of kinase conformations based on the location of the Phe side chain (DFGin, DFGout, and DFGinter or intermediate) and the backbone dihedral angles of the sequence X-D-F, where X is the residue before the DFGmotif, and the DFG-Phe side-chain rotamer, utilizing a density-based clustering algorithm. We have identified eight distinct conformations and labeled them based on the Ramachandran regions (A, alpha; B, beta; L, left) of the XDF motif and the Phe rotamer (minus, plus, trans). Our clustering divides the DFGin group into six clusters including BLAminus, which contains active structures, and two common inactive forms, BLBplus and ABAminus. DFGout structures are predominantly in the BBAminus conformation, which is essentially required for binding type II inhibitors. The inactive conformations have specific features that make them unable to bind ATP, magnesium, and/or substrates. Our structurally intuitive nomenclature will aid in understanding the conformational dynamics of kinases and structure-based development of kinase drugs.

Penicisulfuranol A, a novel C-terminal inhibitor disrupting molecular chaperone function of Hsp90 independent of ATP binding domain

The goal of this study is to explore the mechanism of a heat shock protein 90 (Hsp90) C-terminal inhibitor, Penicisulfuranol A (PEN-A), for cancer therapy. PEN-A was produced by a mangrove endophytic fungus Penicillium janthinellum and had a new structure with a rare 3H-spiro [benzofuran-2, 2′-piperazine] ring system. PEN-A caused depletion of multiple Hsp90 client proteins without induction of heat shock protein 70 (Hsp70). Subsequently, it induced apoptosis and inhibited xerograph tumor growth of HCT116 cells in vitro and in vivo. Mechanism studies showed that PEN-A was bound to C-terminus of Hsp90 at the binding site different from ATP binding domain. Therefore, it inhibited dimerization of Hsp90 C-terminus, depolymerization of ADH protein by C-terminus of Hsp90, and interaction of co-chaperones with Hsp90. These inhibitory effects of PEN-A were similar to those of novobiocin, an inhibitor binding to interaction site for ATP of C-terminus of Hsp90. Furthermore, our study revealed that disulfide bond was essential moiety for inhibition activity of PEN-A on Hsp90. This suggested that PEN-A may be bound to cysteine residues near amino acid region which was responsible for dimerization of Hsp90. All results indicate that PEN-A is a novel C-terminal inhibitor of Hsp90 and worthy for further study in the future not only for drug development but also for unraveling the bioactivities of Hsp90.

Diverse dynamics features of novel protein kinase C (PKC) isozymes determine the selectivity of a fluorinated balanol analogue for PKC\u03b5

Background(−)-Balanol is an ATP-mimicking inhibitor that non-selectively targets protein kinase C (PKC) isozymes and cAMP-dependent protein kinase (PKA). While PKA constantly shows tumor promoting activities, PKC isozymes can ambiguously be tumor promoters or suppressors. In particular, PKCε is frequently implicated in tumorigenesis and a potential target for anticancer drugs. We recently reported that the C5(S)-fluorinated balanol analogue (balanoid 1c) had improved binding affinity and selectivity for PKCε but not to the other novel PKC isozymes, which share a highly similar ATP site. The underlying basis for this fluorine-based selectivity is not entirely comprehended and needs to be investigated further for the development of ATP mimic inhibitors specific for PKCε.ResultsUsing molecular dynamics (MD) simulations assisted by homology modelling and sequence analysis, we have studied the fluorine-based selectivity in the highly similar ATP sites of novel PKC (nPKC) isozymes. The study suggests that every nPKC isozyme has different dynamics behaviour in both apo and 1c-bound forms. Interestingly, the apo form of PKCε, where 1c binds strongly, shows the highest degree of flexibility which dramatically decreases after binding 1c.ConclusionsFor the first time to the best of our knowledge, we found that the origin of 1c selectivity for PKCε comes from the unique dynamics feature of each PKC isozyme. Fluorine conformational control in 1c can synergize with and lock down the dynamics of PKCε, which optimize binding interactions with the ATP site residues of the enzyme, particularly the invariant Lys437. This finding has implications for further rational design of balanol-based PKCε inhibitors for cancer drug development.

Chronic Myeloid Leukemia: Existing Therapeutic Options and Strategies to Overcome Drug Resistance.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused due to translocation between chromosome 9 and 22 leading to a chimeric gene product known as Bcr-Abl. Bcr-Abl fusion protein has constitutively activated Abl tyrosine kinase activity which is responsible for the uncontrolled proliferation in CML The tyrosine kinase inhibitors (TKIs) such as Imatinib, Dasatinib, and Nilotinib are the current first-line treatments approved by the United States Food and Drug Administration (US FDA) for the treatment of the disease. Despite the spectacular progress made over the decade with the TKIs, patients develop resistance to these TKIs. In such cases, stem cell transplant therapy, which is limited by donor availability, is the only proven cure for the patients. This highlights the need for the development of new strategies for CML treatment. The Bcr-Abl point mutations, including the gatekeeper T315I mutations, are the principal cause for the development of resistance to TKIs. However, other mechanisms are also involved in the failure of TKI therapy. This review outlines the Bcr-Abl dependent and independent mechanism of TKIs resistance development and the strategies used to overcome drug resistance, such as the development of ATP site and allosteric site inhibitors. Binding mode and structural elements of Bcr-Abl inhibition are discussed with emphasis on pathways involved in this complex disease to determine alternative strategies and combination therapies.

MTOR kinase inhibition reduces tissue factor expression and growth of pancreatic neuroendocrine tumors

Essentials Tissue factor (TF) isoforms are expressed in pancreatic neuroendocrine tumors (pNET). TF knockdown inhibits proliferation of human pNET cells in vitro. mTOR kinase inhibitor sapanisertib/MLN0128 suppresses TF expression in human pNET cells. Sapanisertib suppresses TF expression and activity and reduces the growth of pNET tumors in vivo. SUMMARY: Background Full-length tissue factor (flTF) and alternatively spliced TF (asTF) contribute to growth and spread of pancreatic ductal adenocarcinoma. It is unknown, however, if flTF and/or asTF contribute to the pathobiology of pancreatic neuroendocrine tumors (pNETs). Objective To assess TF expression in pNETs and the effects of mTOR complex 1/2 (mTORC1/2) inhibition on pNET growth. Methods Human pNET specimens were immunostained for TF. Human pNET cell lines QGP1 and BON were evaluated for TF expression and responsiveness to mTOR inhibition. shRNA were used to knock down TF in BON. TF cofactor activity was assessed using a two-step FXa generation assay. TF promoter activity was assessed using transient transfection of human TF promoter-driven reporter constructs into cells. Mice bearing orthotopic BON tumors were treated with the mTORC1/2 ATP site competitive inhibitor sapanisertib/MLN0128 (3 mg kg-1 , oral gavage) for 34 days. Results Immunostaining of pNET tissue revealed flTF and asTF expression. BON and QGP1 expressed both TF isoforms, with BON exhibiting higher levels. shRNA directed against TF suppressed BON proliferation in vitro. Treatment of BON with sapanisertib inhibited mTOR signaling and suppressed TF levels. BON tumors grown in mice treated with sapanisertib had significantly less TF protein and cofactor activity, and were smaller compared with tumors grown in control mice. Conclusions TF isoforms are expressed in pNETs. Sapanisertib suppresses TF mRNA and protein expression as well as TF cofactor activity in vitro and in vivo. Thus, further studies are warranted to evaluate the clinical utility of TF-suppressing mTORC1/2 inhibitor sapanisertib in pNET management.

Rosmarinic Acid Derivatives' Inhibition of Glycogen Synthase Kinase-3β Is the Pharmacological Basis of Kangen-Karyu in Alzheimer's Disease.

Inhibition of glycogen synthase kinase 3β (GSK-3β) is considered to be the central therapeutic approach against Alzheimer’s disease (AD). In the present study, boiled water extracts of the Kangen-karyu (KK) herbal mixture and its constituents were screened for GSK-3β inhibitory activity. KK is used in traditional Kampo and Chinese medicines for improving cognitive function. The GSK-3β inhibition potential was evaluated by using the Kinase-Glo luminescent kinase assay platform. Furthermore, enzyme kinetics and in silico modeling were performed by using AutoDockTools to demonstrate the mechanism of enzyme inhibition. KK extract significantly inhibited GSK-3β in a concentration-dependent manner (IC50: 17.05 ± 1.14 μg/mL) when compared with the reference drug luteolin (IC50: 2.18 ± 0.13 μM). Among the six components of KK, extracts of Cyperi Rhizoma and Salviae Miltiorrhizae Radix significantly inhibited GSK-3β with IC50 values of 20.68 ± 2.50 and 7.77 ± 1.38 μg/mL, respectively. Among the constituents of the roots of S. miltiorrhiza water extract, rosmarinic acid, magnesium lithospermate B, salvianolic acid A, salvianolic acid B, and salvianolic acid C inhibited GSK-3β with IC50 values ranging from 6.97 to 135.5 μM. Salvianolic acid B was found to be an ATP-competitive inhibitor of GSK-3β and showed the lowest IC50 value (6.97 ± 0.96 µM). In silico modeling suggested a mechanism of action by which the hydrophobic, π–cation, and hydrophilic interactions of salvianolic acid B at ATP and substrate sites are critical for the observed GSK-3β inhibition. Therefore, one of the mechanisms of action of KK against AD may be the inhibition of GSK-3β and one of the active components of KK is the root of S. miltiorrhiza and its constituents: rosmarinic acid, magnesium lithospermate B, and salvianolic acids A, B, and C. Our results demonstrate the pharmacological basis for the use of KK against AD.

Promiscuous attraction of ligands within the ATP binding site of RyR2 promotes diverse gating behaviour

ATP is an essential constitutive regulator of cardiac ryanodine receptors (RyR2), enabling small changes in cytosolic Ca2+ to trigger large changes in channel activity. With recent landmark determinations of the full structures of RyR1 (skeletal isoform) and RyR2 using cryo-EM, and identification of the RyR1 ATP binding site, we have taken the opportunity to model the binding of fragments of ATP into RyR2 in order to investigate how the structure of the ATP site dictates the functional responses of ligands attracted there. RyR2 channel gating was assessed under voltage-clamp conditions and by [3H]ryanodine binding studies. We show that even the triphosphate (PPPi) moiety alone was capable of activating RyR2 but produced two distinct effects (activation or irreversible inactivation) that we suggest correspond to two preferred binding locations within the ATP site. Combinations of complementary fragments of ATP (Pi + ADP or PPi + AMP) could not reproduce the effects of ATP, however, the presence of adenosine prevented the inactivating PPPi effects, allowing activation similar to that of ATP. RyR2 appears to accommodate diverse types of molecules, including PPPi, deep within the ATP binding site. The most effective ligands, however, have at least three phosphate groups that are guided into place by a nucleoside.

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).

Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Janus kinases (JAKs; JAK1 to JAK3 and tyrosine kinase 2) mediate cytokine signals in the regulation of hematopoiesis and immunity. JAK2 clinical mutations cause myeloproliferative neoplasms and leukemia, and the mutations strongly concentrate in the regulatory pseudokinase domain Janus kinase homology (JH) 2. Current clinical JAK inhibitors target the tyrosine kinase domain and lack mutation and pathway selectivity. We sought to characterize mechanisms and differences for pathogenic and cytokine-induced JAK2 activation to enable design of novel selective JAK inhibitors. We performed a systematic analysis of JAK2 activation requirements using structure-guided mutagenesis, cell-signaling assays, microscopy, and biochemical analysis. Distinct structural requirements were identified for activation of different pathogenic mutations. Specifically, the predominant JAK2 mutation, V617F, is the most sensitive to structural perturbations in multiple JH2 elements (C helix [αC], Src homology 2-JH2 linker, and ATP binding site). In contrast, activation of K539L is resistant to most perturbations. Normal cytokine signaling shows distinct differences in activation requirements: JH2 ATP binding site mutations have only a minor effect on signaling, whereas JH2 αC mutations reduce homomeric (JAK2-JAK2) erythropoietin signaling and almost completely abrogate heteromeric (JAK2-JAK1) IFN-γ signaling, potentially by disrupting a dimerization interface on JH2. These results suggest that therapeutic approaches targeting the JH2 ATP binding site and αC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-γ functions. Simultaneously, however, we identified the JH2 αC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.

In silico screening and identification of potential GSK3β inhibitors

Glycogen synthase kinase-3β (GSK3β) has been reported for its impact on multitude biological processes from cell proliferation to apoptosis. The increase in the ratio of active/inactive GSK3β is the major factor associated in the etiology of several psychiatric diseases, diabetes, muscle hypertrophy, neurodegenerative diseases, and some cancers. These findings made GSK3β a promising therapeutic target, and the interest in the discovery, synthesis of novel drugs to effectively attenuate its function with probably no side effects has been increasing in the chronology of GSK3β drug discovery. In the present study, we applied a combination of computational tools on a chemical library for the virtual discovery of their potency to inhibit GSK3β. The chemical library was screened against a set of filters at different levels. Finally, five compounds in the chemical library were found to potentially inhibit GSK3β with no toxic effects. Furthermore, binding mode analysis revealed that all the compounds bound to the ATP site and most of the hydrogen bonding interactions are conserved as in GSK3β structures deposited in PDB.