GMFBI.1 down regulated p38MAPK phosphorylation and NFκB expression essential for proinflammatory response. Further, GMFBI.1 administration at peak of EAE reversed clinical symptoms, immunopathology, proinflammatory cytokine response and up regulated the anti-inflammatory cytokines. Present strategy of substrate inhibition against the key immunomodulatory target has immense therapeutic potential in MS.The concept of substrate inhibition to prevent its phosphorylation has potential in drug discovery and is envisioned to treat the autoimmune disorder multiple sclerosis (MS). Glia maturation factor-β (GMF-β) Ser83 phosphorylation by protein kinase A (PKA) is pivotal in the activation of GMF-β-p38MAPK-NFκB biochemical pathway towards proinflammatory response induction in experimental autoimmune encephalomyelitis (EAE). Using structure-based drug design, we identified the small molecule inhibitor 1-H-indazole-4yl methanol (GMFBI.1) that specifically blocked Ser83 phosphorylation site on GMF-β substrate. Using  Despite different disease-modifying therapies adopted to mitigate the inflammatory milieu of MS using different drugs5, patient’s progress from an acute phase to a stage with considerable neurological disabilities. Current therapies modulate disease differently as they target activated T cells, antigen presenting cells or prevent the egress of T cells into brain. Importantly, none of these drugs control molecules that modulate the proinflammatory response at a fundamental level following infiltration of activated T and B lymphocytes7. Activated lymphocytes undergo clonal expansion assisted by the cytokines produced mainly by astrocytes and microglia and glia maturation factor-β (GMF-β) plays an instrumental role in cytokine induction9. Thus, targeting GMF-β could signify a novel approach in controlling the immune response in the brain.Multiple sclerosis is a chronic autoimmune, demyelinating, neurodegenerative disorder of the central nervous system (CNS) affecting 2.5 million people globally12. In the well-established EAE animal model of MS, GMF-β was over expressed in both brain and spinal cord10. On the other hand GMF-β null mice showed reduced mononuclear cell infiltration with a mild form of EAE compared to the regular EAE animals10. Administration of anti-GMF-β antibody to EAE animals led to reduced inflammation and clinical symptoms13. These studies imply the significance of GMF-β in controlling the specific proinflammatory response in astrocytes leading to the suppression of EAE. Thus, human GMF-β (hGMF-β) could serve as a potential therapeutic druggable target for inflammatory disorders like MS.Over expression of GMF-β in response to immune challenge up regulates p38MAPK and NFκB expression in astrocytes leading to increased GM-CSF production by astrocytes and microglial generation of TNF-α, IL1-β, IL-6 and IFN-γ, augmenting proinflammatory response14. PKA phosphorylates Thr27 and Ser83, while ribosomal S6 kinase, casein kinase and protein kinase C phosphorylates Thr27, Ser53, Ser72 residues respectively14. Among these, Ser83 phosphorylation of hGMF-β by PKA is crucial in inducing p38MAPK phosphorylation and activation of NF-κB leading to copious secretion of GM-CSF through GMF-β-p38MAPK-NFκB axis. Thus, controlling the protein kinases that phosphorylates hGMF-β residue at Ser83 is pivotal in regulating the proinflammatory response14. However, developing inhibitors against these generic kinases involved in the phosphorylation of hGMF-β is highly non-specific and could lead to adverse drug reactions. Thus, a novel molecular mechanistic strategy was conceptualized by which blocking the phosphorylation sites on hGMF-β substrate using small molecule inhibitors prevent the kinase mediated phosphorylation involved in GMF-β induced proinflammatory response.Phosphorylation of GMF-β on residues Thr27, Ser53, Ser72 and Ser83 by different protein kinases is critical in its activation and regulationin vitro and in vivo demonstrates its ability to suppress Ser83 phosphorylation of GMF-β and proinflammatory response leading to reversal of immunopathology in EAE animals. The present discovery shows the therapeutic potential of GMFBI.1 as a lead compound in modulating MS.Here, we report the identification of a naturally occurring small molecule [1-H indazole-4yl methanol] against hGMF-β substrate through structure-based drug design technique and named the compound as GMFBI.1. Further, validation of its inhibitory efficacy both ). Further, the 3D structural stability of hGMF-β for 40 ns molecular dynamics (MD) simulations is shown in Supplementary, Fig. Due to lack of the three dimensional (3D) structure of hGMF-β, homology modelling technique was employed to construct 3D structure of hGMF-β. We obtained murine GMF-β protein (PDB ID: 1V6F) structure solved using NMR technique as the best hit having BLAST score of 274, E-value of 1e-94 and sequence identity of 98%. The hGMF-β homology model development, its 3D structure quality analysis was given in . It consists of key phosphorylating residues Thr27, Ser53, Ser72 and Ser83 of hGMF-β involved in the downstream signalling and proinflammatory response. These important observations further set the platform for structure-based drug design studies on this key neurological drug target.Using SiteMap module, the active site residues of hGMF-β protein was predicted to be at SITE1  strategies are applied to search the chemical space for potential compounds that binds to the active site of the hGMF-β protein by neglecting the false positive hits. Initially, LigFilter module was employed with respect to the physico-chemical properties of the successful CNS drugs. This VS step brought down SPECS database compounds from 961006 to 651217. The second step involves high-throughput VS of 651217 compounds using Glide module which filtered 5758 compounds on the basis of best docking pose and binding energies  of the compound with respect to GMF-β active sites. Further, simple precision (SP) based docking filtered of 335 compounds with BE ≥ −6 kcal/mol and finally extra precision (XP) docking method resulted in 4 best VS compounds and is shown in Fig. Structure based drug design are well established in pre-clinical drug discovery programmes to evaluate the small molecule databases containing millions of compounds in order to identify new lead compounds towards druggable targets21. All the molecular properties predicted by the QikProp module of four VS hits (I-IV) are shown in Fig. in vitro and in vivo validation studies.Next, ADME/T properties of these compounds pertaining to pre-clinical drug discovery stages were analysedin silico binding of GMFBI.1 to hGMF-β, we performed surface plasmon resonance (SPR) based interaction studies. In a steady-state affinity 1:1 binding model, the affinity constant KD value was 19.95 µM for GMFBI.1 towards hGMF-β and the residuals of kinetic data showed a Chi2 value of 0.0041  significantly suppressed GMF-β phosphorylation on Ser83 compared to the vehicle alone control (p < 0.01vs control) as judged by the specificity of the antibody to Ser83 phosphorylated 76–90 GMF-β sequence . MTT toxicity assay of GMFBI.1 at different concentrations on astrocytes [0.1 to 3 mg/mL (670 μM to 0.2 mM)] showed astrocyte viability upto 1 mg/mL (6.70 mM) of GMFBI.1 .In order to further confirm the molecular mechanism of 041 Fig. . Subsequnce Fig. . MTT tox(arrow), (arrow), p = 0.0043 & 0.0001)   Fig. . FurtherI.1 Fig. .Figure 4To further assess whether LPS mediated over expression of GMF-β and the effect of GMFBI.1 on Ser83 phosphorylation suppression can be reproduced in a GMF-β over expressing clone, we generated different stable clones of GMF-β by transfecting the human GMF-β construct into astrocytes and colonies were selected using G418 to make stable GMF-β expressing clones. As shown in Supplementary, Fig. in vitro efficacy of GMFBI.1 in regulating GMF-β activity, we evaluated GMFBI.1 bio-distribution and toxicity in vivo. Intraperitoneal administration of 12 mg/kg of GMFBI.1 to C57BL/6 mice (n = 3) showed high clearance through the renal route and was non-toxic to the liver demonstrated by similar AST and ALT levels to that of control despite administering GMFBI.1 twice daily continuously for 25 days . Importantly, GMFBI.1 could not be detected in any of the organs by 6th hour including brain . Absence of GMFBI.1 in brain prompted us to test the possibility of GMFBI.1 access to brain in EAE animals wherein blood brain barrier (BBB) is compromised22. Thus, we injected 12 mg/kg of GMFBI.1 i.p. in EAE animals with compromised BBB as in MS patients. Within 2 to 3hrs, maximum of 13 μg of GMFBI.1 could be seen in the brains of EAE animals and by 6th hour, we failed to detect any GMFBI.1 in the EAE brains which is similar to the clearance of GMFBI.1 seen from all organs in normal mice as well   Fig. .Figure 5in silico, in vitro and in vivo approach. Blocking the GMF-β substrate Ser83 phosphorylation site rather than inhibiting the kinase involved in the phosphorylation led to down regulation of the key biochemical pathway involved in the propulsion of proinflammatory response and associated clinical symptoms, pathology and proinflammatory cytokine levels at the peak of EAE. Although similar concept of substrate blocking to prevent interaction between signal sequences of proteins with Sec. 61 involved in trafficking has been shown, the protein-protein interactions are of a different kind not involving a kinase15. Here, we developed a small molecule inhibitor GMFBI.1 using structure-based drug design strategy. GMFBI.1 blocked the key phosphorylating residue Ser83 of GMF-β, important in the downstream p38MAPK pathway involved in the propulsion of proinflammatory response . The best site map predicted residues (SITE-1) matched with the phosphorylating residues of GMF-β including the key Ser83 residue established earlier14 confirming the quality of our hGMF-β protein model . Subsequently, using Glide molecular docking methods, we identified four potent inhibitors against hGMF-β with BE ≥ −6 kcal/mol . The ADMET profiles of these compounds were assessed to provide insight into our in vitro/in vivo studies as shown for discovering best lead compounds in the pre-clinical drug discovery program24.Since human and mouse GMF-β phosphorylating residues are conserved, we solved the hGMF-β 3D structure by homology modelling due to the better choice for structure-based drug design studies by considering the potential translational aspects of the present study. Further, 3D structure quality of the hGMF-β model and MD simulation studies provided good structural accuracy making it suitable for structure-based blocker design ues SITE- matched ol Figs. . The ADMin silico  as well as its direct physical interaction of molecules using SPR analysis . Notably, GMFBI.1 establishes direct hydrogen bonding interaction with Ser83 residue  and that at 250 ng/mL (1.68 μM) concentrations, GMFBI.1 was effective in suppressing hGMF-β Ser83 phosphorylation  and remained well within 1 mg/mL of GMFBI.1 . The drop in Ser83 phosphorylation is accompanied by reduced p38MAPK phosphorylation and NFκB down regulation by GMFBI.1 in a concentration dependent manner  to a different site other than GMFBI.1 binding site on hGMF-β  GMFBI.1 concentrations, the astrocytes remained viable ion Fig.  and remaner Fig. . To furtner Fig. . The sup.5 mM GMFin vivo toxicity to liver by measuring AST and ALT levels in mice. Being hydrophilic, GMFBI.1 exhibited high clearance and within three hours following 12 mg/kg i.p injection, majority of the compound was found in urine . Notably, we failed to detect GMFBI.1 in the wild type brain of C57Bl/6 mice suggesting the low BBB penetrability of GMFBI.1 . Interestingly, in EAE animals, approximately 13 µg of GMFBI.1 was found in the brain within three hours following its i.p injection due to the BBB breaching . Notably in our in vitro study, 500 ng/mL (3.36 μM) of GMFBI.1 at 100% bioavailability down regulated p38MAPK phosphorylation and NFκB expression significantly . Since, inhibition of Ser83 phosphorylation of GMF-β is crucial in the activation of proinflammatory cascade, we verified the phosphorylation levels in EAE animals brain treated with and without GMFBI.1. As shown in  EAE animals treated with GMFBI.1 of 12 mg/kg showed significant Ser83 phosphorylation reduction on GMF-β and that the levels of Ser83 phosphorylation remained similar to wild type. This consolidates our in silico and in vitro observations on the molecular mechanism of GMF-β phosphorylation blockage in an in vivo setting.Following induction of EAE in C57BL/6 mice using MOGals Fig.  and S2.  in Fig.  EAE anim28. Interestingly, IL33, a cytokine up regulated in MS was shown to increase GMF-β expression in mast cells26. Thus, GMFBI.1 could modulate the GMF-β activity in mast cells in addition to regulating GMF-β locally in the CNS astrocytes to alter the cytokine response in EAE animals . Indeed, improvements in clinical scores were matched with the reduced inflammation score accompanied by down regulation of proinflammatory cytokines IFN-γ, IL-17A and GM-CSF particularly in the serum . This observation is in agreement with the studies on GMF-β null mice wherein, the levels of mononuclear cell infiltrates were drastically reduced in the brain parenchyma compared to wild type EAE animals10. It is well known that both in MS and EAE, IL17 and GM-CSF play pivotal roles in disease progression and severity during the active phase of the disease. Expression of these key inflammatory cytokines in the periphery plays a major role in the pathology and severity of MS and EAE30. IL-17A contributes immensely to CNS inflammation than the damage caused by Th1 class of cytokines31. Monoclonal antibody secukinumab against IL-17A suppressed EAE and found to attenuate IL-6 mediated proinflammation in primary human astrocyte32. Thus, significant IL-6 and IL-17A down regulation by GMFBI.1 could alter the pathogenesis in the course of the MS disease. Cytokine subsets such as IFN-γ, IL-6, TNF-α and IL1-β induces the activation of macrophage, increases ROS production and drive the homing of T cell into CNS leading to increased levels of IFN- γ, IL-6 and TNF-α transcripts in CSF and CNS tissue of EAE mice and MS patients34. Notably, GMFBI.1 significantly down regulated the levels of IL1-β, IFN- γ, TNF-α and IL-6 following treatment . Although, the exact role of IL1-β in MS remains unclear, elevated levels of IL1-β transcripts are seen in CSF, brain and sera of MS patients35 and that IL-1β stimulate T-helper cells and astrocytes to promote EAE pathogenesis by compromising BBB integrity36. Thus, down regulation of IL-1β following GMFBI.1 treatment in EAE could contribute to the suppression of pathogenesis of EAE. Further, the pleotropic cytokine GM-CSF responsible for the recruitment of peripheral leukocytes to CNS during EAE37 was also down regulated significantly in the sera of GMFBI.1 treated animals  to MS patients led to reduced lesion activity in the CNS39. This cytokine is also responsible for the differentiation of microglia to dendritic cells, a common antigen presenting cells in the CNS. Also, over expression of GMF-β led to increased production of GM-CSF in astrocytes that mediates the release of proinflammatory cytokines by microglia9. Thus, down regulation of GM-CSF mediated by GMFBI.1 have multiple effects on various immune cells which in turn down regulate the overall proinflammatory response in the CNS . These cytokines were down regulated in MS and are important in maintaining an inflammatory balance44. Of note, mice with B cells that do not produce IL-10 did not recover from EAE, indicating the importance of up regulation of IL-10 following treatment with GMFBI.145. Thus, the action of GMFBI.1 in creating a shift in the cytokine profile levels from pro to anti-inflammatory scenario establishes its potential role as an anti-inflammatory agent against inflammatory damage in EAE.In conjunction with the suppression of proinflammatory cytokines, significant up regulation of anti-inflammatory cytokines TGF-β and IL-10 following GMFBI.1 treatment to EAE mice was seen Fig. . These cin vivo by reversal of EAE. Importantly, the integrated approach in developing an inhibitor brings in a novel molecular strategy by which a substrate specific inhibitor was developed without crystal structure and validated its efficacy in suppressing EAE. The identified GMFBI.1 could serve as a promising candidate therapeutic molecule for treating inflammatory disease affecting brain due to its high aqueous solubility and clearance. The oral delivery study of GMFBI.1 is currently underway to translate the finding. The present concept of developing an inhibitor against a substrate of an enzyme than the enzyme itself opens up avenues for developing new therapeutics.In conclusion, using a novel molecular mechanistic approach, we targeted the critical phosphorylation site of GMF-β substrate involved in the proinflammatory response induction in EAE led to identification of a small molecule GMFBI.1 by structure based drug designing. GMFBI.1 binds directly to GMF-β and blocks its Ser83 phosphorylation pivotal in p38MAPK activation and NFκB expression associated with a key biochemical pathway involved in the proinflammatory response particularly mediated by astrocytes in the brain. The efficacy of GMFBI.1 in subduing the proinflammatory response and up regulation of anti-inflammatory response at the peak of the disease is further validated 46, a computer program that models the 3D structure of proteins and their assemblies by satisfaction of spatial restrains was used for building the homology model. The predicted 3D model was further evaluated using PROCHECK and ERRAT programs in SAVES server, to determine the stereo chemical quality of the structure47.The hGMF-β sequence (NP_004115.1) was retrieved from NCBI protein database. The sequence with best sequence identity, blast score and expectation value with that of hGMF-β was retrieved from PDB using BLAST-P program for identifying template for homology modelling. Homology model of hGMF-β was built using MODELLER 9v9 program48. First step include the topologic and coordinate files of the initial structure by LEaP module using ff99SB force field. The system was solvated in a rectangular box of TIP3P water molecules with a minimum solute-wall distance of 8 Å. Thus, the whole simulation system resulted in 10183 water molecules with dimensions of (18.774 × 18.774 × 18.774) Å3. The solvated system was neutralized by adding sodium ions and then subjected to energy minimization in two consecutive steps. The first 500 steps of minimization were performed by the steepest descent method. Conjugate gradient method was subjected for the next 1000 steps of minimization during which the harmonic restraints with a force constant of 500 kcal were applied to the hGMF-β structure. The following system was then subjected to move freely in the second minimization step in which 1000 steps by steepest descent and 2500 steps by conjugate gradient methods were adopted. The system was then subjected to initial equilibration for a time period of 20 ps by heating from 0 to 300 K. The temperature was regulated with the Langevin dynamics with a collision frequency of 1 ps−1 with a pressure relaxation time set to 2.0 ps. The periodic boundary condition was used in the NPT ensemble with Berendsen temperature coupling and P = 1 atm with isotropic molecular-based scaling. SHAKE algorithm was applied to restrain all the covalent bonds involving hydrogen atoms and the Particle-mesh Ewald was used to treat the long-range electrostatic interactions. The integral time step was set as 2 fs and non-bonded cut-off of 10 Å was used. Final step involve the production MD simulation for 40 ns time interval. The dynamic motion of trajectories and energy information were recorded at every 20 ps time interval. Finally, (root Mean Square Deviation (RMSD) of the dynamic atomic positions of hGMF-β protein were evaluated by analysing its MD trajectories. The potential energy of the hGMF-β was also calculated for 40 ns MD simulations.Molecular dynamic (MD) simulation was carried out for the homology modelled structure of hGMF-β using AMBER12 software package in order to understand its dynamic structural and energetic stabilityv9.2 was used for the active site prediction of the developed hGMF-β model49. It uses novel search and analysis to generate information on the character of binding sites by identifying all the possible active sites on the protein. SPECS small compound database having 961006 compounds were used for sequential virtual screening (VS). Initially, compounds were prepared using the LigPrep module of Schrödinger v9.2. LigPrep adds hydrogen atoms, generates tautomers, ionization states, ring conformations, and stereoisomers, and produces the minimized 3D structures using OPLS2005 force field. Possible ionization states were generated at target pH 7.0 +/− 2.0 using EPIK module. The compounds were desalted and no tautomers were generated. For each ligand, single stereoisomers were generated by retaining the specified chiralities. Further, single low energy ring conformation for each compound was generated. The prepared ligands were subjected to VS criteria using the LigFilter module of Schrödinger v9.2. The compounds satisfying the criteria’s such as Molecular Weight ≤400, Number of hydrogen bond (H-bond) donors ≤3, Number of H-bond acceptors ≤7, Number of rotatable bonds ≤8 and LogP ≤5 were filtered as the first stage of VS. This VS criteria employed were based on the attributes of successful CNS drugs reported earlier21.The SITEMAP module of Schrödinger 50. HTVS is used when rapid screening of the large compound databases is required. After HTVS docking, the Dock Scores were analysed, and all the poses showing docking score (or free energy) ≤−5 kcal/mol were exported for SP docking. Ligand poses with high SP docking scores of ≤−6 kcal/mol was used as an input for XP docking. XP docking ensures better VS with negligible false positives filtering using accurate docking simulations. Finally, the QikProp module (version 3.3) was used for absorption, distribution, metabolism, excretion and toxicity (ADMET) prediction in the last stage of our VS procedure by selecting the best XP based high docking score compounds. This program is capable of predicting the physically significant descriptors and pharmaceutically relevant properties of a given set of drug like compounds. These pharmacokinetic properties include (i) predicted central nervous system activity on a –2 (inactive) to +2 (active) scale; (ii) Molecular weight; (iii) QPlogBB- Predicted brain/blood barrier partition coefficient (−3–1.2); (iv) Percent Human-Oral Absorption (PHOA)- Predicted human oral absorption on 0 to 100% scale ; (v) QPlogHERG- Predicted IC50 value for blockage of HERG K+ channels (Cardiotoxicity) (Below −5 not considered); and (vi) QPPCaco- Predicted apparent Caco-2 cell permeability in nm/sec gut blood barrier .The molecular docking was performed using the GLIDE (Grid-based Ligand Docking with Energetics) module of Schrödinger v9.2. Initially, the receptor grid was generated from the Receptor Grid Generation panel of GLIDE module using the OPLS2005 force field. The molecular docking scoring grids were positioned by selecting the centroid of the selected residues from SITE1 predicted region as described above. All the ligands with length less than or equal to 14 was assigned for docking procedure. By default, GLIDE Score (GScore) multi-ligand scoring function was used to score the poses of the ligand to judge its binding affinity (or binding free energy- BE) towards the active site of hGMF-β. GScore has a steric-clash term and added buried polar terms to penalize the electrostatic mismatches between the protein-ligand complexes. GLIDE docking involves three choices of docking precision. Initially, a high-throughput virtual screening (HTVS) was performed followed by the Standard-precision (SP) docking and later Extra-precision (XP) docking procedure to filter best possible candidates2 and U-value.The binding affinity of GMFBI.1 to GMF-β was examined by SPR . GMF-β (50 ug/ml in 10 mM sodium acetate pH5.5 buffer) was immobilized on sensor-chip CM5  using amine coupling protocol with flow rate 10 µL/min for seven minutes GE Healthcare Life Sciences). Among the two flow cells used, one was immobilized with GMF-β protein at ~8000 RU and the other flow cell was used as reference. During immobilization, running buffer used is PBS (1x), pH 7.4 and immobilization buffer used is 10 mM Sodium acetate, pH 5.5. GMF-β protein is immobilized for final response of 3742.7 RU. Interaction of GMFBI.1 to GMF-β is studied in 1X PBS buffer containing 5% DMSO at 25 °C. Five different concentrations  of GMFBI.1 were prepared by 2 fold serial dilution of GMFBI.1 from 50 uM concentration in 1X PBS + 5% DMSO buffer (running buffer). Subsequently, these different concentrations (in increasing order) of GMFBI.1 were passed over the immobilized sensor chip in different cycles (as in multi-cycle kinetics assay format). Surface was regenerated using high flow rate of running buffer (PBS+5%DMSO). The GMFBI.1 was passed for 60 seconds over the sensor surface and dissociated for 60 seconds with buffer at a flow rate of 30 µl/min. A kinetic curve fitting was performed using T200 Evaluation Software version 3.1 . The sensorgrams of test flow cell (FC2) is subtracted from the sensorgrams of reference flow cell (FC1). The subtracted sensorgrams were evaluated using biacore T200 evaluation software version 3.1. The obtained sensorgrams are fitted to the 1:1 binding fit model. The kinetics data are evaluated based on statistical measurements provided by the biacore evaluation software like Chi was purchased from Specs (Netherlands), custom made rabbit anti-phospho Ser83 specific antibodies to GMF-β conserved between mouse, rat and human sequence 76–90 (QHDDGRVS(p)YPLCFIF; Product YZ6909 is from YenZyme, USA), MOG 35–55 (MEVGWYRSPFSRVVHLYRNGK) peptide, hGMF-β construct and EndofectinTM Max transfection reagent from GeneCopoeia (USA), Pertussis toxin from List biological (USA), Mtb H37RA from Difco (USA). GMF-β from Origene (USA). Haematoxylin, Eosin, Chemiluminescent solution Luminata Forte, PKA, Adenosine triphosphate (ATP), LPS [E. coli 0111:B4], complete Freund’s adjuvant and Toluidine blue stain were from Sigma Aldrich-Millipore (USA), DMEM-F12 from Gibco (USA), Fetal Bovine Serum (FBS), Penicillin/Streptomycin, Trypsin (0.01%) were from Invitrogen (USA), protease inhibitor cocktail was from cOmplete, Mini EDTA-free protease inhibitor tablets Roche (Germany), antibodies against GMF-β, NF-κB p65, p38MAPK and Luxol fast blue (LFB) stain kit were from Abcam (UK), β-actin from Santacruz (USA), AST and ALT kit from Aspen (India), mouse Cytokines Multi-Analyte ELISA Array Kit- Qiagen (Germany), Vybrant-MTT cell proliferation assay kit and kinase assay buffer were from Thermo Fisher Scientific (USA).GMFBI.1 compound was characterized by NMR and HPLC with >95% purity 52 and ED50 of GMFBI.1 was calculated from quantified NFκB expression levels against different GMFBI.1 concentrations including 400 and 1000 ng/mL for 48 h and values were plotted using AAT Bioquest® (USA) tool. In vitro phosphorylation of purified hGMF-β using PKA and GMFBI.1 was performed using 1 µg of GMF-β protein and 40U PKA in the presence and absence of 250 ng/mL of inhibitor GMFBI.1 suspended in the kinase assay buffer containing 10 mM ATP at RT overnight in a total of 20 µL assay volume. The kinase assay buffer was used as the diluent for 250 ng/mL of GMFBI.1. The control used include kinase assay buffer alone without the inhibitor. The phosphorylation reaction was stopped by adding SDS running buffer and subjected to SDS-PAGE and immunoblotted using custom made rabbit anti-phospho Ser83 antibody (1:1000) specific to the Ser83 residues of GMF-β and phospho GMF-β levels were quantified using Image J software from NIH, USA.CTX-TNA2 rat brain primary astrocyte cell line  were treated with GMFBI.1 at concentrations 0–3 mg/mL for cell viability assay. p38MAPK and NFκB assay was done on LPS (100 ng/mL) stimulated astrocytes with different GMFBI.1 concentrations  for 48 hours and expression levels were detected by immunoblotting with anti-phospho p38MAPK and anti- NFκB antibody respectively. The band intensity was quantified53. EAE scoring was done on a scale from 0 to 5 as follows: 1- tail weakness; 2-weakness in hind limb; 3- hind limb paralysis; 4- hind limb paralysis with fore limb weakness or paralysis; 5- moribund/deceased53. GMFBI.1 was administered in EAE animals at a stage of clinical score 3. 12 mg/kg of GMFBI.1 was dissolved in saline were administered twice daily, i.p for 25 days. Mice were monitored and scored daily for clinical symptoms and weight changes. For histology analysis, brain and spinal cord of EAE animals treated with GMFBI.1 and saline were harvested, fixed in 10% neutral buffered formalin. 5 μm thick paraffin wax embedded brain and spinal cord tissue sections were analysed after haematoxylin and eosin (H&E), LFB and toluidine blue staining55. Tissue morphology was analysed for mononuclear cell infiltration at the perivascular space and meninges, demyelination and for mast cell infiltration. Mean inflammatory score was plotted by single blind semi-quantitative analysis56. Further, several randomly distributed 20x fields of brain and spinal cord section areas of saline and GMFB1.1 treated mice  were quantified for percentage area of mononuclear cell infiltration (H&E staining) and percentage area of myelination (LFB staining intensity) using ImageJ software.Animal experiments were carried out using female C57BL/6 mice purchased from Tata Memorial Centre - Advanced Centre for Treatment, Research and Education in Cancer (ACTRAC), Mumbai with prior approval from the institutional animal ethical committee AIMS Kochi (Ref. No IAEC:2015/2/8). All animal experiments were carried out in accordance with institutional animal ethics guidelines and regulations. EAE was induced in female C57BL/6 mice (10–12 week old) using 200 µg of myelin oligodendrocyte glycoprotein (MOG) peptide  emulsified in complete Freund’s adjuvant supplemented with heat-inactivated Mycobacterium tuberculosis H37RA (4 mg/ml) subcutaneously. 200 ng pertussis toxin was given i.p. at day 0 and 2 post immunizationsSerum of GMFBI.1 and saline treated animals were analysed for the levels of Th1 & Th17 proinflammatory cytokines  and Th2 anti-inflammatory cytokines (TGF- β and IL-10) after 25 days of treatment using enzyme linked immunosorbent assay according to the manufacturer’s instructions. O.D at 450 nm was read and relative fold changes with respect to saline treated were plotted.Image J software and normalized to the actin bands for each blot to quantify the levels different bands analysed. Statistical differences between groups were evaluated by analysis of variance (ANOVA) with post hoc analysis (Dunnett’s test) and unpaired Students t-test using GraphPad Prism 7. Difference is considered statistically significant when p ≤ 0.05*.Data shown in this study are representations of at least three independent experiments and expressed as mean ± SEM. The band intensity from the blots is measured using The 3D structure of modelled human GMF-β was deposited in Protein Model Database and assigned PMDB id: PM0081347.Supplementary informationSupplementary information2Supplementary information3


The beneficial effects of fatty acids (FAs) on human health have attracted widespread interest. However, little is known about the impact of FAs on the handling of urate, the end-product of human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia, a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway. URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renal urate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly contained in foods or produced in the body. For this purpose, we conducted an in vitro transport assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especially long-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among the tested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0, 14.2, and 15.2 μM, respectively. Although further studies are required to investigate whether the ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as nutritionally important substances influencing human health. Fatty acids (FAs) are physiologically important as energy sources and membrane constituents; moreover, FAs have diverse biological activities that modulate numerous cell/tissue properties in living organisms . AccumulHyperuricemia, which is characterized by elevated serum uric acid (SUA) levels, is a lifestyle-related disease with high prevalence . As hypeURAT1 is the causative gene for renal hypouricemia type 1 -urate with or without test compound at the indicated concentrations , and the cells were further incubated for 20 sec; 1% DMSO was used as a vehicle control. The cells were subsequently washed five times with ice-cold Buffer T2 and then lysed with 500 μL of 0.2 M NaOH on ice with gentle shaking for 1 h. The lysates were neutralized with 100 μL of 1 M HCl. Then, the radioactivity in the lysate was measured using a liquid scintillator . The protein concentration was determined using the PierceTM BCA Protein Assay Kit. The urate transport activity was calculated as the incorporated clearance (μL/mg protein/min): (incorporated level of urate [disintegrations per minute (DPM)/mg protein/min]/urate level in the incubation mixture [DPM/μL]). URAT1-dependent urate transport activity was calculated by subtracting the urate transport activity of mock cells from that of URAT1-expressing cells.The urate uptake assay using URAT1-expressing 293A cells was conducted according to our previous studies ,18 with 50) values. URAT1-mediated transport activities were then expressed as a percentage of control (100%). Based on the calculated values, fitting curves were obtained according to the following formula using the least-squares method with Excel 2019 :max is the maximum effect, EC50 is the half maximal effective concentration, C is the concentration of the test compound, and n is the sigmoid-fit factor. Finally, based on the results, the IC50 was calculated as described previously [Urate uptake was measured in the presence of several concentrations of each test compound to address their half maximal inhibitory concentration (ICeviously .n). Briefly, when analyzing multiple groups, the similarity of variance between groups was compared using Bartlett’s test. When passing the test for homogeneity of variance, a parametric Tukey–Kramer multiple-comparison test for all pairwise comparisons was used. To investigate the inhibitory effect of each FA on URAT1 function (vs. vehicle control indicated as 100%) in the screening stage, one-sample t-test (one-sided) was conducted. Statistical significance was defined as p < 0.05 or 0.01.All statistical analyses were performed using Excel 2019 with Statcel4 add-in software . Different statistical tests were used for different experiments as described in the figure legends, which include the number of biological replicates  (50 of > 100 μM (50 (133 μM); however, LA could inhibit URAT1 at low concentrations (≤1 μM) (Further investigation of the dose-dependent inhibitory effects of the nine unsaturated FAs on URAT1 determined the IC (≤1 μM) g. Second (≤1 μM) a as well (≤1 μM) i strongl> 100 μM . Additio (≤1 μM) . Interes (≤1 μM) , ω-3 FAsIn this study, to the best of our knowledge, we revealed for the first time the inhibitory effects of FAs on URAT1-dependent urate transport in vitro. While further studies are highly warranted to address the pathophysiological impact of our findings in terms of the possible effect on SUA and renal urate excretion in hyperuricemia model animals as well as in humans, the obtained results will open a new avenue for FAs as nutritionally important substances influencing human health.Our findings may extend the potentially beneficial effects of PUFAs of the ω-3 family on reducing the risk of hyperuricemia/gout. In fact, recent studies have shown that a dietary intake of certain ω-3 PUFAs, such as EPA and DHA can, at least partly, reduce a wide number of inflammation-related biological reactions ,4,5,22. The potential effects of the daily consumption of ω-3 PUFA-enriched foods on SUA will also be of interest. While the available information is limited and currently inconclusive, a randomized controlled trial in young healthy subjects showed that daily intake of fish oil  resulted in a significant decrease of SUA after 4 and 8 weeks of supplementation . A similGLUT9 is the causative gene for renal hypouricemia type 2 [There were some limitations to our study. First, the present study could not reveal how the FAs inhibited URAT1 function. Addressing this issue in the future will provide a deeper insight into the latent mechanistic features of URAT1. As an antiporter, URAT1 mediates urate transport in exchange for monocarboxylates such as lactate , which sa type 2 . Given tIn conclusion, we herein found that FAs, especially ω-3 PUFAs such as EPA and DHA, could inhibit URAT1. To gain insight into the potential SUA-lowering effects of certain FAs, further investigations including human studies are required.


The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle. The influenza virus polymerase is a multifunctional enzyme directing viral gene expression and genome replication. Immediately following infection, the polymerase primarily performs transcription to make the viral mRNAs that program the replication cycle. The polymerase then shifts output to produce more copies of the viral genome at later stages of infection. The balance between transcription and replication is critical for successful infection. Here we identify phosphorylation sites within the viral polymerase and describe how these post-translational modifications control polymerase activity. Cellular kinases modify the viral polymerase. We identified a phosphorylation site in the catalytic subunit PB1 that selectively disables transcription, but not replication. We also describe a phosphorylation site in PB1 that disrupts binding to viral RNAs, disabling all activities of the polymerase. These modifications may establish polymerases with specialized function, and help regulate the balance between transcription and replication throughout the viral life cycle. All RNA viruses encode machinery both to express viral transcripts and to replicate genomes. Negative sense RNA viruses must first transcribe using virally-encoded RNA-dependent RNA polymerases (RdRPs) that are packaged into virions. The viral RdRP subsequently replicates the genome, often with the help of protein products from the recently produced mRNA. Regulating the balance and timing of transcription and replication is crucial for successful infection.Orthomyxoviridae do not encode polymerase co-factors and it remains unclear how their polymerases are regulated.Viruses employ diverse strategies to control the abundance of virally-derived RNAs. Many RNA viruses rely on RdRP co-factors whose activity is dynamically regulated by post-translational modifications. For example, the Ebola virus polymerase is regulated by the viral transcription factor VP30. VP30 promotes transcription, whereas phosphorylation of VP30 results in its exclusion from transcription complexes favoring genome replication . A similInfluenza A virus contains eight negative-sense RNA genome segments packaged into virions as ribonucleoprotein (RNP) complexes. RNPs are double helical flexible rod-like structures containing the viral genome coated by oligomeric nucleoprotein (NP) and bound at both ends by the viral polymerase –10. The de novo RNP assembly  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Shin-Ru ShihAssociate EditorPLOS PathogensAndrew PekoszSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The authors have adequately addressed previously raised concerns.Reviewer #2: In this paper Dawson et al. identify multiple phosphorylation sites in the PB1 subunit of influenza A virus polymerase using mass spectrometry. Using RNP reconstitution and primer extension assays the authors demonstrate mechanistic effects of PB1 phosphorylation on the ability of viral polymerase to transcribe and replicate viral RNA. Finally, using viral reverse genetics, the authors also show that some of the discovered phosphorylation sites are required for virus replication in tissue culture.Overall, the presented research supports the authors’ conclusion and presents findings about the modulation of viral genome replication by viral polymerase phosphorylation that will be of interest to the influenza research community.Reviewer #3: The authors use mass spectrometry to identify phosphorylation sites of influenza virus polymerase, either transfected together with active PKCdelta or in infected cells. Three out of 13 sites in PB1 are common to both kinds of experiment. They focus on five sites and by analysing phospho-mimetic and phospho-ablative mutants, in recombinant viruses or transfections, show that the phosphorylation status modulates polymerase activity in diverse ways. Such information is new and beneficial, as a starting point, for improving understanding of polymerase regulation during viral infection. However, in view of the vast amount of structural information now available on influenza polymerase in different functional states, especially during transcription, the discussion of the mechanism of these phosphorylation effects is disappointing and should be further developed. Structural studies show that the polymerase is incredibly flexible, locally and globally, especially in the apo-state, and has multiple interchanging functional conformations. This undoubtedly affects accessibility of certain residues to kinases and phosphatases. Furthermore, many residues have multiple roles. For example, PB1 S673 helps stabilise a particular conformation of the 3’ end in the promoter, but during elongation it is in the outgoing template channel. Similarly, PB1 T570, which the authors unfortunately do not study in detail, is in the newly described secondary 3’ end binding site and its phosphorylation could inhibit RNA binding there. Residues S712 and S720 are both in the interface between the PB1 core and the PA endonuclease domain, but this is not mentioned.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)Reviewer #2: No major comments. Authors have appropriately addressed the previous major from the reviewers and updated their manuscript accordingly.Reviewer #3: 1. The authors make poor use of available structural information to help the reader think about possible effects of the observed phosphorylation sites. Rather than just categorising as catalytic or template entry, they should provide a table with a short description of the structural position of the residue and its possible functional role .2. Figure 2B should be improved to show all identified sites in the global view and the detailed part on top of the global view should be removed. There could be two enlarged view, one of the promoter bound form, one of the transcription elongation form.3. The authors should comment on the accessibility of the individual residues early on (and not only in the discussion), to give the reader an impression how reversible individual modifications might be. The authors should not just say ‘residues are not immediately on the surface’ but calculate solvent accessibility in different polymerase states . The authors might also emphasise that some of the sites  are probably more accessible when the polymerase is RNA free.4. The authors find T223D eliminates vRNA binding and cRNA stabilisation, yet, according to structures, it is not in close contact with viral RNA. On the other hand S673 directly binds the promoter 3’ end and thus S673D would be more expected to effect vRNA binding. The authors should perform the same vRNA and cRNA binding studies on the S673D mutant and try to explain this counter-intuitive phenotypes of these two mutations.5. Lines 275-276. The authors suggest there is evidence that the 3’ end of the template is differently located dependent on whether it is undergoing transcription or replication initiation. Specifically, from its structurally observed position in the active site, the 3’ end appears to need to backtrack by one nucleotide to allow terminal initiation of vRNA to cRNA replication, although using the same reasoning , would apparently not need to do so to allow internal initiation of cRNA to vRNA initiation. The observation that S673D specifically affects transcription but not either sense of replication suggests that, given its key structural role in stabilising the G9-C8 kink  in the observed promoter structure, the template pathway may not be the same and indeed the whole promoter might be different during replication. This is a very significant observation that merits more careful discussion . For instance, another nearby residue, intimately involved in binding the promoter, PA H510, upon mutation also specifically affects transcription :8989‐9001. doi:10.1128/jvi.76.18.8989-9001.2002).**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: No minor comments. Authors have appropriately addressed the previous minor from the reviewers and updated their manuscript accordingly.Reviewer #3: 1. Remind the reader whether PKCdelta is active in the nucleus or cytoplasm or both.2. Is it possible to quantify from the mass-spec analysis the fraction of polymerases that have a particular phosphorylation?3. Expand the phospho-residue identity search to bat influenza A and influenza B. Sites really important for modulation of intrinsic functions are likely to be fully conserved. For instance S384, whose A/D mutations are pretty much like wild-type, is already an aspartate in bat influenza polymerase.4. On lines 268-284 the authors describe the effects of S673 phosphorylation. Indeed, the S673 phosphorylation may influence the behaviour of template RNA in this region. However speculation in lines 281 about charge neutralisation is not supported by the structure organisation of the region.5. The authors speculate that some of the inaccessible sites might be modified already during polymerase translation and are static/irreversible (lane 307). The T223 and S478 are actually an example of inaccessible residues. More over the S478D is completely inactive. How do the authors explain what happens when such a polymerase is incorporated into a newly made RNP (lane 289) thus making it incapable of any further transcription and replication. Such a contradiction should be explained in comparison to positive effect of activation of RNPs in trans (lane 293).6. Line 302-304 unsupported speculation whose relevance is dubious.7. Line 306. This is a very static view of the polymerase, whereas it is actually extremely flexible.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Shin-Ru ShihAssociate EditorPLOS PathogensAndrew PekoszSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments : 28 Aug 2020Dear Dr Mehle,We are delighted to inform you that your manuscript, "Phosphorylation controls RNA binding and transcription by the influenza virus polymerase," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


To the Editor,Allergy is the most common immunologically mediated hypersensitivity disease worldwide affecting upper and lower respiratory tract, the skin as well as the gastrointestinal tract and may cause life‐threatening anaphylaxis.1 antibody specific for the major birch pollen allergen Bet v 1, which had been fused to the human epsilon heavy chain, was expressed and purified from hybridoma cells by affinity chromatography to >95% purity y Figure A. Under y Figure A. IgEmoAy Figure B. Structy Figure C.A/L showing that the dilutions of the IgEmoAb standard were correct down to few nanograms per mL without loss of activity. However, we noted that the ImmunoCAP determination of the concentrations of IgEmoAb was different  from the results obtained by microBCA protein determination which may be due to differences between the ImmunoCAP IgE standard and IgEmoAB (data not shown). Accordingly, a “real conversion factor” of 7 ng/mL = 1 kUA/L for calculating IgE concentrations had to be introduced.Using IgEmoAb and Bet v 1, we next established by ELISA a standard curve allowing to measure and quantify sIgE. A series of dilutions of IgEmoAb ranging from 2.42 to 2420 ng/mL was exposed to ELISA plate‐bound IgE yielding standard curves with a linear range of approximately 2.42‐242 ng/mL IgE depending on the time of assay development Figure . Figure A/l ImmunoCAP results   Tables  and S2. ) Figure . In summA/L) (r = .984, P < .0001, 95% CI 0.956‐0.994; Figure A/L but we were able to detect and quantify Fel d 1‐specific IgE with the IgE ELISA (Table r = .988 P < .0001, 95% CI 0.967‐0.996; Figure Next, we investigated whether the IgEmoAb‐based standard curve is also suitable for the quantification of patients' IgE to an allergen molecule unrelated to Bet v 1. For this purpose, we selected the major cat allergen Fel d 1 which besides Bet v 1 is one of the most frequently recognized allergens in the Russian population.SA Table  were comThe quantitative IgE ELISA assay should be useful for laboratories in less developed countries which cannot afford expensive IgE test systems for routine allergy diagnosis taking advantage of molecular allergy diagnosis.RV has received research grants from HVD Life Sciences, Vienna, Austria and Viravaxx, Vienna, Austria, and serves as consultant for Viravaxx. MvH has received lecture fees from Thermo Fisher Scientific and consultancy fees from Biomay AG, Vienna, Austria, and Hycor Biomedical LLC, CA, US, outside of the submitted work. Other authors have no conflict of interest to declare.Supplementary MaterialClick here for additional data file. Click here for additional data file. Click here for additional data file.


A 42-year-old patient without structural heart disease was referred for catheter ablation of salvos of outflow tract ventricular tachycardia (VT). Activation mapping of the clinical VT (VT1) revealed the earliest ventricular activation site at the right ventricular outflow tract (RVOT). Catheter ablation at this site led to a slight QRS shift of the VT morphology (VT2). Activation mapping of VT2 established the site of origin at the commissure between the right (RCC) and left (LCC) coronary cusps. This case is indicative of the presence of myocardial fibers displaying preferential conduction properties from the RCC–LCC commissure to a breakout site at the RVOT. The QRS morphology was suggestive of a right ventricular outflow tract (RVOT) site of origin (SOO). An electrophysiologic study was performed in a fasting state without sedation. Antiarrhythmic drugs (β-blocker) were stopped at least five half-times before the procedure. High-density activation mapping of the clinical VT (VT1) was performed using a three-dimensional nonfluoroscopic mapping system  via a multipolar catheter . A contact force-sensing catheter  was used for validation of the earliest activation site and ablation (contact force up to 8 g).A 42-year-old patient was referred for catheter ablation of symptomatic salvos of idiopathic ventricular tachycardia (VT). Structural heart disease was excluded by means of transthoracic echocardiography, cardiac magnetic resonance imaging, and exercise stress testing. The electrocardiogram (ECG) during VT displayed a left bundle branch block pattern (LBBB) with an inferior axis, R-wave in lead I, and precordial transition in lead V4  and 2C. The unipolar signal at this site displayed a small initial r-wave with a steep negative dV/dT. Radiofrequency (RF) energy delivery  at this site led to a slight QRS shift of the VT morphology. The ECG morphology of the second VT (VT2) was characterized by LBBB with an inferior axis, a lower-amplitude R-wave in lead I, and a precordial transition in lead V4 . Of note, the most prominent change was seen in lead V1, where the rS pattern shifted to a QS pattern with notching on the downward deflection, which is suggestive of a right coronary cusp (RCC)–left coronary cusp (LCC) commissure SOO. Following the change in QRS morphology, an additional attempt of RF delivery was performed at this site but failed to suppress the arrhythmia. Meticulous activation mapping of the VT2 was subsequently performed at the RVOT, the left ventricular outflow tract (LVOT) including the coronary cusps, and the coronary venous system [great cardiac vein (GCV)]. The new activation mapping attempt revealed the earliest activation site at the RCC–LCC commissure (−25 ms)  and 2C. Catheter ablation  at this site led to suppression of the arrhythmia. Of note, very low voltage and fragmented bipolar signals were recorded at the successful ablation site . On the contrary, the unipolar signal exhibited an initial r-wave with a less steep negative dV/dT as compared with during VT1. VT2 was not inducible by atrial or ventricular stimulation with or without isoproterenol infusion 30 minutes after ablation. The patient remained free from arrhythmias six months after the procedure.Activation mapping of the RVOT revealed the earliest activation site at the posterior septum (−23 ms) This case report is suggestive of the presence of myocardial fibers displaying preferential conduction properties from the RCC–LCC commissure to a breakout site at the RVOT.1 Of 202 patients with monomorphic VT or premature ventricular contractions (PVCs) originating from the outflow tract, six patients (3%) showed changes in QRS morphology following RF energy delivery, requiring an additional RF application at a different site, which was often the LCC. Yamada et al. reported that VAs originating from the coronary cusps display preferential conduction to the RVOT and an insulated myocardial fiber across the ventricular outflow septum may exist in these cases.2 Especially, in 25% of patients with VAs of a coronary cusp origin, pacemapping from the RVOT yielded a closer match of the QRS morphology than pacing from the coronary cusp. In these cases, pacemapping identified the preferential breakout site.2 In the study by Shirai et al., a QRS morphology shift following catheter ablation at the earliest activation site was observed in 4% of cases of idiopathic outflow-tract VAs. The shifted VT or PVC demonstrated a distinct ECG QRS pattern supporting the notion that different preferential QRS exit sites within the outflow tract were operative in these cases. After remapping, the earliest activation site of the second VT or PVC moved to a different anatomical structure adjacent to the initial ablation site in 65% of cases, which was most frequently the LVOT region (coronary cusps).Tada et al. initially investigated the prevalence and characteristics of idiopathic outflow-tract ventricular arrhythmias (VAs) with altered QRS morphology following RF catheter ablation.3 An intramural SOO with preferential conduction channels to specific sites of the outflow tract  may explain this phenomenon.3 The presence of preferential conduction can be unmasked by ablation at the first earliest activation site when the first preferential exit of the VT/PVC is eliminated.3 Unipolar signals displaying an rS pattern at both the earliest activation sites, as seen in our case, favors the presence of an intramural SOO.The complexity of the myocardial network around the outflow tract with the potential for preferential/alternative conduction and multiple exiting sites has been suggested to explain subtle variations in the QRS morphology of idiopathic outflow-tract VAs following RF ablation.In conclusion, a QRS morphology shift following catheter ablation of idiopathic outflow-tract VAs is not an uncommon phenomenon. Meticulous activation mapping usually reveals the true SOO in an adjacent site, which is more commonly the coronary cusps.