Targeting the JAK/STAT Pathway: A Combined Ligand- And Target-Based Approach

Janus kinases (JAKs) are a family of proinflammatory enzymes able to mediate the immune responses and the inflammatory cascade by modulating multiple cytokine expressions as well as various growth factors. In the present study, the inhibition of the JAK-signal transducer and activator of transcription (STAT) signaling pathway is explored as a potential strategy for treating autoimmune and inflammatory disorders. A computationally driven approach aimed at identifying novel JAK inhibitors based on molecular topology, docking, and molecular dynamics simulations was carried out. For the best candidates selected, the inhibitory activity against JAK2 was evaluated in vitro. Two hit compounds with a novel chemical scaffold, 4 (IC50 = 0.81 μM) and 7 (IC50 = 0.64 μM), showed promising results when compared with the reference drug Tofacitinib (IC50 = 0.031 μM).This study was funded by the University of Valencia and Generalitat Valenciana (GVA) through postdoctoral grants no. UVINV_POSTDOC18-785681 and APOSTD/2019/055 (M.G-L.) and by the University of Bologna through research grant no. RFO2019 (P.R., S.C., and M.R.)

Mechanism of sulfur transfer across protein-protein interfaces: The cysteine desulfurase model system

CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking and in iron-sulfur cluster assembly in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface that includes CsdA Cys328 and CsdE Cys61, the two residues known to be involved in the sulfur transfer reaction. Although mechanisms for the transfer of a sulfur moiety across protein-protein interfaces have been proposed based on the IscS-IscU and IscS-TusA structures, the flexibility of the catalytic cysteine loops involved has precluded a high resolution view of the active-site geometry and chemical environment for sulfur transfer. Here, we have used a combination of X-ray crystallography, solution NMR and SAXS, isothermal calorimetry, and computational chemistry methods to unravel how CsdA provides a specific recognition platform for CsdE and how their complex organizes a composite functional reaction environment. The X-ray structures of persulfurated (CsdA) and persulfurated (CsdA-CsdE) complexes reveal the crucial roles of the conserved active-site cysteine loop and additional catalytic residues in supporting the transpersulfuration reaction. A mechanistic view of sulfur transfer across protein-protein interfaces that underpins the requirement for the conserved cysteine loop to provide electrostatic stabilization for the in-transfer sulfur atom emerges from the analysis of the persulfurated (CsdA-CsdE) complex structure.BFU2008-02372/BMC, CSD 2006-23, and BFU2011-22588 to M.C., CTQ2012-36253-C03-03 and CTQ2015-66223-C2 to I.T., CTQ2015-64597-C2-1-P to J.J.B., and BFU2010-22266- C02-02 and CTQ2015-66206-C2-2-R to M.C.V. Further support for this work was obtained from the Generalitat Valenciana (ACOMP/2015/239 to I.T.) and from the European Commission FP7 ComplexINC grant (contract no. 279039) to M.C.V.Peer Reviewe

The mechanism of the transpersulfuration reaction in a cysteine desulfurase-sulfur acceptor model system

Trabajo presentado en las 1as Jornadas Españolas de Biocatálisis, celebradas en Madrid (España) del 02 al 03 de julio de 2015.Escherichia coli CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking, in iron-sulfur cluster assembly, and tRNA hypermodification [1] in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface. Although mechanisms for the transfer of a sulfur moiety across protein-protein interfaces have been proposed based on the IscS-IscU and IscS-TusA structures [2,3], the flexibility of the catalytic Cys loops involved has precluded a high resolution view of the active-site geometry and chemical environment responsible to facilitate sulfur transfer. Here, we have used a combination of X-ray crystallography, solution NMR, biophysical and computational chemistry methods to unravel how CsdA provides a specific recognition platform for CsdE and how their complex organizes a composite functional reaction environment. A mechanistic view of sulfur transfer across protein-protein interfaces emerges from the structural analysis of the CSD system

Catalytic Reaction Mechanism in Native and Mutant COMT from the Adaptive String Method and Mean Reaction Force Analysis.

Catechol-O-Methyltransferase is an enzyme which catalyzes the methylation reaction of dopamine by S-Adenosylmethionine increasing the reaction rate by almost 16 orders of magnitude compared to the reaction in aqueous solution. Here, we combine the recently introduced adaptive string method and the Mean Reaction Force method in combination with structural and electronic descriptors to characterize the reaction mechanism. The catalytic effect of the enzyme is addressed by comparison of the reaction mechanism in the human wild-type enzyme, in the less effective Y68A mutant and in aqueous solution. The influence of these different environments at different stages of the chemical process and the significance of key collective variables describing the reaction were quantified. Our results show that the native enzyme limits the access of water molecules to the active site, enhancing the interaction between the reactants and providing a more favorable electrostatic environment to assist the SN2 methyl transfer reaction

Catalytic Reaction Mechanism in Native and Mutant COMT from the Adaptive String Method and Mean Reaction Force Analysis.

Catechol-O-Methyltransferase is an enzyme which catalyzes the methylation reaction of dopamine by <i>S</i>-Adenosylmethionine increasing the reaction rate by almost 16 orders of magnitude compared to the reaction in aqueous solution. Here, we combine the recently introduced adaptive string method and the Mean Reaction Force method in combination with structural and electronic descriptors to characterize the reaction mechanism. The catalytic effect of the enzyme is addressed by comparison of the reaction mechanism in the human wild-type enzyme, in the less effective Y68A mutant and in aqueous solution. The influence of these different environments at different stages of the chemical process and the significance of key collective variables describing the reaction were quantified. Our results show that the native enzyme limits the access of water molecules to the active site, enhancing the interaction between the reactants and providing a more favorable electrostatic environment to assist the S_N2 methyl transfer reaction

Insights into the inhibited form of the redox-sensitive SufE-like sulfur acceptor CsdE

17 p.-8 fig.Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo.This work was supported by Spanish Instituto de Salud Carlos III (http://www.isciii.es) (PI12/01667 to MCV), Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/portal/site/mineco/) (PET2008_0101, BIO2009-11184, BFU2010- 22260-C02-02, and CTQ2015-66206-C2-2-R to MCV, and CTQ2015-66223-C2-2-P to IT), the Regional Government of Madrid (http://www.madrid.org/) (S2010/BD-2316 to MCV), and the European Commission (Framework Programme 7 (FP7)) (https://ec.europa.eu/research/fp7/index_en.cfm) project ComplexINC (Contract No. 279039) to MCV.Peer reviewe

Trajectory followed by the Cys61 side chain of CsdE as it approaches CsdA for reaction.

<p>Three structures of CsdE are superimposed and represented in cartoons, with helices shown as cylinders. NMR CsdE is colored white, X-ray CsdE is in cyan, and the fully rearranged CsdE observed in the persulfurated (CsdA-CsdE)₂ complex is in green. Helices α6, α7, and, for persulfurated CsdE, α8’ and α8, are labeled in order to facilitate comparisons. The side chains of Cys61 and Glu62 are shown in sticks with carbon colors according to the corresponding CsdE structure and sulfur/oxygen atoms in CPK colors.</p

Structural comparison of CsdE along the proposed conformational change.

<p>(A) Superposition in cartoon with helices shown as cylinders of the CsdE free monomer from X-Ray (overlaid structures colored in green, white and red to highlight the movement, with the Cys61 Cα atom represented as a sphere), the CsdE monomer of dimer of the present study (pale blue) and CsdE monomer from the X-Ray structure of the (CsdA-CsdE)₂ complex (cyan). The CsdE free monomer is depicted over the ensemble-weighted maximally correlated mode contributing to the change in the selected distance (d[Cys61(Cα)–Val88(Cα)]). (B) Insight of the CsdE free monomer’s movement of the loop is shown with Cys61 side chain represented as balls and sticks.</p

Cys61 as an interface hub on CsdE exposed surface.

<p>(A) Opaque molecular surfaces of CsdE (white) with the position of Cys61 mapped out in yellow. From the top right corner and following a clockwise rotation, the following interaction surfaces are shown: TcdA (in pink), CsdA (persulfurated complex, in green), the two non-symmetric CsdE interaction surfaces (in cyan and in blue slate). (B) Close-up on the molecular surface of CsdE around Cys61 (yellow; labeled C61). The outlines of the interaction surfaces shown in (A) are drawn in thick line with the same color code; each area is labeled with the protein that occupies the respective surface area.</p