Freezing the Dynamic Gap for Selectivity: Motion‐Based Design of Inhibitors of the Shikimate Kinase Enzyme

This is the peer-reviewed version of the following article: Prado, V., Lence, E., Thompson, P., Hawkins, A., & González-Bello, C. (2016). Freezing the Dynamic Gap for Selectivity: Motion-Based Design of Inhibitors of the Shikimate Kinase Enzyme. Chemistry - A European Journal, 22(50), 17988-18000, which has been published in final form at https://doi.org/10.1002/chem.201602923. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-ArchivingShikimate kinase (SK), the fifth enzyme of the aromatic amino acid biosynthesis, is a recognized target for antibiotic drug discovery. The potential of the distinct dynamic apolar gap, which isolates the natural substrate from the solvent environment for catalysis, and the motion of Mycobacterium tuberculosis and Helicobacter pylori SK enzymes, which was observed by molecular dynamics simulations, was explored for inhibition selectivity. The results of the biochemical and computational studies reveal that the incorporation of bulky groups at position C5 of 5‐aminoshikimic acid and the natural substrate enhances the selectivity for the H. pylori enzyme due to key motion differences in the shikimic acid binding domain (mainly helix α5). These studies show that the less‐exploited motion‐based design approach not only is an alternative strategy for the development of competitive inhibitors, but could also be a way to achieve selectivity against a particular enzyme among its homologuesSpanish Ministry of Economy and Competiveness. Grant Number: SAF2013-42899-R Xunta de Galicia. Grant Number: GRC2013-041 European Regional Development Fund (ERDF) Spanish Ministry of Economy and Competiveness Xunta de GaliciaS

Structure of Staphylococcus aureus cytidine monophosphate kinase in complex with cytidine 5′-monophosphate

The crystal structure of S. aureus cytidine monophosphate kinase in complex with cytidine 5′-monophosphate has been determined

Synthesis of rigidified shikimic acid derivatives by ring-closing metathesis to imprint inhibitor efficacy against shikimate kinase enzyme

Diverse rigidified shikimic acids derivatives, which are stable mimetics of the high-energy conformation of shikimic acid, have been synthesized to enhance inhibitor efficacy against shikimate kinase enzyme (SK), an attractive target for antibiotic drug discovery. The synthesis of the reported conformationally restricted shikimic acid derivatives was carried out by ring-closing metathesis of allyloxy vinyl derivatives as the key step. The rigidification of the ligand conformation was used to maximize the effectiveness of the substituents introduced in the ether carbon bridge of the scaffold by pre-orienting their interaction with key residues and enzyme domains that are essential for catalysis and enzyme motion. Molecular Dynamics simulation studies on the enzyme/ligand complexes revealed marked differences in the positioning of the ligand substituent in the active site of the two enzymes studied (SK from Mycobacterium tuberculosis and Helicobacter pylori) and this explains their greater efficacy against one of the enzymes. This enhancement is due to the distinct induced-fit motion of the two homologous enzymes. A 20-fold improvement against the H. pylori enzyme was achieved by the introduction of a CH2OEt group in the rigid ether bridge of the reported shikimic acid analogsFinancial support from the Spanish Ministry of Economy and Competiveness (SAF2016-75638-R), the Xunta de Galicia [Centro singular de investigación de Galicia accreditation 2016–2019 (ED431G/09) and ED431B 2018/04], and the European Union (European Regional Development Fund –ERDF) is gratefully acknowledged. MP and EL thank the Xunta de Galicia for their respective predoctoral and postdoctoral fellowshipsS

Mechanistic insight into the reaction catalysed by bacterial type II dehydroquinases

DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymesFinancial support from the Comunidad de Madrid (S2010-BMD-2457 to F.G.), Xunta de Galicia (10PXIB2200122PR and GRC2010/12 to C.G.-B.) and the Spanish Ministry of Science and Innovation (SAF2009-13914-C02-02 to F.G. and SAF2010-15076 to C.G.-B.) is 5076 to CGB and BFU2008-01588/BMC to MJvR) is gratefully acknowledged. C.C. and A.P. thank the Spanish Ministry of Science and Innovation for their respective FPU fellowshipsS

Synthesis of 3-alkyl enol mimics inhibitors of type II dehydroquinase: factors influencing their inhibition potency

Several 3-alkylaryl mimics of the enol intermediate in the reaction catalyzed by type II dehydroquinase were synthesized to investigate the effect on the inhibition potency of replacing the oxygen atom in the side chain by a carbon atom. The length and the rigidity of the spacer was also studied. The inhibitory properties of the reported compounds against type II dehydroquinase from Mycobacterium tuberculosis and Helicobacter pylori are also reported. The binding modes of these analogs in the active site of both enzymes were studied by molecular docking using GOLD 5.0 and dynamic simulations studiesFinancial support from the Xunta de Galicia (10PXIB2200122PR and GRC2010/12) and the Spanish Ministry of Science and Innovation (SAF2010-15076) is gratefully acknowledged. BB, AS and AP thank the Spanish Ministry of Science and Innovation for FPU fellowshipsS

Study of the Phosphoryl‐Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy

This is the peer-reviewed version of the following article: Prado, V., Lence, E., Vallejo, J., Beceiro, A., Thompson, P., Hawkins, A., & González-Bello, C. (2016). Study of the Phosphoryl-Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy. Chemistry - A European Journal, 22(8), 2758-2768, which has been published in final form at https://doi.org/10.1002/chem.201504438. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-ArchivingThe phosphoryl‐transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D 1H and 31P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ‐phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure‐based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymesSpanish Ministry of Economy and Competiveness. Grant Number: SAF2013-42899-R Xunta de Galicia. Grant Number: GRC2013-041 European Regional Development Fund Sara Borrell Programme. Grant Number: CD13/00373 ISCIII General Subdirection of Assesment and Promotion of the Research. Grant Number: PI14/00059S

Exploring the Water-Binding Pocket of the Type II Dehydroquinase Enzyme in the Structure-Based Design of Inhibitors

Structural and computational studies to explore the WAT1 binding pocket in the structure-based design of inhibitors against the type II dehydroquinase (DHQ2) enzyme are reported. The crystal structures of DHQ2 from M. tuberculosis in complex with four of the reported compounds are described. The electrostatic interaction observed between the guanidinium group of the essential arginine and the carboxylate group of one of the inhibitors in the reported crystal structures supports the recently suggested role of this arginine as the residue that triggers the release of the product from the active site. The results of the structural and molecular dynamics simulation studies revealed that the inhibitory potency is favored by promoting interactions with WAT1 and the residues located within this pocket and, more importantly, by avoiding situations where the ligands occupy the WAT1 binding pocket. The new insights can be used to advantage in the structure-based design of inhibitorsFinancial support from the Spanish Ministry of Science and Innovation (Grant SAF2010-15076) and the Xunta de Galicia (Grant GRC2013/041) is gratefully acknowledged. B.B. and A.P. thank the Spanish Ministry of Education for their respective FPU fellowships. A.S. thanks the Spanish Ministry of Economy and Competitiveness for her FPI fellowship. J.M.O. thanks the Xunta de Galicia for a Plan I2C postdoctoral fellowshipS

Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

Targeted irreversible inhibitors bearing electrophiles that become activated towards covalent bond formation upon binding to a specific protein/enzyme is an emerging area in drug discovery. Targeting lysine residues is challenging due to the intrinsically low reactivity of the amino group at physiological pH. Herein we report the first example of a hydroxylammonium derivative that causes a specific covalent modification of an active-site and a sterically inaccessible lysine residue of an enzyme. The described ligands, compounds 1–3, were rationally designed to be activated towards covalent bond formation upon binding to the type I dehydroquinase (DHQ1) enzyme for the development of new anti-virulence agents to combat the widespread resistance to antibiotics. Evidence in atomic detail for the covalent modifications caused by the ligands to the catalytic Lys170 by the formation of a stable secondary amine is provided by the resolution at 1.08–1.25 Å of the crystal structures of DHQ1 from Salmonella typhi enzyme adducts. In addition, the first crystal structure of the addition intermediate adduct at 1.4 Å of a Schiff base formation reaction by using an analog of the natural substrate, compound 4, is also reported. Molecular dynamics simulation studies on non-covalent enzyme/ligand complexes and a two-dimensional QM/MM umbrella sampling simulation study suggested that a direct displacement by Lys170 with the release of NH2OH would be feasible. These studies might open up new opportunities for the development of novel lysine-targeted irreversible inhibitors bearing a methylhydroxylammonium moiety as a latent electrophile.Financial support from the Spanish Ministry of Economy and Competiveness (SAF2016-75638-R), the Xunta de Galicia [Centro singular de investigación de Galicia accreditation 2016-2019 (ED431G/09) and ED431B 2018/04] and theEuropean Union (European Regional Development Fund –ERDF) is gratefully acknowledged. MM and EL thank the Spanish Ministry of Education, Culture and Sport and the Xunta de Galicia for their respective FPU and postdoctoral fellowshipsS

Insights into substrate binding and catalysis in bacterial type I dehydroquinase

Structural, biochemical and computational studies to study substrate binding and the role of the conserved residues of the DHQ1 (type I dehydroquinase) enzyme active site are reported in the present paper. The crystal structure of DHQ1 from Salmonella typhi in complex with (2R)-2-methyl-3-dehydroquinic acid, a substrate analogue, was solved at 1.5 Å. The present study reveals a previously unknown key role for conserved Glu, Phe and Met and Gln, Pro and Ala residues, with the latter three being located in the flexible substrate-covering loop. Glu was shown to be responsible for the folding of this loop and for the dramatic reduction of its flexibility, which triggers active site closure. Glu46 was found to be key in bringing the substrate close to the lysine/histidine catalytic pocket to initiate catalysis. The present study could be useful in the rational design of inhibitors of this challenging and recognized target for the development of novel herbicides and antimicrobial agentsThis work was supported by the Spanish Ministry of Science and Innovation (grant number SAF2010-15076) and via FPU fellowships to M.M. and A.P., the Xunta de Galicia (grant number GRC2013/041) and via postdoctoral fellowships to E.L. and J.M.O., and by the European Regional Development Fund (ERDF)S

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzymeFinancial support from the Spanish Ministry of Science and Innovation (SAF2013-42899-R), Xunta de Galicia (GRC2013-041), and the European Regional Development Fund (ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for his postdoctoral fellowship. A.B. thanks the Miguel Servet Programme ISCIII-FEDER (CP13/00226) and the ISCIIIGeneral Subdirection of Assesment and Promotion of the Research (PI14/00059) for financial supportS