Journal of Enzyme Inhibition and Medicinal Chemistry ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20 Discovery of antimicrobial compounds targeting bacterial type FAD synthetases María Sebastián, Ernesto Anoz-Carbonell, Begoña Gracia, Pilar Cossio, José Antonio Aínsa, Isaías Lans & Milagros Medina To cite this article: María Sebastián, Ernesto Anoz-Carbonell, Begoña Gracia, Pilar Cossio, José Antonio Aínsa, Isaías Lans & Milagros Medina (2018) Discovery of antimicrobial compounds targeting bacterial type FAD synthetases, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254, DOI: 10.1080/14756366.2017.1411910 To link to this article: https://doi.org/10.1080/14756366.2017.1411910 © 2017 The Author(s). Published by Informa View supplementary material UK Limited, trading as Taylor & Francis Group. Published online: 19 Dec 2017. Submit your article to this journal Article views: 523 View related articles View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ienz20 JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018 VOL. 33, NO. 1, 241–254 https://doi.org/10.1080/14756366.2017.1411910 RESEARCH PAPER Discovery of antimicrobial compounds targeting bacterial type FAD synthetases Marıa Sebastiana,b , Ernesto Anoz-Carbonella,b,c, Begona~ Graciac,d, Pilar Cossioe,f, Jose Antonio Aınsab,c,d , Isaıas Lansf and Milagros Medinaa,b aDepartamento de Bioquımica y Biologıa Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; bInstitute of Biocomputation and Physics of Complex Systems (BIFI-IQFR and CBsC-CSIC Joint Units), Universidad de Zaragoza, Zaragoza, Spain; cGrupo de Genetica de Micobacterias, Departamento de Microbiologıa, Medicina Preventiva y Salud Publica. Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain; dCIBER Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain; eDepartment of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt, Germany; fBiophysics of Tropical Diseases, Max Planck Tandem Group, University of Antioquia, Medellın, Colombia ABSTRACT ARTICLE HISTORY The increase of bacterial strains resistant to most of the available antibiotics shows a need to explore Received 17 October 2017 novel antibacterial targets to discover antimicrobial drugs. Bifunctional bacterial FAD synthetases (FADSs) Revised 28 November 2017 synthesise the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These cofactors act in Accepted 28 November 2017 vital processes as part of flavoproteins, making FADS an essential enzyme. Bacterial FADSs are potential KEYWORDS antibacterial targets because of differences to mammalian enzymes, particularly at the FAD producing site. Bacterial FAD Synthetase; We have optimised an activity-based high throughput screening assay targeting Corynebacterium ammo- high-throughput screening; niagenes FADS (CaFADS) that identifies inhibitors of its different activities. We selected the three best Streptococcus pneumoniae; high-performing inhibitors of the FMN:adenylyltransferase activity (FMNAT) and studied their inhibition drug discovery mechanisms and binding properties. The specificity of the CaFADS hits was evaluated by studying also their effect on the Streptococcus pneumoniae FADS activities, envisaging differences that can be used to discover species-specific antibacterial drugs. The antimicrobial effect of these compounds was also eval- uated on C. ammoniagenes, S. pneumoniae, and Mycobacterium tuberculosis cultures, finding hits with favourable antimicrobial properties. Introduction levels, of FMN and FAD lead to the accumulation of apoflavopro- teins, unable to perform the flavin-dependent functions, resulting An important innovation gap in the discovering of antibiotics has in the concomitant death of the cell or the organism13,14. occurred during the last two decades1, with only five new classes – Prokaryotic bifunctional FAD synthetases (FADS) synthesise both available and 51 new antimicrobials in clinical development2 4.In FMN and FAD, being therefore potential new antimicrobial tar- addition, the selection of multi-drug resistant microorganisms5 gets15. Such hypothesis is sustained by several facts; (i) halting the encourages to search for new antimicrobial drugs capable of production of FMN and FAD prevents, from the very beginning, all inhibiting novel protein targets, such as those controlling the bio- pathways that involve flavoproteins and flavoenzymes, (ii) in most synthesis of essential biomolecules. Flavin mononucleotide (FMN) bacteria the only pathway for FMN and FAD biosynthesis occurs and flavin adenine dinucleotide (FAD) are the cofactors of flavo- with bifunctional FADS13,14, (iii) prokaryotic FADSs differ structur- proteins. All living organisms contain a great number of such pro- 6–8 ally and biochemically from the mammalian proteins that trans- teins and many of them are involved in essential functions , – form FMN into FAD16 19, so drugs that target these proteins are including protein folding9, electron transport in the respiratory likely to be selective for bacteria and (iv) the availability of struc- and photosynthetic chains10, b-oxidation of fatty acids11, nucleo- 12 tures of several bacterial FADSs facilitates the design of both tide synthesis or signal transduction , among others. Lack, or low – inhibitory drugs and activity assays20 22. CONTACT Milagros Medina [email protected] Departamento de Bioquımica y Biologıa Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna, 12. 50009 Zaragoza, Spain Supplemental data for this article can be accessed here. ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri- bution, and reproduction in any medium, provided the original work is properly cited. 242 M. SEBASTIAN ET AL. Bacterial FADSs have both ATP:riboflavin kinase (RFK, EC solutions at 50 and 10 mM. The purity of all compounds was 2.7.1.26) and ATP:FMN:adenylyltransferase (FMNAT, EC 2.7.7.2) activ- >95%, as determined by High performance liquid chromatography ities, being the latter reversible (FAD pyrophosphorylase) in some (HPLC), thin layer chromatography (TLC), NMR, IR or basic titration. species17,23. FADSs synthesise FMN and FAD from riboflavin (RF, vita- min B2) through two sequential reactions: RF is first phosphorylated to FMN by the RFK activity, and then the FMNAT activity transfers an Activity-based high-throughput screening for CaFADS adenylyl group from ATP to FMN producing FAD. These catalytic An activity-based HTS was performed on the 1240 compounds of activities are performed by two almost independent modules the Prestwick Chemical LibraryVR . The assays consisted in record- (Supplementary Figure SD1). The C-terminus module produces FMN ing the time dependent decrease in the fluorescence of the iso- from RF (named RFK module), while the N-terminal module trans- alloxazine ring, produced upon transformation of RF and FMN forms FMN into FAD (FMNAT module). The RFK module shows into FAD, as a consequence of the fluorescence quenching in sequence and structural homology with the monofunctional eukary- this later flavin27. When either the RFK or the FMNAT activities otic RFKs, while the FMNAT module does not present neither were inhibited, less FAD was produced and, consequently, the sequence nor structural similarity with the proteins that synthesise fluorescence decrease registered in a specific time interval was FAD in mammals16,21,24,25. Because the enzymes leading to FAD pro- less pronounced. Measurements were carried out using a multi- TM duction in prokaryotes and eukaryotes use different chemistry, and mode microplate reader, Synergy HT Biotek, with BRAND 96- TM belong to different structural families, potential inhibitors that spe- well plates pure Grade . To optimise the assay conditions, a cifically target the FMNAT module of bacterial FADSs are an interest- previous study was performed using constant concentrations of ing option for the novel drug development15. RF, ATP and CaFADS (5, 50 and 0.4 mM, respectively) and – In this work, we have used as a model the FADS from the non- variable concentrations of MgCl2 (0.2 10 mM) and DMSO pathogenic organism Corynebacterium ammoniagenes (CaFADS), (0–12.5% v/v). Optimum conditions were 2.5% DMSO, 10 mM which is the best known model to characterise members of the MgCl2 and sensitivity 70. prokaryotic FADSs family17,24,26–30, in an activity-based high- HTS reaction mixtures contained 5 mM RF, 0.4 mM CaFADS, throughput screening (HTS) assay to find potential inhibitors. The 10 mM MgCl2, in PIPES 20 mM, pH 7.0, 2.5% DMSO, and the corre- HTS hits were assayed to determine their specificity and potency sponding compound of the chemical library at a final concentration for the RFK and FMNAT activities. We also studied the kinetic of 250 mM. Reactions were initiated through addition of 50 mM ATP, inhibition mechanism of the three most potent and selective being the final reaction volume 100 ml. Controls, which contained inhibitors of the FMNAT activity (FMNAT hits), as well as their the reaction mixture but not any chemical from the library, were binding properties. Furthermore,