Full Papers ChemMedChem doi.org/10.1002/cmdc.201900687 1 2 3 Asymmetric Disulfanylbenzamides as Irreversible and 4 5 Selective Inhibitors of Staphylococcus aureus Sortase A 6 [a] [b] [b] [a] 7 Fabian Barthels, Gabriella Marincola, Tessa Marciniak, Matthias Konhäuser, [a] [c] [d, e] [a, f] [d] 8 Stefan Hammerschmidt, Jan Bierlmeier, Ute Distler, Peter R. Wich, Stefan Tenzer, 9 Dirk Schwarzer,[c] Wilma Ziebuhr,[b] and Tanja Schirmeister*[a] 10 11 12 Staphylococcus aureus is one of the most frequent causes of methods allowed the experimentally observed binding affinities 13 nosocomial and community-acquired infections, with drug- and selectivities to be rationalized. The most active compounds 14 resistant strains being responsible for tens of thousands of were found to have single-digit micromolar Ki values and 15 deaths per year. S. aureus sortase A inhibitors are designed to caused up to a 66% reduction of S. aureus fibrinogen attach- 16 interfere with virulence determinants. We have identified ment at an effective inhibitor concentration of 10 μM. This new 17 disulfanylbenzamides as a new class of potent inhibitors against molecule class exhibited minimal cytotoxicity, low bacterial 18 sortase A that act by covalent modification of the active-site growth inhibition and impaired sortase-mediated adherence of 19 cysteine. A broad series of derivatives were synthesized to S. aureus cells. 20 derive structure-activity relationships (SAR). In vitro and in silico 21 22 Introduction target structures.[2] SrtA mediates the attachment of surface 23 proteins to the bacterial cell wall and it was shown that an S. 24 The ongoing spread of antibiotic resistance among Gram- aureus ΔSrtA mutant is clearly attenuated in mouse infection 25 positive bacteria such as Staphylococcus aureus highlights the models compared to the wild type.[3,4] SrtA inhibitors are likely 26 need for new treatment options beyond traditional antibiotics. to interfere with adherence and intercellular communication 27 In this respect, exploring virulence mechanisms as drug targets rather than with bacterial growth, thus imposing a lower 28 might provide novel opportunities to interfere with bacterial selective pressure to promote resistance development.[5] Since 29 pathogenicity.[1] The cysteine transpeptidase sortase A (SrtA) neither genetic deletion[6] nor selective chemical inhibition[7–9] 30 was considered as a putative anti-virulence drug target, which of S. aureus SrtA was found to cause cytotoxic or growth 31 may be addressed also in combination with classical antibiotics’ inhibitory effects on bacterial cells, the enzyme meets the 32 requirements of an anti-virulence target. Microbial surface 33 components recognizing adhesive matrix molecules 34 [a] F. Barthels, M. Konhäuser, S. Hammerschmidt, Jun.-Prof. Dr. P. R. Wich, Prof. Dr. T. Schirmeister (MSCRAMMs) are bacterial surface proteins utilized during 35 Institute for Pharmacy and Biochemistry pathogenesis for adherence to endothelial host cells and 36 Johannes-Gutenberg-University of Mainz playing a role in immune evasion.[10] Many of these virulence- 37 Staudinger Weg 5, 55128 Mainz, Germany E-mail: [email protected] associated proteins are secreted as precursors with C-terminal 38 [b] Dr. G. Marincola, T. Marciniak, Dr. W. Ziebuhr LPXTG-tagged sorting-signals. At the bacterial cell wall, they are 39 Institute for Molecular Infection Biology recognized and cleaved between threonine and glycine by the 40 Julius-Maximilians-University of Würzburg Josef-Schneider-Strasse 2, 97080 Würzburg, Germany membrane-anchored transpeptidase SrtA.[11] Subsequent liga- 41 [c] J. Bierlmeier, Prof. Dr. D. Schwarzer tion to the pentaglycine tail of the peptidoglycan layer yields 42 Interfaculty Institute of Biochemistry the covalent attachment to the bacterial outer surface.[12] In S. 43 Eberhard-Karls-University of Tübingen Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany aureus, approximately 20 surface proteins have been identified 44 [d] Dr. U. Distler, Prof. Dr. S. Tenzer as naturally occurring SrtA substrates, including several factors 45 Institute for Immunology, University Medical Center that are involved in pathogenicity, such as protein A (SpA), 46 Johannes-Gutenberg-University of Mainz Langenbeckstr. 1, 55131 Mainz, Germany fibronectin-binding proteins (FnbpA/B), clumping factors (ClfA/ 47 [e] Dr. U. Distler B), serine-aspartic acid repeat proteins (SdrC/D/E) and staph- 48 Focus Program Translational Neuroscience (FTN), University Medical Center ylococcal surface proteins (Sas).[13,14] 49 Langenbeckstr. 1, 55131 Mainz, Germany [f] Jun.-Prof. Dr. P. R. Wich The eight-stranded β-barrel protein SrtA possesses three 50 School of Chemical Engineering, University of New South Wales conserved residues within the sortase family: His62, Cys126, and 51 Science and Engineering Building, Sydney, NSW 2052, Australia Arg139, each of which cannot be mutated without disrupting 52 Supporting information for this article is available on the WWW under https://doi.org/10.1002/cmdc.201900687 enzymatic functionality.[15] Structural similarities between the 53 © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This transpeptidase SrtA and the papain protease were noticed,[16] 54 is an open access article under the terms of the Creative Commons Attri- however, enzymatic characteristics of the S. aureus SrtA differ 55 bution Non-Commercial NoDerivs License, which permits use and distribu- significantly from most proteases: (i) The catalytic Cys126 is 56 tion in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. “reversely protonated”, which means it does not form a 57 ChemMedChem 2020, 15, 839–850 839 © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Wiley VCH Freitag, 08.05.2020 2010 / 162169 [S. 839/850] 1 Full Papers ChemMedChem doi.org/10.1002/cmdc.201900687 thiolate-imidazolium pair, and thus, only a small fraction (< inhibitors of cysteine proteases,[35–37] thus, we decided to 1 0.1%) of SrtA is competent for catalysis at physiological investigate a disulfide warhead chemotype exchange and 2 pH 7.4.[17] (ii) The active site is predominantly defined by the systematic scaffold optimization to generate more selective and 3 intrinsic flexibility of the β6/7- and β7/8-loops.[18] (iii) The most less cytotoxic disulfide-based SrtA inhibitors (Figure 1C). 4 active form of SrtA is constituted as a homo-dimer with a K = 5 D 55 μM.[19] (iv) The K values for both, the LPXTG- and Gly - 6 M n substrates are exceptionally high (K = 5.5 mM and 0.14 mM), Results and Discussion 7 M probably due to the fact that the enzyme and both substrates 8 are spatially co-localized at the outer membrane yielding high Synthesis of the inhibitors 9 local concentrations.[20] (v) The high redox potential of the 10 catalytic Cys126 (1.27 V) makes SrtA insensitive towards Disulfanylbenzamides were synthesized based on a known 11 oxidation stress contributing to S. aureus phagocytotic procedure for asymmetric disulfides (Scheme 1).[38] Commer- 12 survival.[21] cially available sulfanylbenzoic acids 2a–c were activated at 13 Previous research campaigns investigated competitive-re- À 50 °C with trichloroisocyanuric acid (TCCA) to form electro- 14 versible S. aureus SrtA inhibitors including promising scaffolds philic sulfenyl chlorides in situ. The subsequent conversion with 15 such as 2-morpholinobenzoates,[22] thiadiazoles,[7,23] 2- nucleophilic alkyl thiols provided the disulfanylbenzoic acids 16 phenylthiazoles,[24] macrocyclic peptides,[25] 2- 3b–g. Since methyl mercaptan is gaseous at room temperature, 17 phenylbenzoxazoles,[26] and various other inhibitors.[8,27,28] How- for 2-(methyldisulfanyl)benzoic acid 3a a different strategy was 18 ever, the most active compounds were found to be irreversible employed. S-methyl methanethiosulfonate as thiomethyl-trans- 19 covalent inhibitors containing an electrophilic warhead that ferring reagent was utilized to convert thiosalicylic acid 2a to 2- 20 reacts with the active-site Cys126 of SrtA.[29–33] While having (methyldisulfanyl)benzoic acid 3a.[39] Boc-protected amino acids 21 significant inhibition in the low micromolar range, they typically 4a–l were coupled to various aromatic and aliphatic amines (R1) 22 exhibit poor target selectivity or are cytotoxic such as in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetrameth- 23 quinones,[34] rhodanines,[30] or benzisothiazolinones.[32] Zhulen- ylaminium tetrafluoroborate (TBTU) to provide the inhibitor 24 kovs et al. solved the NMR structure of SrtA in complex with a scaffold precursors 5a–v. The deprotection of the Boc group 25 covalent benzisothiazolinone adduct. Reaction with the active- was achieved by treatment with hydrochloric acid. Finally, the 26 site Cys126 occurred via ring-opening of the isothiazolinone disulfanylbenzoic acids 3a–g were coupled in the presence of 27 moiety yielding a covalent disulfide bond (Figure 1A). TBTU with the appropriate amine hydrochlorides 6a–v to 28 In the corresponding NMR structure (PDB: 2MLM), the ligand provide the desired test compounds 7a–w and 7α–ζ 29 displayed a reasonable fit, mimicking substrate binding in the (Scheme 1). Parent compound 1 was prepared in a one-pot 30 active-site pocket (Figure 1B). Asymmetric disulfides are known procedure from 3a and 6a. Both reactants were coupled by 31 means of TBTU. The subsequent treatment with lithium 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51