Fluoromethylketone-fragment conjugates designed as covalent modifiers of EcDsbA are atypical substrates
Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid
<!>INTRODUCTION<!>RESULTS AND DISCUSSION<!>METHODS<!>Crystallization and structure determination.<!>Macromolecular HSQC NMR experiments.<!>LCMS monitoring of protein species.<!>ASSOCIATED CONTENT<!>Accession codes<!>Corresponding Author
<p>and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.</p><!><p>Disulfide bond protein A (DsbA) is a dithiol-disulfide oxidoreductase enzyme that catalyzes the formation of disulfide bonds in bacterial proteins that enter the periplasm, including toxins and other virulence factors. (1,2) Bacterial strains in which dsbA has been knocked out are avirulent and hence DsbA is a novel antivirulence target (2)(3)(4)(5)(6)(7)(8)(9) . We have previously reported the fragment-based drug design (FBDD) strategy that we used to develop small molecule inhibitors of Escherichia coli DsbA (EcDsbA). (10)(11)(12) Other reported inhibitors of DsbA include peptides based on endogenous sequences that bind to EcDsbA, which have low μM potencies (13) as well as covalent compounds that were targeted at EcDsbB but were also found to bind to EcDsbA. (13) Herein we present our investigation of the development of a phenylthiophene fragment hit into a covalently binding compound. Although the compound initially formed a covalent adduct with EcDsbA, it was unexpectedly found to be turned over. We report our characterization of the mechanism of turnover.</p><p>FBDD has also grown over the last two decades to become a widely accepted method for the development of drugs. (14)(15)(16)(17)(18)(19) While a number of advances in screening methods, library design and subsequent development of fragments have been reported, a major hurdle in the implementation of FBDD is that the initial fragments often have low potency for their target and fragments may have multiple modes of binding. Due to the lack of existing potent inhibitors for EcDsbA, one of our initial approaches to improving potency and providing useful chemical probes was to attach a reactive functional group to a fragment hit to allow selective covalent binding to the target. Covalently modifying compounds has become the subject of increased interest due to their unique properties. (20)(21)(22)(23)(24)(25)(26) In addition, activity-based protein profiling has provided numerous examples of selective covalent compounds being used to investigate the proteome. (27)(28)(29) To generate covalent inhibitors, a highly selective reaction with the target protein is desirable to avoid off-target activity. We hypothesized that this could be achieved by judicious choice of a very low reactivity functional group. Attaching this to a fragment that bound to DsbA would be expected to accelerate the rate and selectivity of reaction with DsbA, by constraining the reactive functional group in close proximity to the active site. A similar approach has been successfully employed with high affinity ligands (30)(31)(32) as well as with reversible covalent linkages. (33,34) A number of examples have also started with covalently reactive compounds for fragment screening strategies. (35)(36)(37)(38) The thiol at the active site of DsbA enzymes possesses a uniquely low pKa. In EcDsbA, this is the thiol of Cys30 which has a reported pKa ~3.5, (39) which dictates that in the reduced protein it exists predominantly in the thiolate anion form at physiological pH. This makes Cys30 of EcDsbA more nucleophilic than most other thiols, which typically have pKa values of 8-9. Hence a weakly thiolatereactive functional group has the potential to generate selectivity for DsbA over other cellular thiols.</p><p>While a number of thiol selective probes are known, (24) we chose to use fluoromethylketone as a covalent warhead for reaction with EcDsbA. Non-aryl fluoromethylketones have been reported as covalent inhibitors for many cysteine hydrolases (e.g. caspases), (40) however the covalent inhibition of p90 ribosomal protein S6 kinase (RSK) (30) by an aryl fluoromethylketone (AFMK) demonstrated the weak and selective reactivity that was desired for the current project. Design and synthesis of a fragment-linked AFMK was expected to generate a compound that could selectively produce a covalently modified EcDsbA in a cellular setting. To allow for rapid variation of fragment-AFMK conjugates, the copper (I) catalyzed azide-alkyne cycloaddition (CuAAC or click) (41,42) reaction was used as the key conjugation step of the synthesis, whilst also forming the aryl unit of the AFMK.</p><!><p>A prior fragment screen against EcDsbA (10,43) resulted in the identification of 8 clusters of structurally similar fragments comprising 26 compounds plus 11 singletons being classified as hits. These were analyzed using X-ray crystallography, biochemical assay and other biophysical assays. One class for which X-ray crystallography yielded structures of the fragment bound to EcDsbA consisted of the amino phenylthiophenes 1, 2 and 3 (Figure 1). Crystal structures were generated by soaking fragments into crystals of EcDsbA in a similar way to our previously reported procedure. (10) The binding mode for 1 and 2 (PDB ID: 6BR4 (9) ) placed the fragment in a hydrophobic groove of EcDsbA below the active site disulfide (Cys30-Cys33). 15 N-1 H HSQC NMR titration data for compound 1 did not reach saturation at the highest assayed concentration, suggesting a K D of >1 mM and a ligand efficiency (LE) of <0.29 kcal mol -1 HAC -1 . Despite the modest ligand efficiency, the fact that complexes of EcDsbA bound to this core were amenable to structural characterization by X-ray crystallography led us to attempt to develop more potent phenylthiophene compounds. Initial development was based on a structure-activity relationships (SAR)by-catalogue approach followed by synthetic elaboration to optimize and then grow the fragments to improve their affinity for EcDsbA (see SI for full details). 15 N-1 H HSQC spectra were used to characterize binding and compounds were ranked based on the number and extent of chemical shift perturbations (CSPs) observed in the spectra according to our previously reported approach. (10) SAR for replacement of the amino substituent (4-17), the expansion of the methyl amino substituent (18-49) and optimization of the aromatic core (50-65) were explored and characterization of their binding using HSQC NMR spectroscopy and X-ray crystallography was conducted. Based on the binding information obtained from this characterization two additional series were synthesized (see SI for full details). These were designed to attach a ring onto the amino substituent, to generate a cyclohexyl (72-78), and a pyridyl (79-81) series, as well as a series amide linked to the core (66-71 and 82-90). 15 N-1 H HSQC data acquired in the presence of these compounds provided evidence of binding, with CSPs being observed for several residues in the hydrophobic groove of EcDsbA (Supplementary Table 1). Analysis of the concentration dependence of the CSP indicated that the data were not consistent with 1:1 binding, with CSP for different residues appearing to saturate at different ligand concentrations (see SI for full details). Nonetheless, the compounds appeared to have residues which showed saturation at 1 mM, indicating an improvement in affinity over the starting phenylthiophenes where no residues had reached saturation. Several of the compounds that bound EcDsbA were soaked into crystals of the protein, although X-ray crystallography revealed different binding modes for compounds within the series (Figure 1). These results were consistent with the compound binding in multiple orientations giving SAR that was difficult to interpret and also a compound series that was difficult to optimize.</p><p>In order to utilize the increase in binding affinity obtained from the analogue series and regain a consistent binding mode in X-ray crystallography we adopted the approach of anchoring the phenylthiophenes in the hydrophobic groove through covalent attachment to the active site. We reasoned that the unusual reactivity of EcDsbA's thiol provided an opportunity to develop selective chemical probes in spite of the relatively low affinity of this series of fragments. Hence, the initial focus was on synthesis of a fragment-FMK conjugate. In order to facilitate development of covalent inhibitors, we implemented a strategy of using the CuAAC reaction of an ethynylFMK and an azide linked fragment for the key linking step to provide a reliable chemistry that was tolerant of the presence of different functional groups and could therefore be used across multiple compound series.</p><p>A previously reported protocol for synthesis of the silyl protected ethynylFMK from ethyl fluoroacetate, (44) gave low yields and aldol dimer byproducts 92a and 92b (Scheme 1). Attempts to optimize this reaction were unsuccessful; hence an alternative synthetic pathway using the Weinreb amide 95 was used to produce the TIPS-protected ethynylFMK 91 in good overall yield (Scheme 1) and no evidence of byproducts 92a and 93b was found by NMR.</p><p>Attention was then turned to the synthesis of a suitable azido-fragment. Based on analysis of the crystal structure of fragment 1 in complex with EcDsbA (Figure 1) an azide linked by two carbons to the amino group of 1 was predicted to place the AFMK within close proximity to the active site Cys30. The azidofragment 96 was prepared using a similar synthetic route to phenylthiophene analogues 18-90 (Scheme 1). Synthesis of the conjugate fragment-AFMK 97 and a control compound 98 having only a benzyl substituent rather than the fragment was achieved by reaction of the respective azido compounds 96 and benzyl azide with the ethynylFMK 91 using in situ TIPS deprotection and CuAAC conditions (Scheme 1).  1). Reaction of the control benzyl-AFMK 98 (1 mM) with dithiothreitol (DTT, 1 mM) or glutathione (1 mM) revealed very slow production of fluoride in phosphate buffer (100 mM phosphate, pH 7.0) at 1 mM and the reaction did not reach completion after 73 h. At pH 12 the reaction with glutathione (1 mM) was complete in less than 20 h as expected due to the higher amount of free thiolate present in solution at higher pH. In the reactions of the AFMK compounds with EcDsbA it was necessary to include a reducing agent (tris-carboxyethylphosphine, TCEP) to prevent aerial oxidation of EcDsbA, which was otherwise observed under the conditions used for the assay. Control experiments with TCEP confirmed the stability of the AFMK to the phosphine reducing agent, indicating that TCEP did not interfere with the reaction. At 100 μM AFMK 98 and 100 μM DTT (100 mM phosphate, pH 7.0), no product was observed after 44 hours, indicating the desired weak reactivity of the AFMK. Conducting the reaction with EcDsbA (100 μM) at pH 6.8 (50 mM HEPES, 50 mM NaCl) however showed ~60% loss of the benzyl-AFMK 98 in 24 h. This confirmed the increased reactivity of EcDsbA to the AFMK compared to DTT suggesting that the compounds did indeed exhibit some degree of selectivity. The fragment-AFMK 97 showed complete conversion within 24 h when added to EcDsbA under the same conditions indicating reaction rate was increased by attaching the phenythiophene fragment, concordant with the AFMK-fragment conjugate binding to EcDsbA and constraining the reactive AFMK group in proximity to the thiol of Cys30.</p><p>The progress of the reaction of EcDsbA with the AFMK was monitored by recording a series of 15 N-HSQC spectra of reduced EcDsbA after addition of fragment-AFMK 97. As expected, the spectra showed progressive loss of cross-peaks consistent with reduced EcDsbA and the appearance of a new set of resonances. However, the new cross peaks were observed at resonance frequencies of oxidized EcDsbA.</p><p>The addition of TCEP at this point reverted the resonance frequencies observed in the 15 N-HSQC spectrum to those of reduced EcDsbA. The appearance of resonances consistent with oxidized EcDsbA suggested that the covalent adduct of AFMK 97 with EcDsbA was unexpectedly being turned over to release oxidized EcDsbA. LCMS analysis of the reaction mixture also revealed a product having a mass consistent with the formation of the desfluoro phenylthiophene 99 in the reaction mixture (Figure 2). These observations led us to propose a mechanism for resolution of the covalent complex whereby the second thiol of EcDsbA (Cys33) attacks the thioether to give an oxidized EcDsbA species and desfluoro 99. This requires the displacement of an enol group, in a process which we hypothesize is catalyzed by a nearby weakly acidic residue (possibly the proximal His32 imidazolium which is thought to stabilize the thiolate form of Cys33), or solvent (Figure 2). Further support for this turnover mechanism was obtained by the synthesis of an authentic sample of the desfluoro 99 which had identical MS fragmentation and HPLC profiles. Desfluoro 99 was synthesized using the same methods as for fragment-AFMK 97 with modified starting materials. The production of an authentic sample also enabled HPLC monitoring of the reaction to investigate the kinetics of the turnover. However, under conditions suitable for this analysis (20 µM EcDsbA, 200-1600 µM fragment-AFMK 97, 50 mM HEPES, 50 mM NaCl, pH 6.8, 25 °C, 0-8-1.6 % DMSO) the rate was too slow to be determined accurately (estimated k cat of 0.035 ± 0.002 h -1 and a K m of 110 ± 30 µM). In an attempt to increase the rate of reaction we investigated the effect of pH. Reactions were monitored by HPLC at pH 5.11, 6.54 and 8.12 in citrate-phosphate buffer (125 mM, 20 μM EcDsbA, 2 mM TCEP, 41-360 mM NaCl, 2% DMSO) at two concentrations of fragment-AFMK 97 (300 and 1000 μM) and showed increased rates at higher pHs at both concentrations (Figure 2).</p><p>The increased rate of turnover between pH 6.54 and 8.12 could not be explained by a higher proportion of Cys30 existing in the thiolate form as at both pHs Cys30 is expected to be >99.9% ionized. The data however could be explained by an increased amount of Cys33 thiolate, proposed as the nucleophile in turnover of the intermediate, although changes in pH may also affect the solubility of 97 and its affinity for EcDsbA.</p><p>The pH dependence was further investigated by LCMS monitoring the relative amount of EcDsbA species using the same reaction conditions at each pH with 1000 μM fragment-AFMK 97 (Figure 2). The reaction profiles at pH 6.54 and 8.12 show the intermediate mono-adduct accumulates relatively quickly, with significant buildup within a few hours. This is consistent with the initial labeling step being of a similar rate in each case, which is not surprising given the expected similar extent of ionization of Cys30 under these conditions. However, at pH 8.12 the adduct is turned over more rapidly to generate oxidized</p><p>EcDsbA, indicating an increased rate of turnover (relative to labeling) consistent with a greater extent of ionization of Cys33 thiolate at this pH. At pH 5.11 the rate of formation of the mono-adduct is relatively slow compared to that observed at pH 6.54 and 8.12. This is also consistent with the expected extent of ionization of Cys30 and is concordant with the reduced rate of product formation observed, due to both a decrease in the rate of labeling as well as a decrease in the rate of turnover. In addition, a relatively low intensity species consistent with a di-adduct was observed with increasing pH, suggesting labeling of Cys33 by the AFMK 97 or a further non-thiol mediated reaction with EcDsbA. The kinetic data fit well to a simple model in which the reduced EcDsbA binds to the fragment-AFMK 97 and undergoes covalent reaction (Figure 2, see SI for full details). This mono-adduct can either turnover (as outlined in Figure 2)</p><p>or EcDsbA can react with a second molecule of AFMK 97 (to account for the observed di-adduct). The oxidized EcDsbA product release on turnover would be expected to react very rapidly with TCEP to regenerate reduced EcDsbA.</p><p>To confirm the Cys33 mediated turnover mechanism and investigate the di-adduct formation, a C33A</p><p>EcDsbA mutant was prepared and monitored by LCMS which showed > 80% mono-adduct formation after 5 hours and no change for a further 7 days. No di-adduct was detected. This provides strong evidence that the second labeling site is Cys33 and further confirms the role of Cys33 in the proposed turnover mechanism as well as the thiol-selective nature of the AFMK.</p><p>Due to the instability of the AFMK labeled EcDsbA we also investigated the reaction with maleimide.</p><p>Although the labeling mechanism is different (Michael addition) the covalent complex that is produced still has an a-thiocarbonyl intermediate, which is the moiety proposed to be required for turnover. The reaction of maleimide with EcDsbA was monitored by 15 N-1 H HSQC; after 20 hours resonances consistent with the presence of oxidized EcDsbA were observed. MS monitoring of a sample for 12 hours showed rapid and complete conversion to the covalent adduct followed by slow conversion back to an apo EcDsbA species, consistent with a similar turnover mechanism to that observed for the AFMK 97. The ability of EcDsbA to turn over the maleimide labeled intermediate should be noted for future investigations of maleimide labeled DsbA enzymes and possibly other dithiol oxidoreductases, which are often presumed to create irreversible covalent linkages. The same mechanism may also exist for other well-known thiol labeling reagents such as iodoacetamide and 1,4-quinone based reactive functional groups.</p><p>In summary, we report the design and synthesis of a clickable alkynyl-FMK 91 for use in production of selective, covalent, thiolate-reactive compounds. Reaction of this species with an azido derivative of a low affinity fragment allowed for rapid synthesis of the fragment-AFMK conjugate 97 reported in this study. Investigations by 19 F NMR revealed an increased rate of formation of covalent adduct with EcDsbA when the AFMK reactive functional group was attached to a low affinity fragment compared to a control benzyl group. In addition, selectivity for EcDsbA over other thiols was observed for the AFMK. Further investigation by MS, 15 N-1 H HSQC NMR and HPLC supports the conclusion that the covalent complex is not stable and can be turned over to give an oxidized EcDsbA and a desfluoro product. Additional investigations suggest that a similar turnover mechanism may operate with maleimide. EcDsbA therefore possesses an ability to turn over a-thioether carbonyl intermediates leading to a formal reductive defluorination of AFMKs and reduction of maleimides to succinimides.</p><!><p>Protein expression and purification. 15 N isotopically enriched EcDsbA was expressed as previously described. (10) A plasmid, pC33A, for the expression of the C33A mutant of EcDsbA (EcC33A), was created by site-directed mutagenesis of the plasmid B0013, using the primers 'C33A_f2' (ctctttcttctgcccgcacgcctatcagtttgaagaagtt) and 'C33A_rev2' (aacttcttcaaactgataggcgtgcgggcagaagaaagag). The plasmid pC33A was used to transform BL21(DE3)-Gold (Agilent) E. coli K12 cells and these were used for protein expression. EcC33A was expressed and purified using the protocol described above for 15 N isotopically-enriched EcDsbA, with the following exceptions: natural isotope abundance NH4Cl was used in the autoinduction broth, and at the final stage of purification, the protein was buffer exchanged into 50 mM Na2HPO4, 50 mM NaCl, pH 6.54.</p><!><p>EcDsbA-compound complexes were crystallized and structures were determined as previously described. (10) Data collection and refinement statistics are summarized in Table S2.</p><!><p>A reference 1 H-</p><!><p>Samples were analyzed on a Shimadzu LCMS-20 system with an SGE ProteoCol C8 HQ1003 150 mm X 2.0 mm ID 3 µm 1000 Å column or SGE ProteoCol C18 HQ303 150 mm X 2.0 mm ID 3 µm 300 Å column monitoring at 214 nm. Buffer A: 99.9 % water, 0.1 % formic acid; buffer B: 99.9 % acetonitrile, 0.1 % formic acid. A gradient of 0-60 % buffer B over 16 mins followed by 1 min isocratic 60 % buffer B, 1 min gradient to 100% buffer A and 2 min of isocratic 100% buffer A at a flow rate of 0.2 mL/min was used as an elution profile. Data was analyzed using LabSolutions v5.31 and mMass v5.0.1. Samples of 20 μM EcDsbA, 1000 μM AFMK 97 in 125 mM citrate-phosphate buffer (at pH 5.11, 6.54 and 8.12), 2 mM TCEP, 41-360 mM NaCl, 2 % DMSO were allowed to react at 25 °C for 7 days. Samples were taken periodically and directly injected and the ratio of apo, mono-adduct and di-adduct determined.</p><!><p>Contains complete synthetic methods and spectra of small molecules, protein expression, NMR studies, MS studies, reaction modeling, synthesis, screening data and X-ray crystallography. This material is available free of charge via the internet at http://pubs.acs.org</p><!><p>The crystallographic coordinates were deposited in the Protein Data Bank under PDB codes 7S1D, 7S1F, 7S1L and 7S1C.</p><!><p>Email: Martin.Scanlon@monash.edu; Jamie.Simpson@monash.edu</p>