Sulfonamido-2-arylbenzoxazole GroEL/ES inhibitors are potent antibacterials against methicillin-resistant Staphylococcus aureus (MRSA)
Extending from a study we recently published examining the anti-trypanosomal effects of a series of GroEL/ES inhibitors based on a pseudo-symmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria \xe2\x80\x93 EC50 values of the most potent analogs were in the 1\xe2\x80\x932 \xc2\xb5M range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC50 values in the 1\xe2\x80\x9310 \xc2\xb5M range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC50 values >20 \xc2\xb5M).
INTRODUCTION:<!>Identifying preliminary SAR of previously developed pseudo-symmetrical compound 1 analogs for antibacterial effects against the ESKAPE pathogens.<!>Synthesis of the two parallel series of 2-chlorothiophene-based analogs.<!>Evaluating compounds for antibiotic effects against the ESKAPE bacteria and inhibition of GroEL/ES-mediated folding of substrate proteins.<!>Characterizing GroEL-inhibitor binding interactions using Isothermal Titration Calorimetry.<!>Counter-screening compounds for inhibition of HSP60/10-mediated refolding of denatured MDH and for cytotoxicity in human cell viability assays.<!>Investigating the potential for MRSA to gain resistance to lead inhibitors.<!>CONCLUSIONS.<!>General Synthetic Methods.<!>35: 5-Chloro-N-(4-hydroxy-3-nitrophenyl) thiophene-2-sulfonamide.<!>36: N-(3-Amino-4-hydroxyphenyl)-5-chlorothiophene-2-sulfonamide.<!>37: 5-Chloro-N-(2-(4-nitrophenyl)benzo[d]oxazol-5-yl)thiophene-2-sulfonamide.<!>38: N-(2-(4-Aminophenyl)benzo[d]oxazol-5-yl)-5-chlorothiophene-2-sulfonamide.<!>General sulfonamidation procedure for the synthesis of 16R-34R.<!>39: Methyl 4-((5-chlorothiophene)-2-sulfonamido)benzoate.<!>40: 4-((5-Chlorothiophene)-2-sulfonamido)benzoic acid.<!>41: 5-Chloro-N-(4-(5-nitrobenzo[d]oxazol-2-yl)phenyl)thiophene-2-sulfonamide.<!>42: N-(4-(5-Aminobenzo[d]oxazol-2-yl)phenyl)-5-chlorothiophene-2-sulfonamide.<!>General sulfonamidation procedure for the synthesis of 16L-34L.<!>Cell information for compound evaluation.<!>Evaluation of compounds for inhibition of bacterial cell proliferation.<!>Protein Expression and purification.<!>Evaluation of compounds in GroEL/ES and HSP60/10-mediated dMDH and dRho refolding assays.<!>Counter-screening compounds for inhibition of native MDH and Rho enzymatic activity.<!>Evaluation of inhibitors 20R and 20L binding to GroEL using Isothermal Titration Calorimetry (ITC).<!>Evaluation of compound effects on HEK 293 and THLE-3 cell viability.<!>Evaluation of MRSA resistance generation against lead inhibitors.<!>Calculation of IC50 / EC50 / CC50 values and statistical considerations.
<p>The persistence of antibiotic resistant pathogens is a significant health and economic burden worldwide. Six of the most problematic drug resistant Gram-positive (Gr+) and Gramnegative (Gr-) bacteria are commonly referred to as the ESKAPE pathogens. This panel of drug resistant bacteria includes Enterococcus faecium (Gr+), Staphylococcus aureus (Gr+), Klebsiella pneumoniae (Gr-), Acinetobacter baumannii (Gr-), Pseudomonas aeruginosa (Gr-), and Enterobacter species (Gr-).1–5 Of particular significance, the CDC recently estimated that ~80,000 individuals are infected with MRSA in the US annually, with ~11,000 deaths – the highest mortality rate amongst all the antibiotic resistant bacteria. S. aureus-associated infections range from mild skin infections to life-threatening endocarditis, osteomyelitis, or bacteremia, which are usually treated with penicillinase-resistant beta-lactams or vancomycin. For more resistant strains, newer classes of antibiotics can be prescribed, such as linezolid, daptomycin, dalfopristin, telavancin, tigecycline, ceftaroline, oritavancin, dalbavancin, or tedizolid.6–8 Unfortunately, despite the availability of several antimicrobials, treating MRSA infections remains a significant challenge owing to off-target toxicities of some antibiotics, lack of efficacy in life-threatening clinical infections such as bacteraemia, endocarditis, etc., and the emergence of pan-drug resistant strains.7 Thus, there is an urgent need for new antibacterial drugs that function against previously unexploited targets and pathways to circumvent predisposed resistance mechanisms.</p><p>Towards developing mechanistically unique antibacterial candidates, we have focused on exploiting bacterial protein homeostasis pathways. A network of molecular chaperones and proteases collectively functions to maintain protein homeostasis by assisting proteins to fold to their native, functional states, or ensuring their proper degradation and recycling.9, 10 Since such quality control mechanisms are vital to cell survival, targeting them with small molecule inhibitors should be an effective antibacterial strategy. While recent studies have investigated targeting DnaK (a molecular chaperone belonging to the HSP70 family) and Clp proteases, targeting of the bacterial GroEL/GroES chaperonin system has gone largely unexplored.11–14
E. coli GroEL, which is the prototypical member of the HSP60 chaperonin family of molecular chaperones, is a homo-tetradecameric protein that forms two, seven-subunit rings that stack back-to-back with one another.15–17 Through a series of events driven by ATP binding and hydrolysis, unfolded substrate polypeptides are bound within the central cavity of a GroEL ring and encapsulated by the GroES co-chaperonin "lid", allowing protein folding within the sequestered chamber.17–21 Since the GroEL/ES chaperonin system is essential for E. coli survival under all growth conditions, as it likely is for other bacteria, it represents an excellent target for antibacterial development.22, 23 Furthermore, GroEL is highly conserved in prokaryotes (generally >50% sequence identity between bacterial species, whether Gram-positive or Gramnegative), thus, targeting this molecular machinery has the potential for broad spectrum applicability. However, since human HSP60 is also highly conserved (48% identity with E. coli GroEL), we also need to consider whether GroEL inhibitors have off-target effects against HSP60 in human cells.</p><p>Towards our goal of exploiting the GroEL/ES chaperonin system as an antibacterial strategy, we previously reported a high-throughput screen that identified 235 small molecule inhibitors of the E. coli GroEL/ES chaperonin system.24 In a subsequent study, we evaluated 22 of these GroEL/ES inhibitor hits for their antibacterial properties against the ESKAPE pathogens.25 In another study, we evaluated a series of compound 1-based GroEL/ES inhibitors for their antibiotic effects against Trypanosoma brucei parasites – the causative agents of African sleeping sickness (Figure 1A).26 As an extension of those two studies, herein, we have explored additional compound 1 analogs for their ability to selectively inhibit the prototypical E. coli GroEL/ES chaperonin system and growth of the ESKAPE pathogens over human HSP60/10 and liver and kidney cells. Overall, this study enabled us to better understand the bioactivity profiles of this molecular scaffold, which provided invaluable SAR to guide future studies to more rationally optimize the pharmacological properties of these antibacterial GroEL/ES chaperonin system inhibitors.</p><!><p>From our previous study exploring the antibacterial effects of our different GroEL/ES inhibitor scaffolds, we found that the parent hit, compound 1 (based on the benzimidazole core, Figure 1A), exhibited no antibacterial effects against any of the ESKAPE pathogens.25 Therefore, before embarking on synthesis of new compound 1 analogs, we tested our previously developed benzoxazole-based GroEL/ES inhibitors (compounds 2-14), which have antitrypanosomal activities, for their antibacterial properties.26 We expected that this initial evaluation would tell us whether or not this scaffold would be worthwhile to pursue for antibacterial development and, if so, allow us to identify which substituents, substitution patterns, and aryl substructures would provide the most potent antibacterial effects. We employed a standard antibacterial efficacy assay in liquid culture as previously reported, with one modification: the media was cation-adjusted by addition of 25 mg/L Ca2+ and 12.5 mg/L Mg2+ to better mimic the concentrations of these divalent cations in vivo, which can alter the antibacterial effects of some compounds (e.g. daptomycin is a more potent inhibitor when media is supplemented with additional Ca2+ and Mg2+ ions).27 Briefly, bacteria were grown at 37°C without shaking (stagnant assay) in media stamped with test compounds. Compounds were first tested at single concentrations of 100 µM, then in dose-response for those that exhibited >50% inhibition of bacterial proliferation (refer to Tables S1A and S2A in the Supporting Information for a tabulation of all EC50 results). After 6–8 h (S. aureus, MRSA, K. pneumonia, E. cloacae, and P. aeruginosa) or 24 h (E. faecium and A. baumannii), the absorbance of each well was read at 600 nm to monitor turbidity from bacterial growth, from which EC50 results were obtained by plotting the dose-response measurements and fitting data with non-linear regression. A detailed protocol for this assay is listed in the Supporting Information.</p><p>Preliminary results from these proliferation assays indicated that all but one compound (2h-p) were inactive against the four Gram-negative KAPE bacterial species, and only three were able to inhibit E. faecium (the ortho-, meta-, and para-substituted analogs of hydroxylated compound 2h); however, several compounds were able to inhibit S. aureus and MRSA proliferation (Figure 1B, and Tables S1A and S2A in the Supporting Information). Furthermore, these compounds were reasonably selective at killing MRSA in relation to their cytotoxicity CC50 values obtained from liver (THLE 3) and kidney (HEK 293) cell viability assays, which were determined in the previous study targeting T. brucei parasites.26 Preliminary SAR revealed that the sulfonamide end-capping thiophene and para-chlorophenyl groups gave the most potent and selective aryl and substituent/substitution patterns, respectively (Figure 1B). Based on these initial results, we decided to combine these two moieties, reasoning that inhibition properties of each could be additive, and developed two parallel series of 2-chlorothiophene-based asymmetrical analogs (Figure 1C, analogs 15–34) containing variable substructures at the R2 positions.</p><!><p>We rationalized that the previously developed pseudo-symmetrical compound 1 analogs (i.e. with the same sulfonamide end-capping substructures on either side of the molecules) may not be truly optimized for binding to and inhibiting the chaperonin system. Thus, to better complement the envisioned asymmetric binding sites, we designed two parallel series of analogs with a variety of alkyl and aryl substructures at the R2 positions on the Right (R-series) and Left (L-series) sides of the 2-phenylbenzoxazole scaffold, while keeping the 2chlorothiophenesulfonamide moiety on the opposite sides (Figure 1C). The same R2-groups were used to create the panel of 19 matched-pairs for the different R- and L-series analogs, with an additional pseudo-symmetrical analog (15) that contained the 2-chlorothiophenesulfonamide on both sides (thus, a total of 39 new compounds were synthesized for evaluation). We expected that these matched pairs of analogs would help us to identify whether or not the directionality of the 2-phenylbenzoxazole core would significantly impact inhibitor effects in the various biochemical and cell-based assays they would be tested in. The general syntheses of these analogs are shown in Scheme 1, with detailed procedures and compound characterizations presented in the Experimental section and Supporting Information. Each of the R- and L-series of analogs were synthesized through 5-step linear protocols employing facile reactions. All final test compounds were characterized by 1H-NMR and LC-MS analyses for structural confirmation, and by two independent sets of HPLC conditions for purity identification (all were >95% pure under both conditions).</p><p>For synthesizing the R-series analogs, 2-chlorothiophenesulfonyl chloride was first coupled to 4-amino-2-nitrophenol in dichloromethane using pyridine as base, giving 35.26, 28, 29 Reduction of the nitro group was accomplished by reacting with tin powder in a 1:10 v/v mixture of hydrochloric acid in glacial acetic acid.28–30 Next, an intermediate Schiff base was formed by refluxing 36 and para-nitrobenzaldehyde with sodium bicarbonate in anhydrous THF, which was then cooled and cyclized to benzoxazole 37 by addition of DDQ.31 The nitro group was again reduced using tin powder in HCl/AcOH, affording intermediate 38, from which the final bissulfonamido analogs (15 and 16R-34R) could be rapidly generated by coupling with a variety of differentially-substituted sulfonyl chlorides (R2-SO2Cl).</p><p>For synthesizing the L-series analogs, 2-chlorothiophenesulfonyl chloride was first coupled to methyl-4-aminobenzoate in dichloromethane using pyridine as base, giving 39. The ester was then hydrolyzed with LiOH in a mixture of H2O/MeOH/THF, affording acid 40.28, 30, 32 The acid was then converted to the acyl chloride, concentrated, and coupled to 2-amino-4-nitrophenol in dichloromethane with pyridine as base, affording the intermediate amide, which was then cyclodehydrated to 41 by refluxing with toluenesulfonic acid in xylenes.29, 33 The nitro group was subsequently reduced with tin powder in HCl/AcOH, affording intermediate 42, from which the final bis-sulfonamido analogs (16L-34L) could be rapidly generated by coupling with a variety of differentially-substituted sulfonyl chlorides (R2-SO2Cl).</p><!><p>Before proceeding with extensive biochemical and biophysical characterization of the new 2-chlorothiophene-based analogs, we first tested for their antibiotic effects on the ESKAPE bacteria using the proliferation assay described above (refer to Tables S3A and S4A in the Supporting Information for tabulations of all EC50 results). Much like the initial testing with the pseudo-symmetrical analogs, all but one of the 2-chlorothiophene analogs were inactive against the four Gram-negative KAPE bacterial species (27R had an EC50 of 37 µM against A. baumannii). However, 14 of the 39 new analogs (36%) exhibited antibacterial effects against E. faecium, compared to 3 out of 50 (6%) of the pseudo-symmetrical compounds. What was particularly impressive was that 27 out of the 39 new analogs (69%) inhibited S. aureus and MRSA, with greater overall potency, compared to the 20 out of 50 (40%) of the pseudosymmetrical compounds (Figure 2A presents a correlation plot of EC50 values for each compound determined in these two assays). To compare assay results between the R- and Lseries analogs, we analyzed the log(EC50) values of each data set using two-tailed, paired t-tests (95% confidence level) and looked at differences between paired values (results are plotted in Figure S1 in the Supporting Information). For the E. faecium EC50 results, we did not see a statistical difference between results from the R- and L-series analogs (Figure S1A). However, there was a statistically significant difference between results of the two series for inhibiting S. aureus and MRSA proliferation, with the L-series generally providing more potent inhibitors of bacterial proliferation for each strain (Figures S1B and S1C). Although exceptions are evident for individual compounds, there was no statistical difference between inhibiting the drug susceptible S. aureus and MRSA strains when the data sets were analyzed for all 89 compounds as a whole (Figure S5A).</p><p>Having ascertained that the majority of the R- and L-series analogs were potent inhibitors of S. aureus and MRSA proliferation, we next evaluated their abilities to inhibit the biochemical function of the GroEL/ES chaperonin system. For this, we employed our previously reported assays that monitor GroEL/ES-mediated refolding of two denatured substrate enzymes, malate dehydrogenase (dMDH) and rhodanese (dRho).25 Since these were coupled assays that monitor chaperonin-mediated refolding of substrates by virtue of the enzymatic activities of the refolded substrates, we further counter-screened against the native MDH and Rho enzymes to ensure that compounds were not simply false-positives of the reporter reactions. Detailed protocols for these assays and tabulation of all IC50 results are presented in the Supporting Information.</p><p>Because the previously developed pseudo-symmetrical analogs (1-14) were not tested in the GroEL/ES-dRho refolding assay, nor the native rhodanese enzymatic reporter reaction counter screen, we also evaluated those compounds in these two assays. When looking at all 89 compounds as a whole, although a correlation is evident, we noticed a statistically significant difference between the GroEL/ES-dMDH and GroEL/ES-dRho refolding IC50 results (Figures 2B and S5B), where compounds were slightly more potent at inhibiting GroEL/ES-mediated refolding of denatured MDH. This difference does not appear to be a result of compounds preferentially inhibiting the native MDH reporter enzyme over rhodanese – while eleven analogs inhibited the native MDH enzymatic reporter reaction, five inhibited native rhodanese, and only to a minor extent. Since only one compound inhibits both native malate dehydrogenase and native rhodanese (28R), and only to a minor extent, this supports that compounds are on-target for inhibiting the GroEL/ES-mediated folding cycle.</p><p>For the purposes of categorizing inhibitor potencies in the GroEL/ES-mediated refolding assays, we consider compounds with IC50 values >100 µM to be inactive, 30–100 µM to be weak inhibitors, 10–30 µM moderate inhibitors, 1–10 µM potent inhibitors, and <1 µM very potent and acting near stoichiometrically since the concentration of GroEL tetradecamer is 50 nM during the refolding cycle (i.e. 700 nM of GroEL subunits). Upon further dissection of the GroEL/ES-dMDH refolding assay IC50 results, we observed that the asymmetric R- and L-series analogs are generally more potent than the previously developed pseudo-symmetric analogs: 28 out of 38 (74%) of the asymmetric R- and L-series analogs (16-34) have IC50 values less than 10 µM, whereas only 16 out of 51 (31%) of the pseudo-symmetric analogs (1-15) have IC50 values less than 10 µM. These results are not entirely surprising since the R- and L-series analogs were hypothesized to better complement the envisioned asymmetric binding sites than the pseudosymmetric compounds. However, we caution on over-interpreting these results since the analog groupings did contain different alkyl and aryl substructures on the sulfonamide end-caps. When comparing the R- and L-series analogs with each other, though, we do not see any statistically significant differences in either the GroEL/ES-dMDH or GroEL/ES-dRho refolding assay results (Figure S2A and S2B), suggesting that the orientation of the 2-phenylbenzoxazole core scaffold does not play a significant factor in these compounds binding to and inhibiting the GroEL/ES chaperonin system.</p><p>When comparing IC50 results for compounds tested in the biochemical GroEL/ES-dMDH refolding assay with EC50 results for testing in the S. aureus proliferation assay (Figure 2C and Tables S3A-B and S4A-B), we note that no compounds are active against bacteria unless they are able to inhibit GroEL. In general, the more potent compounds are at inhibiting the GroEL/ES chaperonin system, the more potent their antibacterial effects, in particular for the L-series inhibitors. However, several exceptions are evident where potent GroEL/ES inhibitors are poorly effective, or ineffective, against bacteria, which may owe to poor cell wall permeability and/or efflux, as we had previously observed for other GroEL/ES inhibitor scaffolds.25 Also, while correlations between biochemical IC50 and cell-based EC50 are only suggestive of mechanisms of action in cells, they are not confirmatory. Thus, whether compounds are "ontarget" for GroEL within bacteria is still unknown and warrants further investigation. These studies are ongoing and will be reported in the future.</p><!><p>To further interrogate inhibitor mechanisms of action against GroEL, we used isothermal titration calorimetry (ITC) to identify the thermodynamic parameters, binding affinities, and binding stoichiometries of compounds 20R and 20L. While these two compounds were only moderately active at inhibiting S. aureus and MRSA proliferation, they were strong inhibitors of GroEL/ES-mediated folding functions, and thus likely had high binding affinities. Furthermore, because they bore primary amines that would be charged under physiological conditions, they were much more soluble than other inhibitors and thus more amenable to ITC analysis, which requires high concentrations of both protein and ligands in matched aqueous buffers. We performed ITC analyses by titrating 400 µM GroEL (monomer concentration) into solutions containing either 50 µM 20R or 20L (detailed protocols are presented in the Experimental section). Two representative isotherms for the binding of 20R and 20L to GroEL are presented in Figure 3. After subtraction of background heats of mixing and dilution, plots of the integrated heats from compound binding fit well to a single-site binding model.34 Averaged results for the various binding parameters (Kd, n, ∆H, ∆S, and ∆G) obtained from replicate analyses (six replicates for 20R, and five replicates for 20L) are presented in Table 1.</p><p>From the averaged results, we found that 20R had a Kd of 36 nM for binding to E. coli GroEL, while the Kd of 20L was about four-fold higher (150 nM), which corresponds reasonably well with the relative IC50 values for inhibiting GroEL/ES-mediated refolding of dMDH and dRho – the IC50 values for 20L were typically two- to four-fold higher than for 20R. For both compounds, binding was predominantly enthalpically driven, with minor entropic contributions to affinity. What was particularly interesting from these analyses was the stoichiometry of binding of each compound to GroEL. Since GroEL consists of 14 identical subunits, we anticipated that compounds could bind with a stoichiometry of 14, or potentially 7 if there is negative cooperativity between the two GroEL rings (also assuming that for inhibitor binding there is no negative cooperativity between subunits within a ring). Since IC50 values were previously found to correlate between GroEL/ES-mediated refolding and ATPase assays for this inhibitor scaffold, the most likely binding sites may be the ATP pockets, of which there are 1 per GroEL subunit (14 per oligomer).26 However, 20R bound with a stoichiometry of roughly 18 molecules per GroEL tetradecamer, and 20L with 23 molecules, which could indicate more than one potential binding site per GroEL subunit, and potentially an unknown site outside of the ATP pockets. We are currently pursuing X-ray crystallographic studies to identify specific inhibitor binding sites, which we will report on in future studies.</p><!><p>Having established that the R- and L-series analogs were potent GroEL/ES chaperonin system inhibitors with anti-staphylococcal properties, we next investigated their selectivity profiles compared to the human HSP60/10 chaperonin system. For this, we evaluated compounds in our previously reported HSP60/10-dMDH refolding assay, which is analogous to that used for determining inhibition of bacterial GroEL/ES, but alternatively employs the human chaperonin system. A detailed protocol and tabulation of all IC50 results for this assay are presented in the Supporting Information. We previously reported that the pseudo-symmetrical analogs (1-14) were highly selective for inhibiting E. coli GroEL/ES over human HSP60/10 (Figure 3A and S3A).26 However, for the asymmetric compounds developed in this study, selectivity for inhibiting GroEL/ES over HSP60/10 was statistically significant for the R-series analogs (Figure S3C), but was lost for the L-series analogs (Figure S3B). Correspondingly, differences in IC50 values between the R- and L-series inhibitors in the HSP60/10-dMDH refolding assay were statistically significant (Figure S4A), which suggests that directionality of the 2-phenylbenzoxazole core does have an effect on compounds binding to and inhibiting the human chaperonin system, unlike the situation for E. coli GroEL/ES.</p><p>We next evaluated the cytotoxicity of our compounds to human liver (THLE 3) and kidney (HEK 293) cell lines in a well-established, 48 h, Alamar Blue-based cell viability assay. Detailed protocols and tabulation of all cytotoxicity CC50 results for these assays are presented in the Supporting Information. Intriguingly, even though the L-series analogs are more potent HSP60/10 inhibitors than the R-series, we observed no statistically significant differences between the L- and R-series CC50 values in the HEK 293 kidney cell viability assay (Figure S4B). Furthermore, we observed an opposing trend where the L-series was less cytotoxic to the THLE 3 liver cells compared to the R-series analogs, although the differences were small (Figure S4B & C). These results suggest that the compounds may be unable to penetrate the mitochondrial matrix to engage with the HSP60/10 chaperonin system, and instead may be interacting with other targets in the cytosol or other sub-cellular compartments; however, we have yet to confirm this hypothesis. What is particularly promising with these results is that the cytotoxicity of lead analogs has not concomitantly increased as with antibacterial potency. Compared to the previously developed pseudo-symmetrical analogs, several lead asymmetrical analogs have appreciably higher Selectivity Indices (SI) for inhibiting MRSA proliferation over cytotoxicity in the liver cell viability assay (Figure 4C). The biochemical and cell-based IC50, EC50, and CC50 results for the top four lead inhibitors of the pseudo-symmetrical, R-series, and L-series analogs are presented in Table 2, ranked based on their selectivity indices. Notably, compounds 24L and 25L have selectivity indices of 31 and 46, respectively.</p><!><p>While selectivity indices (and therapeutic windows with regards to in vivo evaluation) are important criteria to consider for antibacterial development, it is also important to determine whether or not bacteria will be able to rapidly develop resistance to lead candidates, especially if they function through new mechanisms of action. To gauge the efficacy of our GroEL/ES inhibitors for eluding acute antibacterial resistance mechanisms, we performed a step-wise, liquid culture resistance assay by consecutively passaging the MRSA strain (ATCC BAA-44) for 12 cycles over 12 consecutive days in the presence of serially-diluted inhibitors 20R, 28R, and vancomycin for comparison (a detailed protocol is presented in the Experimental section).35, 36 At the end of each cycle (i.e. each 24 h passage), EC50 values were determined for the test compounds, with the assumption that EC50 values would increase over time if MRSA was able to generate acute resistance. A plot of test compound EC50 values over time is presented in Figure 5A. Despite the fact that 28R was a potent MRSA growth inhibitor (initial EC50 value of 1.7 µM), the bacteria rapidly developed resistance to compound concentrations in excess of 100 µM. However, after culturing in the absence of inhibitors for two days (24 h on agar, and another 24 h in liquid media), bacteria regained sensitivity to 28R (Figure 5B), suggesting resistance is reversible, potentially from up-regulation of efflux pumps. Most importantly, we found that the parent MRSA strain was unable to generate resistance against 20R over the 12 day passage, even though this analog was only a moderate inhibitor of MRSA proliferation (EC50 = 19 µΜ). These results suggest that pan-resistance to these GroEL inhibitor analogs may be difficult to develop, which supports the continued optimization of lead antibacterial candidates based on this molecular scaffold.</p><!><p>From exploratory screening of the antibacterial effects of our previously reported pseudosymmetrical compound 1 analogs,26 we were able to develop two parallel R- and L-series of 2chlorothiophene-based asymmetrical analogs with significantly improved antibacterial efficacy profiles against MRSA. While there were no statistical differences between either the R- or Lseries analogs for inhibiting in the GroEL/ES-mediated refolding assays, the L-series analogs were found to be more potent at inhibiting S. aureus and MRSA proliferation – i.e. preferred from an antibiotic development perspective (Table 3). While a general trend is observed between inhibitor potencies in the biochemical GroEL/ES-dMDH refolding assay and the MRSA proliferation assay (in particular for the L-series analogs), further studies are needed to conclusively determine whether or not inhibitors are on-target in bacteria. Using ITC to characterize the binding interactions between GroEL and inhibitors 20R and 20L, we found that inhibitor binding was predominantly enthalpically driven. Furthermore, we observed that >14 molecules were bound per GroEL tetradecamer (i.e. more than one molecule bound per GroEL subunit), suggesting that inhibitors could be binding to unknown sites outside of the ATP pockets. Current studies are underway to identify and characterize these putative binding sites so that more rigorous structure-based optimization of lead candidates can be conducted. Even though the R-series analogs were overall less potent at inhibiting HSP60/10, the L-series was less cytotoxic against the human liver cells tested, and is likely preferential for further antibacterial optimization going forward. However, before pursuing in vivo development of this series, further medicinal chemistry optimization is warranted to increase the selectivity indices for killing MRSA over cytotoxicity to human cells.</p><!><p>Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification. Reaction progress was monitored by thin-layer chromatography on silica gel 60 F254 coated glass plates (EM Sciences). Flash chromatography was performed using a Biotage Isolera One flash chromatography system and eluting through Biotage KP-Sil Zip or Snap silica gel columns for normal-phase separations (hexanes:EtOAc gradients), or Snap KP-C18-HS columns for reverse-phase separations (H2O:MeOH gradients). Reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using a Waters 1525 binary pump, 2489 tunable UV/Vis detector (254 and 280 nm detection), and 2707 autosampler. For preparatory HPLC purification, samples were chromatographically separated using a Waters XSelect CSH C18 OBD prep column (part number 186005422, 130 Å pore size, 5 µm particle size, 19×150 mm), eluting with a H2O:CH3CN gradient solvent system. Linear gradients were run from either 100:0, 80:20, or 60:40 A:B to 0:100 A:B (A = 95:5 H2O:CH3CN, 0.05% TFA; B = 5:95 H2O:CH3CN, 0.05% TFA. Products from normal-phase separations were concentrated directly, and reverse-phase separations were concentrated, diluted with H2O, frozen, and lyophilized. For primary compound purity analyses (HPLC-1), samples were chromatographically separated using a Waters XSelect CSH C18 column (part number 186005282, 130 Å pore size, 5 µm particle size, 3.0×150 mm), eluting with the above H2O:CH3CN gradient solvent systems. For secondary purity analyses (HPLC-2) of final test compounds, samples were chromatographically separated using a Waters XBridge C18 column (either part number 186003027, 130 Å pore size, 3.5 µm particle size, 3.0×100 mm, or part number 186003132, 130 Å pore size, 5.0 µm particle size, 3.0×100 mm), eluting with a H2O:MeOH gradient solvent system. Linear gradients were run from either 100:0, 80:20, 60:40, or 20:80 A:B to 0:100 A:B (A = 95:5 H2O:MeOH, 0.05% TFA; B = 5:95 H2O:MeOH, 0.05% TFA). Test compounds were found to be >95% in purity from both RP-HPLC analyses. Mass spectrometry data were collected using either an Agilent analytical LC-MS at the IU Chemical Genomics Core Facility (CGCF), or a Thermo-Finnigan LTQ LC-MS in-lab. 1H-NMR spectra were recorded on either Bruker 300 MHz or 500 MHz spectrometers. Chemical shifts are reported in parts per million and calibrated to the d6-DMSO solvent peaks at 2.50 ppm. We previously synthesized compounds 1–14 (including 2a-m-o/m/p)26 and re-synthesized where necessary due to stock depletion. Synthesis and characterization of intermediates 35–42 are presented below. General sulfonamide coupling steps are presented for analogs 15 and 16R-34R and 16L-34L below, with compound characterizations for each analog presented in the Supporting Information.</p><!><p>To a stirring mixture of 4-amino-2-nitrophenol (5.34 g, 24.6 mmol) in anhydrous CH2Cl2 (50 mL) was added 5-chlorothiophene-2-sulfonyl chloride (4.21 g, 27.3 mmol) and pyridine (2.40 mL, 29.4 mmol). The reaction was allowed to stir at room temperature for 18 h and was then diluted with hexanes and the precipitate was filtered, rinsed with 1 M HCl and water, collected, and dried to afford 35 as a reddish-brown solid (7.70 g, 94% yield). 1H-NMR (300 MHz, d6-DMSO) δ 10.98 (br s, 1H), 10.62 (br s, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.39 (d, J = 4.1 Hz, 1H), 7.30 (dd, J = 8.9, 2.7 Hz, 1H), 7.22 (d, J = 4.1 Hz, 1H), 7.10 (d, J = 8.9 Hz, 1H); MS (ESI) C10H6ClN2O5S2 [M-H]-
m/z expected = 332.9, observed = 332.8; HPLC-1 = >99% (RT = 8.1 min).</p><!><p>Tin powder (8.73 g, 73.6 mmol) was added slowly to a stirring mixture of 35 (8.11 g, 24.2 mmol) in a 1:10 mixture of HCl:AcOH (24 mL). The reaction was allowed to stir at R.T. for 18 h, then diluted with EtOAc and H2O, neutralized with NaHCO3, and filtered. The filtrate was extracted with EtOAc and the organics dried over Na2SO4, filtered, and concentrated. The residue was diluted in a 50% mixture of DCM in hexanes and the precipitate was filtered, rinsed with hexanes, collected, and dried to afford 36 as a brown powder (6.08 g, 82% yield). 1H-NMR (300 MHz, d6-DMSO) Î´ 9.81 (br s, 1H), 8.99 (br s, 1H) 7.28 (d, J = 4.1 Hz, 1H), 7.18 (d, J = 4.1 Hz, 1H), 6.49 (d, J = 8.3 Hz, 1H), 6.41 (d, J = 2.5 Hz, 1H), 6.13 (dd, J = 8.3, 2.6 Hz, 1H), 4.64 (br s, 2H); MS (ESI) C10H10ClN2O3S2 [M+H]+
m/z expected = 305.0, observed = 304.9; HPLC-1 = 97% (RT = 6.0 min).</p><!><p>Compound 36 (2.94 g, 9.65 mmol), 4-nitrobenzaldehyde (2.03 g, 13.4 mmol), NaHCO3 (2.08 g, 24.8 mmol), and Na2SO4 (3.35 g) were stirred in THF (40 mL) for 4 h at reflux (under Ar), then cooled to R.T. DDQ (2.85 g, 12.6 mmol) was then add portion-wise and the reaction was left to stir for 2 h, then filtered. The filtrate was extracted into EtOAc, rinsed with saturated NaHCO3, 1 M HCl, and brine. The organics layer was dried over Na2SO4, filtered, and concentrated. The residue was diluted in a 25% mixture of DCM in hexanes and the precipitate was filtered, rinsed with hexanes, collected, and dried to afford 37 as a brown powder (4.01 g, 95% yield). 1H-NMR (300 MHz, d6-DMSO) δ 10.74 (s, 1H), 8.37–8.48 (m, 4H), 7.82 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 1.9 Hz, 1H), 7.43 (d, J = 4.0 Hz, 1H), 7.25 (dd, J = 8.8, 2.1 Hz, 1H), 7.19 (d, J = 4.1 Hz, 1H); MS (ESI) C17H9ClN3O5S2 [M-H]-
m/z expected = 434.0, observed = 433.8; HPLC-1 = 91% (RT = 7.7 min).</p><!><p>Tin powder (2.74 g, mmol) was added slowly to a stirring mixture of 37 (2.43 g, 5.58 mmol) in a 1:10 mixture of HCl:AcOH (15 mL). The reaction was allowed to stir at R.T. for 18 h, then diluted with EtOAc and H2O, neutralized with NaHCO3, and filtered. The filtrate was extracted with EtOAc and the organics dried over Na2SO4, filtered, and concentrated. The residue was chromatographed over silica (hexanes:EtOAc gradient) and concentrated to afford 38 as an orange solid (936 mg, 41% yield). 1H-NMR (300 MHz, d6-DMSO) δ 10.54 (s, 1H), 7.78–7.86 (m, 2H), 7.60 (d, J = 8.7 Hz, 1H), 7.35–7.40 (m, 2H), 7.18 (d, J = 4.1 Hz, 1H), 7.04 (dd, J = 8.6, 2.2 Hz, 1H), 6.64–6.71 (m, 2H), 6.04 (br s, 2H); MS (ESI) C17H13ClN3O3S2 [M+H]+
m/z expected = 406.0, observed = 405.9; HPLC-1 = 97% (RT = 7.6 min).</p><!><p>To a stirring mixture of compound 38 (1 eq.) in anhydrous CH2Cl2 (5 mL) was added the respective R2 sulfonyl chloride (1.2 eq.) followed by anhydrous pyridine (1.2 eq.). The reaction was allowed to stir at room temperature for 18 h and was then chromatographed over silica (hexanes:EtOAc gradient) and concentrated. If necessary, the product was further purified by preparatory RP-HPLC (H2O:CH3CN gradient), concentrated, and lyophilized. Refer to the Supporting Information for individual compound characterization data.</p><!><p>To a stirring mixture of methyl-4-aminobenzoate (2.76 g, 18.3 mmol) in anhydrous CH2Cl2 (50 mL) was added 5-chlorothiophene-2-sulfonyl chloride (4.78 g, 22.0 mmol) and pyridine (1.80 mL, 22.1 mmol). The reaction was allowed to stir at room temperature for 18 h and was then diluted with hexanes and acidified with 1 M HCl. The precipitate was then filtered, rinsed with 1 M HCl and water, collected, and dried to afford 39 as a pinkish-orange solid (5.94 g, 98% yield). 1H-NMR (300 MHz, d6-DMSO) δ 11.16 (br s, 1H), 7.86–7.93 (m, 2H), 7.56 (d, J = 4.1 Hz, 1H), 7.25–7.31 (m, 2H), 7.22 (d, J = 4.1 Hz, 1H), 3.80 (s, 3H); HPLC-1 = 99% (RT = 6.2 min).</p><!><p>LiOH•H2O (7.02 g, 167 mmol) was added to a stirring mixture of 39 (5.67 g, 17.1 mmol) in THF (20 mL), MeOH (20 mL), and H2O (20mL). The reaction was allowed to stir at room temperature for 2 days and was then diluted with 1M HCl. The precipitate was filtered, washed with H2O, collected, and dried to afford 40 as an off-white solid (5.20 g, 96% yield). 1H-NMR (300 MHz, d6-DMSO) δ 12.85 (br s, 1H), 11.09 (br s, 1H), 7.83–7.90 (m, 2H), 7.54 (d, J = 4.1 Hz, 1H), 7.19–7.28 (m, 3H); HPLC-1 = >99% (RT = 6.7 min).</p><!><p>Compound 40 (2.96 g, 9.31 mmol) was stirred in SOCl2 (10 mL) for 6 h at 60°C, then was concentrated to a solid. This was refluxed with 2-amino-4-nitrophenol (2.96 g, 19.2 mmol) and pyridine (1.50 mL, 18.4 mmol) in anhydrous CH2Cl2 (50 mL) for 6 h, then stirred at R.T. for 3 days. The reaction was then extracted into EtOAc and rinsed with 1 M HCl and brine, dried over Na2SO4, filtered, and concentrated. This amide intermediate was then refluxed with TsOH•H2O (3.61 g, 19.0 mmol) in xylenes (50 mL) using a Dean-Stark apparatus to remove the residual H2O. After 18 h, the reaction was cooled, the xylenes decanted off, the sludge extracted with EtOAc, and the combined organics dried over Na2SO4, filtered, and concentrated. Flash chromatographic purification over silica (hexanes:EtOAc gradient) afforded 41 as a peach solid (1.81 g, 45% yield). 1H-NMR (300 MHz, d6-DMSO) δ 11.25 (br s, 1H), 8.64 (br s, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 8.1 Hz, 2H), 8.01 (d, J = 8.7 Hz, 1H), 7.60 (br s, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.24 (br s, 1H); MS (ESI) C17H11ClN3O5S2 [M+H]+
m/z expected = 436.0, observed = 436.1; HPLC-1 = 88% (RT = 5.5 min).</p><!><p>Tin powder (1.53 g, 12.9 mmol) was added slowly to a stirring mixture of 41 (1.78 g, 4.08 mmol) in a 1:7 mixture of HCl:AcOH (8 mL). The reaction was stirred and heated at 60°C for 3 h, then cooled to R.T., diluted with EtOAc and H2O, neutralized with NaHCO3, and filtered. The filtrate was extracted with EtOAc and the organics dried over Na2SO4, filtered, and concentrated. The residue was diluted in a 10% mixture of DCM in hexanes and the precipitate was filtered, rinsed with hexanes, collected, and dried to afford 42 as a pale-yellow powder (1.59 g, 96% yield). 1H-NMR (300 MHz, d6-DMSO) δ 8.01–8.09 (m, 2H), 7.55 (d, J = 4.1 Hz, 1H), 7.30–7.41 (m, 3H), 7.22 (d, J = 4.1 Hz, 1H), 6.83 (d, J = 2.0 Hz, 1H), 6.64 (dd, J = 8.7, 2.2 Hz, 1H); MS (ESI) C17H13ClN3O3S2 [M+H]+
m/z expected = 406.0, observed = 406.1; HPLC-1 = 86% (RT = 6.8 min).</p><!><p>To a stirring mixture of compound 42 (1 eq.) in anhydrous CH2Cl2 (5 mL) was added the respective R2 sulfonyl chloride (1.2 eq.) followed by anhydrous pyridine (1.2 eq.). The reaction was allowed to stir at room temperature for 18 h and was then chromatographed over silica (hexanes:EtOAc gradient) and concentrated. If necessary, the product was further purified by preparatory RP-HPLC (H2O:CH3CN gradient), concentrated, and lyophilized. Refer to the Supporting Information for individual compound synthesis and characterization data.</p><!><p>The ESKAPE bacteria were purchased from the American Type Culture Collection (ATCC): E. faecium (Orla-Jensen) Schleifer and Kilpper-Balz strain NCTC 7171 (ATCC 19434); S. aureus subsp. aureus Rosenbach strain Seattle 1945 (ATCC 25923); Multi-drug resistant S. aureus (MRSA) subsp. aureus Rosenbach strain HPV107 (ATCC BAA-44); K. pneumonia, subsp. pneumoniae (Schroeter) Trevisan strain NCTC 9633 (ATCC 13883); A. baumannii Bouvet and Grimont strain 2208 (ATCC 19606); P. aeruginosa (Schroeter) Migula strain NCTC 10332 (ATCC 10145); E. cloacae, subsp. cloacae (Jordan) Hormaeche and Edwards strain CDC 442–68 (ATCC 13047). HEK 293 kidney cells (ATCC CRL-1573) and THLE-3 liver cells (ATCC CRL-11233) were used for compound toxicity assays.</p><!><p>All compounds were evaluated for inhibiting the proliferation of each of the ESKAPE bacteria as per previously reported procedures with one minor modification: Liquid growth media was cation-adjusted by addition of 25 mg/L Ca2+ and 12.5 mg/L Mg2+ to reflect the free concentrations of these divalent cations in vivo.27 Detailed protocols for bacterial growth assays are presented in the Supporting Information.</p><!><p>E. coli GroEL and GroES, and human HSP60 and HSP10, were expressed and purified as previously reported.25 Detailed protocols for these protein purifications are presented in the Supporting Information.</p><!><p>All compounds were evaluated for inhibiting E. coli GroEL/ES and human HSP60/10mediated refolding of the denatured MDH and denatured Rho reporter enzymes as per previously reported procedures.26 Detailed protocols for these assays are presented in the Supporting Information.</p><!><p>All compounds were counter-screened for inhibiting the enzymatic activity of the native MDH and native Rho reporter enzymes as per previously reported procedures.25, 26 Detailed protocols for the assays are presented in the Supporting Information.</p><!><p>All ITC experiments were performed on a MicroCal VP-ITC system. To minimize background heats of dilution, matched solutions of GroEL and compounds 20R or 20L were prepared in buffer containing 20 mM HEPES, pH 7.4, 50 mM KCl, 10 mM MgCl2, and 1% DMSO. All buffers were freshly prepared from stock solutions on the day of use and degassed under vacuum. For each analytical run, a solution of 400 µM GroEL (monomer concentration) in the syringe was titrated into 50 µM solutions of either compound 20R or 20L in the sample cell (equilibrated at 20°C for 300 seconds): the first injection was 0.4 µL (discarded during analysis) and subsequent injections (2–20) were each 2.0 µL. Each injection was made over 4 s durations with 3-min intervals between subsequent injections. The reference power of the experiment was set to 6 µcal/s and a 5 s filter period. Sample stirring was set at 600 rpm for all measurements. Six independent replicates were conducted to analyze compound 20R binding, and five replicates for 20L. Raw thermograms from each replicate were analyzed independently using the ORIGIN instrument software (MicroCal Inc., version 7.0). In each experiment, the upper baseline was collected after the binding reaction was saturated, and the terminal 7–8 points in the linear region were fit to a straight line and subtracted from the entire data set to remove contributions from background heats of mixing and dilution. The ∆H was obtained by non-linear least-squares fitting of the plot of ∆H (mol of injectant)−1 versus molar ratio using a single site binding model.34 The ∆H, ∆S, Kd (KA= 1/ Kd), and binding stoichiometries (n) in each titration were obtained, and ∆G was calculated using the Gibbs equation at a temperature of 20°C (293.15 K). Standard deviations were determined based on results from each of the replicate analyses.</p><!><p>Evaluation of compound cytotoxicities to HEK 293 kidney and THLE-3 liver cells were performed using Alamar Blue-based viability assays. HEK 293 cells were maintained in MEM medium (Corning Cellgro, 10–009 CV) supplemented with 10% FBS (Sigma, F2242). THLE-3 cells were maintained in Clonetics BEBM medium (Lonza, CC-3171) supplemented with the BEGM bullet kit (Lonza, CC-3170) and 10% FBS. All assays were carried out in 384-well plates (BRAND cell culture grade plates, 781980). Cells at 80% confluence were harvested and diluted in growth medium, then 45 µL of the HEK 293 cells (1,500 cells/well) or THLE-3 cells (1,500 cells/well) were dispensed per well, and plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and incubated at 37°C, 5% CO2, for 24 h. The following day, 1 µL of the compound stocks (10 mM to 4.6 µM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 25 µL of the relevant growth mediums. Then, 15 µL aliquots of the diluted compounds were added to the cell assay plates to give inhibitor concentration ranges of 100 µM to 46 nM during the assay (final DMSO concentration of 0.1% was maintained during the assay). Plates were sealed with "Breathe Easy" oxygen permeable membranes and incubated for an additional 48 h at 37°C and 5% CO2. The Alamar Blue reporter reagents were then added to a final concentration of 10%, the plates incubated at 37°C and 5% CO2, and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader (readings taken between 4–24 h of incubation so as to achieve signals in the 30–60% range for conversion of resazurin to resorufin). Cell viability was calculated as per vendor instructions (Thermo Fisher - Alamar Blue cell viability assay manual). Cytotoxicity CC50 values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism 6 and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of CC50 values obtained from at least triplicate experiments.</p><!><p>To identify potential resistance toward inhibitors, a liquid culture, 12-day serial passage assay was employed as per procedures reported by Kim. S et al., using the MRSA strain (ATCC BAA-44).35, 36 Briefly, MRSA bacteria were streaked onto a Tryptic Soy agar plate and grown overnight at 37°C. A fresh aliquot of Tryptic Soy Broth (TSB) was inoculated with a single bacterial colony and the cultures were grown overnight at 37°C with shaking (250 rpm). The overnight culture was then sub-cultured (1:5 dilution) into a fresh aliquot of media and grown at 37°C for 1 h with shaking, then diluted into fresh media to achieve a final OD600 reading of 0.01. Aliquots of the diluted culture (200 µL) were dispensed to 96 well plates along with addition of 2 µL of test compounds in DMSO (20R, 28R, and vancomycin as a control). The inhibitor concentration range during the resistance assay was 100 µM to 48.8 nM (2-fold dilution series). Plates were sealed with "Breathe Easy" oxygen permeable membranes (Diversified Biotech) and left to incubate at 37°C without shaking (stagnant assay). OD600 readings were taken at the 24 h time point to monitor for bacterial growth. A second set of baseline control plates were prepared analogously, without any bacteria added, to correct for possible compound absorbance and/or precipitation, as well as plate and media baseline effects. For inoculations on subsequent days, bacteria from the wells with the highest drug concentration where the OD600 was >0.2 were diluted with fresh media to OD600 of 0.01 and dispensed into a new 96-well plate. Test compounds were added, and the bacteria propagated again as described above. This procedure was repeated each day for a total of 12 days to observe changes in EC50 values over each passage. EC50 values for the test compounds were obtained by plotting the OD600 results from each passage in GraphPad Prism 6 and analyzing by non-linear regression using the log(inhibitor) vs. response (variable slope) equation. Results presented represent the averages of EC50 values obtained from at triplicate experiments.</p><!><p>All IC50 / EC50 / CC50 results reported are averages of values determined from individual dose-response curves in replicate assays as follows: 1) Individual I/E/CC50 values from replicate assays were first log-transformed and the average log(I/E/CC50) values and standard deviations (SD) calculated; 2) Replicate log(I/E/CC50) values were evaluated for outliers using the ROUT method in GraphPad Prism 6 (Q of 10%); and 3) Average I/E/CC50 values were then backcalculated from the average log(IC50) values. To compare statistical differences between log(I/E/CC50) values between the matched R- and L-series of analogs, two-tailed, paired t-tests were performed using GraphPad Prism 6 (95% confidence level) and looking at differences between paired values (results are plotted in Figures S1-S5 in the Supporting Information). For compounds where log(I/E/CC50) values were greater than the maximum compound concentrations tested (i.e. >1.8 and >2.0, or >63 and >100 µM, respectively), results were represented as 0.1 log units higher than the maximum concentrations tested (i.e. 1.9 and 2.1, or 79 and 126 µM, respectively), so as not to overly bias comparisons because of the unavailability of definitive values for these inactive compounds.</p>