Screening and Evaluation of Small Organic Molecules as ClpB

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Screening and evaluation of small organic molecules as ClpB inhibitors and potential antimicrobials Ianire Martin, Jarl Underhaug, Garbiñe Celaya, Fernando Moro, Knut Teigen, Aurora Martinez, and Arturo Muga J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400499k • Publication Date (Web): 20 Aug 2013 Downloaded from http://pubs.acs.org on August 21, 2013

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Journal of Medicinal Chemistry

Revised ms: jm-2013-00499k.R2

Screening and evaluation of small organic molecules as ClpB inhibitors and potential antimicrobials

Ianire Martin†, Jarl Underhaug‡, Garbiñe Celaya†, Fernando Moro†, Knut Teigen‡, Aurora Martinez‡* and Arturo Muga†,*

†

Biophysics Unit (CSIC-UPV/EHU) and Department of Biochemistry and Molecular

Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 48080 Bilbao, Spain.

‡

Department of Biomedicine, University of Bergen, 5009 Bergen, Norway

*

Corresponding authors

1 Environment ACS Paragon Plus


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ABSTRACT Inhibition of ClpB, the bacterial representative of the heat-shock protein 100 family that is associated with virulence of several pathogens, could be an effective strategy to develop new antimicrobial agents. Using a high-throughput screening method, we have identified several compounds that bind to different conformations of ClpB, and analyzed their effect on the ATPase and chaperone activities of the protein. Two of them inhibit these functional properties as well as the growth of Gram negative bacteria (E. coli), displaying antimicrobial activity under thermal or oxidative stress conditions. This activity is abolished upon deletion of ClpB, indicating that the action of these compounds is related to the stress cellular response in which ClpB is involved. Moreover, their moderate toxicity in human cell lines suggests that they might provide promising leads against bacterial growth.


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INTRODUCTION ClpB is a member of the Hsp100/ClpB family of hexameric AAA+-ATPases1,2. This family comprises bacterial, fungal, and plant Hsp100 ATPases, ClpB being the bacterial representative3. This molecular chaperone collaborates with the Hsp70 system in protein disaggregation, a crucial process for cell survival under stress conditions4-6. During the infection process, bacterial pathogens encounter stress conditions generated by the host defense to eliminate them, and respond by increasing the synthesis of heat shock and other stress proteins7. In this context, ClpB has been described as an essential factor for acquired thermotolerance8 and for the virulence and infectivity of eukaryotes9,10 and several Gram-negative and Gram-positive pathogenic bacteria, such as Staphylococcus aureus11, Francisella turalensis12, Listeria monocytogenes13, Yersinia enterocolitica14, and Salmonella thyphimurium15. Therefore, the development of specific inhibitors of ClpB might be useful not only to understand the molecular mechanism of this chaperone, but also to develop new antimicrobial compounds to fight the rapid emergence of bacterial strains that are resistant to current antibiotics. Other chaperone families, such as Hsp7016 and Hsp9017, have been used as targets for the identification of antimicrobial and anticancer compounds, respectively. ClpB might also be a useful target to develop antimicrobial compounds since mammal cells do not have an Hsp100 homolog other than mitochondrial Hsp7818. An important factor usually considered to estimate the potential use of a protein as a target is its ability to bind small molecules, often known as “ligandability”19. Binding of these compounds might modulate the activity of the target protein, which in turn could have a therapeutic effect. If this happens, the target protein can be considered as druggable19. Therefore, we first screen for compounds that interact with ClpB. The screening for potential hits can be conventional, using chemically diverse, drug-like



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compound libraries, as employed here, or alternatively, a structure-guided design of a ligand with the desired drug properties might be a more efficient strategy when the 3D structure of the target is known. In this work, we have used the first experimental strategy because the 3D structure of the functional hexameric ClpB assembly remains as yet unknown. The available structural information of the Hsp100 family comes from Xray studies of Thermus thermophilus ClpB (TClpB), which unfortunately crystallized in a trimeric spiral in which monomer-monomer interactions might differ from those of the functional hexamer20. Although there is a lack of general consensus about the domain organization within the functional hexameric molecular machine21,22, each TClpB monomer contains an N-terminal domain followed by the first nucleotide binding domain (NBD), i.e. NBD1, a middle domain also known as the M domain that is inserted toward the C-terminal end of NBD1, and a second NBD, the NBD2. Both NBDs adopt a canonical AAA+ fold and bind and hydrolyze ATP, which drives ClpB through a functional cycle in which the protein adopts different conformations with distinct affinities for protein partners and substrates. The development of inhibitors targeting the conserved ATP binding sites has been an effective strategy because of the ease of assay design and likelihood of finding hits23. As an example, the development of kinase inhibitors has focussed on ATPcompetitive small molecules24. Although some examples of highly selective ATPcompetitive inhibitors of kinases24 and chaperones such as Hsp9025 exist, in many cases this type of inhibitors are troubled by the similarity of the ATP binding sites of many ATPases that could lead to toxicity through the undesired inhibition of their activities26. We describe here a protocol for the assay of ClpB ligands that preferentially bind to sites different to the nucleotide binding pockets and the discovery of two compounds


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that suppress bacterial growth. They display an antimicrobial activity under thermal or oxidative stress, which depends on ClpB since deletion of the chaperone abolishes it.



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RESULTS High-throughput screening (HTS) and hit structures. Selection of ClpB ligands was based on their effect on the thermal stability of ClpB measured as an up-shift of the midpoint denaturation temperature (Tm) value by differential scanning fluorimetry (DSF)27. To validate the use of DSF-based HTS to detect changes in the thermal stability of ClpB due to ligand binding, the experimental DSF and differential scanning calorimetry (DSC) traces were compared. Thermal unfolding of ClpB analyzed by DSC is a complex process28 and, as shown in this work, it is sensitive to the presence of nucleotides. The thermogram of the apo-protein shows two overlapped peaks at 49.0 and 58.2 °C (Fig. 1A), in agreement with published data28. As seen by DSF, there seems to be some interactions of the hydrophobic fluorescent dye with the native state of ClpB at low temperatures, which is expected since the chaperone interacts with (partly) unfolded proteins that expose hydrophobic patches to the solvent (Fig. 1B). A further increase in temperature unfolds the protein in two steps associated with exposure of new hydrophobic areas and therefore with enhanced fluorescent probe binding (Fig. 1B). The derivative of the temperature profile of the apo-protein in 2% DMSO yielded two events at 53.6±0.5 °C and 59.2±0.4 °C (Fig. 1C), values comparable to those obtained by DSC. DMSO (2%) did not affect the thermal denaturation profile of the protein. Nucleotide (ADP or ATPγS) binding to the apo-protein resulted in one major endotherm at 60.7 °C as seen by DSC (Fig. 1A), and in the merging of the two peaks into one at 63.2±0.2 °C by DSF (Figs. 1B and 1C). The assignment of each transition to the unfolding of specific protein domains is not straightforward, although it has been proposed that the high-temperature transition arises, at least in part, from the unfolding of the N-terminal domain28. A deeper interpretation of these results requires a further characterization of the protein thermal unfolding under different experimental


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conditions, an aim beyond the scope of this work. Nevertheless, these data demonstrate that DSF results compare well with DSC data in terms of Tm values for ClpB denaturation, thus validating further DSF-based detection of ligand-binding by measuring the effect of the compounds on ClpB stability. To screen for putative small molecular weight inhibitors, we first selected compounds that bind to ClpB and increased its stability. Thus, binders were experimentally detected by comparing the Tm values of the protein in the absence and presence of ligands. Ten thousand chemical compounds (MyriaScreen Diversity Library) were screened by DSF, and those that stabilized the apo-protein were also measured in the presence of 5 mM ADP, 5 mM ATP, or 1 mM ATPγS. The compounds that increased the denaturation temperature of some of the ClpB states (△Tm) by > 2.0 °C were considered potential hit compounds. Out of the 67 hits from the primary screening (Fig. 1D), 47 stabilized only the apo-state, 9 the apo and some of the nucleotide-bound conformations, and 11 apparently increased the stability of all protein conformations (see Fig. 1E-G for a representative example). The criterion used to restrict the number of compounds for further analysis was that they must bind to the apo and at least one of the nucleotide-bound conformations that the protein samples during its functional cycle, namely the ADP- or ATP-states. This would favour binding of the ligand to the target protein since ClpB, as an ATPase, spends most of the time in its ADP- or ATP-bound state. Additionally, if a compound binds to at least one nucleotide-bound conformation, it would be likely that its binding site(s) will be other than the nucleotide binding pocket(s), and therefore it will interact less efficiently with other AAA+ ATPases essential for cell viability. Following these arguments and the commercial availability of the ligands, 12 stabilizing compounds were selected for in vitro testing of their effect on the functional properties of ClpB. The



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name and structures of these compounds are presented in Table S1 and Figure 2. Seven of them (compounds 1, 2, 4, 5, 6, 8 and 10; Fig. 2) interacted with the apo-protein and at least one, but not all, nucleotide-bound conformation and five (compounds 3, 7, 9, 11 and 12; Figure 2) with all chaperone conformations. The analysis of the structure of the selected hit compounds (Fig. 2) reveals that several have features characteristic of pan assay interference compounds (PAINS)29. Compounds 1, 2, 5, 7, 9 and 12 are rhodanines or related heterocycles, which are abundant in the MyriaScreen library (212 structures with rhodanine scaffold and 38 with thiazolidenedione scaffold). These are compounds with a low selectivity and broad spectrum of biological activity29,30. The remaining compound hits from the primary screening also contain reactive functional groups, which are recurrent in diversity compound libraries despite being not recommended for screening29,30. However, promiscuity and polypharmacy have also been associated to successful drug optimization and discovery, since lead optimization might still make use of inherent high biological activity31.

In vitro testing of ligands for modifications of the ATPase activity of ClpB. Binding of the selected compounds to ClpB might modulate its activity, which as a chaperone is related to its ability to reactivate protein aggregates1-6. This disaggregase activity requires ATP hydrolysis at both nucleotide binding sites of the protein monomer, and thus relies on the energy provided by nucleotide hydrolysis to extract unfolded protein substrate molecules from the aggregate for their subsequent refolding. Therefore, we first characterized the effect of the selected compounds on the essential ATPase activity of ClpB (Figure 3), and grouped them as follows: i) those that have a slight, if any, effect on the basal and substrate-activated ATP hydrolysis (compounds 4, 7, 8, 9, and


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12; Fig. 3A); ii) those that activate (compounds 1, 2, 5, 11; Fig. 3B) or inhibit (compound 10; Fig. 3C) free and/or substrate–bound ClpB, and iii) those that inhibit activation of the ATPase activity of ClpB by α-casein (compounds 3 and 6 in Figs. 3D and 3E). The rational behind this classification is not straightforward, since ClpB is a hexamer with two NBDs per monomer that bind and hydrolyze ATP. Changes in the ATPase activity could be due to a direct interaction of the compounds with residues at the NBDs, or to an indirect effect through an interaction with a structural element involved in the allosteric regulation of their hydrolytic activity1-3. Furthermore, nonspecific inhibition of enzyme activity can be promoted by formation of colloidal aggregates, as found for some small organic molecules32,33, which can adsorb proteins causing partial denaturation. To test if colloidal aggregation could affect the ATPase activity of ClpB, we repeated the same experiments in the presence of detergents that disrupt colloid formation34. Tween 80 was chosen since it was the only surfactant that above its critical micellar concentration did not significantly diminish the concerted reactivation activity of the DnaK-ClpB bichaperone network (see below; Fig. S1). The surfactant did not affect the basal ATPase activity of ClpB and slightly reduced its substrate-induced activation in the absence of compounds (Fig. 3). The ligand-induced modulation of the chaperone ATPase activity was preserved in the presence of Tween 80 for compounds 2-12, suggesting that their effect was not due to colloid formation (Fig. 3, empty symbols; Table 1). In the case of compound 1 (Fig. 3B), the activation detected at concentrations above 60 µM could be related to its aggregation, since it was not observed in the presence of detergent (Fig. 3). The fact that increasing casein concentrations reverted the inhibitory effect of compound 3 but not 6 (Fig. 4A) suggests that the first one reversibly competes with substrate binding to the chaperone. Supporting this hypothesis is the fact that casein-



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induced activation is observed at higher substrate concentrations for compound 3, as compared with results obtained in the absence of ligands or in the presence of compound 6. The activation factor, i.e., the casein-mediated increase in the chaperone ATPase activity, is also remarkably higher in the presence of compound 3, indicating that it might also modify the communication between nucleotide binding sites that regulate the ATPase activity of ClpB35. The effect of these compounds on the affinity of the chaperone for α-casein was analyzed using a trap mutant of ClpB (ClpBtrap) that binds but does not hydrolyze ATP36 (Fig. 4B). The ATP-bound state of this ClpB variant, which forms stable complexes with protein substrates, showed a 4-fold decrease in the affinity in the presence of compound 3 (Kd = 2.4±0.1 µM vs 0.62±0.04 µM in the absence of the ligand) and a similar affinity in the presence of compound 6 (Kd = 0.83±0.05 µM) (Fig. 4B). Moreover, titration of a preformed ClpB:α-casein complex (20:1 molar ratio) with compound 3, but not with 6 or other ligands, progressively dissociated the complex (Fig. 4B; inset), so that the anisotropy of the sample at 500 µM compound 3 became similar to that of free α-casein. The higher affinity of ClpB for casein (Kd = 0.62 µM; Fig. 4B), as compared with compound 3 (Kd ⋍ 12 µM), explains why significant displacement of the substrate protein occurred only above 100 µM compound. An alternative effect that will also result in a decreased affinity of ClpB for substrates is that compound 3 could modify the oligomerization state of the chaperone. To rule out this possibility, the association equilibrium of ClpB in the presence of compound 3 was analyzed by sedimentation equilibrium measurements (Fig. S2). The average molecular masses obtained were 593±5 (control; ClpB+DMSO), 599±4 (ClpB+compound 3), and 590±3 (ClpB+ADP+compound 3) kDa, demonstrating that compound 3 did not dissociate the ClpB hexamer. Taken together, these data suggest that compound 3 and protein substrates interact with the same region of ClpB, while 10 Environment ACS Paragon Plus

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compound 6 could allosterically hamper the conformational change that links substrate binding and enhancement of the ATPase activity, e.g. compound 6 might behave as an allosteric inhibitor37. This molecular mechanism of inhibition must always be kept in mind with allosteric chaperones38. Direct binding of compounds 3 and 6 to the chaperone was corroborated by monitoring the stabilization of ClpB by concentration-dependent DSF (Fig. 5A), a method that also estimates the affinity for the compounds 39 (Kd ⋍ 12 µM and ⋍ 23 µM for compounds 3 and 6, respectively). The interaction was also analyzed by surface plasmon resonance (SPR) with ClpB immobilized in CM5 (Fig. 5B). At the same concentration, the RU values for compound 6 were higher than those of compound 3, as expected from the lower molecular weight of the latter. Titration with compound 3 reached saturation (Fig. 5B, inset), in contrast to compound 6 that under the SPR experimental conditions precipitated above 200 µM (at concentrations of DMSO up to 5%). Good fittings to the simple Langmuir 1:1 were however obtained for the binding responses of both compounds, which revealed different association kinetics and provided Kd values of 12.9 ± 3.6 µM and 19 ± 3 µM for compounds 3 and 6, respectively, in good agreement with DSF results.

Effect of compounds on the coordinated chaperone activity of the DnaK-ClpB network. To reactivate protein aggregates ClpB needs, besides the energy coming form ATP hydrolysis, to cooperate with the DnaK system. Therefore, we next analyzed the effect that the selected compounds exerted on the coordinated ability of the DnaK system (DnaK, DnaJ and GrpE) and ClpB to chaperone protein aggregates (Fig. 6). It should be noted that ClpB by itself shows no disaggregase activity, in contrast to the DnaK system that is able to reactivate small aggregates of some protein substrates.



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Reactivation of stable aggregates becomes strictly ClpB-dependent and requires the concerted action of both chaperone systems40-42. Therefore, to assign a putative effect of a given compound on the chaperone activity of ClpB it has first to be ruled out any effect on the DnaK system. This is necessary since the current model of protein disaggregation considers that the DnaK system extracts polypeptides or remodels aggregates at the initial stages of the disaggregation process, presenting their unstructured regions to ClpB41,42. Therefore, a detrimental effect on the initial DnaK action would negatively affect or abolish the subsequent activity of ClpB. To clarify this point, the ability of the DnaK system alone to reactivate aggregates of luciferase previously unfolded in GndHCl (Fig. 6; circles) was compared with the reactivation of malate dehydrogenase (MDH) aggregates that requires the action of both chaperones (DnaK+ClpB; Fig. 6; triangles), in the presence of different ligand concentrations. Among the compounds, some inhibited these activities below 100 µM to a different extent, with distinct half maximal inhibitory concentration (IC50) values (Table 1). Six compounds did not affect the DnaK system and had a small effect on the activity of the bichaperone network (compounds 7, 8, 9, 11 and 12; Fig. 2; Fig. 6A and Table 1). Six (compounds 1, 2, 4, 5, 6 and 10; Fig. 2; Fig. 6B and Table 1) display a selective inhibitory activity against ClpB, since they inhibit less efficiently the DnaK-mediated refolding of denatured and aggregated luciferase. Finally, compound 3 inhibits the DnaK system and therefore the subsequent action of ClpB (Fig. 6C). As mentioned above, to discard the possibility that the observed inhibition could be due to colloid formation, the same reactivation measurements were performed in the presence of 0.025 % Tween 80, the only detergent that did not disrupt the weak interaction between DnaK and ClpB (Kd=10-30 µM40,43; Fig. S1). However, the surfactant decreased the reactivation efficiency of the DnaK system and hence of the bichaperone network form


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80 to 60%. Results shown in Fig. 6 and Table 1 indicate that the trend observed for each compound in the absence of detergent was essentially maintained in its presence and, therefore, that inhibition was not related to colloid formation by these organic molecules. Compound 3 inhibited the chaperone action of the DnaK system (Fig. 6, empty symbols). This system contains besides DnaK another two proteins, the co-chaperone DnaJ which carries protein substrates to DnaK and enhances its ATPase activity, and the nucleotide exchange factor GrpE. In order to be productive, the functional cycle of DnaK requires a sequential interaction of the chaperone with these partners and with protein substrates driven by nucleotide binding and hydrolysis44. Thus, the observed inhibition might be due to an effect of compound 3 on the ATPase activity of DnaK, and/or on the interaction of the chaperone with protein substrates or with its cochaperones DnaJ and GrpE. Therefore, the effect of compound 3 on the interaction of DnaK with a peptide substrate (F-APPY) and with its two co-chaperones was investigated (Fig. 7). In contrast to the interaction of ClpB with casein, the apparent affinity of DnaK for this peptide substrate was not significantly modified by the ligand (Kd = 0.40±0.06 and 0.71±0.14 µM in the absence and presence of compound 3, respectively; Fig. 7A). The lack of effect on peptide binding was also revealed by the substrate-induced enhancement of the DnaK ATPase activity, which was around four times regardless of the presence of compound 3 (Fig. 7B). However, this compound reduced to one third the basal ATPase activity of the chaperone, and, notably, induced a 7-fold reduction of the co-chaperone (DnaJ and GrpE) induced activation of the ATPase activity of the DnaK-substrate complex (Fig. 7B). These data suggest that compound 3 precludes the functional interplay between DnaK and its co-chaperones.



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Inhibition of bacterial growth. We next wanted to find out which ligands were able to inhibit growth of two different strains of E. coli. To this end, the effect of these compounds on bacterial growth was analysed under different conditions (Table 2). These conditions include elevated temperatures (42 and 47 ºC) and presence of reactive oxygen species to mimic the heat and oxidative stress conditions elicit by the host of pathogenic bacteria45,46. Oxidative stress is physiologically relevant, since in the host environment macrophages generate reactive oxygen species to eliminate bacteria, which react inducing a global response that includes an increased synthesis of ClpB47. Two compounds, 3 and 6, were able to inhibit growth of E. coli strains MC4100 (Fig. 8A-E) and BL21(DE3) (Fig. S3) under stress conditions. Their effect on cell growth becomes stronger with increasing temperatures (Figs. 8A-C and S3), specially in the presence of paraquat (Figs. 8D, E and S3), indicating that the antimicrobial activity of the compounds is more potent when thermal and oxidative stresses are combined. The minimum inhibitory concentration (MIC) values obtained at 42 ºC in the presence of paraquat are 60 and 60-100 µM for compound 6 and 3, respectively. To investigate how growth inhibition was related to bacterial viability we performed plating experiments (Figs 8H-I). Compounds 3 and 6 inhibited bacterial growth at 37 ºC in the presence of paraquat (Fig. 8G) and at 42 °C in its absence (Fig. 8H), as revealed by a reduction of the colony-forming units (CFU) count by 2-3 log units over 24 h of exposure. Remarkably, bacterial viability is lost in the presence of both compounds at 42 ºC under oxidative stress (Fig. 8H) or under severe heat stress conditions, i.e., 47ºC (Fig. 8I). None of the effects on bacterial growth (Fig. 8A-E; open circles) and viability (not shown) are observed upon deletion of the chaperone, as seen with the MC4100∆clpB strain, indicating that the specific interaction of these compounds with ClpB is essential


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to inhibit bacterial growth. It is important to note that deletion of the target protein, ClpB, only causes a slower growth rate at 44 °C and reduced cell survival at 50 °C8. Furthermore, deletion of TolC, a component of the bacterial efflux system, lowered the MIC values of both compounds to 5-25 µM (Fig. S4), pointing out that the discrepancy between the concentrations that inhibit ClpB in vitro and those that inhibit bacterial growth might be due to drug efflux. As a control, ampicillin, a well-known antibiotic, displayed this activity against all the bacterial strains used in this work with MIC values (2-5 µM; see Fig. 8F for MC4100 and MC4100∆clpB strains) similar to those previously reported48.

Toxicity of the inhibitors in human cell lines. The cytotoxicity of the compounds that inhibited bacterial growth (compounds 3 and 6) was assayed using HeLa and CHO/K1 cells (Fig. 9). Compound concentrations were chosen to be around the estimated MIC values at moderately high temperature (42 ºC) under oxidative stress. Results show cell viability percentage values around 50-70% for CHO and HeLa cells, respectively,



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DISCUSSION Bacteria often encounter stressing environmental conditions, which in the case of pathogens are generated by their hosts in response to infection, including febrile episodes49 and the host oxidative immune response50. These conditions induce a myriad of adaptive and protective responses in bacteria that result in a different gene expression pattern and susceptibility to antimicrobials. Among them, some involve an increased synthesis of chaperones such as ClpB, which have been linked to antimicrobial resistance45, therefore suggesting that stress responses could be considered as potential therapeutic targets. In this context, resistance to both thermal and oxidative stress has been related to virulence of pathogenic bacteria47, although the way these properties are linked is far from being understood. Taking advantage of this relationship, we use in this work thermal and oxidative stresses to mimic the unfavorable conditions generated in the host environment during infection49,50. The involvement of ClpB in virulence reflects its ability to help bacteria to cope with these stressful conditions. Although it remains unknown whether this activity is related to the general chaperone function of ClpB or to its involvement in specific processes essential for pathogenic development13, it is clear that ClpB is important for virulence and long-term persistence of pathogenic bacteria in host organisms. ClpB deletion reduces the ability of bacteria to adapt to infection, one of the most unfavorable conditions that bacteria encounter. Our data indicate that the antimicrobial activity of compounds 3 and 6 relies on ClpB, since deletion of the chaperone abolishes it. Furthermore, the fact that it is only observed under thermal and/or oxidative stress strongly suggests the involvement of ClpB in the ligand-induced inhibition of bacterial growth. Interestingly, our results put forward that inhibition of ClpB by these ligands is more pernicious than its deletion, suggesting that specific alterations of physiological events in which ClpB participates to allow a better


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adaptation to stress conditions might cause compound-induced inhibition of bacterial growth. Thus it seems that the antimicrobial action of these compounds does not only rely on the general chaperone function of ClpB, but rather in the modulation of its interplay with other partners under stress conditions. Compound 3 inhibits the chaperone activity of ClpB by competing with substrate binding and modifying the ATPase activity of the protein in the presence of substrate proteins, and that of the DnaK system by interfering with the productive interaction between the chaperone and its cochaperones DnaJ and GrpE. However, the effect of inhibiting the DnaK system is not observed in vivo, since in the absence of ClpB (MC4100∆clpB strain) cells grow at stress temperatures despite the fact that DnaK is required for bacterial growth under these conditions51. Extrapolation of in vitro data to in vivo conditions is not straightforward, especially in the case of chaperones with overlapping activities that might be regulated by other factors, and that interact with many still unknown partners. A comparative analysis of the active hits structures towards the other binders and other compounds in the library can provide some leads to identify structural determinants for the antimicrobial activity of these compounds. Compound 3 is a salicylaldehyde derivative over a benzylbenzene scaffold (Fig. S5A). It is the only salicylaldehyde derivative in the MyriaScreen library, while a benzylbenzene scaffold is shared by 228 other compounds, none of them being ClpB binders. A benzylbenzene with a nitrous acid in meta-position (R1; Fig. S5A) is unique to compound 3, but the aryl-nitro functional group is found in 963 compounds of the library, including compounds 4 and 8 (Fig. 2). These compounds also share with compound 3 the ring with ortho-substituted chlorine, further pointing to the specific substitutions in the salicylaldehyde ring (the hydroxyl oxygen at R2 and the acetaldehyde at R3; Fig. S5A)



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as possible determinants for the inhibitory activity of compound 3. Aldehydes are considered potentially undesirably reactive groups29 and this group might be targeted in a posterior derivatization of compound 3. Compound 6 binding to ClpB hampers the substrate-induced enhancement of its ATPase activity, an effect that cannot be restored with increasing substrate concentrations despite the fact that the affinity of the protein for the substrate is not significantly altered. A reasonable explanation for this behavior is that ligand binding blocks the conformational change that couples substrate binding and the enhancement of the ATPase activity of the chaperone. Although this structural transition has so far not been characterized in detail, it is clear that, as found in other chaperones, allosteric communication between different ClpB domains is necessary for the chaperone activity of the protein35. This mode of action on ClpB aids in gaining specificity, since the chaperone activity of the DnaK system is slightly reduced at compound concentrations, i.e., 60 µM, that virtually abolish the chaperone activity of ClpB. Compound 6 is a longitudinal molecule with two lipophilic anchors (anilines) and a flexible relatively long (6n) linker, resulting in a fairly high element of entropy. There are ten other analogues in the MyriaScreen library that share this feature, including compound 11 (Fig. 2), but lack substituents in the aniline rings. This might indicate that the chlorine substituents and a more flexible linker might be determinant for the inhibitory activity of compound 6. This compound appears as an attractive hit in an alternative class to compound 3, and with a different binding site in ClpB, although its reactive thiocarbamate group in the linker should be taken into account for derivatization. Interestingly, the compound concentration required for in vitro inhibition of ClpB (around 10 and 20 µM for compounds 3 and 6, respectively) is lower than that


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necessary to inhibit cell growth. This difference depends on the stress conditions used to challenge the cells, being less pronounced under a combination of thermal and oxidative shocks. The finding that it disappears upon deletion of a component of the multidrug efflux system (TolC) in E. coli, suggests that pumping out of the otherwise harmful compounds 3 and 6 is one of the factors responsible for the observed differences between the in vitro and in vivo effects of these ClpB inhibitors.

CONCLUSIONS This study points to ClpB as a useful target to develop antimicrobial compounds. Several ClpB ligands have been identified, using a high-throughput screening method, and their effects on the biological properties of ClpB and bacterial growth have been characterized. Two of these chemically unrelated compounds inhibited bacterial growth under either thermal or oxidative stress conditions. The fact that their antibacterial activity was suppressed upon deletion of ClpB, indicates that the specific interaction of the compounds with the chaperone is essential for their antimicrobial action. Their moderate cytotoxicity suggests that these two compounds could be used as leads for the development of new antimicrobials. Moreover, our data also could help to understand the link between stress and antimicrobial resistance, and point out that some stressinduced effectors might be used as valuable therapeutic targets.



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EXPERIMENTAL SECTION Materials and structure validation of compounds 3 and 6. The MyriaScreen Diversity Library from Sigma Aldrich/TimTec (Newark, DE) was used for screening. It consists of 10000 compounds stored at -20 oC at 2 mg/ml in 10 mM DMSO. The hit compounds from the screening (12 compounds; Fig. 2 and Table S1) were consecutively ordered from TimTec and Sigma Aldrich, prepared at concentrations of 10 mM in 100% DMSO and stored at -20 oC. The purity of these compounds is on average >95%, and in any case for all the batches used in this work >90%, as determined by LCMS. The structures of compounds 3 (ST012549) and 6 (ST034398) from TimTec were verified using high-resolution nuclear magnetic resonance (NMR) spectroscopy (Fig. S6). They were dissolved in deuterated DMSO (DMSO-d6) to a concentration of 15.3 mM and 5.8 mM for compound 3 and 6, respectively, and 1H NMR spectra were acquired at 314 K on a Bruker Avance DRX 600 spectrometer. NMR spectra were also acquired on the compounds taken directly from the library and diluted in DMSO-d6 (0.14 mM and 0.10 mM for compound 3 and 6, respectively). The chemical shifts were compared with theoretical shifts (ChemBioDraw Ultra 13.0, Perkin Elmer). The 1H NMR spectrum of Compound 3 (10.27 (s), 8.14 (d, 2 Hz), 7.86 (d, 2 Hz), 7.48 (dd, 1.5 Hz, 7,5 Hz), 7.45 (dd, 1.5 Hz, 7,5 Hz), 7.35 (dt, 1.5 Hz, 7.5 Hz), 7.32 (dt, 1.5 Hz, 7.5 Hz) , 4.15 (s)) fits well with the theoretical spectrum (14.88 (s), 10.19 (s), 8.23 (d, 1.5 Hz), 7.91 (d, 1.5 Hz), 7.64 (dd, 1.5 Hz, 7.5 Hz), 7.18 (dt, 1.5 Hz, 7.5 Hz), 7.17 (dt 1.5 Hz, 7.5 Hz), 7.12 (dd, 1.5 Hz, 7.5 Hz), 3.99 (s)). There are no impurities and the sample is stable at room temperature. The spectrum of compound 6 (10.75 (s), 9.72 (s), 7.85 (d, 2 Hz), 7.45 (m), 7.28 (mt,

Screening and Evaluation of Small Organic Molecules as ClpB

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