A Small Molecule Substrate Mimic to Inhibit Bacterial Hsp70 Chaperone Function

Abstract

Given the rapid spread of antibiotic resistance worldwide, it is imperative to develop new strategies against pathogenic bacteria. Here, we report a small‐molecule inhibitor of bacterial heat shock protein 70 (Hsp70), called DnaK, a central component of the bacterial protein chaperone network. Screening of an in‐house pyridylamide‐based peptidomimetic library identified M7 as an inhibitor of DnaK ATPase and substrate refolding activity with intrinsic fluorescence properties. Mechanistic studies revealed that M7 likely engages the substrate‐binding pocket of DnaK. Treatment of bacterial cells with M7 led to the accumulation of σ‐32, without affecting protein translation, consistent with impaired DnaK function. Microscopy images of Escherichia coli AS19 cells treated with M7 revealed filamentous growth, a phenotypic defect consistent with compromised DnaK function. Moreover, M7 effectively inhibited the growth of E. coli AS19 and some Gram‐positive bacteria, including Staphylococcus aureus and Bacillus cereus. These findings highlight a promising class of peptidomimetic DnaK inhibitors as potential antibacterial agents, as well as a chemical biology tool to interrogate the broader network of chaperone‐mediated protein folding.

Antibiotic resistance has reemerged as a global threat necessitating the development of novel antibacterial targets. Here, we describe the development of a substrate mimetic inhibitor for bacterial heat shock protein 70 (Hsp70), which is a central component of bacterial protein homeostasis. The molecule effectively inhibits the growth of E. coli AS19 and some Gram‐positive bacteria. Our findings highlight a promising class of peptidomimetic DnaK inhibitors as potential antibacterial agents and as tools to interrogate chaperone‐mediated protein folding.

1. Introduction

Antibiotic resistance is a global threat affecting human health and livestock. Thus, it is imperative to understand the mechanisms involved in this process and to identify previously unexplored biochemical pathways in bacteria for antibacterial therapy. The chaperone Hsp70, known as DnaK in bacteria, is a well‐studied system that plays a central role in protein homeostasis. It regulates several housekeeping functions in the cell, including de novo protein folding, protein trafficking, protein disaggregation, proteosome‐mediated protein degradation, and is also involved in assisting bacteria in overcoming antibiotic stress [1, 2].

This 70‐kDa protein includes a nucleotide‐binding domain (NBD) and a substrate‐binding domain (SBD) (Figure 1a), connected by a linker for coordinated chaperone function and is assisted by co‐chaperones DnaJ and GrpE (Figure 1b) [1, 3]. DnaJ interacts with the ATP‐bound form of DnaK, delivers the unfolded substrate, and stimulates its ATPase activity. GrpE serves as a nucleotide‐exchange factor by exchanging ADP to ATP and also assists in substrate release. DnaK is highly conserved in prokaryotes (50%–70% sequence identity) and ensures proper protein folding and stress response, making it crucial for the survival of many pathogens [1, 4]. However, its structural flexibility and complex functional cycle make it challenging to target, and existing inhibitors primarily targeting the NBD encounter limitations due to cross‐reactivity with mammalian Hsp70 or cell impermeability [5, 6]. Peptide‐based inhibitors, for example, proline‐rich antimicrobial peptides (PrAMPs) bind to DnaK in vitro, but DnaK is not their primary intracellular target [7, 8, 9, 10, 11], making them less suitable for the mechanistic studies of DnaK. A few small‐molecule inhibitors, like 2‐phenylethynesulfonamide (PES), have shown the ability to interfere with DnaK–client protein interactions [12]. However, these molecules were not pursued further due to off‐target effects arising from their covalent reactivity.

FIGURE 1.

(a) The structure of full‐length DnaK (PDB ID: 2KHO) showing the nucleotide‐binding domain (DnaK NBD) and the substrate‐binding domain (DnaK SBD) connected with a linker. The regions involved in ATP binding (red) and substrate binding (blue) are shown. The image was generated with UCSF Chimera. (b) The DnaK chaperone cycle in bacteria. DnaK interacts with the co‐chaperones DnaJ (pink) and GrpE (red) to maintain protein homeostasis in the cell. (c) Schematic depiction of screening of an in‐house library to identify small molecules that target DnaK SBD and inhibit DnaK‐mediated chaperone activity. (d) Representative substrate‐mimetic small molecules tested in this study (see Figure S1 for molecule structures).

Here, we report a pyridylamide‐based small‐molecule library to target the DnaK SBD, as shown in the scheme (Figure 1c). We hypothesized that the SBD of DnaK, which binds unfolded peptides, might be a good target for pyridylamide‐based peptidomimetic small molecules. Molecules based on such scaffolds can faithfully mimic peptide side‐chain projection from the backbone and have been highly successful as protein–protein interaction inhibitors with improved resistance to degradation [13, 14]. We set out to investigate whether these abiotic peptide mimics impact DnaK function and eventually bacterial growth. By screening an in‐house library of small‐molecule substrate mimics, we identified M7 as an inhibitor of DnaK. M7 binding disrupts DnaK ATPase and luciferase refolding activity in vitro. It also disrupts DnaK chaperone function in Escherichia coli cells, as shown by the accumulation of transcription factor σ‐32, a known client of DnaK, leading to impaired bacterial growth. M7 possesses intrinsic fluorescence and can serve as a surrogate for fluorescent peptide substrates to probe ligand binding in DnaK SBD. Overall, these findings establish M7 as a promising peptidomimetic bacterial Hsp70 inhibitor and a chemical biology tool to interrogate the broader network of chaperone‐mediated protein folding.

2. Results and Discussion

2.1. Identification of M7 as a Potent In Vitro DnaK Inhibitor From an In‐House Small Molecule Screen

To screen for DnaK inhibitors, we explored a small in‐house library of oligopyridylamide‐based peptide mimics with various hydrophobic or aromatic side chains (Figures 1d and S1, see Materials and Methods for synthetic procedures) [15, 16, 17]. The advantage of using such molecules is that their hydrophobic side chains mimic peptide residues and can potentially bind to the DnaK SBD, while the lack of a canonical peptide backbone provides stability against proteases [14]. We first measured the inhibition of DnaK ATP hydrolysis activity using a malachite green‐based colorimetric assay in the presence of co‐chaperones DnaJ and GrpE (Figure 2a). As a positive control, we used myricetin (Figure 2b), which inhibits DnaK–DnaJ interaction in vitro by binding to DnaK NBD. Although chemically distinct from our library, myricetin served as a starting point for initial benchmarking [18]. In the primary screening, one of the molecules called M7 inhibited the DnaK ATPase activity similar to myricetin (Figure 2a,c).

FIGURE 2.

(a) Primary screening study depicting ATPase activity of DnaK‐K (0.7 µM) with DnaJ‐J (0.5 µM) and GrpE‐E (1.2 µM). Small molecules (50 µM) and 1 mM ATP were used for screening. Myricetin (Myr) was used as a positive control. (b) The structure of myricetin and (c) M7 (d) dose dependent inhibitory effect of M7 on ATPase activity of DnaK. DnaK‐K (0.7 µM), DnaJ‐J (0.5 µM), GrpE‐E (2.5 µM), and ATP (1 mM). (e) Dose‐dependent effect of M7 on luciferase refolding activity of DnaK; DnaK‐K (0.8 µM), DnaJ‐J (0.16 µM), GrpE‐E (0.4 µM), and 2 mM ATP were used. Inset shows luciferase refolding kinetics at different concentrations of M7. All data are representative of three independent experiments.

The inhibitory effect of M7 on DnaK‐mediated ATP hydrolysis was dose‐dependent, with an IC₅₀ = 8.4 ± 0.4 μM (Figure 2d), comparable to myricetin (IC₅₀ ~ 14 μM) [18] and some known peptide inhibitors of DnaK [8]. In the ATPase cycle, adding GrpE stimulates the ATPase activity of the DnaK–DnaJ complex. Interestingly, we did not observe significant inhibitory effect of M7 on DnaK ATPase activity in the absence of GrpE (Figure S2a), suggesting that M7 likely interferes with GrpE‐mediated stimulation of DnaK. Varying the concentration of the other co‐chaperone, DnaJ, in the presence of M7 had no significant effect on DnaK ATPase activity (Figure S2b), indicating that M7 does not disrupt the DnaK–DnaJ interaction.

Next, we tested the inhibitory effect of M7 on DnaK‐mediated protein folding of chemically denatured luciferase. M7 inhibited DnaK‐mediated substrate refolding with an IC₅₀ = 2.0 ± 0.4 μM (Figure 2e), which is better than most known peptide‐based inhibitors [8, 9]. These data support the conclusion that M7 effectively inhibits DnaK function by altering its substrate‐binding activity. A structure–activity relationship study showed that the entire M7 molecule was necessary for activity (Figure S2c), and none of the analogs tested were as effective as M7 in inhibiting DnaK ATPase activity. Our findings align with established reports that DnaK ATPase function is allosterically modulated upon binding of M7 to the DnaK SBD, as with some known peptide substrates [19]. In contrast, the NR‐peptide, a known substrate of DnaK, does not inhibit its ATPase activity (Figure S2d) [20, 21]. This suggests that unlike M7, the NR peptide does not disrupt the allosteric communication between the substrate‐binding and NBDs required for efficient ATP hydrolysis.

2.2. M7 Binds to the Substrate‐Binding Pocket of DnaK

A recent report from our group has shown that M7 is highly effective in inhibiting amyloid formation by disrupting protein–protein interactions [22]. Amyloidogenic intermediates typically present exposed hydrophobic regions that closely resemble the consensus motifs recognized by DnaK SBD. We hypothesized that M7 likely functions as a substrate‐mimetic inhibitor and binds to DnaK at its SBD. To identify the nature and strength of interactions between M7 and DnaK, we first performed molecular docking to identify the possible binding site of M7 at the SBD of DnaK. The docked structure was overlayed with a crystal structure of DnaK bound to NR substrate peptide (PDB ID: 4R5I) (Figure 3a). The NR peptide binds in a pocket formed between the loops of DnaK SBDβ and involves residues M404, A429, and V436 [23]. The docked structure of M7 occupies a binding site in the loop similar to the NR peptide (Figure S2e), suggesting that M7 might bind at the substrate‐binding site in DnaK SBD.

FIGURE 3.

(a) The structure of M7 (orange) docked in DnaK‐SBD (structure used for overlay PDB ID: 4R5I, NR‐peptide is in green). The residues that interact with the NR‐peptide are also involved in M7 binding (blue balls). (b) Change in M7 (2 μM) fluorescence upon titration with DnaK: 0–100 μM. (c) Equilibrium titrations between M7 (2 μM) with DnaK and mutant DnaK‐V436F (inset). (d) Fluorescence quenching of M7 (10 μM) bound with DnaK (5 μM) in the presence of 0.3 mM NR‐peptide. Bars represent mean ± standard deviation. p values were determined using unpaired two‐tailed Student’s t‐tests. *p = 0.011. Data are representative of three independent experiments.

Incidentally, M7 exhibited intrinsic fluorescence, with excitation (λ _ex) at 340 nm and an emission peak at >400 nm. We checked if this property was due to aggregation by monitoring the fluorescence of different batches of M7 as a function of concentration. We observed a linear increase in fluorescence until 25 μM (Figure S3a), indicating that fluorescence is an intrinsic property of M7 and not due to aggregation of M7 at the concentrations tested. Further, the fluorescence of M7 increased in response to DnaK addition (Figures 3b and S3b), allowing us to monitor the binding between the two (Figure 3c). Using M7 fluorescence as a readout, we determined its binding affinity for DnaK to be ~20 μM (K _d).

To confirm whether M7 indeed bound to the substrate‐binding pocket, we used a DnaK mutant V436F, which is reported to be deficient in substrate binding, as the mutation blocks the hydrophobic substrate‐binding pocket [24, 25]. The purified mutant did not show any conformational defects (Figure S3c), consistent with prior reports [23, 26]. Titration of mutDnaK‐V436F with M7 did not show any significant change in fluorescence (Figure 3c inset and Figure S3d), supporting our hypothesis that M7 bound to DnaK in a substrate‐mimetic manner. We did not observe any nonspecific binding between M7 and GrpE that could indirectly affect DnaK function (Figure S3e). A control M7 analog, M7a, which was also fluorescent, did not give any measurable dose‐dependent binding with DnaK (data not shown).

To verify that M7 binds specifically to the DnaK‐SBD, we conducted a chase experiment using the NR peptide. An excess of NR peptide was added to a preformed complex of M7 and DnaK. The addition of the peptide resulted in a decrease in M7 fluorescence to no DnaK levels, implying that the NR peptide competitively displaced M7 from DnaK (Figure 3d). The NR peptide alone did not affect M7 fluorescence. Taken together, these results suggest that both the NR peptide and M7 compete for binding to DnaK SBD.

2.3. M7 Disrupts DnaK Function in E. coli AS19 Cells

To confirm the in vivo specificity of M7 in inhibiting DnaK‐mediated pathways, we quantified the cellular level of the heat shock transcription factor σ‐32 (also known as RpoH), a known client of DnaK. Reports suggest that DnaK regulates the proteolysis of σ‐32 via the protease FtsH, resulting in higher levels of σ‐32 in ΔdnaK cells than in wild‐type bacterial cells [27, 28, 29]. Thus, if M7 prevented the binding of DnaK to its client proteins, we would see increased σ‐32 levels in the cell. Here, we used the Gram‐negative E. coli AS19 strain,as it allows for enhanced permeability of small molecules [30]. Indeed, the levels of σ‐32 in E. coli AS19 cells, as measured by western blot, were ~5–7 times higher in M7‐treated cells than in untreated cells (Figures 4a,b and S4a). There was no difference in expression of DnaK and another housekeeping protein, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), consistent with the interpretation that M7 does not disturb de novo protein biosynthesis. For comparison, we also measured σ‐32 levels in the presence of Myr, a known DnaK inhibitor, and M7a, a negative control that showed no effect on ATPase activity. We observed an increase in σ‐32 levels in the presence of Myr (Figure S4b), consistent with earlier reports [31]. However, there was no change in σ‐32 levels in the presence of M7a (Figure S4c). These data further support our hypothesis that, like other DnaK‐specific inhibitors, M7 inhibits the DnaK chaperone pathway without adversely affecting other cellular pathways, including protein translation.

FIGURE 4.

(a) Western blot showing the cellular levels of σ‐32, DnaK, and GAPDH in E. coli AS19 cells on treatment with 5 μM M7. (b) Band intensities in panel (a) were normalized relative to protein levels in untreated E. coli AS19 cells. Bars represent mean ± standard deviation. p‐values were determined using unpaired two‐tailed Student’s t‐tests. *p = 0.027. (c) Bacterial growth inhibition of E. coli AS19 cells with M7 at 37 °C. (d) Scanning electron micrographs of 50 μM M7‐treated E. coli AS19 cells showing a filamentous phenotype at the site of cell division (black arrows). (e) Confocal fluorescence microscopy images of M7 (cyan) in live E. coli AS19 cells stained with the membrane‐specific FM4‐64 dye (red). Images show the zoomed version of untreated or M7‐treated cells (also see Figure S5). M7 excitation/emission = 405/415–450 nm; FM4‐64X red fluorescence excitation/emission = 561/565–650 nm. Scale bar: 3.8 μm. All images were processed using ImageJ. (f) The difference in cell length after M7 treatment is shown by a box plot. The p‐values were determined using ordinary one‐way ANOVA and Dunnett’s multiple comparisons; ****p < 0.0001 (n = 44 cells for each condition).

2.4. M7 Causes Phenotypic Defects in Bacteria

DnaK plays an essential role in bacterial growth and cell division, and any defect in the DnaK chaperone machinery results in delayed growth and a filamentous phenotype [32, 33, 34]. We reasoned that inhibition of the cellular DnaK chaperone activity by M7 would disrupt bacterial growth. We monitored the effect of M7 on the growth of the E. coli ATCC 25 922 and E. coli AS19 strains. E. coli AS19 strains allow enhanced penetration of small molecules into these Gram‐negative cells. We observed a dose‐dependent inhibitory effect of M7 on bacterial growth (Figure 4c) at 37 °C, with complete inhibition at 18 ± 2 μM (Figure S4e) but no growth defect in E. coli ATCC 25 922 (Figure S4g), likely due to impermeability of M7 into these cells. Moreover, we observed a small increase in MIC to 33 ± 8 μM for M7 at 42 °C (Figure S4d,e). The increase in MIC at 42 °C may indicate that enhanced chaperone expression partially compensates for the inhibitory effect of M7, supporting DnaK as a possible cellular target. The MIC values indicate superior cellular inhibitory activity of M7 compared with reported peptide‐based DnaK inhibitors [8, 35].

Myr and the monomeric form of M7 (M7c), both of which showed DnaK ATPase inhibition comparable to M7 (Figure S2), showed no observable effect on the growth of E. coli AS19 at concentrations of 50–100 µM (Figure S4h,i). These observations align with previous reports describing Myr as a weak inhibitor with no significant growth inhibition of E. coli [31]. Molecule M7a, which has the same side chains as M7 but is coupled at the carboxylate end, rather than the amino end, did not inhibit bacterial growth (Figure S4j), further validating the specificity of M7. No significant M7‐related cytotoxicity was observed on HEK293T and Vero cells (CC₅₀ > 100 μg/mL) (Figure S4f,g).

The growth defect in the E. coli AS19 strain was visualized by SEM imaging, which showed filamentous M7‐treated cells compared to the DMSO control (Figure 4d). We took advantage of the intrinsic fluorescence of M7 to identify the cellular localization of the molecule. We performed live‐cell fluorescence microscopy imaging for M7‐treated AS19 cells and used FM4‐64 dye for cell membrane staining (Figure 4e, untreated; Figure S5a). M7 could permeate the cells and was observed as three major nonexclusive fluorescence patterns: (i) puncta formations localized at the polar site, (ii) presence at the membrane or near the division site, and (iii) diffusion throughout the cytoplasm (Figure 4e, M7‐treated, Figure S5a–e). Puncta formation at polar sites has been reported in E. coli to be associated with defects in the protein folding and degradation machinery [33]. We measured the differences in size of the M7‐treated E. coli AS19 cells with the untreated cells and observed a statistically significant increase in cell size with an increase in M7 concentration (Figure 4f). The phenotypic defects displayed by M7‐treated bacterial cells are consistent with the role of DnaK in cell division and parallel those reported for inhibition of the DnaK pathway [31, 34].

The chaperone DnaK also regulates antibiotic resistance in pathogenic bacteria through various defense mechanisms, including biofilm formation [31]. To test biofilm inhibition by M7, we evaluated growth and biofilm formation in the Gram‐positive bacterium Bacillus cereus. We observed an MIC of 1.1 ± 0.3 μM for B. cereus (Figure S6e) and inhibition of biofilm formation in B. cereus (Figure S6g,h). We further tested the inhibitory effects of M7, M7c, and Myr against several clinically relevant bacterial strains, including a few from the ESKAPE family [36], a group of six highly pathogenic and drug‐resistant bacteria. M7 inhibited Staphylococcus aureus (MTCC 1430) growth with an MIC of 3.5 ± 0.3 μM (Figure S6a,b,f), and drug‐resistant S. aureus (nine MRSA and three VRSA) strains showed MIC values of ~8.7 μM (Figure S6a). The sequence alignment analysis indicates that residues predicted to interact with M7 in E. coli, likely residues M404, A429, and V436 that interact with NR‐peptide [37], are also present in B. cereus and S. aureus DnaK (Figure S8). Myr and the monomeric form of M7 (M7c) induced a modest growth delay in S. aureus and no observable effect on the growth of E. coli AS19 or B. cereus at concentrations of 50–100 µM (Figure S7a–c). Although we lack direct evidence of M7 interacting with DnaK in S. aureus, we surmise that this may result from misregulation of membrane proteins, either directly or indirectly through DnaK‐client proteins, and warrants further investigation. All four Gram‐negative strains tested: Acinetobacter baumannii BAA1605, Klebsiella pneumoniae BAA1705, Pseudomonas aeruginosa ATCC 27853, and E. coli ATCC 25922 showed no growth defect (Figure S6f), likely due to impermeability of M7 in these cells.

3. Conclusions

Heat shock protein DnaK, a highly conserved protein, is a key component of the proteostasis network and is also involved in multidrug resistance in bacteria. The nonredundant essential role of DnaK in pathogenic bacteria, including S. aureus and mycobacteria, suggests that inhibiting DnaK would be lethal to cells and makes it a promising antibacterial target. DnaK supports heritable antimicrobial resistance mechanisms, and fitness defects are observed in Mycobacterium sps in the absence of dnaK [2]. Thus, targeting DnaK, a conditionally essential protein, with small molecules might be an effective approach for clinically relevant multiple drug‐resistant strains with a lower impact on selective pressure‐induced resistance. Moreover, such inhibitors also have great potential as adjuvants to increase the bioactivity of existing antibiotics.

This study presents a pyridylamide‐based peptidomimetic small molecule as a disruptor of DnaK‐mediated protein folding. Our data support the conclusion that the lead compound M7 binds the DnaK SBD, blocks GrpE‐mediated ATPase stimulation, and likely traps DnaK in a substrate‐bound state. This interference results in a significant reduction in luciferase refolding activity, indicating that M7 is a substrate‐mimetic molecule that engages the DnaK substrate‐binding pocket and competitively inhibits client binding. The intrinsic fluorescence of M7 allowed us to probe ligand binding in the DnaK SBD and was supported by the V436F DnaK SBD mutant and a competitive peptide displacement assay. Molecular docking of M7 with the peptide‐bound (closed) DnaK SBD indicated that M7 fits into the substrate‐binding pocket. We propose that M7 disrupts ATP‐driven conformational cycle of DnaK by blocking substrate exchange and the conformational changes required for interactions with the co‐chaperones DnaJ and GrpE. Additionally, the peptide displacement results support that M7 binds to the DnaK SBD. This model offers a plausible explanation for inhibition of DnaK activity by M7. However, further structural studies are needed to determine whether M7 indeed binds to the closed conformation of DnaK.

In bacteria, the DnaK chaperone system has been shown to negatively regulate the heat shock response by affecting the synthesis, activity, and stability of σ‐32(RpoH) [29, 38, 39, 40]. It is well established that general stress conditions, such as heat or osmotic stress, can cause σ‐32 accumulation [41, 42, 43]. Also, any mutation that alters the function of DnaK, DnaJ, or GrpE results in a longer half‐life of σ‐32. M7 disrupts DnaK‐mediated σ‐32 degradation, impairing protein homeostasis in E. coli AS19 cells and inhibiting its growth. We leveraged the intrinsic fluorescence of M7 for imaging E. coli AS19. The microscopy data confirmed that M7 caused phenotypic defects, such as filamentation and increased cell size, making them resemble an isogenic dnaK deletion mutant. Given the essential role of DnaK in regulating cell division and maintaining surface integrity in bacteria, these observations are consistent with impaired DnaK function contributing to the observed cellular defects.

Our study showcases M7, a peptidomimetic molecule, as an inhibitor of the DnaK chaperone pathway with enhanced avidity and improved occupancy at the DnaK SBD, demonstrating its potential as a lead antibacterial candidate. Although we did not observe significant M7‐related cytotoxicity on mammalian cells, direct biochemical evidence for selectivity over mammalian Hsp70 will be necessary for practical applications. Nonetheless, M7 will serve as a valuable tool for investigating the broader network of chaperone‐mediated protein folding mechanisms in bacteria.

Funding

This work was supported by Science and Engineering Research Board and Indian Institute of Science Education and Research, Bhopal.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Upadhyay Tulsi, Kumari Reshma, Gangarde Yogesh M., Potteth Upasana S., Roychowdhury Rishij, Das Anirban, Saxena Deepanshi, Maitra Rahul, Chopra Sidharth, Saraogi Ishu, A Small Molecule Substrate Mimic to Inhibit Bacterial Hsp70 Chaperone Function, ChemBioChem 2026, 27, e70518. 10.1002/cbic.70518

Data Availability Statement

The data supporting this manuscript has been uploaded separately as supporting information.