Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2025 Mar 18;11(4):998–1007. doi: 10.1021/acsinfecdis.4c01045

Identification of a New FtsZ Inhibitor by Virtual Screening, Mechanistic Insights, and Structure–Activity Relationship Analyses

Pietro Sciò †, Viola Camilla Scoffone ‡, Anastasia Parisi †, Marianna Bufano †, Martina Caneva ‡, Gabriele Trespidi ‡, Samuele Irudal ‡, Giulia Barbieri ‡, Lisa Cariani §, Beatrice Silvia Orena §, Valeria Daccò ∥, Francesco Imperi ⊥, Silvia Buroni ‡, Antonio Coluccia †,*
PMCID: PMC11998009  PMID: 40100965

Abstract

graphic file with name id4c01045_0010.jpg

Antimicrobial resistance (AMR) poses a major threat to human health globally. Approximately 5 million deaths were attributed to AMR in 2019, and this figure is predicted to worsen, reaching 10 million deaths by 2050. In the search for new compounds that can tackle AMR, FtsZ inhibitors represent a valuable option. In the present study, a structure-based virtual screening is reported, which led to the identification of derivative C11 endowed with an excellent minimum inhibitory concentration value of 2 μg/mL against Staphylococcus aureus. Biochemical assays clarified that compound C11 targets FtsZ by inhibiting its polymerization process. C11 also showed notable antimicrobial activity against S. aureus cystic fibrosis isolates and methicillin-resistant S. aureus strains. Derivative C11 did not show cytotoxicity, while it had a synergistic effect with methicillin. C11 also showed increased survival in the Galleria mellonella infection model. Lastly, structure–activity relationship and binding mode analyses were reported.

Keywords: virtual screening, FtsZ, antibiotics, antimicrobial resistance, drug discovery

Introduction

Antimicrobial resistance (AMR) is a serious global health threat. In 2019, it was estimated that around 5 million deaths were attributed to AMR, and this situation is getting worse with an estimation of 10 million deaths annually by 2050.1 This phenomenon is mainly due to the extensive misuse of antibiotics which leads to the selection of resistant bacteria.2 Although the development of new antibiotics with novel mechanisms of action is crucial to tackle this issue, only 50 new antibacterial drugs are facing clinical evaluation.3 Furthermore, only 32 of these are potentially effective against World Health Organization (WHO) priority pathogens.3 Among these bacteria, the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) group is particularly worrisome. S. aureus, for instance, can persist and adapt to different hostile niches and is equipped with many virulence factors. Methicillin-resistant S. aureus (MRSA) strains represent a particular threat to immunocompromised subjects and people with cystic fibrosis.4

In the search for new antibiotics with unrelated mechanisms of action, the bacterial cell division machinery represents a valuable target. The division of the parent cell into daughter cells is mainly orchestrated by FtsZ (temperature-sensitive mutant Z). This protein polymerizes, through GTP hydrolysis, to form a single strained filament, linked to the cytoplasmic membrane, leading to a ring-like structure (Z-ring) located in the middle of the parent cell.5 The Z-ring serves as a scaffold for the recruitment of other downstream effectors for cell division,5 and last, it leads to the final division through the septum constriction along with its invagination and the biosynthesis of peptidoglycan.5 The crucial role played by FtsZ makes it a very attractive target. Furthermore, FtsZ is highly conserved in most prokaryotic organisms,6 and it is widely demonstrated that its inhibition drives bacteriostatic or bactericidal effects.7 Tubulin is the eukaryotic analogue of FtsZ; these proteins share a common structure, nevertheless, the sequence alignment shows that the two proteins have a low sequence identity (less the 20%) and conserved residues localized only in the nucleotide-binding site.8

All these points confirm the validity of targeting FtsZ: the inhibitors are very effective, and they may have a broad spectrum of activity and are expected to have no cytotoxicity toward eukaryotes. At the state of the art, different FtsZ inhibitors have been reported, including peptides, natural products, and small molecules.9,10 Peptides like MciZ (49 residues long)11 mainly target the C terminal domain of FtsZ but none of the reported derivatives continued to preclinical or clinical evaluation.9 Natural compounds, like Sanguinarine and Berberine, showed promising antimicrobial activity, but they also had significant toxicity toward mammalian cells mainly because of the inhibition of tubulin polymerization.12−14 Among the small molecules, three chemical classes showed the most favorable results (Figure 1): (i) the arene-diol digallates (UCM53) targeted the nucleotide-binding site with promising activity toward Gram-positive bacteria but with some cytotoxicity to eukaryotic cells;15−17 (ii) the benzimidazole exhibited excellent antibacterial activity against clinical Mycobacterium tuberculosis (minimal inhibitory concentration of 0.5–15 μM),18 and (iii) benzamide derivatives.19 The latter class is exemplified by TX707, which had potent bactericidal activity against several Gram-positive bacteria, including Bacillus subtilis, MRSA, and other multidrug resistant (MDR) S. aureus in vitro and in vivo. TX709, a prodrug of TX707, is in the clinical phase of evaluation.19−22

Figure 1.

Figure 1

Open in a new tab

Small molecule FtsZ inhibitors.

Despite the effort in the field, the urgency of a new therapeutic armamentarium for MDR strains has made the search for new FtsZ inhibitors a very current topic.

In this scenario, a structure-based virtual screening (VS) campaign was carried out, resulting in the identification of compound C11 with a promising minimum inhibitory concentration (MIC) of 2 μg/mL against S. aureus. Further biological evaluation confirmed that compound C11 can impair FtsZ polymerization without being cytotoxic to eukaryotic cells.

The compound was effective against S. aureus biofilms, and it also showed a notable antimicrobial activity against cystic fibrosis S. aureus clinical isolates and MRSA strains. Moreover, the combination of C11 with Meropenem disclosed a synergistic effect that restored methicillin susceptibility to MRSA strains. The compound can also increase the survival of Galleria mellonella infected with S. aureus. A small group of commercially available C11 analogues (12 derivatives) was evaluated in vitro and in silico to outline a structure–activity relationship (SAR) and to define the pharmacophoric moieties within the scaffold.

Results and Discussion

Screening

To identify new FtsZ inhibitors, a structure-based VS was carried out. FtsZ is characterized by two main binding pockets: (i) the nucleotide binding pocket, bound by GTP/GDP and localized at the polymerization interface, and (ii) an interdomain cleft located at the central core of FtsZ (Figure S1). This pocket has no catalytic activity, and it is not directly involved in polymerization but plays a pivotal role in this process. Thus, this pocket in the S. aureus FtsZ protein (FtsZSa; PDB: 4DXD)23 was targeted by a docking and pharmacophore-based VS campaign. A database of commercially available compounds (about 5.000.000 molecules) was prefiltered to retain those matching Lipinsky drug-like properties24 and to remove unsuitable chemical moieties displaying promiscuous and cytotoxic effects.25 The obtained database was docked at the allosteric site of FtsZSa by Glide,26 and a pharmacophore model was generated by Phase27 merging the information on TX707 SAR.19−23 The resulting model included nine features: one H-bond acceptor, two H-bond donors, three hydrophobic, and three aromatic features (Figure S2). This model was used to filter out the docking proposed conformations. In the end, the conformers with the best fitting to the pharmacophore model (a total of 5000) were visually inspected,28 and 12 structurally unrelated molecules were purchased for biological evaluation.

Evaluation of Biological Activities of the Selected Compounds on S. aureus FtsZ

To verify whether the selected compounds retained biological activity against bacteria and, in particular, against FtsZSa, the inhibition of bacterial growth, the impairment of the GTPase activity, and, eventually, the inhibition of FtsZ polymerization were assayed (Table S1).

Three compounds (C3, C8, and C11) showed promising inhibitory activity toward FtsZSa GTPase activity. Derivatives C3 and C8 showed good inhibition of the GTPase activity with an IC50 of 14.6 and 8.4 μM, respectively (Table S1 and Figure S3). Unfortunately, both compounds had MIC values of ≥256 μg/mL when assayed against S. aureus ATCC25923 growth (Table S1). On the other hand, C11 showed a significant inhibition of S. aureus ATCC25923 growth with a MIC of 2 μg/mL, but it did not affect the FtsZSa GTPase activity (Table S1). Since FtsZ is characterized by both GTPase and polymerization activity, a sedimentation assay was performed to evaluate the ability of compound C11 to inhibit FtsZSa polymers formation (Figure 2 and Figure 3A).

Figure 2.

Figure 2

Open in a new tab

C11 structure and biological activity.

Figure 3.

Figure 3

Open in a new tab

FtsZSa polymerization in the presence of C11. (A) Sedimentation assay of FtsZSa in the presence of 100 μM of C11. GDP was used as a negative control of the reaction. (P: pellet fraction; S: supernatant fraction). (B) Right-angle light scattering of FtsZSa in the presence of increasing concentration of C11. GTP was added after 75 s of incubation. (C) Dose-response curve of FtsZSa residual polymerization activity with C11. Data are the mean ± SD of the results from three different replicates. (D) Typical micrograph of FtsZSa at cryo-electron microscope in the absence (left, CT) or in the presence of 100 μM of C11 (right). The scale bar represents 50 nm. Data and images are representative of the results from three different replicates.

Similar to what was previously described for other FtsZ inhibitors, like C109,29C11 had an inhibitory effect on FtsZSa polymerization (Figure 3A). To further evaluate this effect, the kinetics of FtsZSa polymerization was studied using a 90° light scattering measurement. C11 inhibited the polymerization in a concentration-dependent manner, with an IC50 of 47.97 μM (Figure 3B,C). Of note, the presence of C11 decreased the extent of FtsZSa polymerization and bundling, in a concentration-dependent manner like other previously described inhibitors30,31 and decreasing the compound concentration however had an effect on polymer stability (Figure 3B). Moreover, the decrease of polymer formation was also confirmed by cryo-electron microscopy analysis. At a concentration of 100 μM, C11 significantly impaired the quantity, stability, and bundling of FtsZSa polymers (Figure 3D).

C11 Biological Effect and FtsZ Localization

A time–kill curve was constructed using concentrations ranging from 1/2 to 2 times the MIC value (Figure 4A) to assess whether C11 exerted a bacteriostatic or bactericidal effect on S. aureus cells. Bacteriostatic effect was observed using 1- or 2-fold the MIC of C11, while a slight regrowth was observed when cells were treated with an amount of compound equal to half of the MIC (Figure 4A). Single-cell time-lapse microscopy of S. aureus ATCC25923 demonstrated that the area of the cells is significantly larger in the sample exposed to 2 × MIC of C11 compound for 5 h compared to untreated cells (Figure 4B). To study FtsZ localization, we used the S. aureus TD276 strain expressing the FtsZ-mCherry fusion protein.32,33 Against this strain, C11 retained a MIC of 2 μg/mL. As shown in Figure 4C, in untreated cells, FtsZ was localized in the middle of the cell and formed the Z-ring. In the treated sample, cells were larger and FtsZ was mislocalized, forming different foci which were not functionally competent for cell division (Figure 4C). Moreover, staining of endogenous DNA with Hoechst demonstrated that DNA segregation is not impaired in the treated cells since DNA segregates currently in both treated and untreated cells (Figure 4C). This experiment confirmed once again that FtsZ polymerization is likely the target of the C11. Since bacterial growth might be interrupted by the stress conditions imposed by time-lapse microscopy, treated and untreated cells were analyzed for their FtsZ localization in 10 fields. Results showed that the percentage of cells in which FtsZ was delocalized increased over time, and it was significantly higher in C11-treated samples as compared to untreated cells (60% vs 4%) (Figure 4D).

Figure 4.

Figure 4

Open in a new tab

C11 effect on S. aureus ATCC25923 growth. (A) Time-killing curve of S. aureus ATCC25923 exposed to C11. A S. aureus ATCC25923 culture in the exponential phase of growth was split and concentrations of C11 corresponding to 1/2-, 1-, and 2-fold the MIC value were added. The viable counts were determined at 37 °C over 24 h. (B) Measurements of cell areas of S. aureus ATCC25923 untreated and treated with 4 μg/mL of C11. Statistically significant differences are indicated (unpaired t-test, ****p < 0.0001). (C) Fluorescence microscopy images of S. aureus TD276, carrying the FtsZ-mCherry (red) construct, untreated (CT) and treated with 4 μg/mL of C11. Cells were stained also with Hoechst (1 μg/mL) (blue). The scale bar corresponds to 2 μm. (D) Graphical representation of the percentage of cells showing FtsZ delocalization (left y axis-bars), during growth (right y axis-lines) in the absence (black), or in the presence (violet) of 4 μg/mL C11, in time-lapse experiments. Data and images are representative of the results of at least three different experiments.

C11 Activity on Biofilm Formation

S. aureus can readily form biofilms enhancing its drug resistance and inducing life-threatening infections in different body compartments.34 Hence the ability of C11 to block biofilm formation and to eradicate formed biofilms was tested using the S. aureus ATCC25923 strain. First, the inhibitory activity of the compound was evaluated by CFU counting (Figure 5A), showing that concentrations of 2- or 4-fold the MIC (4–8 μg/mL) were enough to almost completely inhibit biofilm formation (Figure 5A). The structure of the biofilm formed in the presence of increasing concentrations of C11 was analyzed using confocal laser scanning microscopy (CLSM) and COMSTAT2. These analyses confirmed that C11 at 2 μg/mL only slightly affected biofilm formation, while it almost completely abolished it at 4 and 8 μg/mL (Figures 5BC and S4). Then the biofilm eradication was also assayed by CFU counting and CLSM. The assays showed that even the lower dose of C11 (2 × MIC, 4 μg/mL) could reduce the number of viable cells in S. aureus ATCC25923 biofilms (Figure S5A); increasing concentrations of C11 had a strong impact on the biofilm, eradicating the majority of the attached cells (Figure S5B) producing a significant alteration of the biofilm (Figures S5C and S6).

Figure 5.

Figure 5

Open in a new tab

C11 effect on biofilm inhibition. (A) Bacterial biofilms of S. aureus ATCC25923 were grown in the presence of increasing concentrations of C11. The results are expressed as CFU/ml recovered after 24 h of incubation. (B) CLSM images of S. aureus ATCC25923 biofilms grown in “μ-Slide 4 Well Ibidi treated”. Cells were grown overnight at 37 °C in TSB + 1% glucose with no C11 (CT), 2, 4, or 8 μg/mL C11. Planes at equal distances (0.3 μm) along the Z-axis of the biofilm were imaged by CLSM. These 2D images were the maximum projection of the planes. Scale bar represents 15 μm. (C) Analysis of biofilm properties by COMSTAT 2. Measures of the total biomass of the biofilms in the presence of increasing concentrations of C11. Data are the mean ± SD of the results from three independent replicates. Images are representative of at least three different experiments. *p < 0.05, ***p < 0.001 (one-way ANOVA test).

C11 Combination with Currently Used Antibiotics

Antibiotic therapies frequently require the combination of multiple antimicrobial drugs to increase efficacy and to overcome antibiotic resistance.35 To assess the interaction of C11 with other compounds, checkerboard assays with currently used antibiotics with different mechanisms of action (Ceftazidime, Erythromycin, Linezolid, Meropenem, Rifampicin, and Tetracycline) were performed. The panel was combined with C11 and tested against S. aureus ATCC25923. While the combination of C11 with Erythromycin, Linezolid, and Tetracycline had an additive effect, the combination of C11 with Ceftazidime, Meropenem, and Rifampicin significantly increased their efficacy, showing a synergistic effect (Table S2).

The derivative C11 was also tested against a panel of clinical isolates obtained from cystic fibrosis patients showing relevant activity against all strains, with MIC values ranging from 2 to 4 μg/mL for the vast majority of them (Tables S3 and S4).

Since some of the tested strains were MRSA, showing resistance to Meropenem (Table S4), the effect of the Meropenem/C11 combination was assayed against these strains. Results showed that the combination had a strong synergistic effect against the most resistant strains to Meropenem (BG9 and BG10, Table S5 and Figure S7A,B). These data suggest that C11 has a synergistic effect with β-lactam, as already described for other FtsZ inhibitors, such as PC190723.22,23 Moreover, a synergistic effect with Rifampicin was previously described in the case of compounds that induced FtsZ depletion and resulted to be useful to eradicate persistent cells in a chronic biofilm infection.36

C11 Cytotoxicity Studies

FtsZ inhibitors might exert a certain toxicity due to their potential inhibition of human tubulin. C11 cytotoxicity was assayed on mammalian tubulin in vitro and against different human cellular lines.37 Compound C11 decreased tubulin polymerization of 20% at the highest tested concentration (25 μM, 9.6 μg/mL) (Table S6). C11 cytotoxicity was evaluated by an MTT assay on human A549 pulmonary epithelial cells. In the presence of 25 μM of C11, A549 cells showed a viability higher than 90% after 6 h of incubation and the percentage of live cells remained higher than 70% after 24 h (Figure 6). C11 cytotoxicity was also assayed using CFBEo– cells, mimicking cystic fibrosis lung tissue. In this cell line, with 12.5 μM of compound (4.8 μg/mL) the percentage of live cells was higher than 90% after 6 h of incubation (Figure S8), while in the presence of 25 μM of C11 the viability decreased to 65% after 6 h of incubation and to 25% after 24 h (Figure S8).

Figure 6.

Figure 6

Open in a new tab

Cytotoxicity of C11 on A549 using MTT assay. Different C11 concentrations (3.125–25 μM) and different times of incubation (3, 6, or 24 h) were assayed. Data are presented as mean value ±SD calculated on triplicate experiments. *p < 0.05, **p < 0.01 (unpaired t-test).

Efficacy of Compound C11 in a G. mellonella Infection Model

A proof-of-concept of the in vivo potential of C11 in eradicating S. aureus infections was given using the well-assessed G. mellonella infection model. The larvae were infected with S. aureus ATCC25923 and after 2 h treated with 40 mg/kg (4-fold the MIC) of C11. Three different groups were used as control: not infected and not treated (CT), injected with saline solution and treated with 40 mg/kg of C11, or injected with saline solution and treated with 8% of DMSO. First, results highlighted that C11 was not toxic for G. mellonella since the moths not infected but treated with C11 did not show a significant decrease in their survival rate (Figure 7). After infection with S. aureus ATCC25923, in the group treated with DMSO, 80% of the larvae died after 6 days. Larvae infected and treated with C11 (40 mg/kg) had a survival rate of 77% (vs 46%) 2 days postinfection. Three days postinfection, the survival rate was 58% (vs 32%), and after 6 days, the survival rate was still significantly higher than the mock-treated group (Figure 7).

Figure 7.

Figure 7

Open in a new tab

Kaplan–Meier survival curve of G. mellonella larvae infected with S. aureus ATCC25923 and treated with 8% DMSO (violet) or C11 (yellow). As a control, larvae were treated with saline solution and C11, saline solution and 8% DMSO (red), or untreated (black). The experiment was performed three times. Statistically significant differences are indicated (Significance level for each time point was evaluated with Fisher’s test: 48 h p < 0.001, 72 h p < 0.01, and 144 h p < 0.05).

C11 Activity against Other ESKAPE Pathogens

The intriguing results prompted us to further investigate the activity of C11 against a panel of Gram-negative bacteria. The compound was tested against P. aeruginosa PAO1, K. pneumoniae ATCC13883, and A. baumannii ATCC19606. Unfortunately, the measured MIC values were ≥128 μg/mL for all of the tested strains. Consequently, to determine whether C11 can be a substrate of efflux pumps,38 the MIC was evaluated in the presence of different concentrations of the efflux pump inhibitor PaβN.39 Indeed, the presence of PAβN conferred to compound C11 excellent biological activity, with MIC values of 1–16 μg/mL (Table S7).

Since FtsZSa and the FtsZ of P. aeruginosa (FtsZPa) share 47% sequence identity, to better understand the results highlighted by the MIC, FtsZPa was expressed, purified, and tested for its activities in the presence of C11. Results showed that, as already observed for FtsZSa, C11 did not interfere with the GTPase activity of FtsZPa, while it can prevent FtsZPa polymerization in a concentration-dependent manner with an IC50 of 34 μM (Figure S9A,B).

Overall, these results strongly suggest that, although C11 may be also active against the FtsZ protein of Gram-negative bacteria, it is extruded out of the cell by efflux pump before it can reach its target.

Binding Mode Analyses

The proposed docking binding mode of C11 was inspected with the aim of highlighting the key interactions with the target. Overall, the binding with FtsZ was observed to rely largely on hydrophobic contacts: the terminal benzyl ring was involved in interactions with Ile197, Met218, Met226, and Ile311; the oxazole ring had contacts with Leu200, Leu261, and Thr309; and the benzothiophene dioxo ring had hydrophobic interactions with Val203, Leu200, Leu209, and Val297. Polar contacts were also observed involving the sulfone moiety, namely, with the calcium cation and with Leu209 backbone through a H-bond (Figure 8).

Figure 8.

Figure 8

Open in a new tab

Proposed binding mode of C11. The enzyme is reported as blue cartoon, C11 is depicted as gray sticks. The residues involved in interaction are reported as white lines, polar interactions are reported as yellow dotted lines.

SAR Study

A series of commercially available analogues of C11 was purchased and tested to draw a SAR. Their ability to inhibit polymerization (Figures S10A,B and S11A–E) and S. aureus growth (Table 1) was evaluated. Also, the IC50 values against FtsZSa polymerization were determined for the most promising compounds, i.e., C11.2, C11.5, C11.7, and C11.8. Compound C11.1 was selected as a control according to the mild activity on FtsZSa polymerization (Table 1 and Figure S10AB).

Table 1. C11 Derivatives and Their Activitiesa.

graphic file with name id4c01045_0009.jpg

Open in a new tab
a

Inhibition of FtsZSa polymerization; nd, not determined.

First, it was observed that the methylene bridge between the terminal phenyl ring and the 5-member ring heterocycle could be modified without affecting the binding to the enzyme. Indeed, planar and shortened derivatives without this bridge atom (C11.2, C11.5, C11.7, and C11.8) showed lower IC50 values than the lead compound C11, and derivative C11.2 had the lowest IC50 value of 23.73 μM (Table 1, Figures S10A,B and S11A–E). Another interesting observation was that the sulfur atom bridging the two aromatic rings could be changed to nitrogen with marginal effect on the inhibition of the polymerization (compare C11 and C11.5). Also the introduction of a nitrogen atom in the thiophane dioxo ring did not affect the IC50. However, none of the assayed derivatives had MIC values lower than those of the lead compound C11, despite the improved inhibition of FtsZ polymerization. Only derivatives C11.2 and C11.7 retained the ability to inhibit S. aureus growth in the same range of concentrations of the lead compound.

Lastly, the introduction of a short alkyl ramification at the thiazole ring (compare derivatives C11.5 and C11.7) improved the biological activity. Indeed, compound C11.7 had a slightly better inhibition of the FtsZ polymerization than C11.5 (IC50 29.39 μM and IC50 34.61 μM, respectively) but a marked improvement of the antibacterial activity (MIC 8 and ≥ 128 μg/mL). This result might be related to an increased membrane permeability of the compound.40

Conclusions

The lack of new solutions against the spread of antimicrobial resistance prompts the research toward different strategies to identify new active molecules, with the exploration of new and essential pathways being a valuable option for this aim. In this work, a structure-based VS on the essential cell division protein FtsZ led to the identification of derivative C11, with a notable antimicrobial activity against S. aureus, cystic fibrosis S. aureus clinical isolates, and MRSA strains. C11 mechanism of action is based on impairing FtsZ polymerization. Furthermore, this molecule has very low toxicity. The efficacy of C11 was further studied in the G. mellonella infection model, confirming that it can increase the survival of larvae infected with S. aureus. A series of C11 analogues were assayed to outline a SAR and together with a docking experiment, essential interactions with the enzyme were highlighted. Taken together, the reported results indicate that C11 is a promising drug candidate with great potential to be further structurally optimized in order to improve its efficacy and to broaden the spectrum of action.

Methods

All details of the molecular modeling, chemistry, and biological assays are reported in the Supporting Information.

Acknowledgments

We gratefully acknowledge the service provided by the Cryo-Electron Microscopy Laboratory (Andrea Alfieri), Optical Microscopy Laboratory (Amanda Oldani and Patrizia Vaghi) (PASS-BioMed Imaging - Centro Grandi Strumenti - Università di Pavia) for sample preparation and image acquisitions. We thank Professor Suzanne Walker for kindly providing the S. aureus strain TD276 (HG003 pDT72). A.C. acknowledges the CINECA award under the ISCRA initiative (ISCRA C HP10C3DQ2H) for the availability of high-performance computing resources and support. P.S.` and A.P. thank Project ECS 0000024 Rome Technopole—CUP B83C22002820006, NRP Mission 4 Component 2 Investment 1.5, funded by the European Union—NextGenerationEU″.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.4c01045.

  • FtsZ domains and binding sites; proposed pharmacophore model; list and inhibitory activity of the compounds selected from the virtual screening; dose-response curves of residual FtsZSa GTPase activity in the presence of C3, C8, or C11; analysis of biofilm inhibition by COMSTAT2; C11 effect on biofilm eradication; analysis of biofilm eradication by COMSTAT2; MIC values of different antibiotics and C11, their combination, and FICI values; bacterial strains used in this study; MIC of C11 for cystic fibrosis clinical isolates; MIC values of Meropenem and C11, their combination and the FICI values, against different MRSA clinical isolates; tubulin polymerization assay; checkerboard assay of BG9 and BG10 strains; cytotoxicity of C11 to CFBE41o- using MTT assay; MIC of P. aeruginosa, K. pneumoniae, and A. baumannii against C11 in combination with PaBN; FtsZPa polymerization assay in the presence of the C11 derivatives; FtsZSa sedimentation assay densiometric analyses of the C11 derivatives; dose-response curves of FtsZSa residual polymerization activity in the presence of C11 analogues; and 1H NMR spectrum and 13C NMR spectrum of compound C11 (PDF)

Author Contributions

# P.S. and V.C.S. contributed equally to the work. A. Coluccia, S. Buroni: Writing, Conceptualization, Funding acquisition. P. Sciò and M. Bufano: Software, Investigation, Data curation; A. Parisi: Formal analysis, Data curation. V.C. Scoffone: Writing, Conceptualization, Investigation. M. Caneva, G. Trespidi, S. Irudal, G. Barbieri: Validation, Methodology, Formal analysis, Data curation. L. Cariani, B.S. Orena, V. Daccò, F. Imperi: Formal analysis, Data curation.

This work was supported by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN) 2020, project code 20208LLXEJ_005 and by Italian Cystic Fibrosis Research Foundation grant: FFC#6/2023 (adopted by Delegazione FFC Ricerca di Campiglione Fenile; Delegazione FFC Ricerca Valle Scrivia Alessandria; Delegazione FFC Ricerca di Vigevano; Delegazione FFC Ricerca di Boschi Sant’Anna Minerbe “Alla fine esce sempre il sole”; Delegazione FFC Ricerca di Lecco Valsassina; Delegazione FFC Ricerca di Padova).

The authors declare no competing financial interest.

Supplementary Material

id4c01045_si_001.pdf (1.4MB, pdf)

References

  1. Naghavi M.; Mestrovic T.; Gray A.; Gershberg Hayoon A.; Swetschinski L. R.; Robles Aguilar G.; Davis Weaver N.; Ikuta K. S.; Chung E.; Wool E. E.; et al. Global burden associated with 85 pathogens in 2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Infect. Dis. 2024, 24 (8), 868–895. 10.1016/S1473-3099(24)00158-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ruggieri F.; Compagne N.; Antraygues K.; Eveque M.; Flipo M.; Willand N. Antibiotics with novel mode of action as new weapons to fight antimicrobial resistance. Eur. J. Med. Chem. 2023, 256 (256), 115413. 10.1016/j.ejmech.2023.115413. [DOI] [PubMed] [Google Scholar]
  3. Beyer P.; Paulin S. The antibacterial research and development pipeline needs urgent solutions. ACS Infect Dis 2020, 6 (6), 1289–1291. 10.1021/acsinfecdis.0c00044. [DOI] [Google Scholar]
  4. Ribeiro C. M. P.; Higgs M. G.; Muhlebach M. S.; Wolfgang M. C.; Borgatti M.; Lampronti I.; Cabrini G. Revisiting Host-Pathogen Interactions in Cystic Fibrosis Lungs in the Era of CFTR Modulators. Int. J. Mol. Sci. 2023, 24 (5), 5010. 10.3390/ijms24055010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Du S.; Lutkenhaus J. Assembly and activation of the Escherichia coli divisome. Mol. Microbiol. 2017, 105 (2), 177–187. 10.1111/mmi.13696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Casiraghi A.; Suigo L.; Valoti E.; Straniero V. Targeting Bacterial Cell Division: A Binding Site-Centered Approach to the Most Promising Inhibitors of the Essential Protein FtsZ. Antibiotics (Basel). 2020, 9 (2), 69. 10.3390/antibiotics9020069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan F. Y.; Sun N.; Leung Y. C.; Wong K. Y. Antimicrobial activity of a quinuclidine-based FtsZ inhibitor and its synergistic potential with β-lactam antibiotics. J. Antibiot. (Tokyo) 2015, 68 (4), 253–258. 10.1038/ja.2014.140. [DOI] [PubMed] [Google Scholar]
  8. Nogales E.; Downing K. H.; Amos L. A.; Löwe J. Tubulin and FtsZ form a distinct family of GTPases. Nat. Struct. Biol. 1998, 5 (6), 451–458. 10.1038/nsb0698-451. [DOI] [PubMed] [Google Scholar]
  9. Han H.; Wang Z.; Li T.; Teng D.; Mao R.; Hao Y.; Yang N.; Wang X.; Wang J. Recent progress of bacterial FtsZ inhibitors with a focus on peptides. FEBS J. 2021, 288 (4), 1091–1106. 10.1111/febs.15489. [DOI] [PubMed] [Google Scholar]
  10. Carro L. Recent Progress in the Development of Small-Molecule FtsZ. Inhibitors as Chemical Tools for the Development of Novel. Antibiotics 2019, 8 (4), 217. 10.3390/antibiotics8040217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Araújo-Bazán L.; Ruiz-Avila L. B.; Andreu D.; Huecas S.; Andreu J. M. Cytological Profile of Antibacterial FtsZ Inhibitors and Synthetic Peptide MciZ. Front. Microbiol. 2016, 3 (7), 1558. 10.3389/fmicb.2016.01558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Beuria T. K.; Santra M. K.; Panda D. Sanguinarine blocks cytokinesis in bacteria by inhibiting FtsZ. assembly and bundling. Biochemistry 2005, 44 (50), 16584–16593. 10.1021/bi050767+. [DOI] [PubMed] [Google Scholar]
  13. Croaker A.; King G. J.; Pyne J. H.; Anoopkumar-Dukie S.; Liu L. Sanguinaria canadensis: Traditional Medicine, Phytochemical Composition, Biological Activities and Current Uses. Int. J. Mol. Sci. 2016, 17 (9), 1414. 10.3390/ijms17091414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wolff J.; Knipling L. Antimicrotubule properties of benzophenanthridine alkaloids. Biochemistry 1993, 32 (48), 13334–13339. 10.1021/bi00211a047. [DOI] [PubMed] [Google Scholar]
  15. Kunal K.; Tiwari R.; Dhaked H. P. S.; Surolia A.; Panda D. Mechanistic insight into the effect of BT-benzo-29 on the Z-ring in Bacillus subtilis. IUBMB Life 2020, 72 (5), 978–990. 10.1002/iub.2234. [DOI] [PubMed] [Google Scholar]
  16. Ruiz-Avila L. B.; Huecas S.; Artola M.; Vergoñós A.; Ramírez-Aportela E.; Cercenado E.; Barasoain I.; Vázquez-Villa H.; Martín-Fontecha M.; Chacón P.; López-Rodríguez M. L.; Andreu J. M. Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ. ACS Chem. Biol. 2013, 8 (9), 2072–2083. 10.1021/cb400208z. [DOI] [PubMed] [Google Scholar]
  17. Artola M.; Ruíz-Avila L. B.; Ramírez-Aportela E.; Martínez R. F.; Araujo-Bazán L.; Vázquez-Villa H.; Martín-Fontecha M.; Oliva M. A.; Martín-Galiano A. J.; Chacón P.; López-Rodríguez M. L.; Andreu J. M.; Huecas S. The structural assembly switch of cell division protein FtsZ probed with fluorescent allosteric inhibitors. Chem. Sci. 2017, 8 (2), 1525–1534. 10.1039/C6SC03792E. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kumar K.; Awasthi D.; Lee S. Y.; Zanardi I.; Ruzsicska B.; Knudson S.; Tonge P. J.; Slayden R. A.; Ojima I. Novel trisubstituted benzimidazoles, targeting Mtb FtsZ, as a new class of antitubercular agents. J. Med. Chem. 2011, 54 (1), 374–381. 10.1021/jm1012006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haydon D. J.; Stokes N. R.; Ure R.; Galbraith G.; Bennett J. M.; Brown D. R.; Baker P. J.; Barynin V. V.; Rice D. W.; Sedelnikova S. E.; Heal J. R.; Sheridan J. M.; Aiwale S. T.; Chauhan P. K.; Srivastava A.; Taneja A.; Collins I.; Errington J.; Czaplewski L. G. An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 2008, 321 (5896), 1673–1675. 10.1126/science.1159961. [DOI] [PubMed] [Google Scholar]
  20. Andreu J. M.; Schaffner-Barbero C.; Huecas S.; Alonso D.; Lopez-Rodriguez M. L.; Ruiz-Avila L. B.; Núñez-Ramírez R.; Llorca O.; Martín-Galiano A. J. The antibacterial cell division inhibitor PC190723 is an FtsZ polymer-stabilizing agent that induces filament assembly and condensation. J. Biol. Chem. 2010, 285 (19), 14239–14246. 10.1074/jbc.M109.094722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Elsen N. L.; Lu J.; Parthasarathy G.; Reid J. C.; Sharma S.; Soisson S. M.; Lumb K. J. Mechanism of action of the cell-division inhibitor PC190723: modulation of FtsZ assembly cooperativity. J. Am. Chem. Soc. 2012, 134 (30), 12342–12345. 10.1021/ja303564a. [DOI] [PubMed] [Google Scholar]
  22. Lepak A. J.; Parhi A.; Madison M.; Marchillo K.; VanHecker J.; Andes D. R. Vivo Pharmacodynamic Evaluation of an FtsZ Inhibitor, TXA-709, and Its Active Metabolite, TXA-707, in a Murine Neutropenic Thigh Infection Model. Antimicrob. Agents Chemother. 2015, 59 (10), 6568–6574. 10.1128/AAC.01464-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tan C. M.; Therien A. G.; Lu J.; Lee S. H.; Caron A.; Gill C. J.; Lebeau-Jacob C.; Benton-Perdomo L.; Monteiro J. M.; Pereira P. M.; Elsen N. L.; Wu J.; Deschamps K.; Petcu M.; Wong S.; Daigneault E.; Kramer S.; Liang L.; Maxwell E.; Claveau D.; Vaillancourt J.; Skorey K.; Tam J.; Wang H.; Meredith T. C.; Sillaots S.; Wang-Jarantow L.; Ramtohul Y.; Langlois E.; Landry F.; Reid J. C.; Parthasarathy G.; Sharma S.; Baryshnikova A.; Lumb K. J.; Pinho M. G.; Soisson S. M.; Roemer T. Restoring methicillin-resistant Staphylococcus aureus susceptibility to β-lactam antibiotics. Sci. Transl. Med. 2012, 4 (126), 126ra35. 10.1126/scitranslmed.3003592. [DOI] [PubMed] [Google Scholar]
  24. Lipinski C. A.; Lombardo F.; Dominy B. W.; Feeney P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv Rev. 2001, 46 (1–3), 3–26. 10.1016/S0169-409X(00)00129-0. [DOI] [PubMed] [Google Scholar]
  25. Bruns R. F.; Watson I. A. Rules for identifying potentially reactive or promiscuous compounds. J. Med. Chem. 2012, 55 (22), 9763–9772. 10.1021/jm301008n. [DOI] [PubMed] [Google Scholar]
  26. Yang Y.; Yao K.; Repasky M. P.; Leswing K.; Abel R.; Shoichet B. K.; Jerome S. V. Efficient Exploration of Chemical Space with Docking and Deep Learning. J. Chem. Theory Comput. 2021, 17 (11), 7106–7119. 10.1021/acs.jctc.1c00810. [DOI] [PubMed] [Google Scholar]
  27. Dixon S. L.; Smondyrev A. M.; Knoll E. H.; Rao S. N.; Shaw D. E.; Friesner R. A. PHASE: a new engine for pharmacophore perception, 3D QSAR model development, and 3D database screening: 1. Methodology and preliminary results. J. Comput. Aided Mol. Des 2006, 20 (10–11), 647–671. 10.1007/s10822-006-9087-6. [DOI] [PubMed] [Google Scholar]
  28. Fischer A.; Smieško M.; Sellner M.; Lill M. A. Decision Making in Structure-Based Drug Discovery: Visual Inspection of Docking Results. J. Med. Chem. 2021, 64 (5), 2489–2500. 10.1021/acs.jmedchem.0c02227. [DOI] [PubMed] [Google Scholar]
  29. Hogan A. M.; Scoffone V. C.; Makarov V.; Gislason A. S.; Tesfu H.; Stietz M. S.; Brassinga A. K. C.; Domaratzki M.; Li X.; Azzalin A.; Biggiogera M.; Riabova O.; Monakhova N.; Chiarelli L. R.; Riccardi G.; Buroni S.; Cardona S. T. Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ. Antimicrob. Agents Chemother. 2018, 62, 012311-18. 10.1128/AAC.01231-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kaul M.; Mark L.; Zhang Y.; Parhi A. K.; Lavoie E. J.; Pilch D. S. An FtsZ-targeting prodrug with oral antistaphylococcal efficacy in vivo. Antimicrob. Agents Chemother. 2013, 57 (12), 5860–5869. 10.1128/AAC.01016-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kaul M.; Zhang Y.; Parhi A. K.; Lavoie E. J.; Pilch D. S. Inhibition of RND-type efflux pumps confers the FtsZ-directed prodrug TXY436 with activity against Gram-negative bacteria. Biochem. Pharmacol. 2014, 89 (3), 321–328. 10.1016/j.bcp.2014.03.002. [DOI] [PubMed] [Google Scholar]
  32. Monteiro J. M.; Pereira A. R.; Reichmann N. T.; Saraiva B. M.; Fernandes P. B.; Veiga H.; Tavares A. C.; Santos M.; Ferreira M. T.; Macário V.; VanNieuwenhze M. S.; Filipe S. R.; Pinho M. G. Peptidoglycan synthesis drives an FtsZ-treadmilling-independent step of cytokinesis. Nature 2018, 554 (7693), 528–532. 10.1038/nature25506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Do T.; Schaefer K.; Santiago A. G.; Coe K. A.; Fernandes P. B.; Kahne D.; Pinho M. G.; Walker S. Staphylococcus aureus cell growth and division are regulated by an amidase that trims peptides from uncrosslinked peptidoglycan. Nat. Microbiol 2020, 5 (2), 291–303. 10.1038/s41564-019-0632-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jean-Pierre V.; Boudet A.; Sorlin P.; Menetrey Q.; Chiron R.; Lavigne J. P.; Marchandin H. Biofilm Formation by Staphylococcus aureus in the Specific Context of Cystic Fibrosis. Int. J. Mol. Sci. 2023, 24 (1), 597. 10.3390/ijms24010597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Cacace E.; Kim V.; Varik V.; Knopp M.; Tietgen M.; Brauer-Nikonow A.; Inecik K.; Mateus A.; Milanese A.; Mårli M. T.; Mitosch K.; Selkrig J.; Brochado A. R.; Kuipers O. P.; Kjos M.; Zeller G.; Savitski M. M.; Göttig S.; Huber W.; Typas A. Systematic analysis of drug combinations against Gram-positive bacteria. Nat. Microbiol. 2023, 8 (11), 2196–2212. 10.1038/s41564-023-01486-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Conlon B. P.; Nakayasu E. S.; Fleck L. E.; LaFleur M. D.; Isabella V. M.; Coleman K.; Leonard S. N.; Smith R. D.; Adkins J. N.; Lewis K. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 2013, 503 (7476), 365–370. 10.1038/nature12790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pradhan P.; Margolin W.; Beuria T. K. Targeting the Achilles Heel of FtsZ: The Interdomain Cleft. Front. Microbiol. 2021, 12 (12), 732796. 10.3389/fmicb.2021.732796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nikaido H.; Takatsuka Y. Mechanisms of RND multidrug efflux pumps. Biochim. Biophys. Acta 2009, 1794 (5), 769–781. 10.1016/j.bbapap.2008.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pagès J. M.; Masi M.; Barbe J. Inhibitors of efflux pumps in Gram-negative bacteria. Trends Mol. Med. 2005, 11 (8), 382–389. 10.1016/j.molmed.2005.06.006. [DOI] [PubMed] [Google Scholar]
  40. Richter M. F.; Drown B. S.; Riley A. P.; Garcia A.; Shirai T.; Svec R. L.; Hergenrother P. J. Predictive compound accumulation rules yield a broad-spectrum antibiotic. Nature 2017, 545 (7654), 299–304. 10.1038/nature22308. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

id4c01045_si_001.pdf (1.4MB, pdf)

Articles from ACS Infectious Diseases are provided here courtesy of American Chemical Society

RESOURCES