A dual-function probe for inhibition and rapid detection of Mycobacterium tuberculosis

Abstract

We introduce a dual-purpose solvatochromic naphthalimide-benzothiazinone (BTZ) conjugate with theranostic potential that enables specific and rapid detection of Mycobacterium tuberculosis by targeting the DprE1 enzyme, while also exhibiting antimicrobial activity. The photoinduced electron transfer (PeT)-based fluorophore incorporates into the mycobacterial cell wall, producing detectable fluorescence within 15 minutes and thus, highlighting its suitability for rapid, point-of-care TB detection in low-resource settings.

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Tuberculosis (TB) remains a grave concern for global health, causing an estimated 10.8 million cases and 1.25 million deaths worldwide in 2023.¹ Early detection of the causative agent, Mycobacterium tuberculosis, and timely intervention can help prevent tuberculosis. To date, microbial cell culture remains the gold standard for diagnosing Mtb infection.² However, this technique is time-consuming and labor-intensive because of the exceptionally slow growth rate of the Mtb and the requirement for specialized facilities. While rapid diagnostic tests exist, they come with significant drawbacks. PCR-based diagnostics, for instance, are costly and require trained technicians for operation. Microscopy-based methods, such as sputum smear tests with fluorescent Auramine-O or colored Ziehl–Neelsen stain, are low-cost with a shorter time frame but have a low sensitivity of 50–60% in confirmed pulmonary TB cases.³ Both these tests are based on the propensity of the unique mycobacterial membrane to bind and retain hydrophobic dyes.^4,5 However, the procedure involves multiple wash steps to minimize nonspecific background signals. The Ziehl–Neelsen (ZN) test requires a stringent counterstaining process to make the stained Mtb cells visible under a microscope. Fluorogenic probes that require minimal processing are more suitable for the detection of TB. Over the last decade, there has been some progress in imaging techniques that allow rapid detection of Mtb. Recently, a microfluidic system was employed to detect Mtb using a dual enzyme-targeting sensitive probe, where the β-lactamase (BlaC) enzyme activates the fluorophore.⁶ Kamariza et al. describe two dye-trehalose conjugates that allow the detection of Mtb.^7,8 Hence, continuous efforts are necessary to replace the century-old staining technique with robust, specific, cost-effective point-of-care detection probes against Mtb.

The present study introduces an innovative conjugated naphthalimide-based solvatochromic fluorophore, NA-H, engineered for the rapid, real-time detection of Mtb within minutes. This probe operates as a “first-in-class” dual-action molecule, combining diagnostic capability with potent bacteriocidal activity to simultaneously detect and inhibit Mtb. By merging diagnostics and therapeutics into a supramolecular sensor, our design addresses the unmet need for point-of-care tuberculosis detection and opens an opportunity for novel theranostic strategies for tackling antimicrobial resistance. The NA-H comprises three components: the fluorophore, the linker, and the benzothiazinone (BTZ) group. Naphthalimide-based fluorophores provide excellent characteristics for diagnostics, such as large Stokes shift, high thermal and oxidation stability, high electron affinity, and versatile photophysical properties.^9–12 The BTZs are known to be promising anti-TB drugs, among which BTZ043 and PBTZ169 exhibited potent antimicrobial activity and advanced to clinical trials. They target DprE1, a crucial periplasmic epimerase enzyme conserved among actinobacteria and essential for cell wall biosynthesis in Mtb.¹³ DprE1, in complex with DprE2 (decaprenylphosphoryl-D-2-keto erythro pentose reductase), catalyzes the epimerization of decaprenylphosphoryl-β-D-ribose (DPR) into its arabinose isomer, decaprenyl-phospho-β-D-arabinose (DPA)—a key precursor in the synthesis of arabinogalactan, a significant component of the mycobacterial cell wall. BTZs are suicide inhibitors of DprE1. The enzyme reduces its nitro group to a nitroso intermediate, forming a semi-mercaptal covalent adduct with the thiol side chain of Cys387 within the DprE1 active site. This irreversible interaction inhibits the enzymatic activity and disrupts cell wall synthesis. The nitro group is essential for this mode of action, as substitutions eliminate antimicrobial efficacy.^14,15

We sought to modify the 4-amino position on the naphthalimide scaffold, as it enhances fluorescence emission through intramolecular charge transfer (ICT), resulting in an increased quantum yield.¹⁶ Next, we explored variations in linker length with consideration for optimal hydrophobicity, flexibility, and the consequent membrane permeability. We found C7 to be the most suitable linker, offering increased fluorescence intensity and improved prospects for membrane permeability. The BTZ scaffold was synthesized in three steps as previously described^17–19 with a few modifications. The fluorophore was conjugated with BTZ in a one-step nucleophilic aromatic substitution reaction in ethanol under reflux (Fig. 1a). The detailed synthetic route, procedures, and characterization are described in the ESI.†

Fig. 1 (a) Synthetic scheme for NA-H. 1. (i) H₂SO₄, HNO₃, 90 °C, (ii) SOCl₂, DMF, 80 °C; NH₃, CH₃CN, 0 °C, (iii) NaOH, CS₂, CH₃I, 0 °C. 2. 1,7-Diaminoheptane, 80 °C. 3. EtOH, reflux. (b) Fluorescence polarization assay using NA-H for binding studies with the DprE1 protein, (c) fluorescence lifetime experiment to assess the local environment of the protein upon binding of the probe, and (d) DprE1 enzyme inhibition by NA-H.

Owing to the covalent interaction between the BTZ scaffold and the catalytic cysteine residue (Cys387) of the DprE1 enzyme, the appended fluorophore undergoes activation via a photoinduced electron transfer (PeT) ‘on’ mechanism upon specific binding within the polar mycobacterial cellular environment. The probe achieves rapid, wash-free visualization of Mtb within a few minutes, offering critical advantages for point-of-care diagnostics in resource-limited settings. Beyond its diagnostic utility, the probe suppresses Mtb growth by targeting and inhibiting the essential enzyme DprE1, thus positioning it as a useful theranostic agent with dual diagnostic and therapeutic functionality.

To elucidate its solvatochromic behaviour, we checked the fluorescence spectra of the NA-H in dioxane–water mixtures. Upon excitation at 405 nm, the emission intensity increased markedly with higher dioxane ratios. This increased intensity was marked by a bathochromic shift (500–550 nm) with the rise in solvent polarity, confirming the PeT-based environmental response of fluorescence modulation (Fig. S1, ESI†).

Structural and biochemical analyses have shown that the BTZ core ring is positioned within the active site of the DprE1 enzyme, where it forms a covalent bond with the Cys387 residue.¹⁵ Meanwhile, the substituent functional groups on the BTZ molecule engage in limited interactions with other residues in DprE1. We hypothesized that the presence of the linker and fluorophore would ensure proper orientation and binding within the target site. We aimed to explore the potential of the dye to bind to the DprE1 enzyme, assessing its ability to interfere with its activity. To evaluate the DprE1 binding affinity of the probe NA-H, the fluorescence polarization (FP) assay²⁰ revealed a concentration-dependent increase in polarized emission, bolstering a stable protein–ligand complexation (Fig. 1b). Complementarily, time-correlated single photon counting (TCSPC) demonstrated a drastic reduction in protein fluorescence lifetime (2.7 ns to 186.9 ps) upon ligand binding (Table S1, ESI†). This reflects changes in the local environment of tryptophan residues of DprE1. A shorter lifetime correlates with increased solvent exposure or the changed polarity around the fluorophore, suggesting structural perturbations in DprE1 upon ligand binding. The observed lifetime reduction and altered lifetime components suggest microenvironmental heterogeneity, which confirms stable complex formation and altered protein conformation. The larger magnitude of lifetime shortening also indicates large-scale conformational shifts and thus provides direct experimental evidence of induced fit binding mechanisms.²¹ The lifetime data complements FP results by offering a time-resolved quantitative metric of the induced fit binding mechanism of the probe and DprE1. Thus, by integrating TCSPC, we were able to prove the structural and temporal consequences of probe DprE1 binding, establishing a mechanism-resolved binding interaction of the probe to DprE1 (Fig. 1c). Next, we investigated whether NA-H disrupts the DprE1 enzymatic activity. The DprE1-enzyme assay was performed as reported previously.¹⁵ The NA-H inhibited DprE1 with a modest IC₅₀ of 0.8 μM (Fig. 1d).

We initially evaluated the labeling potential and conditions of the NA-H in another member of the Mycobacterium genus, Mycobacterium smegmatis (Msmeg), a model organism for Mycobacterium tuberculosis because of its nonpathogenic nature, fast-growth, and genetic similarities. Briefly, the cells were cultured until they reached an optical density (OD₆₀₀) of 0.5, then incubated with the probe for 1 hour at 37 °C to allow for labeling, washed, and evaluated using confocal microscopy and flow cytometry with 488/525 nm as excitation/emission filter sets. We observed that the probe labels Msmeg cells successfully (Fig. 2a). To ensure applicability in the field, a probe labeling procedure should adhere to a straightforward protocol to ensure ease of execution. We aimed to assess whether a wash step was necessary for Mycobacterium labeling by evaluating the background signal level during the procedure. Interestingly, we observed minimal background signal while skipping the wash step, rendering additional washing steps redundant. Interestingly, the arabinogalactan, peptidoglycan, and mycolic acid layer are synthesized at the mycobacterial cell poles. As expected, the poles of the mycobacterial cells exhibited enhanced fluorescence intensity (Fig. 2a).

Fig. 2 (a) Confocal microscopy analysis of Msmeg (Ms) with and without washing, Mtb, S. aureus (Sa), and E. coli (Ec), (b) flow cytometry analysis of Msmeg, S. aureus, and E. coli, and (c) flow cytometry analysis of Mtb labelled with NA-H after incubation for 15 and 60 minutes.

Next, we aimed to evaluate how rapidly the dye-conjugate NA-H can label the Mycobacterium tuberculosis H37Rv cells. For this purpose, Mtb cells were grown to OD₆₀₀ = 0.5, and incubated with 0.1 mM of dye-conjugate for 15 and 60 minutes at 37 °C. Following this, the cells were washed three times, fixed, and analyzed using flow cytometry and confocal microscopy. Remarkably, fluorescent labeling was evident as early as 15 minutes and persisted at 60 minutes, as observed by confocal microscopy (Fig. 2a). Consistent with this, flow cytometry analysis revealed a ∼24-fold increase in fluorescence at 15 minutes and a ∼25-fold increase at 60 minutes, with no significant difference between the two time points (Fig. 2c). Lastly, to evaluate the specificity of NA-H toward the Actinobacteria suborder, we labelled Gram-positive (S. aureus) and Gram-negative bacteria (E. coli), and performed FACS and microscopy analyses. Both bacteria exhibited minimal labeling, characterized by an approximate two-fold increase in fluorescence intensity (Fig. 2a and b).

Finally, as the probe inhibited DprE1 enzyme function, it was pertinent to evaluate the molecule for possible antimicrobial potency against Mycobacterium tuberculosis H37Rv. The anti-TB potency was evaluated using the broth microdilution assay as per the EUCAST guidelines. The MIC of the NA-H dye-conjugate was observed to be 6 μg ml⁻¹. This dual functionality-enzyme inhibition and the bacteriocidal action positions the NA-H probe as a promising theranostic agent capable of bridging diagnosis and therapy in TB management.

Tuberculosis persists as a global health crisis, exacerbated by the absence of rapid, affordable diagnostics deployable in disease-heavy endemic zones. To address this unmet medical need, we present a solvatochromic naphthalimide-based fluorophore conjugate that enables Mtb detection in 15 min with no additional wash steps, unlike previously reported probes, which require post-labeling wash steps and longer incubation for optimal signal.^22,23 Furthermore, the probe exhibited anti-tubercular activity by targeting DprE1, the cell wall-anchored enzyme to which the conjugate binds, offering an additional notable advantage over previously reported probes that lack this therapeutic potential.^6–8 The naphthalimide-based probe is robust, highly stable at room temperature, pH independent across the physiological pH range, and cost-effective, with a straightforward one-pot synthetic route. By integrating diagnostic immediacy with therapeutic potential, this technology transcends conventional approaches and thus offers a pragmatic solution for TB in endemic regions. Therefore, besides the performance of labeling and detection, the stability, cost-effectiveness, and ease of access of the dye-conjugates will play crucial roles in the TB diagnosis setup in low-income and resource-poor environments. Given the promising performance in pure cultures, future studies will evaluate the probe in clinical sputum samples.

We thank the DST and CSIR-IMTech for the financial grants (SB/SO/BB/023/2014 and OLP0186, respectively) for funding and CSIR for the Senior Research Fellowship to S. C. The Table of Contents was created in BioRender, PARKESH, R. (2025). The M. tuberculosis (H37Rv) was kindly provided by Dr. Pawan Gupta, CSIR-Institute of Microbial Technology, Chandigarh – 160036, India.

Conflicts of interest

The findings of the study are part of the patent WO2025032611, with R. P. and S. C. are listed as inventors.

Data availability

The data supporting this article have been included as part of the paper and in the ESI† of the article.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5cc02317c

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