TZD-Based Hybrid Molecules Act as Dual Anti-Mycobacterium tuberculosis and Anti-Toxoplasma gondii Agents

Two distinct intracellular pathogens, namely Mycobacterium tuberculosis (Mtb) and Toxoplasma gondii (Tg), cause major public health problems worldwide. In addition, serious and challenging health problems of co-infections of Tg with Mtb have been recorded, especially in developing countries. Due to this fact, as well as the frequent cases of resistance to the current drugs, novel anti-infectious therapeutics, especially those with dual (anti-Tg and anti-Mtb) modes of action, are needed. To address this issue, we explored the anti-Tg potential of thiazolidinedione-based (TZD-based) hybrid molecules with proven anti-Mtb potency. Several TZD hybrids with pyridine-4-carbohydrazone (PCH) or thiosemicarbazone (TSC) structural scaffolds were more effective and more selective than sulfadiazine (SDZ) and trimethoprim (TRI). Furthermore, all of these molecules were more selective than pyrimethamine (PYR). Further studies for the most potent TZD-TSC hybrids 7, 8 and 10 and TZD-PCH hybrid molecule 2 proved that these compounds are non-cytotoxic, non-genotoxic and non-hemolytic. Moreover, they could cross the blood–brain barrier (BBB), which is a critical factor linked with ideal anti-Tg drug development. Finally, since a possible link between Tg infection and the risk of glioblastoma has recently been reported, the cytotoxic potential of TZD hybrids against human glioblastoma cells was also evaluated. TZD-PCH hybrid molecule 2 was found to be the most effective, with an IC50 of 19.36 ± 1.13 µg/mL against T98G cells.


Introduction
Two distinct intracellular pathogens, namely Mycobacterium tuberculosis (Mtb) and Toxoplasma gondii (Tg), cause major public health problems worldwide; Mtb is the human etiologic agent of tuberculosis (TB), while the zootonic parasite Tg causes serious health problems in immunocompromised people and developing fetuses. Both infections share many characteristics; they can cause acute disease, or they can be latent and asymptomatic. Latent infections represent the majority of the cases after infection with either of these two pathogens. Latency could be explained by the capacity of Mtb and Tg pathogens to survive or even replicate within macrophages or other target cells, and their capacity to shield themselves from the immune response by residing within a non-fusogenic vacuole inside the macrophage. However, in immunocompromised people, both infections manifest as acute disease [1]. As per the estimation of the World Health Organization, Mtb infection is the thirteenth leading cause of death and the second leading cause of mortality from infectious diseases after COVID-19; in 2020, a total of 10 million people fell ill with TB and Figure 1. TZD-PCH (1-6) and TZD-TSC (7)(8)(9)(10)(11)(12) hybrid molecules with promising anti-Mtb [43][44][45]. For the numbers used to identify the hybrid molecules (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), see Table 1. Table 1. Cytotoxicity (CC30), anti-Tg activity (IC50Tg), and selectivity index (SI) of TZD molecules (1-12) a .
With respect to the cytotoxic effect against the host cells, all TZD hybrids (1-12) displayed negligible cytotoxicity towards the normal human foreskin fibroblast Hs27 cell line and all of them were not toxic to the host cells at anti-parasitic concentrations. Further calculation of the SI indexes showed that these compounds are promising for their anti-Tg activity, as indicated by a marked increase in their SI values, compared to SDZ, PYR, and TRI. Among them, molecule 7 with an SI > 148.28, 2 with an SI > 118.93, 8 with an SI > 110.67, and 12 with an SI > 94.59 are the most effective. Thus, it might be concluded that TZD hybrid compounds present comparably or even better anti-Tg potency compared to those recently reported hybrid compounds with thiazolidinone [50,51], TSC [52,53] or PCH [54] structural scaffolds.

In Vivo Toxicity Tests and Hemolytic Activity and Cytotoxic Effects on Host Cells
To assess the cytotoxic effect of the most potent TZD hybrid molecules (2, 7, 8 and 10) on living organisms, the increasingly popular zebrafish model was used. Rapid development of Danio rerio embryos and the transparency of the larvae allow the assessment of drug-induced changes without the use of invasive techniques. The concentrations used for in vivo testing were selected based on the IC 50Tg values (5 µg/mL and 10 µg/mL). The results showed that in the therapeutic concentration range, the compounds 2, 7, 8 and 10 were not toxic to the zebrafish embryos and larvae within 96 h of observation. In the next step, higher concentrations were tested to determine the TZD hybrids' effect on Danio rerio viability and development. Zebrafish embryos were exposed to the concentrations of 15, 25, 50, 75 and 100 µg/mL. The results showed that the compounds 7 and 8 had the smallest effect on the larvae viability (Figure 4). At the same time, the impact of these compounds on the occurrence of deformations was noticeable at a concentration of 25 µg/mL (i.e., 2.8-4.6-times higher than IC 50Tg ) (Figures 4 and 5).
Finally, after the red blood cell treatment of molecules 2, 7, 8, and 10, no statistically significant increase in the level of free hemoglobin released to the medium was observed (Table 2), thereby indicating that the tested TZD hybrids at the concentration equal to IC 50Tg showed no specific hemolytic activity. of observation. In the next step, higher concentrations were tested to determine the TZD hybrids' effect on Danio rerio viability and development. Zebrafish embryos were exposed to the concentrations of 15, 25, 50, 75 and 100 µg/mL. The results showed that the compounds 7 and 8 had the smallest effect on the larvae viability (Figure 4). At the same time, the impact of these compounds on the occurrence of deformations was noticeable at a concentration of 25 µg/mL (i.e., 2.8-4.6-times higher than IC50Tg) (Figures 4 and 5).  Finally, after the red blood cell treatment of molecules 2, 7, 8, and 10, no statistically significant increase in the level of free hemoglobin released to the medium was observed (Table 2), thereby indicating that the tested TZD hybrids at the concentration equal to IC50Tg showed no specific hemolytic activity. Each value is expressed as the mean ± SD (n = 3). The results were designated as statistically significant (ANOVA with post-hoc Dunnett's test), when p < 0.05 (vs. untreated cells). Compounds 2, 7, 8 and 10 were tested at the concentrations that were equal to their IC 50Tg values.

Cytotoxic Effect against Human Glioblastoma T98G Cells
As a possible link between Tg infection and the risk of glioblastoma has recently been suggested [46], it was also of interest to characterize the cytotoxic potential of TZD hybrid molecules against human glioblastoma cells. For these studies, the most potent molecules (2, 7, 8 and 10) with experimentally confirmed ability to cross the blood-brain barrier ( Table 3) were selected. According to the results presented in Table 4, compound 2 was found to be active against T98G glioblastoma cells with an IC 50 of 19.36 ± 1.13 µg/mL. Although it demonstrates at least 5-fold higher activity than that of temozolomide, i.e., the anti-cancer agent routinely used in glioblastoma treatment, this concentration of 2 caused an impairment in the viability and functionality of living organisms, as shown in the zebrafish model.

Clinical Significance of the Findings
This article describes pilot studies that explored the possible dual anti-infectious mode of action (anti-Tg and anti-Mtb) of TZD-based hybrid molecules. The most potent compounds were found to be more effective and more selective for Tg and Mtb pathogens than the reference drugs. Further studies for the selected candidates proved that they are non-cytotoxic, non-genotoxic, and non-hemolytic. Moreover, they could cross the BBB, which is a critical factor linked with ideal anti-Tg drug development. General conclusions from our studies suggest that within a series of TZD-based hybrid molecules, we are able to find new starting points for developing them into effective medicines to treat co-infections of Tg with Mtb.

Chemistry
TZD-PCH (1-6) and TZD-TSC (7-12) hybrid molecules were resynthesized by previously reported procedures [43][44][45]. All commercial reactants and solvents were purchased from either Alfa Aesar (Kandel, Germany) or Tokyo Chemical Industry Co. (Tokyo, Japan) with the highest purity and were used without further purification. Melting points (Gallenkamp MPD 350.BM 3.5 apparatus; Sanyo, Japan) and NMR spectra (Bruker Avance 300 MHz instrument with DMSO-d 6 as a solvent and TMS as an internal standard) were found in accordance with the literature data.

General procedure for the synthesis of TZD-PCH (1-6) hybrid molecules
To the mixture of 0.001 mol of corresponding formylphenyl (2,4-dioxothiazolidin-5yl/ylidene)acetate and 0.001 mol of isonicotinic acid hydrazide, 8 mL of anhydrous ethanol was added. The mixture was heated under reflux until the substrates dissolved (15 min). After this, the mixture was cooled to room temperature and the precipitate was filtered off, dried and crystallized using butanol.

hybrid molecules
To the mixture of 0.001 mol of corresponding formylphenyl (2,4-dioxothiazolidin-5yl/ylidene)acetate and 0.001 mol of thiosemicarbazide, 5 mL of ethanol was added. The reaction mixture was heated under reflux for 15 min. After cooling, the precipitate was filtered off and washed with ethanol. After drying, the precipitate was recrystallized using the appropriate solvents (butanol or acetic acid).

In Vitro Anti-Tg Assay
The influence of the hybrid molecules 1-12 and drugs on Tg RH-GFP proliferation was analyzed as follows: 1 × 10 4 per well of Hs27 cells were seeded on black, 96-well, tissue culture-treated plates with optical bottoms (Corning ® ) in DMEM cell culture medium. After 72 h of incubation, the medium was removed, and then 1 × 10 5 per well of tachyzoite strains (RH-GFP) were added to the cell monolayers in the parasite culture medium. One hour later, the compounds and drug dilutions in the parasite culture medium were added to the Hs27 cells with Tg. The concentration of the compounds < CC 30 were as follows: sulfadiazine, 0-10,000 µM; trimethoprim and pyrimethamine, 0-200 µM and atovaquone, 0-10 µM. After a subsequent 96 h of incubation, the plates were read with covered lids, and both excitation (475 nm) and emission (509 nm) values from the bottoms were obtained using the multi-mode microplate reader SpectraMax ® i3 (Molecular Devices, San Jose, CA, USA). The results were expressed as the mean fluorescent intensity (MFI) and transformed to the percentage of viability compared to the untreated cells. Finally, the inhibitory concentrations for 50% inhibition of Tg proliferation (IC 50 ) were calculated. All experiments were performed in triplicate.

Cytotoxicity Assay
Cytotoxicity assay was performed according to international standards (ISO 10993-5:2009(E)), using tetrazolium salt (MTT, Sigma-Aldrich) and mouse, as well as human, fibroblasts. For this assay, DMEM w/o phenol red (21063, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS, 100 I.U./mL of penicillin and 100 µg/mL of streptomycin was used. Briefly, 1 × 10 4 /well of Hs27 cells were placed into 96-well plates and incubated for 24 h in 37 • C and 5% CO 2 . Afterwards, an old culture medium was replaced by 100 µL of the compounds or drug dilutions in culture medium and the cells were treated for 24 h. The concentration ranges of the molecules were as follows: hybrid molecules 1-12: 0-2500 µM, sulfadiazine: 0-25,000 µM, trimethoprim, pyrimethamine and atovaquone: 0-250 µM, and DMSO as the solvent: 0-20%. Then, 50 µL of 1 mg/mL of MTT solution in DMEM w/o phenol red was added to each well and incubated for 2 h (37 • C; 10% CO 2 ). Following this, the cell culture medium was aspirated carefully, 150 µL of DMSO was added to each well and the plates were gently mixed. Then, 25 µL of 0.1 M glycine buffer (pH 10.5) (Sigma-Aldrich) was added. The optical density at 570 nm on the multi-mode microplate reader SpectraMax ® i3 was read. The results were expressed as a percentage of viability compared to the untreated cells. All experiments were performed in triplicate.

Danio Rerio Culture and Fish Embryo Toxicity Test (FET)
Danio rerio (Experimental Medicine Centre, Medical University of Lublin, Poland) were maintained at 28 ± 0.5 • C, under a 14/10 h light/dark cycle with standard aquaculture conditions. After mating, the fertilized eggs were collected within 30 min. Embryos were reared in E3 embryo medium (pH 7.1-7.3; 17.4 µM NaCl, 0.21 µM KCl, 0.12 µM MgSO 4 and 0.18 µM Ca(NO 3 ) 2 ) in an incubator (IN 110 Memmert GmbH, Germany) at 28 ± 0.5 • C. The eggs were examined to remove the unfertilized, coagulated and damaged samples. The FET test was performed based on the OECD Guidelines for the Testing Chemicals, Test No. 236. TZD hybrids were weighted, dissolved in DMSO as stock solutions, and diluted in E3 embryo medium to the indicated treatment concentrations. The stock of TZD hybrids and dilutions in E3 embryo medium were freshly prepared before testing. Embryos were incubated at each concentration (5,10,15,25,50,75; 100 µg/mL) of TZD hybrids. The final DMSO concentration had no detectable effects on zebrafish development. The test was conducted in 24-well plates, with 5 embryos per well and 10 per group, in triplicate. The covered plates were kept at 28 ± 0.5 • C under the light/dark conditions (12 h/12 h). Embryonic viability, and the malformation rates of each treatment group were recorded at 24, 48, 72, and 96 hpf. All experiments were conducted in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the European Community Council Directive for the Care and Use of Laboratory Animals (2010/63/EU). For the experiments on larvae up to 5 dpf, the agreement of the Local Ethical Commission is not required.

Hemolytic Activity Determination
Human red blood cell (RBC) concentrate was obtained from the Regional Blood Donation and Transfusion Centre (Lublin, Poland). The RBC concentrate (5 mL) was washed three times with sterile PBS and centrifuged at 500× g for 3 min. The obtained pellet was resuspended using sterile PBS in order to obtain a 2% suspension of RBCs, which was subsequently mixed with 1 mL of different concentrations (i.e., 5, 10 and 35 mg/mL) of 2, 7, 8, and 10. The mixtures were incubated at 37 • C for 30 min and centrifuged at 1400× g for 10 min. The amount of free hemoglobin in the supernatants was measured spectrophotometrically at 405 nm. Negative and positive controls were performed by incubating RBCs with sterile PBS and 0.1% Triton-X, respectively. Each experiment was run in triplicate.

PAMPA-BBB Assay
BBB permeability of 2, 7, 8, and 10 was investigated using a parallel artificial membrane permeability assay (PAMPA) method. The PAMPA system, which consisted of a 96-well microfilter plate, was divided into the following two chambers: a donor at the bottom and an acceptor at the top, separated by a 120 µm thick microfilter disc coated with BBB lipid solution (Pion, Inc., Billerica, MA, USA). The solutions of 2, 7, 8, and 10 were prepared in dimethylsulfoxide (DMSO) at 4 mg/mL concentration, and then diluted with Prisma buffer (pH = 7.4) to obtain the donor drug solution with the final nominal concentration of 20 µg/mL. Then, 180 µL of the donor solution was added to the donor wells. Subsequently, each filter membrane of the top plate was coated with 5 µL of BBB-1 lipid solution (Pion Inc., Billerica, MA, USA) and the acceptor well was filled with 200 µL of brain sink buffer (BSB). The acceptor plate and the donor plate were sandwiched together and incubated at 37 • C for 180 min.
The permeability coefficient value (Pe) was calculated using the following equation: where V D -donor volume, V A -acceptor volume, C equilibrium -equilibrium concentration, , C D -donor concentration, C A -acceptor concentration, Smembrane area and t-incubation time (in seconds).

Cytotoxicity against Human Glioblastoma T98G Cells
The T98G glioblastoma cells (ATTC, CRL-1690, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose) (Sigma Aldrich, St. Louis, MO, USA), supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 U/mL) (Sigma Aldrich, St. Louis, MO, USA). Cells were maintained in a humidified atmosphere of 5% CO 2 and 95% air at 37 • C. Stock solutions of the investigated compounds (2, 7, 8 and 10, and temozolomide) were prepared by dissolving solid substances in sterile DMSO. T98G glioblastoma cells were seeded into 96-well sterile plates at a density of 4 × 10 4 cells/mL. After 24 h of incubation, the medium was removed from each well, and then the cells were incubated for the next 24 h with different concentrations of the investigated compounds (10,25,35,50, and 100 mg/mL) in the medium that contained 2% FBS. Viability of the T98G cells was evaluated using MTT assay. In brief, after 24 h of incubation of the cells with varying concentrations, 15 µL of MTT solution (5 mg/mL) was added to each well. After 3 h of incubation, 100 mL (per well) of 10% SDS buffer solution was added. After overnight incubation, the absorbance was measured at 570 nm using a microplate reader (Epoch, BioTek Instruments, Winooski, VT, USA). Experiments were repeated three times, and the measurements in each experiment were run in quadruplicate. Viability of the investigated cells was expressed as % of the viability of the untreated cells. DMSO in the concentrations present in the dilutions of the stock solutions did not influence the viability of the tested cells.

Conclusions
We report herein the anti-Tg characterization of TZD hybrid molecules that incorporated PCH (1-6) or TSC (7-12) structural fragments. By comparison of the biological data of TZD-PCH hybrids (1-6) with those of 7-12, it emerges that TSC could be an important pharmacophoric requirement for anti-Tg activity. All the tested TZD-TSC hybrids (7)(8)(9)(10)(11)(12) were more effective and more selective than SDZ and TRI. In addition, all of the hybrids were more selective than PYR. Further studies for the most potent TZD-TSC hybrids 7, 8 and 10 and TZD-PCH hybrid 2 proved that the compounds at the concentrations equal to IC 50Tg are non-cytotoxic, non-genotoxic and non-hemolytic. Moreover, they could cross the BBB, which is a critical factor linked with ideal anti-Tg drug development. Finally, the anti-proliferative evaluation of 2, 7, 8, and 10 disclosed the anti-cancer effect of 2 against human glioblastoma cells. Thus, the most potent TZD-based hybrid molecules could serve as a template for the development of novel therapeutics with dual anti-infectious (anti-Tg and anti-Mtb) modes of action and anti-glioma potency.