The chlorinated derivative AB15 containing 2-aminooxazole shows promising attributes for a drug candidate

In our previous study published by Juhás et al., 2022 [26], we investigated the physico-chemical and some biological properties in a series of substituted N-oxazol-2-yl and N-thiazol-2-yl carboxamides. It has generally been concluded that oxazole-containing compounds have more favorable physico-chemical and biological properties. Based on our findings associated with some essential attributes for drug candidates such as solubility, in vitro cytotoxicity, sufficient stability, and promising antibacterial activity, we decided to subject the compound AB15 (2-chloro-N-(oxazol-2-yl)isonicotinamide; in the previous study designated as 6b, Fig. 1) to further investigation. This advanced study was intended to recognize whether this compound can be legitimately selected as a potential candidate adjuvant molecule for antibiotic therapy. Within the evaluation of a favorable profile for drug-likeness criteria by in silico approach, it can be stated that all the commonly applied physico-chemical rules were met e.g., the Lipinski, Veber, and Muegge rules (Table S2, Supplementary Information). AB15 also fulfills stricter lead-likeness criteria (Oprea), allowing the further structural modifications (Table S2). In silico prediction of pharmacokinetic parameters by SwissADME [36] (Table S2) indicates high gastrointestinal absorption and blood–brain barrier penetration based on the boiled-egg method [37]. AB15 is a small molecule with balanced numbers of polar and non-polar functional groups. Such fragments are valuable in medicinal chemistry instead of bulky and greasy molecules.Many successful antibacterial compounds tend to be more polar and less lipophilic, facilitating penetration through bacterial cell envelopes and avoiding efflux. This trend is especially important for compounds targeting Gram-negative bacteria [38].

Fig. 1

The chemical structure of the studied compound AB15

AB15 is expected to be metabolically stable since the carboxamidic linker is sterically protected. This is confirmed by the metabolism prediction by BioTransformer 3.0 [39] (Phase I CYP450, combined model), which suggests mild oxidation reactions, mainly the N-oxidation of the pyridine nitrogen or hydroxylation of the heteroaromatic rings, but not the hydrolysis of the amidic linker (Table S3).

In an attempt to find similar compounds and their application, we performed a search of the existing literature. Surprisingly, no biological activity was reported for N-oxazol-2-yl)pyridinecarboxamides, or compounds with analogous simple (hetero)aromatic rings in the acyl part (benzene, thiophene, furane etc.). The acyl part of similar compounds with reported biological activity was either alicyclic (Fig. 2, compounds A, B, C) or larger heteroaromatic (Fig. 2D).

Fig. 2

Examples of compounds bearing N-(oxazol-2-yl) moiety described in the literature

Compound A was investigated for antibacterial and antitumor activity [40], compound B was investigated as an antitumor agent [41], compound C was investigated for eliminating invertebrate pests [42], and compound D was investigated as a metabotropic glutamate receptor ligand [43].

Several benzoxazole analogues, that is N-(benzoxazol-2-yl)carboxamides (Fig. 3), were investigated for antimicrobial activity. N-(Benzoxazol-2-yl)benzamide [44], N-(benzoxazol-2-yl)furan-2-carboxamide [45, 46], and N-(benzoxazol-2-yl)thiophene-2-carboxamide [45] were investigated as antimycobacterial compounds, often with activity against MDR and extensively drug-resistant (XDR) Mycobacterium (M.) tuberculosis strains.

Fig. 3

Examples of N-(benzoxazol-2-yl)carboxamides described in the literature

Interestingly, N-(benzoxazol-2-yl)pyridine-2-carboxamide, similar in its acyl part to our compound, was tested as an apolipoprotein A-I expression stimulator [47]. Based on our literature search, we concluded that neither our compound nor a structurally similar one has been studied for antibiofilm properties.

The promising activity of AB15 against some medically relevant bacteria from the ESKAPE group

As shown in our previous study [26] within the antibacterial activity screening of AB15 against four selected Gram-positive and four Gram-negative reference bacterial strains, the highest activity (after 24 h incubation) corresponding to 62.5 µM was revealed against MRSA (ATCC 43300), Staphylococcus epidermidis (ATCC 12228), E. coli (ATCC 25922) and A. baumannii (ATCC 19606) (Table S4, Supplementary Information). A significantly more pronounced antimycobacterial activity of AB15 corresponding to a concentration range of 13.95–27.90 µM was registered against a broad spectrum of mycobacteria (Mycolicibacterium smegmatis, Mycolicibacterium aurum, M. avium, M. kansasii, M. tuberculosis H37Ra, M. tuberculosis H37Rv and multidrug-resistant M. tuberculosis IZAK, M. tuberculosis MATI (Table S5, Supplementary Information). The highest antifungal activity of AB15 was registered against the yeast strain Candida albicans (ATCC 2443), corresponding to 31.25 µM, and filamentous fungus Lichtheimia corymbifera (Czech Collection of Microorganisms–CCM 8077), corresponding to 62.5 µM (Table S6, Supplementary Information).

A follow-up study of the antibacterial activity of AB15—the introduction of Gram-positive and Gram-negative clinical bacterial isolates

Based on the results mentioned above, a set of highly medically relevant clinical bacterial isolates with determined antibiogram profiles was employed to provide a deeper insight into antibacterial action of AB15. Specifically, four Staphylococcus and twelve Gram-negative bacterial isolates were selected. As shown in Table S7 (Supplementary Information), the activity of AB15 against four Staphylococcus strains, including vancomycin-resistant ones, showed MICs ranging from 62.5 to > 250 µM. Members of the ESKAPE group with high resistance and MDR profiles were placed in an extended study of AB15 antibacterial action against Gram-negative bacteria. The susceptibility/resistance profiles of these strains, listed in Table S8 (Supplementary Information), were determined by disc diffusion and microdilution broth methods employed according to the recommendations of EUCAST. As it is shown in Table 1, the MIC ranged from 15.63 to > 500 µM. The highest activity (15.63 µM) was recognized against MDR A. baumannii (20/21), and the lowest against MDR Pseudomonas aeruginosa (26/21) (> 500 µM). The registered moderate activity of AB15 against A. baumannii, the bacterial agent mentioned as being among the most threatening in the list of antibiotic-resistant “priority pathogens” supported the strong premise of continuing in a more comprehensive characterization of other antibacterial activity attributes and for making easier and more justified conclusions about the appropriateness of categorizing this compound as a candidate adjuvant for antibiotic therapy. In addition, the anti-Acinetobacter activity of oxazole-type compounds was recently proven by Trush et al., 2020. In all tested derivatives included in this study, a high level of growth inhibition of MDR A. baumannii clinical isolates was also revealed [48].

Table 1 Antibacterial activity of AB15 against Gram-negative clinical bacterial isolates compared to the reference internal quality control strain, Escherichia coli (ATCC 25922)

ATCC, American Type Culture Collection; ID No., internal laboratory identification number; GEN, gentamicin; MIC, minimum inhibitory concentration. Determined by the microdilution broth method according to EUCAST recommendations. MIC was evaluated by visual inspection via the metabolic indicator Alamar Blue, and by spectrophotometric measurement. The results were read after 24 h of incubation at 37 °C.

AB15 shows bactericidal action against Acinetobacter baumannii clinical isolate

As presented in Table S4 (Supplementary Information), only a slight (one-step) shift in the MIC of AB15 was registered in the reference strain, A. baumannii (ATCC 19609), evaluated after both 24 and 48 h. This only slight decrease in activity recognized after 48 h could indicate the bactericidal mode of the action of AB15. To determine the cidal activity of AB15, the microdilution method with a subsequent spread plate technique and the clinical isolate A. baumannii (20/21) were employed. After the exposure of A. baumannii to AB15 at a concentration level corresponding to fourfold MIC (250 µM), the percentage reduction in the number of CFU/mL corresponded to 99.983–99.984%, compared to the initial inoculum (Table S9, Supplementary Information). According to the mentioned criterion in the Materials and Methods section (Distinguishing between the bactericidal and static effect of AB15), AB15 appears to be a bactericidal compound. The cidal effect of a thiazole-based compound was also recently revealed by Haroun et al., 2021, against MDR Gram-positive and Gram-negative bacteria [49]. The bactericidal or bacteriostatic effects of oxazole derivatives do not appear to have been extensively studied yet. Nevertheless, in a study conducted by Azzali et al., 2020 [50], it was suggested that the activity of 2-aminooxazoles maintain similar cidal action compared to their 2-aminothiazole isosteres.

Insight into the mechanism of the action of AB15

This study employed a macromolecular biosynthesis assay and a membrane depolarization assay for the recognition of bacterial biosynthesis pathway and/or cellular structures targeted by the compound AB15.

Macromolecular biosynthesis assay was performed according to study published by Nowakowska, et al., 2013 with slight modifications [29]. This method is based on the exposure of bacteria to a tested compound and subsequent measurement of the percentage of [3H] radiolabeled precursors incorporated into newly synthesized biomacromolecules. After the measurement, data were compared with results obtained after exposing bacteria to commercial antibacterial drugs with known different mechanisms of action (inhibitors of DNA, RNA, peptidoglycan, and protein synthesis). When evaluating bacterial cytoplasmic membrane as a potential target of the tested compound, a voltage-sensitive dye, 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)), CHX as the positive control and fluorometric measurement were employed.

As shown in Fig. 4, the action of AB15 resulted in a statistically significant decrease in the incorporation of radiolabeled precursors participating in the protein synthesis pathway (Fig. 4D) of the reference bacterial strain MRSA (ATCC 43300) (used according to a protocol described in Nowakowska et al., 2013) [29]. Therefore, it can be concluded that the protein synthesis pathway is primarily affected by the action of AB15. In addition, as a matter of fact, protein synthesis is recognized as a universally conserved macromolecular biosynthetic pathway in prokaryotic microorganisms, therefore, the same target can be expected in other bacterial species. In addition, acquired data from the membrane depolarization assay, shown in Fig. 5, revealed only a slight increase in bacterial membrane depolarization after treatment with 4 × MIC of AB15 (represented in purple), compared to the negative control (black). Thus, this finding suggests that the cytoplasmic membrane is not the target of AB15's effective activity.

Fig. 4

A–D Results of macromolecular biosynthesis assay. Inhibition of biosynthetic pathway is indicated by lower incorporation of radioactively labeled precursors. A [3H] N-acetylglucosamine (peptidoglycan synthesis), B [3H] uridine (RNA synthesis), C [3H] thymidine (DNA synthesis) and D [3H] leucine (protein synthesis) in methicillin-resistant Staphylococcus aureus (ATCC 43300) strain, treated for 2 h with 4 × MIC of VAN, RIF, CIP, CHL, CHX, and AB15. Results are expressed as the percentage of biomolecule incorporation related to untreated controls. The values shown are means of two independent experiments prepared in duplicates ± SEM. Significant reduction in biosynthetic pathway compared to control is indicated by p-value, where p < 0.05 was accepted as statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001), determined by nonparametric one-way (ANOVA) test (Kruskal–Wallis test)

Fig. 5

Membrane depolarization assay. Membrane potential was monitored using the fluorescence dye, DiSC3(5), (λEx = 620 nm, λEm = 680 nm). Methicillin-resistant Staphylococcus aureus (ATCC 43300) was stained with 0.5 mM DiSC3(5). After 10 min of measurement (black arrow), AB15, positive control represented by chlorhexidine (CHX), in final concentrations corresponding to 4 × MIC were added. The graph depicts the mean of six replicates and the standard deviation

Focus on the in vitro cytotoxicity of AB15, and employment of invertebrate animal model, Galleria mellonella, for evaluating in vivo toxicity of AB15

Newly synthesized compounds can be classified as candidate molecules for advanced studies and potential candidate molecules for antibiotic therapy fulfilling basic premises such as low or non-cytotoxicity expressed against eukaryotic cell lines. As was reported in our previous study [26], the cytotoxic potential recognized in AB15 against the employed standard hepatic cell line, human hepatoma cell line (HepG2), was negligible—the cytotoxicity parameter (IC50) corresponded to 664.1 µM [26]. Subsequently, to gain deeper insight into the cytotoxic and toxic potential of AB15, the standard renal cell line human kidney 2 (HK-2), and the alternative animal model, Galleria mellonella, were employed. Figure 6 demonstrates the cytotoxic effect of AB15 against the HK-2 cell line, with the IC50 > 1000 µM. This result indicates the lack of cytotoxic potential of AB15 against this employed cell line.

Fig. 6

In vitro cytotoxicity of AB15 determined using the standard renal cell line HK-2 and expressed as the standard toxicological parameter IC50. IC50 was calculated by nonlinear regression from a semi-logarithmic plot of incubation concentrations versus the percentage of absorbance related to untreated controls using GraphPad Prism 9.0.0 (GraphPad Software, Inc., USA)

The compound efficacy in vitro can be expressed through the parameter known as selectivity index (SI). SI is calculated as the ratio of IC50/MIC. Compounds with SI above 10 are considered promising drug candidates since they present sufficient effectiveness and safety. As it was published in our previous study [26], after employing the HepG2 cell line, AB15 displayed a range of SI = 42.5–5.31 (calculated with MIC values for different strains). The employment of the HK-2 cell line led to a recognition of SI ranging from > 64 to > 16.

To assess the in vivo toxicity of AB15, an invertebrate model, Galleria mellonella larvae, was included. Galleria mellonella is a frequent substitute for animal models such as mice and rats in pathogenesis and toxicity studies. This model seems to be fully appropriate for distinguishing toxic and non-toxic chemicals. In addition, the 3Rs conditions (Replacement, Reduction, and Refinement), which are recognized as a framework for high-quality science in the academic sector, are fulfilled [51,52,53].

The larvae were divided into groups according to the final volume of administered compound per kg of the animal's body weight. The highest administered doses corresponded to 500 mg/kg of body weight, while the lowest doses had a final concentration of 5 mg/kg of body weight of larvae. Two ways of administration of the tested compound were employed, per oral administration by the force-feeding method, and injection into the hemocoel through the last left proleg, which mimics the intravenous route of administration in mammals and reflects systemic toxicity. As shown in Table S10 and Fig. S3 (Supplementary Information), the median lethal dose leading to the death of 50% of the animals (LD50) was not reached after the intra-hemocoel administration of AB15 in any group. Therefore, it can be concluded that LD50 of AB15 is higher than 500 mg/kg of body weight of the larvae. The highest mortality was registered in group one (500 mg/kg of body weight), corresponding to 12.5%. However, no mortality was registered in the other included groups (administered concentration ranged from 250 mg/kg to 5 mg/kg of body weight). Regarding toxicity after per oral administration, as shown in Table S11 and Fig. S4 (Supplementary Information), LD50 was not reached either. In addition, no larval deaths were recorded in any group at any inspection time interval. Therefore, it can be concluded that the recognized in vivo toxicity of AB15 after per oral administration corresponds to > 500 mg/kg. Based on these findings, AB15 could be categorized within the GHS (Globally Harmonized System) as class 4, which represents non/low toxic compounds [53].

After the administration of AB15 by both mentioned routes, no statistically significant effect on the survival of larvae was registered in a data analysis of all included groups via the pairwise Log-rank Mantel-Cox curve comparison test (Supplementary Information, Tables S12, S13).

In summary, the results from in vitro cytotoxicity, and in vivo toxicity evaluations indicate the promising ability of AB15, and this compound should be at least considered a possible adjuvant for antibiotic therapy.

The mutual interaction of AB15 with selected, commercially available antibacterial drugs and its impact on antibacterial activity

In view of fact that AB15 showed promising activity against Gram-negative bacteria and showed low/non-toxic potential, it was further studied, what benefit the compound AB15 could have within the potential combination treatment strategy. Ideally, within antibiotic combination therapy, greater antibacterial activity of drugs in combination should be registered compared to the antibacterial action of drugs alone. Furthermore, reduced toxicity and mitigated adverse effects should be registered. In addition, the combination of antibacterial drugs, especially those with different mechanisms of action, reduces the risk of resistance development and spreading, which could alleviate the current advancing AMR crisis. To determine the mutual effect of AB15 with commercially available drugs, six different, preferentially selected, antibiotics were employed in this study, namely, CIP, GEN, TGC, SXT, CHL and CST. For preliminary insight, the bacterial strain E. coli (ATCC 25922) was employed. According to FICI values gathered in Tables S14–S19 (Supplementary Information), an additive effect was recognized at specific concentration ratios of AB15 in combination with GEN, CST, and CHL. The values of several FICI in combinations mentioned above were recognized to be very close to the categorization of synergy. For example, at some concentration ratios of AB15 + GEN, the FICI values corresponded to 0.516 and 0.531, respectively (the criterion to categorize the mutual interaction of two compounds as synergetic is FICI ≤ 0.5). Indifferent effect, revealed at other concentration ratios, indicates that the actions of compounds in combination are not mutually influenced. Finally, in all combinations of AB15 with selected antibiotic drugs at all tested concentration ratios, no antagonistic effect was recognized. Therefore, AB15 can be considered a valid candidate adjuvant molecule for supporting existing selected antibiotics.

For better clarity, the results of checkerboard assays are also presented in form of heat maps (Fig. 7B–G). Each figure illustrates the percentual growth inhibition of the reference bacterial strain E. coli after being exposed to AB15 in combination with selected antibiotic drugs. The percentage of growth inhibition is illustrated using a color scheme (Fig. 7A), where the darkest shade represents the lowest inhibition and, therefore, the highest growth of the employed bacterial strain, while the lightest shade indicates the highest achieved inhibition and, consequently, the lowest growth of Escherichia coli. As was seen within the combination AB15 + CIP and AB15 + CHL (Fig. 7B and G), there was no distinct contribution of AB15 to the activity of these two commercial drugs. Nevertheless, the AB15 + GEN, and AB15 + CST combinations led to the increased AB15 activity (Fig. 7C and F). The combination of AB15 + SXT appeared to be disadvantageous at some concentration ratios (Fig. 7E).

Fig. 7

A–G Two-color heat maps of checkerboard MIC assays. The percentage of growth inhibition (compared to positive growth control) of bacterial strain, Escherichia coli (ATCC 25922), after 20 h of exposition at 37 °C to AB15 in combination with selected antibiotic drugs, expressed by color spectrum. A color spectrum, B AB15 in combination with ciprofloxacin (CIP), C gentamicin (GEN), D tigecycline (TGC), E trimethoprim-sulfamethoxazole (SXT), F colistin (CST) and G chloramphenicol (CHL). The black horizontal (selected antibacterial drug) and vertical (AB15) lines denote the MIC of each compound alone

AB15 has an additive effect with colistin against the MDR Acinetobacter baumannii strain

Our study explored the activity of AB15 against the most feared pathogen with regard to its multidrug resistance and limited possibilities of available effective antibiotic treatment, the clinical bacterial isolate, MDR A. baumannii (20/21). CST is considered an antibacterial drug of last resort, primarily reserved for the treatment of serious infections caused by MDR Gram-negative bacterial strains. Its mechanism of action involves targeting lipopolysaccharides in the outer membrane of these bacteria. However, in recent years, a rapidly increasing number of reports describing emerging cases of bacterial resistance to CST have been published [54,55,56].

Studies focused on the use of CST within combination therapy have been widely discussed in recent years. For example, in a study published by Brennan-Kohn et al., 2018, the authors focused on various combinations of antibiotics with CST. In 18 out of 19 combinations, a synergistic effect against more than two bacterial clinical isolates from the Enterobacteriaceae family was registered [57]. Furthermore, in a study conducted by Almutairi et al., 2022, the synergistic interaction of two drug combinations of CST with VAN, aztreonam, ceftazidime, and imipenem was revealed against clinical isolates of colistin-resistant A. baumannii [58].

CST seems to be the last relevant antibiotic, which is available to treat severe infections caused by MDR A. baumannii. Clinicians often choose polymyxins in combination with other antibiotics. Nevertheless, the relevance of only some of them can be proven by published studies. For example, Khalil et al., 2019, demonstrated the beneficial action of CST in combination with trimethoprim-sulfamethoxazole [59]. On the other hand, some studies have pointed out that the efficacy of CST monotherapy compared to combination therapy with CST of infections caused by A. baumannii is comparable [60,61,62]. It should be mentioned that the limitations of CST monotherapy lie in its undesirable neurotoxicity [63] and nephrotoxicity [64].

The additive effect of the AB15 + CST combination revealed against E. coli was subsequently evaluated against the clinical isolate MDR A. baumannii (20/21). As illustrated in Table 2, the synergistic potential at concentration ratios corresponding to 0.054 µM CST + 31.25 µM AB15 was revealed. The concentration ratio 0.108 CST µM + 7.813 µM AB15 was recognized as being very close to meeting the FICI criterion for classifying the interaction as synergistic (FICI = 0.563). Additivity was revealed at four other concentration ratios. In addition, no antagonistic interaction was registered within the evaluated ratios. concentration ratios. In addition, no antagonistic interaction was registered within the evaluated ratios. Similarly to the checkerboard studies including E. coli, the antibacterial activity of the CST and AB15 in combination is graphically illustrated in a heat map (see Fig. 8A, B). Generally, the antibacterial action of CST is potentiated by the presence of AB15, and vice versa, the presence of AB15 is potentiated by CST.

Table 2 Total fractional inhibitory concentration indexes (FICI) determined by checkerboard assay with a combination of colistin (CST) and AB15. The clinical bacterial isolate, Acinetobacter baumannii (20/21) was included in the assay. The minimum inhibitory concentration (MIC) of the compounds alone was: MICCST = 0.216 µM, MICAB15 = 125 µMFig. 8

A, B Two-color heat map of the checkerboard MIC assay. The percentage of growth inhibition (compared to the positive growth control) of the bacterial strain, Acinetobacter baumannii (20/21) after 20 h of exposure at 37 °C to AB15 in combination with colistin (CST), expressed by the color spectrum. The minimum inhibitory concentration (MIC) of compounds alone corresponds to: MICCST = 0.216 µM, MICAB15 = 125 µM. A color spectrum, B AB15 in combination with colistin (CST)

FIC, Fractional Inhibitory Concentration, FICI, Fractional Inhibitory Concentration Index, FIC(CST, AB15) = MIC of the combination/MICCST, AB15 alone, FICI = FICCST + FICAB. The effect was interpreted as follows: synergy when FICI ≤ 0.5, an additive effect when 0.5 < FICI ≤ 1, an indifferent effect when 1 < FICI ≤ 4, and an antagonistic effect when FICI > 4.

AB15 potentiates the antibiofilm effect of CST by suppressing the dissemination of bacteria from biofilm consortia formed by a clinical isolate of Acinetobacter baumannii

The ability to form biofilms strongly contributes to the A. baumannii broad spectrum of virulence and resistance factors. As mentioned above, A. baumannii thrives in hospital environments and is capable of creating biofilm consortia on numerous medical devices and surfaces. Biofilm formation promotes the resistance of A. baumannii cells and plays a crucial role in its pathogenesis. A. baumannii is the causative agent of many life-threatening infections, such as catheter-related infections, meningitis, urinary tract infections, ventilator-associated pneumonia, etc., whose treatment is highly challenging [65,66,