Preliminary Investigation of the Antibacterial Activity of Antitumor Drug 3-Amino-1,2,4-Benzotriazine-1,4-Dioxide (Tirapazamine) and its Derivatives

Round 1

Reviewer 1 Report

Within the article Antibacterial activity of antitumor drug 3-amino-1,2,4-benzo-triazine-1,4-dioxide (tirapazamine) and its derived compounds E. Polmickaite-Smirnova and co-authors presented interesting research on re-purposing applicability of anticancer agent, tirapazamine, as antibiotic by designing the chemistry of its functional groups.

Extensive set of TPZ – derivatives has been synthesized and comprehensively investigated for antimicrobial activity against Gram+ and Gram- bacterial strains. Although very interesting improvements in activity depend on the functional groups has been observed (through variation in MIC values and synergic activity of tested components with some antibiotics), there is not enough data that can lead to effective discussion and major conclusions. A list of very important questions left open, including:

1. Solubility in water: testing was performed from DMSO stock probably due to limited solubility in water (it should be commented in the text). The change of solubility in water that might appear with substitution of side functional groups should be experimentally tested since it can provide valuable information about activity and availability of drugs during antimicrobial testing. One should also comment on applicability of the drug with limited solubility (potential for its limited bioavailability, accumulation, degradation, elimination etc.). Please provide information on experimentally determined water solubility of TPZ and its derivatives.
2. ROS generation: TPZ works through ROS-induced DNA damage. It is essential to experimentally test quantitative ROS generation induced by TPZ and its most and least active derivatives. It will explain or antimicrobial results either results from computer simulations. Please add ROS generation data of investigated TPZ derivatives.
3. Anaerobic conditions: similar to cancer, anaerobic conditions might be developed also for some bacterial infections (gut, biofilms, etc) and they are critical for efficacy of antimicrobial treatment. It is the point where TPZ might be particularly effective. I suggest introducing results of testing for at least one anaerobic strain.
4. Toxicity: even very small changes in structure or chemistry can trigger large changes in physiochemical properties and even larger changes in interactions with living surroundings. Therefore testing in at least one healthy cell line for toxicity and determination of selectivity index is essential before suggesting any drug –derivate for therapeutic applications. Please add these data at least for the most effective TPZ derivate.
5. Other: introduction part should include more specific details about the idea of pre-purposing TPZ and formation of its derivatives for treatment of bacterial infections (i.e. anaerobic conditions).

Upon providing the relevant information and detailed discussion the manuscript could be suggested for publication.

Author Response

Answers to comments and suggestions of referee 1#

 

Comment 1: Solubility in water: testing was performed from DMSO stock probably due to limited solubility in water (it should be commented in the text). The change of solubility in water that might appear with substitution of side functional groups should be experimentally tested since it can provide valuable information about activity and availability of drugs during antimicrobial testing. One should also comment on applicability of the drug with limited solubility (potential for its limited bioavailability, accumulation, degradation, elimination etc.). Please provide information on experimentally determined water solubility of TPZ and its derivatives.

Answer:    We understand that the Reviewer's comment about the tested compounds solubility is important. In this respect it should be noted that we had a relatively rich experience in working with the current chemical group of 1,2,4-benzotriazine derivatives as well as other redox active agents (such as quinones, aromatic N-oxides and nitroaromate compounds (including conventional antibiotic nitrofurantoin and related structure agents) which have been used for studies of their enzymatic reactivity and cytotoxic activity.

   In order to facilitate their solubility in aqueous solutions, their stock solutions are typically prepared in DMSO as aliphatic water soluble solute. It should be noted that DMSO is a polar aprotic non-toxic solvent with a median lethal dose being higher than those of ethanol and MeCN. In our current work, this  solute was chosen as a testing compound vehicle for the stock solutions in  in vitro experiments. DMSO was most preferable for benzo-1,2,4-triazine 1,4-dioxides due to their very good solubility in this solvent. DMSO application might be optimal in achieving the required sample concentration in water. Note that we have applied the remaining minor concentrations of DMSO (1-2 %) which virtually did not affect the viability of bacteria strains used in the current work  (it has been tested by separate blank experiments).

   We have pre-selected only the compounds appropriate as antibacterial agents,  possessing sufficient solubility. So, before starting the experiments, we had excluded molecules with hydrophobic substituents, i.e., TPZs bearing long acyl-/ aliphatic chains or aromatic acyl derivatives.  Note that TPZ itself is a hydrophilic substance with a solubility of 2450 µg/mL in deionizied water (23 C) (it can be even recrystallized from the water).  Prior to the experiments, the solubility of other TPZs used in our studies had been measured by inserting their DMSO stock solutions into the stirred water until supernatant was observed by means of spectrophotometrical measurement.  Solubility of TPZs  was estimated from 1700 µg/mL in the case of acetyl substituent (compound 2) to the lowest 380 µg/mL in the case of the least active analogue (compound 10). TPZs bearing alkoxy groups were substantially more soluble than their alkylacyl analogs. In general, all the compounds tested (1-10) were sufficiently soluble in water for the experimental conditions (setup).

   Comment 2: ROS generation: TPZ works through ROS-induced DNA damage. It is essential to experimentally test quantitative ROS generation induced by TPZ and its most and least active derivatives. It will explain or antimicrobial results either results from computer simulations. Please add ROS generation data of investigated TPZ derivatives.

   Answer: The action of HNOX compounds (including TPZ) through ROS-induced DNR damage has been proven in previous works. As we have described in the Introduction of our article, "...transcriptomic and proteomic studies of E. coli strains have provided clear evidence that HNOXs induce ROS generation accompanied by DNA cleavage, involving a substantial contribution of putative reductases in bacteria [12,13]" (page2, lines 61-64).

Comment 3: Anaerobic conditions: similar to cancer, anaerobic conditions might be developed also for some bacterial infections (gut, biofilms, etc) and they are critical for efficacy of antimicrobial treatment. It is the point where TPZ might be particularly effective. I suggest introducing results of testing for at least one anaerobic strain.

Answer: We highly appreciate the Reviewer’s comment on this issue. Regarding this, it should be noted that all the strains used in our work as model organisms are not obligate aerobes. They can be classified as facultative anaerobes, i. e. under aerobiosis, ATP is generated during aerobic respiration, while in the absence of oxygen, they can be switched to anaerobic respiration. E. coli and S. enterica strains cause intestinal infections where the environment is more anaerobic, suggesting that the action of TPZs may partly be related to their reductive activation producing free anion radical species accompanied by ROS generation.

      Comment 4: Toxicity: even very small changes in structure or chemistry can trigger large changes in physiochemical properties and even larger changes in interactions with living surroundings. Therefore testing in at least one healthy cell line for toxicity and determination of selectivity index is essential before suggesting any drug –derivate for therapeutic applications. Please add these data at least for the most effective TPZ derivate.

     Answer: We highly appreciate the Reviewer's comment on this aspect. Regarding this, it should be noted that the tolerated dose of TPZ drug has previously been reported to be 390 mg m−2 (Shah et al., and references therein) corresponding to ~ 85 µM (~ 15 µg/mL) which is twice as high as the doze estimated in our current work towards the Gram-negative bacteria strains and it is close to that with respect to the Gram-positive strain. The same probably applies to TPZ analogues. Furthermore, the combined study carried out for TPZs coupled with conventional antibiotics (which is an important aspect of our work) revealed the additive and enhanced (partial or complete synergistic) effects, which decreased the concentrations of TPZs. This should allow for the use of a lower dosage of the compounds thereby reducing the risk of their toxic and other side effects. This is described in Conclusion (page 17, lines 449-460).

 

     Comment 5: Other: introduction part should include more specific details about the idea of pre-purposing TPZ and formation of its derivatives for treatment of bacterial infections (i.e. anaerobic conditions). Upon providing the relevant information and detailed discussion the manuscript could be suggested for publication.

Answer: We completely agree that some important questions of our work remain open as the study described in our manuscript is the starting point for further (more extensive) research. Highly appreciating the Reviewer’s suggestions, we are going to implement them in our next (proceeding) work.              

 

 

 

 

Reviewer 2 Report

The paper entitled "Antibacterial activity of antitumor drug 3-amino-1,2,4-benzo-triazine-1,4-dioxide (tirapazamine) and its derived compounds" deals with the antibacterial activity of the anti-tumor drug tirapazamine and its derivatives. The study is very interesting and very well designed and explained in the manuscript. Generally, the paper is well written and only few minor changes are required.

Minor language change:

• lane 71, 78, 414, 415 it is not clear what means DNR,
• lane 76 delete "-" before antibiotic-resistant,
• lane 235 the authors wrote highest activity, whereas ciprofloxacin had the highest activity. it's better to specify highest between the tirazapamide derivatives or something like this.
• lane 237 add the μM unit after MICs of nitrofurantoin
• lane 243 and 253 delete one of the two "))"
• lane 245 and 246 delete "=" to uniform with the others
• lane 325 please replace "in contrast" with "By contrast"
• lane 423 please add even the number of the compound after their names.

References:

• lane 492 and 589 add the lane page number 
• lane 529 add the year
• lane 534, 546 and 596 the year has to be in bold letters

In the methods section, the authors have to specify the software that used to perform the analysis of drug combination assay.

In the results section at lane 232, the authors wrote "significant activity" but it is not clear if they performed a statistical analysis.

In the legend of Table 3 I would have added the extended names of substances.

In the table 2 specify the unit eV or A² for each parameters since now it is not clear.

In Table 3 the author have to change putting in the first line the name of the strains  and specify for each columns MIC (μg/mL), Fold potentiation and  FICI because now the table is not promptly understandable.

 

Author Response

Answers to comments and suggestions of Reviewer 2#

Comment 1: lane 71, 78, 414, 415 it is not clear what means DNR.

Answer: throughout the manuscript, DNR is corrected and written as DNA (i.e., desoxyribonucleic acid).

Comment 2: lane 76 delete "-" before antibiotic-resistant,

Answer: it has been corrected

 

Comment 3: lane 235 the authors wrote highest activity, whereas ciprofloxacin had the highest activity. it's better to specify highest between the tirazapamide derivatives or something like this.

Answer: The sentence (in lines 241) has been corrected by writing " Of the whole set of compounds tested, TPZ analogues (compounds 2-6) exhibited the highest activities towards all the strains..." .

  

Comment 4: lane 237 add the μM unit after MICs of nitrofurantoin

Answer: it has been corrected (line 244).

 

Comment 5: lane 243 and 253 delete one of the two "))"

Answer: we have corrected

 

Comment 6: lane 245 and 246 delete "=" to uniform with the others.

Answer: they have been deleted and uniformed with others (see lines 253 and 254).

 

Comment 7: lane 325 please replace "in contrast" with "By contrast"

Answer: it has been replaced (line 334).

 

Comment 8: lane 423 please add even the number of the compound after their names.

Answer: it has been corrected.

 

Comment 9: References: lane 492 and 589 add the lane page number;  lane 529 add the year, lane 534, 546 and 596 the year has to be in bold letters.

Answer: All references were corrected as noted by Reviewer (lines 505, 544, 549, 561, 582, 604).

 

Comment 10: In the methods section, the authors have to specify the software that used to perform the analysis of drug combination assay.

Answer: The software package performed for analysis of drug combination is specified by writing " The isobolograms of combined action of TPZs with conventional antibiotics were generated by using SigmaPlot 2000 (SPSS Inc., Version 6.10) software package." (lines 228-230).

Comment 11: In the results section at lane 232, the authors wrote "significant activity" but it is not clear if they performed a statistical analysis.

Answer: As described in the section of Materials and Methods (page 6, lines 218-219), " For each concentration of the compounds, experiments were performed in triplicate on at least two independent occasions."

 

Round 2

Reviewer 1 Report

Most of the issues observed in first version remained in the second version of the manuscript. Consequently, some of the critical conclusions, regarding main purpose of using the drug as antimicrobial (no testing under anaerobic conditions), safety and selectivity (without any experimental data) and mechanism of action (without any experimental data), remained non-explained. The initial idea of this work is very interesting, however according to my opinion the work is not ready for publication.   

Author Response

 

Answer to comments and suggestions of Referee 1

   Comment:

Most of the issues observed in first version remained in the second version of the manuscript. Consequently, some of the critical conclusions, regarding main purpose of using the drug as antimicrobial (no testing under anaerobic conditions), safety and selectivity (without any experimental data) and mechanism of action (without any experimental data), remained non-explained. The initial idea of this work is very interesting, however according to my opinion the work is not ready for publication.

   Answer:    

Dear reviewer, thank you so much for your comments.

   As we have already noted, the present work is the starting stage for further (more extensive) research. In order to keep a rational context of the work, the additional experiments should be done not only on TPZs as single compounds but also in combination with conventional drugs which is an important aspect of our work. And most important, we must stress that currently, due to corona virus lockdown, it is virtually impossible to conduct the additional experiments required by the Referee. The pause of our research activities of our team may last at least up to the summer or longer. 

 

Round 3

Reviewer 1 Report

I am suggesting authors to add a clear statement in every relevant part of the text including abstract, discussion and conclusions that presented research is preliminary and that further research regarding toxicity, mechanism of action and selectivity is urgently needed before accepting this platform seriously for novel antimicrobial therapy. "Preliminary" should be added in title, too. In this format I agree with publication of this work.   

Author Response

Thank you very much for taking the trouble to review our manuscript and providing an opportunity to improve it. We have made the corrections in relevant parts of the manuscript such as the title, abstract and conclusion, hence noting that our current research is preliminary. Could you please find the text supplementations highlighted in green of yellow background. In addition, the editorial corrections of the text are marked in green.