PREreview of Identification and evaluation of besifloxacin as repurposed antifungal drug in combination with fluconazole againstCandida albicans

Identification and evaluation of besifloxacin as repurposed antifungal drug in combination with fluconazole against Candida albicans

1. Summary and Overall Impression

Recommendation: Major revisions are required. 

The investigators explore besifloxacin (BS), an FDA-approved antibiotic, as a potential antifungal agent against Candida albicans. Fungal infections represent a significant clinical challenge, particularly in immunocompromised patients, with limited therapeutic options and increasing antimicrobial resistance. Drug repurposing offers an efficient pathway to identify new antifungal treatments by leveraging existing safety data from approved medications. This preprint investigates the potential antifungal activity of besifloxacin, a fluoroquinolone antibiotic, against C. albicans, both alone in vivo and in combination with fluconazole in vitro. Through computational screening targeting conserved proteins in C. albicans, the researchers identified BS as a candidate for antifungal activity. In vitro testing demonstrated that BS inhibited C. albicans growth with an IC50 of 15.6 mg/L and showed synergistic effects when combined with fluconazole, reducing fluconazole's MIC from 2 mg/L to 0.5 mg/L. The researchers also evaluated BS in a mouse model of systemic candidiasis, where intravenous administration at 100 mg/kg/day reduced fungal burden in kidneys by 83% compared to controls. The findings suggest that BS may inhibit fungal topoisomerase II, similar to its mechanism against bacterial pathogens. The observed synergy with fluconazole indicates potential utility in combination therapy to combat resistance. This research exemplifies how antibacterial agents might be repurposed to address fungal infections, particularly in cases of concurrent bacterial and fungal infections where a dual-action agent could be advantageous. However, the authors note that new intravenous formulations would need development before clinical application, as the current vehicle (DMSO/saline) is unsuitable for human use. The study would benefit from direct experimental evidence demonstrating besifloxacin's interaction with the proposed target in C. albicans. While the paper mentions the potential binding of fluoroquinolones to topoisomerase II based on previous in silico studies, no direct target validation experiments were conducted to confirm this mechanism in C. albicans. The findings suggest that BS displays a fungistatic effect, synergy with fluconazole, and a reduction in fungal burden in vivo. The overall research design is promising and addresses a significant clinical need, however, the conclusions are overstated with respect to the data presented, especially because the investigators did not test the synergistic effects of BS/FLC combination in vivo despite highly synergistic effects in vitro. Statistical reporting is inconsistent, and mechanisms of BS drug targets remain speculative. Major revisions are needed to support the manuscript’s claims and improve clarity regarding the scientific rigor of the experiments. If these concerns are addressed, this work could meaningfully contribute to the expanding antifungal arsenal, having important implications for future studies in combination therapy and dual-pathogen treatment. 

1. Section-by-Section Review

Abstract

Major issues:

1. Line 21: The statement that besifloxacin reduced fungal load by 83% in mice is accurate, but the synergy with fluconazole was not evaluated in vivo. To avoid implying in vivo synergy, please rephrase to emphasize that BS without combination with an antifungal showed 83% reduced fungal load, but synergistic effects were not evaluated in vivo. 

2. Line 22: The phrase “proposing its use in combination therapy…” should be changed to “proposing its potential for utility in combination therapy” or similar vernacular to reflect the early-stage nature of these experiments. A single C. albicans laboratory reference strain (ATCC MYA-2876) was used rather than clinical isolates, though antifungal susceptibility patterns can vary significantly among clinical isolates and Candida species, with different resistance mechanisms and inherent susceptibilities.  

Minor issues:

1. Line 19: “BS was able to reduce the MIC of FLC from 2mg/L to 0.5 mg/L…”. Add a space between “2” and “mg/L”. 

Introduction

Major issues:

1. The central hypothesis that besifloxacin may inhibit fungal topoisomerase through evolutionary conservation is only introduced later (line 184 onward), but should be explicitly stated earlier in the Introduction. 

2. Please state all other relevant hypotheses in the Introduction section.

3. Lines 33-47: The discussion of antifungal classes is useful, but no mention is made of current resistance rates or clinical limitations of fluconazole. Please add brief data to support the rationale for investigating a drug combination using antibiotics and antifungals to treat systemic candidiasis. 

Minor issues:

1. Lines 33-35: Please insert a reference for this sentence.

2. Lines 63-65: Please insert a reference for this sentence. 

3. The given rationale for combination therapy is compelling in this section, but it would strengthen your contextualization of the issue to cite specific challenges with current antifungals (e.g., resistance rates or failure rates) to strengthen the argument for drug repurposing. 

Methodology

Subheading: Screening for homologs in Candida proteome

Major issues: 

1. The computational screening methodology requires substantial enhancement in transparency and detail to enable proper assessment of the target selection process. The paper cites using "70 shortlisted targets" from a previous publication (Chakraborty et al. 2023) without explaining these targets' relevance to antifungal discovery or their selection criteria. Please provide a comprehensive report of the 70 initial target proteins, including their functions, relevance to antifungal activity, and selection rationale. Detail the exact results of the homology screening, reporting the number of C. albicans proteins identified with significant alignment, their UniProt identifiers, and putative functions. Provide a comprehensive table listing the 70 initial target proteins, including their functions, relevance to antifungal activity, and selection rationale.

2. The progression from homology identification to besifloxacin selection lacks clear documentation, creating a significant gap in the logical flow of the experimental design. Clearly explain the decision-making process that led from computational screening to selecting besifloxacin for testing, including any intermediate steps and selection criteria.

3. Please specify a justification for choosing e-value cut-off < 0.0001 as the homology threshold.

4. Line 99 states “Drugs targeting these conserved domains were used for further validation in vitro”. According to the methodology and findings presented in the manuscript, it appears that no molecular-based assays were performed to confirm that the drugs tested actually target these conserved domains. Adjust this statement to reflect this by saying, “Drugs believed to target these conserved domains based on screening for homologs in the Candida proteome were used for further validation in vitro.” 

Subheading: In vitro evaluation of besifloxacin

Major issues:

1. The study lacks essential components of standardized antifungal susceptibility testing, including appropriate quality control strains with known MIC values, which are crucial for validating the testing system. Include reference quality control strains in all susceptibility tests and report their MIC determination. 

2. Additionally, the reporting of technical and biological replicates is absent, making it difficult to assess the reliability and reproducibility of the MIC determinations. Clearly state the number of replicates performed for each experiment, presenting data with appropriate measures of variability. 

3. Detail the calculation method used for IC50 determination including software, curve-fitting approach, and statistical parameters.

4. The paper reports a single synergy score (δ = 29.58) without providing confidence intervals, p-values, or statistical significance assessment of this value. The justification for selecting the Loewe additivity model over other commonly used methods for antifungal combinations is not provided, limiting the contextual interpretation of the synergy claims. Please specify the exact concentration matrix design and individual concentration points tested for both drugs. Perform biological replicates of the synergy experiments, and report variability in synergy scores with appropriate statistical methods. Also, please justify the selection of the Loewe additivity model and discuss its theoretical assumptions as they relate to the drug mechanisms.

5. Consider elaborating on why gemifloxacin (GM) performed may have performed poorly in vitro compared to besifloxacin (BS). Any discussion of molecular differences between GM and BS, the docking model with the topoisomerase, or the antibacterial IC50s in the intended targets of the two would help the reader understand how successful applying a similar methodology to identify new targets could be.    

Minor issues:

1. Line 107: Change “till” to “until”

Subheading: In vivo evaluation of besifloxacin for systemic candidiasis

Major issues: 

1. The animal study design requires enhancement through the inclusion of a positive control group to properly contextualize the efficacy of besifloxacin treatment. The current design includes untreated and vehicle controls but lacks a standard antifungal treatment group. The authors justify this omission by referring to the previously documented efficacy of standard antifungals, but inter-experimental variability in infection models makes direct comparison within the same experiment crucial for valid conclusions. Include a positive control group treated with a standard antifungal agent at clinically relevant doses to provide direct comparative efficacy data. 

Minor issues:

1. Line 149-150: Please state the rationale for why 2 x 105 cell inoculum was chosen for this experiment. 

Subheading: Statistical analysis

Major issues:

1. Please justify why non-parametric one-way ANOVA was used over parametric alternatives, including results of normality tests or other assessments that guided this decision. Please report post-hoc analyses, or justify why none were performed. 

2. The small sample sizes (n=5-6 per group) raise concerns about statistical power, particularly without formal power calculations or sample size justifications.  Include a power analysis or sample size calculation (even retrospectively) to assess whether the study was adequately powered to detect biologically meaningful effects. 

Results and Discussion

Major issues:

1. Report confidence intervals or variance measures from SynergyFinder. 

2. Please justify in the Discussion or in the Methodology why the in vivo study did not utilize the drug combination of BS/FLC despite synergistic findings in vitro.

Minor issues:

1. Line 194: Add a period to end the second to last sentence of the paragraph. 

2. Line 199-202: This sentence is not grammatically correct. 

3. When reporting percentages, please report coinciding standard deviations. See lines 207 and 208. 

4. Lines 209-213: This explanation of the principle of Loewe Additivity should be moved to the Methodology section. 

5. Line 219: Change “quantitated” to “quantified”.

6. Consider splitting Results and Discussion into subheadings that coincide with a subheading of the Methodology section for clarity. 

Conclusion

Major issues:

1. Line 251-252: Change “...besifloxacin was observed to prevent the spread of infection in the kidneys of mice” to “...reduce the spread of infection to the kidneys” to reflect the findings of an 83% reduction of fungal burden rather than total clearance.  

2. Line 256: “...broadening treatment options” is an overstatement and should be replaced with a conclusion that is grounded in the data and findings of this study. Reframe as “...providing preliminary evidence to support further investigations into utility of BS in combination with FLC to treat systemic candidiasis” or a similar conclusion. 

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they used generative AI to come up with new ideas for their review.