Stress-Activated Protein Kinase Pathways as Potential Targets for the Development of New Antifungals
Round 1
Reviewer 1 Report
The authors presented an interesting review about the topic of HOG dependent signal cascade as targets for the development of new antifungals. They also showed that the pathway is connected with azole and amphotericin B response.
Figure 1 is a very informative figure but should be better connected with the following text.
Lines 140-150. The overall description of the two-component system should be better connected to Figure 1A. If I understand the authors correctly, the osmosensor represents the histidine kinase genes.
Figure 1B is very impressive, but I miss a chapter about C. auris. Therefore, this part of the figure is not explained within the text.
Lines 240-247. The HOG pathway repressed the ergosterol biosynthesis and if deleted more ergosterol is formed. I do not understand the molecular mechanism, why the increase of ergosterol lead to a higher susceptibility against amphotericin B. May be the authors know some literature to enlighten this problem. Overexpression of Erg11 genes is a simple mechanism for azole resistance. In this respect what is the reason that itraconazole behave different than fluconazole and ketoconazole? May be the authors could enlighten also this problem. Are there changes in the protein structure of Erg11?
Finally, the authors should include an abbreviation list. Sometimes, the text is difficult to understand.
Author Response
“Figure 1 is a very informative figure but should be better connected with the following text. “Lines 140-150. The overall description of the two-component system should be better connected to Figure 1A. If I understand the authors correctly, the osmosensor represents the histidine kinase genes.”
We appreciate this relevant reviewer’s insight and agree that the text required revision to more accurately reflect the information depicted in the Figure 1. As you will see, the text has been revised as stated in the following epigraph as well as in distinct parts of the manuscript.
(Lines 167-174). “This phosphorelay system is initiated with the autophosphorylation of a Histidine residue in the histidine kinase, acting as sensor. Then, phosphate is sequentially transferred to Aspartic also in the Histidine Kinase, to be later transferred to an acid aspartic residue in an intermediate phosphorelay (phosphotransfer) protein. Finally, phosphate is transferred to a histidine residue in a response regulator protein [39]. In the presence of stimuli, autophosphorylation is prevented, Dephosphorylation of the response regulator protein is an potent signal to activate the MAPK module [39][36].
Figure 1 and the figure caption have also been changed”.
Figure 1B is very impressive, but I miss a chapter about C. auris. Therefore, this part of the figure is not explained within the text.
We really thank the reviewer’s observation. The section on C. auris must have been inadvertently omitted throughout the various versions elaborated of the manuscript. This important clue has now been included in this definitive edition.
“Lines 240-247. The HOG pathway repressed the ergosterol biosynthesis and if deleted more ergosterol is formed. I do not understand the molecular mechanism, why the increase of ergosterol lead to a higher susceptibility against amphotericin B. May be the authors know some literature to enlighten this problem….”
Thank you for raising this important mechanistic point. The increased susceptibility to amphotericin B under conditions of elevated ergosterol level is consistent with its mechanism of action, as amphotericin B binds directly to ergosterol in the fungal membrane to form pores and disrupt membrane integrity. Thus, increased ergosterol content enhances the availability of binding sites, potentiating amphotericin B–mediated toxicity (PMID: 22308411; PMID: 31745151). In contrast, azoles inhibit lanosterol 14-α-demethylase (Erg11), leading to reduce ergosterol synthesis. Overexpression of the ERG11 gene has been associated with diminished azole efficacy by partially compensating for enzyme inhibition (PMID: 40583995).
This significant information, together with a new paragraph, has been included:
(lines 304-319) “These differential susceptibilities are consistent with the mechanisms of action of AMB and azoles. While AMB binds directly to ergosterol, forming pores and disrupting membrane integrity, azoles inhibit Cyp51A, thereby reducing ergosterol synthesis. Therefore, higher ergosterol levels potentiate AMB toxicity by increasing target availability [22,69], whereas they attenuate azole efficiency by partially compensating for the enzymatic blockade induced by these drugs [70]. Furthermore, differences between FLC, FTC, and ITC may be attributable to species-specific differences in Erg11 protein sequence. For example, A. fumigatus Cyp51A enzyme (Erg11 ortholog) contains an isoleucine at position 301, where C. albicans has a threonine (T315). This single amino acid substitution substantially reduces FLC and KTC binding, explaining A. fumigatus's intrinsic resistance to those drugs. By contrast, ITC retains its antifungal activity due to its bulkier structure and stronger binding affinity, which are less affected by this variation [71]. Likewise, in C. neoformans, a Y145F substitution in Erg11 abolishes the FLC and VRC activity, but paradoxically enhances susceptibility to ITC and PSZ, highlighting the existence of drug-specific interactions within the binding pocket [72]”.
-Finally, the authors should include an abbreviation list.
- In fact, a small list has been included in the original manuscript. However, it omitted some terms which appear abbreviated in the text; this has now been remedied by means of their replacement with a new extended list.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Line 13: Define WHO
Line 27: Mention these four fungal priority pathogens.
Line 34: Define WHO
Lines 37-38: Reference
Line 51: Mention some examples of these strains and what antifungal drugs.
Lines 61-63: References
I suggest placing the base chemical structures of polyenes, azoles, and echinocandins.
Line 77: Mention the species of these microorganisms.
Lines 84-85: Reference
Line 103: mention some examples.
Lines 107-109: References
Line 110-113: References.
Lines 162-163: References
Line 189: Reference
Line 216: Define CWI
Lines 220-222: References
Lines 222-224: References
Line 245: (imidazole), What exactly do you mean by this?
Line 248: Define CNS
Lines 248: 253: Reference
Although you mention references that expand on or delve deeper into the mechanism of action of polyene antifungals, azoles, and echinocandins, briefly describe the mechanism of action of each group in the corresponding section, with their respective references.
Lines 81-83: Please mention the fungi on which these experiments were conducted and their respective references.
Throughout the text, there are several incomplete ideas. To mention one example (line 103): “Different compounds, directed mainly to invasive mycoses, are under clinical trials.” Here, neither the compounds nor the type of mycosis are mentioned.
lines 107-109: Although there are similarities, there is a difference between the pathogens referred to in the review and the hosts. I am referring to the cell wall; it is necessary to comment on this and discuss how feasible it is to use this difference, and then continue with what relates to the SAPK pathway.
Lines 157-159. The manuscript should discuss genetic deletion and susceptibility or resistance in greater detail.
Author Response
Reviewer #2
“In general, Fig. 1 needs improvement. The caption mentions the secondary signaling branch identified in S. cerevisiae, but the figure does not show what it is. What is mentioned in lines 157-159”. Regarding genetic deletion and susceptibility or resistance, you should mention it a little more extensively in the manuscript, with the corresponding references…"
This central query is shared by the two Reviewers. Indeed, we have already addressed this question in our response to Rev. #1. We think these explanations are also valid for this Reviewer.
In addition, both the figure and the figure caption have been improved, we expect the information is clearer now.
Line 13: Define WHO…; Line 34: Define WHO
The reviewer recommends the complete denomination of WHO should be included various times in the text. Although this might be useful, the majority of potential readers are habituated to this terminology and we think that after the first indication, further repetitions are not necessary.
“Line27: Mention these four fungal priority pathogens.”
The names of these relevant fungi have been included.
Lines 37-38: Reference
The reference is the same as in the previous sentence. We consider that it is not required to duplicate it.
Line 51: Mention some examples of these strains and what antifungal drugs.
This information will be mentioned in the manuscript. Therefore, a reference reporting this information has been included.
Lines 61-63: References
The reference Campoy et al. Antifungals. Biochem. Pharmacol. 133 (2017), has been included.
I suggest placing the base chemical structures of polyenes, azoles, and echinocandins.
The chemical structure of these antifungals has unequivocally been established long time ago, and we remark this fact in the new version to focus the readers’ attention on the important number of available revisions [4, 14-16].
Line 77: Mention the species of these microorganisms.
The sentence has been rephrased as follows for accuracy (line 80-86): This group of cyclic organic molecules contains the largest number of antifungals. In particular, the former triazoles (i.e., fluconazole (FLC) and itraconazole (ITC), and those of the second generation (i.e., voriconazole (VRC) or posaconazole) show high efficacy against opportunistic mycosis, and are applied for prophylaxis of invasive infections caused by Candida spp. Azoles are also used to treat aspergillosis caused mainly by A. fumigatus [11,23], although VRC and ITC-resistant strains of A. fumigatus have been isolated in different European countries [24]
Detailed comments
-Although you mention references that expand on or delve deeper into the mechanism of action of polyene antifungals, azoles, and echinocandins, briefly describe the mechanism of action
The reviewer is right. The mechanism of action is crucial for the antifungal activity and a brief description should be adequate. We have carried out this for each family of compounds directly in the text as follows:
(l.68 71): Polyenes specifically bind to the ergosterol present in the fungal plasma membrane, causing a loss of permeability and metabolic alterations in the host. Additionally, AMB induces the production of Reactive Oxygen Species (ROS), contributing to its fungicidal effect.
(l.86-89 ): Azoles inhibit the fungal cytochrome P450-dependent enzyme lanosterol 14-α-demethylase, causing an ergosterol deficit, which impairs the correct structure of the cell membrane and accumulation of toxic sterol precursors.
(l.104-107 ): Echinocandins act as non-competitive inhibitors of the β-(1,3)-D-glucan synthase that catalyzes the formation of β-glucan polymers, impeding the biosynthesis of essential components of the fungal cell wall.
Lines 81-83: Please mention the fungi on which these experiments were conducted
Some information has been provided to respond to the reviewer´s suggestion: These types of resistance mechanisms have been identified in the four priority fungal pathogens. The presence of active biofilms or alterations of metabolic pathways leading to a reduction or loss of function are also additional ways to acquire resistance in C. albicans and other fungi [15].
“Throughout the text, there are several incomplete ideas. To mention one example (line 103): “Different compounds, directed mainly to invasive mycoses, are under clinical trials.” Here, neither the compounds nor the type of mycosis are mentioned”.
The number of antifungal agents under clinical trials or in vitro investigation is too long to be summarized in a paragraph. Therefore, we have added the following sentence that we hope will respond to reviewer's suggestion (l. 120-125): The emerging antifungals display diverse mechanisms of action and interact with different components. Compounds acting on traditional targets such as cell wall polymer biosynthesis, specific plasma membrane elements (e.g. ergosterol), or intracellular metabolism are currently under study. Alternative strategies, such as biofilm or mycelium development and adjuvant immunotherapy, are also being investigated. (see [29] for more details). ).
“lines 107-109: Although there are similarities, there is a difference between the pathogens referred to in the review and the hosts. I am referring to the cell wall; it is necessary to comment on this and discuss how feasible it is to use this difference, and then continue with what relates to the SAPK pathway”.
We have included a short sentence at the beginning of the paragraph to focus the statement: “The antifungals in clinical use are directed against specific metabolic or biosynthetic fungal pathways. Nevertheless, a major challenge in antifungal chemotherapy...”.
Lines 157-159. The manuscript should discuss genetic deletion and susceptibility or resistance in greater detail.
More information has been included in the manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
I thank the authors for taking the time to address my comments on their review. At this time, I have no further suggestions or comments.
N/A
