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Dual action antifungal small molecule modulates multidrug efflux and TOR signaling
Open accessAbstract
There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. We establish two activities of the natural product beauvericin, which potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens, blocks the emergence of drug resistance, and renders resistant pathogens responsive to treatment in mammalian infection models. Harnessing genome sequencing of beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for fungal infectious disease that evades resistance.
Overview
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Written from the paper’s full text.
TL;DR
Beauvericin potentiates azole antifungals against major fungal pathogens by dual inhibition of multidrug efflux and TORC1 signaling, leading to CK2 activation and Hsp90 impairment. This combination blocks resistance emergence and shows therapeutic benefit in mammalian infection models.
Key findings
- Beauvericin had no antifungal activity alone but enhanced azole efficacy against *S. cerevisiae*, *C. albicans*, *C. neoformans*, and *A. fumigatus*; against *C. albicans*, fluconazole–beauvericin synergy gave a fractional inhibitory concentration index (FICI) of <0.1 and converted fungistatic azole activity into a fungicidal combination.
- Beauvericin blocked emergence of azole resistance: plating 1×10⁵ *C. albicans* cells on 32 μg/ml fluconazole with 20 μg/ml beauvericin prevented resistant colonies.
- Beauvericin-resistant mutants selected at 100 μg/ml beauvericin in transporter-deficient backgrounds had mutations in *PDR5* (five mutants) or *CKB1*/*CKB2* (ten mutants, six independent events); Pdr5 substitutions enabled beauvericin efflux while impairing azole efflux.
- Affinity purification with biotinylated beauvericin identified 12 proteins at FDR ≤1%, including Cka2 and Tor2; beauvericin inhibited TORC1-dependent Sch9 phosphorylation, activated CK2, impaired Hsp90 clients, and in mice infected with 5×10⁶ *C. albicans*, beauvericin (4 mg/kg) plus fluconazole (0.5 mg/kg) gave potent therapeutic benefit; in a rat catheter biofilm model, the combination sterilized catheters.
Methods
- Drug combination and resistance assays used azole Etest strips, checkerboard matrices, broth microdilution MIC testing, and spotting assays with *S. cerevisiae*, *C. albicans*, *C. neoformans*, and *A. fumigatus*, including *C. albicans* clinical isolates.
- Target identification combined selection of beauvericin-resistant mutants in ABC-transporter-deficient backgrounds, whole-genome sequencing, Sanger validation, and affinity purification coupled to mass spectrometry with a biotinylated beauvericin analog, analyzed by SAINTexpress.
- Mechanistic assays included radiolabeled [³H]-fluconazole accumulation, rhodamine-6G transport and accumulation, Pdr5 ATPase activity, CK2 kinase assays with Sic1 phosphorylation, qRT-PCR, Sch9/Ypk1 and Hog1 western blots, β-galactosidase reporters, and murine/rat infection models.
Limitations
- The paper does not state a dedicated limitations section.
- Evident from the text: the ABC16 selection experiment was performed once, and the murine disseminated-infection model used no blinding.
- Some target conclusions are indirect: excess unmodified beauvericin did not compete the biotinylated analog from most proteins, including Cka2, and beauvericin did not alter recombinant human CK2 activity, leaving fungal-specific or indirect effects unresolved.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“inhibits the global regulator TORC1 kinase”
- Also stated in: § Beauvericin activates CK2 and inhibits TORC1 · plain
“Beauvericin caused a significant reduction in Sch9 phosphorylation and a modest reduction in Ypk1 phosphorylation (Fig. 4e, Supplementary Fig. 10).”
- Also stated in: § Discussion · plain
“beauvericin inhibits TORC1”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Also stated in: § Beauvericin activates CK2 and inhibits TORC1 · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Whole genome sequencing of ten resistant mutants revealed that each harboured a mutation in either CKB1 or CKB2, encompassing six independent mutational events (Fig. 3a).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Mutations in CK2 subunits confer beauvericin resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We used a dose response matrix (checkerboard) to corroborate the synergy between the azole fluconazole and beauvericin against C. albicans, with a fractional inhibitory concentration index (FICI) of <0.1 (Fig. 1b).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole efficacy and blocks resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Beauvericin reduced resistance of all isolates (Fig. 1c), with cidality of the combination evident at higher azole concentrations”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole efficacy and blocks resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Inclusion of beauvericin in the medium at a concentration that did not impair growth (20 μg/ml) blocked the emergence of resistance (Fig. 1d).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole efficacy and blocks resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“deletion of either CKA1 or CKA2 from the ABC16 strain confers partial resistance to beauvericin (Supplementary Fig. 6)”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Mutations in CK2 subunits confer beauvericin resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Transferring cells from the checkerboard onto drug-free rich medium revealed that beauvericin transforms azole fungistatic activity into a fungicidal combination (Fig. 1b).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole efficacy and blocks resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Beauvericin increased Sic1 phosphorylation, suggesting that it activates CK2 (Fig. 4b and Supplementary Fig. 8a).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin activates CK2 and inhibits TORC1
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Tor2 was identified in all three experiments”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Biotinylated beauvericin interacts with Cka2 and Tor2
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesA result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens”
- Also stated in: § Beauvericin enhances azole efficacy and blocks resistance · plain
“Beauvericin had no antifungal activity alone, but enhanced azole efficacy against the model yeast Saccharomyces cerevisiae and fungal pathogens C. albicans, C. neoformans, and A. fumigatus (Fig. 1a).”
- Also stated in: § Introduction · plain
“Beauvericin enhances azole efficacy against the leading human fungal pathogens, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Abstract
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passes- Rests on an assumption: The mechanism of beauvericin action (inhibition of Pdr5, TORC1, CK2, and Hsp90) identified in S. cerevisiae and C. albicans is conserved in other human fungal pathogens such as A. fumigatus and C. neoformans.
- Also stated in: § Beauvericin enhances azole efficacy and blocks resistance · plain
- It may state two separate claims as one. 13
- Probably not a substantial claim. 20
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Both the increase in fluconazole accumulation and the drug synergy were blocked in an S. cerevisiae strain lacking Pdr5 (Supplementary Fig. 5 and Fig. 2d).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole intracellular accumulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Beauvericin inhibited Pdr5 ATPase activity and transport of rhodamine-6G (Fig. 2e).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole intracellular accumulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“expression of wild-type CKB1 in the ABC16 ckb1Δ strain restored beauvericin susceptibility, while resistance was maintained upon expression of CKB1E220* (Fig. 3b).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Mutations in CK2 subunits confer beauvericin resistance
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
In the paper
“The interaction between beauvericin and rapamycin was abrogated by deletion of genes encoding FKBP12 (FPR1 in S. cerevisiae, RBP1 in C. albicans)25 and impervious to deletion of PDR5 (Supplementary Fig. 9), confirming the effects are mediated via TOR.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin activates CK2 and inhibits TORC1
What the sources say
- Not checked yetcites [25] Cruz, M.C. et al. Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR. Antimicrob. Agents Chemother. 45, 3162–3170 (2001). source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Beauvericin caused a significant reduction in Sch9 phosphorylation and a modest reduction in Ypk1 phosphorylation (Fig. 4e, Supplementary Fig. 10).”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin activates CK2 and inhibits TORC1
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Thus, CK2 and TOR may be binding targets of beauvericin through which it exerts antifungal activity.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Biotinylated beauvericin interacts with Cka2 and Tor2
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Thus, beauvericin enhances azole efficacy through inhibition of Pdr5, thereby causing increased azole accumulation within the fungal cell.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin enhances azole intracellular accumulation
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“qRT-PCR reactions were performed in triplicate for biological duplicate experiments.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Quantitative reverse transcription (RT)-PCR
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Together, these results suggest that beauvericin activates CK2 by binding to Tor2 and inhibiting TORC1 function.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin activates CK2 and inhibits TORC1
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“Inhibition of Hsp90 abrogates drug resistance in diverse human fungal pathogens, rendering resistant infections responsive to treatment7–9.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Introduction
What the sources say
- Not checked yetcites [7] Cowen, L.E. et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc. Natl. Acad. Sci. USA 106, 2818–2823 (2009). source
- Not checked yetcites [8] Robbins, N. et al. Hsp90 governs dispersion and drug resistance of fungal biofilms. PLoS Pathog. 7, e1002257 (2011). source
- Not checked yetcites [9] Cowen, L.E. & Lindquist, S. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309, 2185–2189 (2005). source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim. It may state two separate claims as one.
In the paper
“it was found to be well tolerated by human cells and to potentiate azole efficacy against Candida parapsilosis in a systemic infection model by a mechanism that remains enigmatic12.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Introduction
What the sources say
- Not checked yetcites [12] Zhang, L. et al. High-throughput synergy screening identifies microbial metabolites as combination agents for the treatment of fungal infections. Proc. Natl. Acad. Sci. USA 104, 4606–4611 (2007). source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesIt may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Experiments were performed in biological duplicates; plates were incubated at 30°C for five days and photographed each day.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Selection experiments
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“No blinding was done.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Murine model of disseminated candidiasis
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“In these conditions, recovered cells were still in late log phase.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Radiolabelled fluconazole accumulation assay
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“The difference of both assays corresponds to the OM-sensitive ATPase activity of Pdr550.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > ATPase Activity Assay
What the sources say
- Not checked yetcites [50] Ernst, R. et al. A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5. Proc. Natl. Acad. Sci. USA 105, 5069–5074 (2008). source
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What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“The average coverage of each genome was sufficient for confident variant detection and is listed in Supplementary Table 3.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Whole genome sequencing
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In the paper
“AP-MS was performed in three independent experiments.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Affinity purification coupled to mass spectrometry with biotinylated beauvericin
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“CKB1 and CKB2 encode the non-redundant beta regulatory subunits of protein kinase CK2, which is an essential serine/threonine protein kinase that phosphorylates over 300 substrates, including Hsp9017.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Mutations in CK2 subunits confer beauvericin resistance
What the sources say
- Not checked yetcites [17] Miyata, Y. Protein kinase CK2 in health and disease: CK2: the kinase controlling the Hsp90 chaperone machinery. Cell. Mol. Life Sci. 66, 1840–1849 (2009). source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“Pdr5 transports a diverse set of structurally unrelated compounds including azoles, cycloheximide, and rhodamines15.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Pdr5 substitutions enable beauvericin efflux
What the sources say
- Not checked yetcites [15] Prasad, R. & Goffeau, A. Yeast ATP-binding cassette transporters conferring multidrug resistance. Annu. Rev. Microbiol. 66, 39–63 (2012). source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“All strains were assessed in biological triplicate experiments with technical duplicates.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Drug susceptibility and checkerboard assays
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What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“No growth defect was observed in comparison to media without methanol.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Selection experiments
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“In S. cerevisiae and C. albicans, CK2 phosphorylates Hsp90, thereby modulating chaperone function27,28.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin impairs Hsp90 function
What the sources say
- Not checked yetcites [27] Mollapour, M. et al. Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity. Mol. Cell 41, 672–681 (2011). source
- Not checked yetcites [28] Diezmann, S., Michaut, M., Shapiro, R.S., Bader, G.D. & Cowen, L.E. Mapping the Hsp90 genetic interaction network in Candida albicans reveals environmental contingency and rewired circuitry. PLoS Genet. 8, e1002562 (2012). source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
None of the sources cited for this claim have been checked yet.
In the paper
“Hsp90 regulates cellular responses required to survive drug-induced stress, and inhibition of Hsp90 transforms azoles from fungistatic to fungicidal7,9.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Results > Beauvericin impairs Hsp90 function
What the sources say
- Not checked yetcites [7] Cowen, L.E. et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc. Natl. Acad. Sci. USA 106, 2818–2823 (2009). source
- Not checked yetcites [9] Cowen, L.E. & Lindquist, S. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309, 2185–2189 (2005). source
Tests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“The selection experiment with the ABC16 strain was performed once, while the selection experiment with the yor1Δ strain was done in biological triplicates.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Selection experiments
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Control experiments were performed in which the biotinylated-beauvericin was omitted.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Affinity purification coupled to mass spectrometry with biotinylated beauvericin
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Mutations identified in CKB1, CKB2, and PDR5 were validated by Sanger sequencing.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Whole genome sequencing
Load-bearingStrength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✓ Load-bearing: passes✓ Based: passesProbably not a substantial claim.
In the paper
“Hsp90, an essential molecular chaperone that stabilizes diverse cellular regulators6.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Introduction
What the sources say
- Not checked yetcites [6] Leach, M.D., Klipp, E., Cowen, L.E. & Brown, A.J.P. Fungal Hsp90: a biological transistor that tunes cellular outputs to thermal inputs. Nat. Rev. Microbiol. 10, 693–704 (2012). source
Strength:plain· not yet calibratedCausal:direct· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Infected mice were randomly chosen for each group.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Murine model of disseminated candidiasis
Strength:plain· not yet calibratedTests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.
In the paper
“Survival data for all of the tested mice were included in the statistical analysis.”
§ Dual Action Antifungal Small Molecule Modulates Multidrug Efflux and TOR Signaling > Online Methods > Murine model of disseminated candidiasis
Strength:plain· not yet calibratedn = 10Tests✓ Asserted: passes✓ Checkable: passes✗ Load-bearing: fails✓ Based: passesProbably not a substantial claim. The paper’s conclusions may not depend on it.
The paper relies on this without support.
The mechanism of beauvericin action (inhibition of Pdr5, TORC1, CK2, and Hsp90) identified in S. cerevisiae and C. albicans is conserved in other human fungal pathogens such as A. fumigatus and C. neoformans.
“potentiates the activity of the most widely deployed class of antifungal against the leading human fungal pathogens”
“Thus, dual targeting of multidrug efflux and TOR signaling provides a powerful, broadly effective therapeutic strategy for fungal infectious disease that evades resistance.”
The paper relies on this without support.
The therapeutic benefit of the beauvericin-fluconazole combination in mammalian infection models is mediated by the same dual targeting of multidrug efflux and TOR signaling.
“As a combination, there was potent therapeutic benefit of beauvericin and fluconazole (Fig. 6a).”
“Our findings reveal that dual targeting of multidrug efflux and TOR signaling provides a powerful strategy to enhance antifungal efficacy, impede drug resistance, and render resistant pathogens responsive to treatment.”
The paper relies on this without support.
The Pdr5 substitutions that enable beauvericin efflux and impair azole efflux are representative of resistance mechanisms that would emerge in clinical settings.
“Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“The natural product beauvericin, which potentiates azole activity via both inhibition of TOR and multidrug efflux, is effective against diverse fungal pathogens including C. albicans, C. neoformans, and A. fumigatus87.”
From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [87]
In the citing paper
“The natural product beauvericin, which potentiates azole activity via both inhibition of TOR and multidrug efflux, is effective against diverse fungal pathogens including C. albicans, C. neoformans, and A. fumigatus87.”
From Molecular mechanisms governing antifungal drug resistance (2023) · cites this paper as [87]
In the citing paper
“Combination therapy is an alternative approach to overcoming the growing microbial infection resistance [23,24].”
From Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris (2024) · cites this paper as [23]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
AP-MS buffer conditions counted inconsistently
MinorThe Results state that AP-MS used three distinct buffer conditions, but the Methods describe only two: experiments 1 and 2 used the same NP-40 buffer, while experiment 3 used Triton X-100 buffer.
“AP-MS was performed under three distinct buffer conditions with biological duplicates and negative controls.”
“Experiments 1 and 2 were performed using 0.1% NP-40 buffer, and experiment 3 was performed using 1% Triton X-100 buffer to allow for efficient extraction of membrane proteins.”
Citations
Cites
Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples
Cibulskis, K., Lawrence, M. S., Carter, S. L., Sivachenko, A., Jaffe, D. B., Sougnez, C. L., Gabriel, S., Meyerson, M. M., Lander, E. S., & Getz, G. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2514 · Cited by 4,965
Hidden Killers: Human Fungal Infections
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Targets for Cell Cycle Arrest by the Immunosuppressant Rapamycin in Yeast
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Self-citationSynergistic drug combinations tend to improve therapeutically relevant selectivity
Lehár, J., Krueger, A., Avery, W., Heilbut, A. M., Johansen, L. M., Price, E. R., Rickles, R. J., Short, G. F., Staunton, J. E., Jin, X., et al. (2009) · Nature Biotechnology · doi:10.1038/nbt.1549 · Cited by 959
The promise and peril of chemical probes
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SAINT: probabilistic scoring of affinity purification–mass spectrometry data
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Self-citationSch9 Is a Major Target of TORC1 in Saccharomyces cerevisiae
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Self-citationHsp90 Potentiates the Rapid Evolution of New Traits: Drug Resistance in Diverse Fungi
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Self-citation
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BindingDB Entry 50021197: Selenization of Small Molecule Drugs: A New Player on the Board.
BindingDB (2025) · UC San Diego · doi:10.7270/q2m331cq
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Metadata
- Authors
- Shekhar-Guturja, T., Gunaherath, G. M. K. B., Wijeratne, E. M. K., Lambert, J.-P., Averette, A. F., Lee, S. C., Kim, T., Bahn, Y.-S., Tripodi, F., Ammar, R., Döhl, K., Niewola-Staszkowska, K., Schmitt, L., Loewith, R. J., Roth, F. P., Sanglard, D., Andes, D., Nislow, C., Coccetti, P., Gingras, A.-C., Heitman, J., Gunatilaka, A. A. L., & Cowen, L. E.
- Affiliations
- University of Arizona (US)
- Mount Sinai Hospital (CA)
- Duke Medical Center (US)
- Yonsei University (KR)
- University of Milano-Bicocca (IT)
- University of Toronto (CA)
- Heinrich Heine University Düsseldorf (DE)
- University of Geneva (CH)
- Centre Hospitalier Universitaire Vaudois (CH)
- University of Wisconsin–Madison (US)
- University of British Columbia (CA)
- Corresponding author
- Cowen, Leah E
- ORCID
- Gunaherath, G M Kamal B
- Wijeratne, E M Kithsiri
- Lambert, Jean-Philippe
- Averette, Anna F
- Lee, Soo Chan
- Kim, Taeyup
- Bahn, Yong-Sun
- Tripodi, Farida
- Ammar, Ron
- Schmitt, Lutz
- Loewith, Robbie J
- Roth, Frederick P
- Sanglard, Dominique
- Andes, David
- Nislow, Corey
- Coccetti, Paola
- Gingras, Anne-Claude
- Heitman, Joseph
- Gunatilaka, A A Leslie
- Cowen, Leah E
- Venue
- Nature Chemical Biology
- Published
- 2016-08-29
- Volume
- 12
- Issue
- 10
- Pages
- 867-875
- ISSN
- 1552-4450 (ISSN-L)
- 1552-4469
- PMC
- PMC5030160
- OpenAlex
- W2509038340
- Semantic Scholar
- 703e3c9f64b33ff7226b5d45e13b72dc9ef9243c
- Cited by
- 101
- References
- 57
- Citations by year
- 2026: 5 · 2025: 10 · 2024: 11 · 2023: 9 · 2022: 15 · 2021: 8 · 2020: 13 · 2019: 10 · 2018: 9 · 2017: 9 · 2016: 2
- FWCI
- 9.24
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Chemical Biology (publishedVersion)
- PubMed (publishedVersion)
- Archive ouverte UNIGE (University of Geneva) (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- SERVAL (Université de Lausanne) (acceptedVersion, other-oa)
- IRIS (submittedVersion)
- BOA (University of Milano-Bicocca) (submittedVersion)
- Archive ouverte UNIGE (University of Geneva) (submittedVersion, cc-by)
- Funders
- TD Bank
- Genome Canada
- Ministry of Trade, Industry and Energy (N0001720)
- University of Toronto
- National Institutes of Health (5r01ai112595-04)
- Canadian Institutes of Health Research (119520_1, MOP-86452)
- Natural Sciences and Engineering Research Council of Canada (462167, 06261)
- National Cancer Institute (R01CA090265)
- National Institute of General Medical Sciences (P41GM094060)
- National Institute of Allergy and Infectious Diseases (R01-AI112595, P01AI104533, R56AI112595)
- Topics
- Health Sciences › Medicine › Infectious Diseases › Antifungal resistance and susceptibility
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
- Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Microbial Natural Products and Biosynthesis
- Keywords
- beauvericin, antifungal resistance, multidrug efflux, TOR signaling, casein kinase 2, HSP90 inhibition, invasive fungal infections, natural products, drug repurposing
- MeSH terms
- Antifungal Agents, Fungi, Microbial Sensitivity Tests, Mycoses, Signal Transduction, Drug Resistance, Multiple, ATP-Binding Cassette Transporters, HSP90 Heat-Shock Proteins, Drug Resistance, Fungal, Saccharomyces cerevisiae Proteins, Protein Kinase Inhibitors, Depsipeptides, Small Molecule Libraries, TOR Serine-Threonine Kinases
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
A language model drew the claims from the paper on 2026-10-09, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).
A second model labelled each claim on 2026-10-09 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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