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Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

Butts A, Martin JA, DiDone L, Bradley EK, Mutz M, Krysan DJ - PLoS ONE (2015)

Bottom Line: Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity.Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules.Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14642, United States of America.

ABSTRACT
Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

No MeSH data available.


Related in: MedlinePlus

Effect of tamoxifen-derivatives on the temperature-induced, calmodulin-dependent nuclear localization of the transcription factor Crz1.As described in the materials and methods, C. neoformans cells harboring Crz1-mCherry and the nuclear marker Nop1-GFP were shifted to 37°C in the presence or absence of the indicated tamoxifen derivative (see Figs 2–4 for structures corresponding to the molecule numbers) at ¼ of minimum inhibitory concentration (MIC). The percentage of cells with co-localized mCherry and GFP signals was determined for a minimum of 100 cells with at least two independent biological samples. The bars indicate mean percentage of cells with nuclear Crz1 and error bars indicate standard deviation. All compounds except those with an asterisk (*) above the bar gave values that were significantly different than the untreated control (P< 0.05, Student’s unpaired, two-tailed t test). MIC values are taken from data in Figs 2–4.
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pone.0125927.g005: Effect of tamoxifen-derivatives on the temperature-induced, calmodulin-dependent nuclear localization of the transcription factor Crz1.As described in the materials and methods, C. neoformans cells harboring Crz1-mCherry and the nuclear marker Nop1-GFP were shifted to 37°C in the presence or absence of the indicated tamoxifen derivative (see Figs 2–4 for structures corresponding to the molecule numbers) at ¼ of minimum inhibitory concentration (MIC). The percentage of cells with co-localized mCherry and GFP signals was determined for a minimum of 100 cells with at least two independent biological samples. The bars indicate mean percentage of cells with nuclear Crz1 and error bars indicate standard deviation. All compounds except those with an asterisk (*) above the bar gave values that were significantly different than the untreated control (P< 0.05, Student’s unpaired, two-tailed t test). MIC values are taken from data in Figs 2–4.

Mentions: Tamoxifen and its derivatives are well-characterized inhibitors of calmodulin function [21] and we have shown that calmodulin inhibition is an important contributor to the mechanism of the antifungal activity of triphenylethylenes [12, 18]. Based on these results, we hypothesized that tamoxifen derivatives with good antifungal activity (MIC 2–8 μg/mL) would inhibit calmodulin function while those with poor activity (MIC 32–64 μg/mL) would not. To test this hypothesis, we used a cell-based reporter assay developed previously in our laboratory that allows us to monitor the localization of the transcription factor Crz1p [12]. Under conditions of heat stress in C. neoformans, calmodulin binds to, and activates, the protein phosphatase calcineurin [22]. Calcineurin, in turn, dephosphorylates Crz1p which triggers its translocation to the nucleus were it is responsible for stress related gene expression [23]. The assay is based on a C. neoformans reporter strain that expresses both Crz1p-mCherry and Nop1p-GFP, a nucleolar protein, as a nuclear marker. When C. neoformans is incubated at 30°C, Crz1p is predominately localized in the cytoplasm with 38% of cells showing nuclear localization (Fig 5). Upon shifting cultures to 37°C, the proportion of cells with nuclear Crz1p increases to 87% (Fig 5). We have previously shown that this process is blocked by calmodulin antagonists [12] including the well-characterized calmodulin inhibitor trifluopromazine (Fig 5).


Structure-activity relationships for the antifungal activity of selective estrogen receptor antagonists related to tamoxifen.

Butts A, Martin JA, DiDone L, Bradley EK, Mutz M, Krysan DJ - PLoS ONE (2015)

Effect of tamoxifen-derivatives on the temperature-induced, calmodulin-dependent nuclear localization of the transcription factor Crz1.As described in the materials and methods, C. neoformans cells harboring Crz1-mCherry and the nuclear marker Nop1-GFP were shifted to 37°C in the presence or absence of the indicated tamoxifen derivative (see Figs 2–4 for structures corresponding to the molecule numbers) at ¼ of minimum inhibitory concentration (MIC). The percentage of cells with co-localized mCherry and GFP signals was determined for a minimum of 100 cells with at least two independent biological samples. The bars indicate mean percentage of cells with nuclear Crz1 and error bars indicate standard deviation. All compounds except those with an asterisk (*) above the bar gave values that were significantly different than the untreated control (P< 0.05, Student’s unpaired, two-tailed t test). MIC values are taken from data in Figs 2–4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446328&req=5

pone.0125927.g005: Effect of tamoxifen-derivatives on the temperature-induced, calmodulin-dependent nuclear localization of the transcription factor Crz1.As described in the materials and methods, C. neoformans cells harboring Crz1-mCherry and the nuclear marker Nop1-GFP were shifted to 37°C in the presence or absence of the indicated tamoxifen derivative (see Figs 2–4 for structures corresponding to the molecule numbers) at ¼ of minimum inhibitory concentration (MIC). The percentage of cells with co-localized mCherry and GFP signals was determined for a minimum of 100 cells with at least two independent biological samples. The bars indicate mean percentage of cells with nuclear Crz1 and error bars indicate standard deviation. All compounds except those with an asterisk (*) above the bar gave values that were significantly different than the untreated control (P< 0.05, Student’s unpaired, two-tailed t test). MIC values are taken from data in Figs 2–4.
Mentions: Tamoxifen and its derivatives are well-characterized inhibitors of calmodulin function [21] and we have shown that calmodulin inhibition is an important contributor to the mechanism of the antifungal activity of triphenylethylenes [12, 18]. Based on these results, we hypothesized that tamoxifen derivatives with good antifungal activity (MIC 2–8 μg/mL) would inhibit calmodulin function while those with poor activity (MIC 32–64 μg/mL) would not. To test this hypothesis, we used a cell-based reporter assay developed previously in our laboratory that allows us to monitor the localization of the transcription factor Crz1p [12]. Under conditions of heat stress in C. neoformans, calmodulin binds to, and activates, the protein phosphatase calcineurin [22]. Calcineurin, in turn, dephosphorylates Crz1p which triggers its translocation to the nucleus were it is responsible for stress related gene expression [23]. The assay is based on a C. neoformans reporter strain that expresses both Crz1p-mCherry and Nop1p-GFP, a nucleolar protein, as a nuclear marker. When C. neoformans is incubated at 30°C, Crz1p is predominately localized in the cytoplasm with 38% of cells showing nuclear localization (Fig 5). Upon shifting cultures to 37°C, the proportion of cells with nuclear Crz1p increases to 87% (Fig 5). We have previously shown that this process is blocked by calmodulin antagonists [12] including the well-characterized calmodulin inhibitor trifluopromazine (Fig 5).

Bottom Line: Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity.Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules.Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, NY 14642, United States of America.

ABSTRACT
Cryptococcosis is one of the most important invasive fungal infections and is a significant contributor to the mortality associated with HIV/AIDS. As part of our program to repurpose molecules related to the selective estrogen receptor modulator (SERM) tamoxifen as anti-cryptococcal agents, we have explored the structure-activity relationships of a set of structurally diverse SERMs and tamoxifen derivatives. Our data provide the first insights into the structural requirements for the antifungal activity of this scaffold. Three key molecular characteristics affecting anti-cryptococcal activity emerged from our studies: 1) the presence of an alkylamino group tethered to one of the aromatic rings of the triphenylethylene core; 2) an appropriately sized aliphatic substituent at the 2 position of the ethylene moiety; and 3) electronegative substituents on the aromatic rings modestly improved activity. Using a cell-based assay of calmodulin antagonism, we found that the anti-cryptococcal activity of the scaffold correlates with calmodulin inhibition. Finally, we developed a homology model of C. neoformans calmodulin and used it to rationalize the structural basis for the activity of these molecules. Taken together, these data and models provide a basis for the further optimization of this promising anti-cryptococcal scaffold.

No MeSH data available.


Related in: MedlinePlus