Novel Macrocyclic Amidinoureas: Potent Non-Azole Antifungals Active against Wild-Type and Resistant Candida Species

Novel macrocyclic amidinourea derivatives <b>11</b>, <b>18</b>, and <b>25</b> were synthesized and evaluated as antifungal agents against wild-type and fluconazole resistant Candida species. Macrocyclic compounds <b>11</b> and <b>18</b> were synthesized through a convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds <b>25</b> were obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida strains showing in many cases minimum inhibitory concentration values lower than voriconazole

Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT <i>C. albicans</i> cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.

<p>Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT <i>C. albicans</i> cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.</p

<i>HSF1</i> conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous in presence of BER.

<p><i>HSF1</i> conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous in presence of BER.</p

TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of <i>HSF1</i> (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.

<p>TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of <i>HSF1</i> (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.</p

Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and <i>HSP90</i> gene deleted to evaluate BER MIC₅₀, (b) end point comparative RTPCR of genes involved in CW integrity in WT <i>C. albicans</i> cells in presence and absence of BER, (c) and in <i>HSF1</i> conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.

<p>Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and <i>HSP90</i> gene deleted to evaluate BER MIC₅₀, (b) end point comparative RTPCR of genes involved in CW integrity in WT <i>C. albicans</i> cells in presence and absence of BER, (c) and in <i>HSF1</i> conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.</p

BER treatment results in dysfunctional mitochondria (a) growth of <i>C. albicans</i> cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in <i>C. albicans</i> WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous strains in presence and absence of BER.

<p>BER treatment results in dysfunctional mitochondria (a) growth of <i>C. albicans</i> cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in <i>C. albicans</i> WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous strains in presence and absence of BER.</p

Model depicting pathways affected by BER treatment in <i>C. albicans</i>.

<p>Model depicting pathways affected by BER treatment in <i>C. albicans</i>.</p

Antifungal potential of BER (a) Growth curve of WT <i>C. albicans</i> cells at 100, 150 and 200 µg/ml, (b) serial dilution assays in solid (left panel) and liquid medium for testing BER susceptibility of <i>C. albicans</i> and non albicans species.

<p>(c) Serial dilution assays of <i>CDR1</i> (Gu5) and <i>MDR1</i> (F5) overexpressing and (d) their deletions strains in presence of BER.</p

Estimation of the quality of the mapping onto KEGG maps by performing a re-prediction of the annotation of <i>S. pombe</i> proteome through intermediary data set consisting of one, two, three, or 18 fungal proteomes.

<p>The KO - <i>S. pombe</i> association pairs obtained by “blasting” an intermediary data set were evaluated <i>a posteriori</i> as true positive (TP) or false positive (FP) according to the KO - <i>S. pombe</i> mapping which is provided by KEGG. Those missed KO - <i>S. pombe</i> pairs existing in KEGG were taken as false negatives (FN). The overall quality of the obtained mapping can be expressed in terms of precision TP/(TP+FP) and recall TP/(TP+FN).</p

Number of enzymes dedicated to the biosyntheses of amino acids identified in <i>P. carinii</i> and <i>S. pombe</i>^a.

a<p>The reference gene numbers of <i>S. pombe</i> are those obtained from KEGG.</p>b<p>The four enzymes are the same for Ile and Val syntheses.</p>c<p>One of the enzymes is also involved in Ile and Val syntheses.</p