Synthesis and antifungal evaluation of novel triazole derivatives bearing a pyrazole-methoxyl moiety.

Life-threatening invasive fungal infections pose a serious threat to human health. A series of novel triazole derivatives bearing a pyrazole-methoxyl moiety were designed and synthesized in an effort to obtain antifungals with potent, broad-spectrum activity that are less susceptible to resistance. Most of these compounds exhibited moderate to excellent in vitro antifungal activities against Candida albicans SC5314 and 10,231, Cryptococcus neoformans 32,609, Candida glabrata 537 and Candida parapsilosis 22,019 with minimum inhibitory concentration (MIC) values of ≤0.125 µg/mL to 0.5 µg/mL. Use of recombinant Saccharomyces cerevisiae strains showed compounds 7 and 10 overcame the overexpression and resistant-related mutations in ERG11 of S. cerevisae and several pathogenic Candida spp. Despite being substrates of the C. albicans and Candida auris Cdr1 drug efflux pumps, compounds 7 and 10 showed moderate potency against five fluconazole (FCZ)-resistant fungi with MIC values from 2.0 µg/mL to 16.0 µg/mL. Growth kinetics confirmed compounds 7 and 10 had much stronger fungistatic activity than FCZ. For C. albicans, compounds 7 and 10 inhibited the yeast-to-hyphae transition, biofilm formation and destroyed mature biofilm more effectively than FCZ. Preliminary mechanism of action studies showed compounds 7 and 10 blocked the ergosterol biosynthesis pathway at Erg11, ultimately leading to cell membrane disruption. Further investigation of these novel triazole derivatives is also warranted by their predicted ADMET properties and low cytotoxicity.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 µg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

From Synergy to Monotherapy: Discovery of Novel 2,4,6-Trisubstituted Triazine Hydrazone Derivatives with Potent Antifungal Potency In Vitro and In Vivo.

Invasive fungal infections pose a serious threat to public health and are associated with high mortality and incidence rates. The development of novel antifungal agents is urgently needed. Based on hit-to-lead optimization, a series of 2,4,6-trisubstituted triazine hydrazone compounds were designed, synthesized, and biological evaluation was performed, leading to the identification of compound 28 with excellent in vitro synergy (FICI range: 0.094-0.38) and improved monotherapy potency against fluconazole-resistant Candida albicans and Candida auris (MIC range: 1.0-16.0 µg/mL). Moreover, 28 exhibited broad-spectrum antifungal activity against multiple pathogenic strains. Furthermore, 28 could inhibit hyphal and biofilm formation, which may be related to its ability to disrupt the fungal cell wall. Additionally, 28 significantly reduced the CFU in a mouse model of disseminated infection with candidiasis at a dose of 10 mg/kg. Overall, the triazine-based hydrazone compound 28 with low cytotoxicity, hemolysis, and favorable ADME/T characteristics represents a promising lead to further investigation.

Late consolidation of rRNA structure during co-transcriptional assembly in E. coli by time-resolved DMS footprinting.

The production of new ribosomes requires proper folding of the rRNA and the addition of more than 50 ribosomal proteins. The structures of some assembly intermediates have been determined by cryo-electron microscopy, yet these structures do not provide information on the folding dynamics of the rRNA. To visualize the changes in rRNA structure during ribosome assembly in E. coli cells, transcripts were pulse-labeled with 4-thiouridine and the structure of newly made rRNA probed at various times by dimethyl sulfate modification and mutational profiling sequencing (4U-DMS-MaPseq). The in-cell DMS modification patterns revealed that many long-range rRNA tertiary interactions and protein binding sites through the 16S and 23S rRNA remain partially unfolded 1.5 min after transcription. By contrast, the active sites were continually shielded from DMS modification, suggesting that these critical regions are guarded by cellular factors throughout assembly. Later, bases near the peptidyl tRNA site exhibited specific rearrangements consistent with the binding and release of assembly factors. Time-dependent structure-probing in cells suggests that many tertiary interactions throughout the new ribosomal subunits remain mobile or unfolded until the late stages of subunit maturation.