Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.

A series of indazole analogs, derived from the B,C-ring-truncated scaffold of deguelin, were designed to function as C-terminal inhibitors of heat shock protein 90 (HSP90) and investigated as novel antitumor agents against HER2-positive breast cancer. Among the synthesized compounds, compound 12d exhibited substantial inhibitory effects in trastuzumab-sensitive (BT474) and trastuzumab-resistant (JIMT-1) breast cancer cells, with IC50 values of 6.86 and 4.42 μM, respectively. Notably, compound 12d exhibited no cytotoxicity in normal cells. Compound 12d markedly downregulated the expression of the major HSP90 client proteins in both cell types, attributing its cytotoxicity to the destabilization and inactivation of HSP90 client proteins. Molecular docking studies using the homology model of an HSP90 homodimer demonstrated that inhibitor 12d fit nicely into the C-terminal domain, boasting a higher electrostatic complementary score than ATP. In vivo pharmacokinetic study indicated the high oral bioavailability of compound 12 d at F = 66.9 %, while toxicological studies indicated its negligible impact on hERG channels and CYP isozymes. Genotoxicity tests further confirmed its safety profile. The findings collectively position compound 12d as a promising candidate for further development as an antitumor agent against HER2-positive breast cancer.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

The mechanistic target of rapamycin (mTOR) is a master regulator of cell growth and metabolism, integrating environmental signals to regulate anabolic and catabolic processes, regulating lipid synthesis, growth factor-induced cell proliferation, cell survival, and migration. These activities are performed as part of two distinct complexes, mTORC1 and mTORC2, each with specific roles. mTORC1 and mTORC2 are elaborated dimeric structures formed by the interaction of mTOR with specific partners. mTOR functions only as part of these large complexes, but their assembly and activation require a dedicated and sophisticated chaperone system. mTOR folding and assembly are temporarily separated with the TELO2-TTI1-TTI2 (TTT) complex assisting the cotranslational folding of mTOR into a native conformation. Matured mTOR is then transferred to the R2TP complex for assembly of active mTORC1 and mTORC2 complexes. R2TP works in concert with the HSP90 chaperone to promote the incorporation of additional subunits to mTOR and dimerization. This review summarizes our current knowledge on how the HSP90-R2TP-TTT chaperone system facilitates the maturation and assembly of active mTORC1 and mTORC2 complexes, discussing interactions, structures, and mechanisms.

Tumors develop in an oxidative environment characterized by peroxynitrite production and downstream protein tyrosine (Y) nitration. We showed that tyrosine nitration supports schwannoma cell proliferation and regulates cell metabolism in the inheritable tumor disorder NF2-related Schwannomatosis (NF2-SWN). Here, we identified the chaperone Heat shock protein 90 (Hsp90) as the first nitrated protein that acts as a metabolic switch to promote schwannoma cell proliferation. Doubling the endogenous levels of nitrated Hsp90 in schwannoma cells or supplementing nitrated Hsp90 into normal Schwann cells increased their proliferation. Metabolically, nitration on either Y33 or Y56 conferred Hsp90 distinct functions; nitration at Y33 (Hsp90NY33) down-regulated mitochondrial oxidative phosphorylation, while nitration at Y56 (Hsp90NY56) increased glycolysis by activating the purinergic receptor P2X7 in both schwannoma and normal Schwann cells. Hsp90NY33 and Hsp90NY56 showed differential subcellular and spatial distribution corresponding with their metabolic and proliferative functions in schwannoma three-dimensional cell culture models. Collectively, these results underscore the role of tyrosine nitration as a post-translational modification regulating critical cellular processes. Nitrated proteins, particularly nitrated Hsp90, emerge as a novel category of tumor-directed therapeutic targets.

Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90\'s apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.

The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors.
OBJECTIVE: The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.

Glioblastoma multiform (GBM) is categorized as the most malignant subtype of gliomas, which comprise nearly 75% of malignant brain tumors in adults. Increasing evidence suggests that network pharmacology will be a novel method for identifying the systemic mechanism of therapeutic compounds in diseases like cancer. The present study aimed to use a network pharmacology approach to establish the predictive targets of sciadopitysin against GBM and elucidate its biological mechanisms. Firstly, targets of sciadopitysin were obtained from the SwissTargetPrediction database, and genes associated with the pathogenesis of GBM were identified from the DiGeNET database. Sixty-four correlative hits were identified as anti-glioblastoma targets of sciadopitysin. Functional enrichment and pathway analysis revealed significant biological mechanisms of the targets. Interaction of protein network and cluster analysis using STRING resulted in two crucial interacting hub genes, namely, HSP90 and AKT1. Additionally, the in vitro cytotoxic potential of sciadopitysin was assessed on GBM U87 cells. The findings indicate that the pharmacological action of sciadopitysin against GBM might be associated with the regulation of two core targets: HSP90 and AKT1. Thus, the network pharmacology undertaken in the current study established the core active targets of sciadopitysin, which may be extensively applied with further validations for treatment in GBM.

BACKGROUND: The surplus cytokines remaining after use in the early stages of the inflammatory response stimulate immune cells even after the response is over, causing a secondary inflammatory response and ultimately damaging the host, which is called a cytokine storm. Inhibiting heat shock protein 90 (Hsp90), which has recently been shown to play an important role in regulating inflammation in various cell types, may help control excessive inflammatory responses and cytokine storms.
METHODS: We discovered an anti-inflammatory compound by measuring the inhibitory effect of CD86 expression on spleen DCs (sDCs) using the chemical compounds library of Hsp90 inhibitors. Subsequently, to select the hit compound, the production of cytokines and expression of surface molecules were measured on the bone marrow-derived DCs (BMDCs) and peritoneal macrophages. Then, we analyzed the response of antigen-specific Th1 cells. Finally, we confirmed the effect of the compound using acute lung injury (ALI) and delayed-type hypersensitivity (DTH) models.
RESULTS: We identified Be01 as the hit compound, which reduced CD86 expression the most in sDCs. Treatment with Be01 decreased the production of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β) in BMDC and peritoneal macrophages stimulated by LPS. Under the DTH model, Be01 treatment reduced ear swelling and pro-inflammatory cytokines in the spleen. Similarly, Be01 treatment in the ALI model decreased neutrophil infiltration and lower levels of secreted cytokines (IL-6, TNF-α).
CONCLUSIONS: Reduction of CD80 and CD86 expression on DCs by Be01 indicates reduced secondary inflammatory response by Th1 cells, and reduced release of pro-inflammatory cytokines by peritoneal macrophages may initially control the cytokine storm.

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.