We conducted a phase Ib/II clinical trial to evaluate the safety, feasibility, and clinical activity of combining pembrolizumab (anti-PD-1) with XL888 (Hsp90 inhibitor) in patients with advanced colorectal cancer (CRC). We hypothesized that this regimen would modulate soluble and cellular immune mediators and enhance clinical outcomes. The trial employed a 3 + 3 open-label design, with an expansion cohort at the recommended phase II dose (RP2D) in treatment-refractory, mismatch repair-proficient CRC patients. Comprehensive analyses of plasma cytokines, peripheral blood mononuclear cells (PBMCs), and spatial immune cell patterns in liver biopsies were performed to identify unique immune signatures resulting from the combined therapy. The combination of pembrolizumab and XL888 proved to be safe and feasible, with a subset of patients achieving stable disease, although no objective responses were observed in this heavily pre-treated population. Correlative studies revealed immunomodulatory effects in tumors and circulation, including a reduction in IL6+ cells and macrophages (CD68+) within metastatic liver tissue, alterations in blood CD3+ cells, and upregulation of numerous inflammatory plasma cytokines. These findings suggest local and systemic immune activation by the combination of pembrolizumab and XL888. While clinical activity was modest in treatment-refractory CRC patients, there were notable effects on the tumor immune environment and systemic immune modulation.

Cancer stem cells (CSCs) represent a distinct subpopulation of cancer cells that orchestrate cancer initiation, progression, metastasis, and therapeutic resistance. Despite advances in conventional therapies, the persistence of CSCs remains a major obstacle to achieving cancer eradication. Nanomedicine-based approaches have emerged for precise CSC targeting and elimination, offering unique advantages in overcoming the limitations of traditional treatments. This review systematically analyzes recent developments in nanomedicine for CSC-targeted therapy, emphasizing innovative nanomaterial designs addressing CSC-specific challenges. We first provide a detailed examination of CSC biology, focusing on their surface markers, signaling networks, microenvironmental interactions, and metabolic signatures. On this basis, we critically evaluate cutting-edge nanomaterial engineering designed to exploit these CSC traits, including stimuli-responsive nanodrugs, nanocarriers for drug delivery, and multifunctional nanoplatforms capable of generating localized hyperthermia or reactive oxygen species. These sophisticated nanotherapeutic approaches enhance selectivity and efficacy in CSC elimination, potentially circumventing drug resistance and cancer recurrence. Finally, we present an in-depth analysis of current challenges in translating nanomedicine-based CSC-targeted therapies from bench to bedside, offering critical insights into future research directions and clinical implementation. This review aims to provide a comprehensive framework for understanding the intersection of nanomedicine and CSC biology, contributing to more effective cancer treatment modalities.

Targeted protein degradation (TPD) technology is a promising strategy for drug development, while the on-target off-tumor risks of current TPD technologies were intractable. Herein, a series of (HSP90)-mediated targeting chimeras (HEMTACs) based WEE1-target degraders were designed to enhance the efficiency and decrease off-tumor risks. Among them, 8b and 9c could effectively degrade cellular WEE1 protein and exhibited superior anti-proliferative activity in MV-4-11 cells by inducing cell cycle arrest in G2/M phase. Meanwhile, 8b and 9c exhibited high selectivity to primary AML cells over normal cells. Furthermore, 3 mg/kg of 9c demonstrated superior anti-cancer activity than 5 mg/kg AZD1775 in an AML PDX model. And most importantly, 9c exhibited lower hematotoxicity than equimolar AZD1775 in mice safety evaluation, suggesting that 9c is a promising degrader for AML target therapy, comfirming that HSP90-based HEMTACs is a valid strategy to reduce off-tumor risks.

Targeting Hsp90 is an effective strategy for cancer therapy. TAS-116 has been approved for the treatment of gastrointestinal stromal tumors. Our previous studies identified a series of pyrazole derivatives as covalent Hsp90 inhibitors that allosterically disrupt the Hsp90-Cdc37 interaction. Here, through systematic structure-activity relationship (SAR) optimization, compound 39 (DDO-6691) with a new covalent warhead was developed, which demonstrates improved ADME properties and significantly enhanced antitumor activity. Notably, parental HCT-116 cells exhibited markedly greater sensitivity to compound 39 (IC50 > 50 μM) compared to their Cdc37-knockout counterparts. Importantly, compound 39 displayed potent tumor growth inhibition in HCT-116 xenograft mouse models. These collective findings underscore the therapeutic promise of covalent Hsp90-targeted disruption of the Hsp90-Cdc37 complex, offering a novel mechanistic approach to cancer treatment.

The threat of drug-resistant pathogens continues to rise and underscores the need for new antimicrobial and antifungal strategies. Diverse chemical scaffolds have been shown with high affinity to bind the human heat-shock protein Hsp90. Orthologous proteins are present in microbial pathogens and have been shown to be particularly abundant in these organisms, suggesting they may serve as therapeutic targets. Here, we examine the potency and selectivity of human Hsp90 ligands for their capacity to bind to the nucleotide binding domain of Hsp90 from the pathogenic fungi, Candida albicans. Using a series of biochemical, structural, and fragment and in silico screening investigations, we define key chemical features that lead to effective C. albicans Hsp90 (CaHsp90) binding. We support these studies with crystal structures of five diverse human Hsp90 ligands in complex with CaHsp90, as well as the structure of this protein with a nonhydrolyzable ATP analog. We demonstrate the structural basis for the selectivity of the human Hsp90 inhibitor TAS116 for CaHsp90, features that may be exploited in the future development of improved CaHsp90 inhibitors.

Members of the heat shock protein 90 family (HSP90s) are evolutionarily conserved and play crucial roles in protein transport, immune regulation and antigen presentation. In this study, five hsp90s were identified from the genome of large yellow croaker (Larimichthys crocea) and analyzed using bioinformatics. All five identified hsp90s encode proteins with HATPase_c and HSP90 domains, and are mainly localized in the cytoplasm, mitochondria and endoplasmic reticulum. Chromosomal mapping revealed their distribution across three distinct chromosomes. Quantitative real-time PCR (qPCR) analysis showed differential expression patterns of the five hsp90s in 11 tissues. Additionally, their expression dynamics in the liver, spleen, head kidney, gill and blood were analyzed at 3 h, 12 h, 24 h and 48 h post thermal stress, Vibrio parahaemolyticus infection or under a combination of these two stressors. Results showed that the L. crocea hsp90s exhibited distinct expression patterns in response to the above three stimuli in different immune tissues. Notably, hsp90s in the spleen were most responsive. This study systematically clarified for the first time the gene structure characteristics, tissue expression patterns, and environmental stress response mechanisms of the HSP90 family in L. crocea. It confirmed that hsp90s show significant functional differentiation and synergy in response to biotic (pathogen infection) and abiotic (thermal stress) stresses, and provides important clues for a deeper understanding of the genetic basis of environmental adaptation in L. crocea.

This review summarizes the structure, function, expression, and inhibitors of HSP90, the chaperone, in cancers. It systematically investigates the effects of HSP90 inhibitors, including AUY922, B11B021, CCT-018159, D7-gedunin, geldanamycin, and gedunin, across a range of cancer cell lines (HCC151, HT29, MCF7, PC3, VCAP, and A375) and a normal HA1E cell line, using data from the CLUE database. Our analysis reveals that treatment with these HSP90 inhibitors induces significant stress responses in tumor cells, initiating intrinsic and extrinsic apoptotic pathways. The HSP90AA1, HSP90AB1, HSP27, HSP70, VEGF, and NOTCH exhibited notable upregulation at 24 h post-treatment compared to 6 h, indicating a time-dependent increase in cellular stress (heat shock response) and activation of pro-survival signaling mechanisms. Additionally, the study highlights a significant upregulation of immune-related pathways, including those involving IL10, IL3, and IL7, following HSP90 inhibition, indicating that these inhibitors not only directly affect tumor cell viability but also modulate the tumor microenvironment by enhancing immune cell activation and cytokine release. The elevated levels of IL10 point to a dual role, where immune suppression mechanisms are also at play, potentially facilitating immune evasion by the tumor. The findings suggest that HSP90 inhibitors exhibit varying mechanisms of action across different cancer cell lines despite the presence of some common targets. These insights highlight the need for further investigation into the precise mechanisms of HSP90 inhibitors to optimize their therapeutic potential in different cancers.

Immune checkpoint inhibitors (ICIs) have revolutionized colon cancer treatment, but their efficacy is largely restricted by the limited presence of CD8+ cytotoxic T lymphocytes (CTLs). However, the specific genetic alterations that impact the CD8+ CTL infiltration in colon cancer remain poorly understood. Here, we analyzed clinical and multi-omics data from the Memorial Sloan-Kettering Cancer Center (MSKCC) ICIs-treated and The Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) cohorts to screen the key mutations that may influence the efficacy of immunotherapy. We found that patients with NCOA3 mutations exhibit better response to immunotherapy and higher CD8+ CTL infiltration. In vitro and in vivo experiments revealed that mutant NCOA3 increases the efficacy of anti-PD-L1 and CD8+ CTL recruitment by upregulating C-X-C motif chemokine ligand 9 (CXCL9), which is dependent on its impaired intrinsic histone acetyltransferase activity. Mechanistically, wild-type NCOA3 as histone acetyltransferase upregulates Heat shock protein 90 alpha (HSP90α) by enhancing histone H3 lysine 27 acetylation (H3K27ac) at its promoter region. Increased HSP90α stabilizes Enhancer of zeste homolog 2 (EZH2), which then increase the histone H3 lysine 27 trimethylation (H3K27me3) at the CXCL9 promoter region, thereby suppressing the expression of CXCL9. Targeted inhibition of NCOA3 by small molecular inhibitor SI-2 improves the efficacy of PD-L1 blockade therapy. NCOA3 could serve as a novel biomarker and potential target to improve the efficacy of immunotherapy.

Triple-negative breast cancer (TNBC) is a rare and highly metastatic form of cancer. Honokiol (HNK), a biphenolic compound, has been utilized in TNBC treatment, though its specific targets remain unclear. This study aimed to elucidate the effects of HNK on TNBC by combining network pharmacology predictions and experimental validation to uncover its mechanisms. MDA-MB 231 and MDA-MB 468 cells were pre-treated with varying doses of HNK for 24 h. Cell viability, proliferation, and apoptosis were assessed using CCK8 and FACS assays, whereas a wound healing assay was used to evaluate cell migration. A tubule formation assay was used to assess blood vessel formation in HUVECs. Additionally, in vivo activity was confirmed using a zebrafish xenograft model. Network pharmacology and molecular docking predicted active ingredients, key targets, and potential mechanisms of HNK against TNBC. Results indicated that HNK induces apoptosis in MDA-MB 231 and MDA-MB 468 cells and inhibits their migration and proliferation. Furthermore, HNK suppressed blood vessel formation. Zebrafish xenograft experiments validated HNK's inhibitory effect on TNBC cells in vivo. Network pharmacology identified 36 potential HNK targets against TNBC, including HSP90AA1, AKT1, EGFR, ERBB2, HSP90AB1, PGR, MDM2, HDAC1, NR3C1, and MAPK14. Key signaling pathways such as PI3K-Akt, MAPK, Rap1, Ras, and FoxO were implicated in HNK's anti-TNBC mechanism. Molecular docking demonstrated spontaneous interactions between HNK and the targeted proteins. In conclusion, HNK may reduce angiogenesis by blocking the EGFR and HSP90AB1 pathways thereby decreasing proliferation and increasing apoptosis in TNBC cells.

The complexity of disease biology extends beyond mutations or overexpression, encompassing stress-induced mechanisms that reshape proteins into pathological assemblies. Epichaperomes, stable and disease-specific assemblies of chaperones and co-chaperones, exemplify this phenomenon. This review emphasizes the critical structural and functional distinctions between epichaperomes and canonical chaperones, highlighting their role in redefining therapeutic strategies. Epichaperomes arise under stress conditions through post-translational modifications that stabilize these assemblies, enabling them to act as scaffolding platforms that rewire protein-protein interaction networks and drive the pathological phenotypes of complex diseases such as cancer and neurodegeneration. Chemical biology has been instrumental in uncovering the unique nature of epichaperomes, with small molecules like PU-H71 elucidating their biology and demonstrating their therapeutic potential by dismantling pathological scaffolds and restoring normal protein-protein interaction networks. By targeting epichaperomes, we unlock the potential for network-level interventions and personalized medicine, offering transformative possibilities for diseases driven by protein-protein interaction network dysregulation.

Mitochondria perform multiple functions within the cell, including the production of ATP and a great deal of metabolic intermediates, while also contributing to the cellular stress response. The majority of mitochondrial proteins are encoded by nuclear genomes, highlighting the importance of mitonuclear communication for sustaining mitochondrial homeostasis and functional. As a crucial part of the intracellular signalling network, mitochondria can impact stem cell fate determinations. Considering the essential function of stem cells in tissue maintenance, regeneration and aging, it is important to understand how mitochondria influence stem cell fate. This review explores the significant roles of mitonuclear communication and mitochondrial proteostasis, highlighting their influence on stem cells. We also examine how mitonuclear interactions contribute to cellular homeostasis, stem cell therapies, and the potential for extending lifespan.

The beta isoform of 90 kDa heat shock protein (Hsp90β) plays a critical role in maintaining cellular proteostasis by assisting in the folding and refolding of proteins, which is essential for both normal cellular function and stress response. It is constitutively expressed in mammalian cells, differentiating it from the inducible Hsp90α isoform. Hsp90β's involvement in diverse cellular processes, such as signal transduction, cell cycle control, and apoptosis, underscores its significant role in various diseases, including cancer and neurodegenerative disorders. The isoform-specific functions of Hsp90β and its interaction with unique client proteins make it a promising target for therapeutic intervention, particularly in the development of selective inhibitors that avoid the adverse effects observed with pan-Hsp90 inhibitors. This review delves into the structural and functional intricacies of Hsp90β, its role in disease, and the potential for selective drug development.

Low-temperature second near-infrared region (NIR-II) photothermal therapy (PTT) has shown significant potential in minimizing damage to normal tissues and reducing inflammation. However, it still faces challenge of insufficient immune response. Thus, a multifunctional phototheranostic nanoparticle (BDPB/Pt/Fe@P[5]) is developed by co-loading BDPB, CDHPt, and Fe2⁺ with a pH-sensitive lipid DSPE-PEOz2K. The carboxylatopillar[5]arene (CP[5]) used to construct this nanoparticle exhibits strong host-guest recognition with pyridine salts, alleviating aggregation caused quench (ACQ) effect and enhancing the NIR-II emission of the donor-acceptor-donor (D-A-D)-type organic small molecule (BDPB). CP[5] provides suitable vehicles for encapsulating platinum (IV) prodrugs (CDHPt) and Fe2⁺ ions via metal coordination for controllable reactive oxygen species (ROS) release. Under low-intensity NIR-II laser irradiation and an acidic tumor microenvironment, the nanoparticles degrade, releasing CDHPt and Fe2⁺ ions for platinum-based therapy and chemodynamic therapy (CDT). CDHPt facilitates the direct production of superoxide anions (O₂·⁻) from O₂ and partially converts it into the highly cytotoxic hydroxyl radicals, thereby promoting the Fenton reaction process. The therapeutic efficacy is further synergized by immunogenic cell death (ICD) effect.

Hepatoblastoma, the most common malignant liver tumor in pediatric patients, is characterized by a remarkably low mutation rate, thereby impeding targeted therapies. Current treatment regimens rely on conventional cytotoxic agents that often cause severe adverse effects, necessitating the search for novel, less toxic therapeutic approaches.

In this study, we explored the anti-tumor potential of heat shock protein 90 (HSP90) inhibitors using a unique collection of hepatoblastoma in vitro models.

Among the five tested inhibitors, we identified ganetespib as the most effective, significantly suppressing tumor cell growth while sparing healthy, non-tumor cells. Ganetespib treatment at low nanomolar concentrations markedly reduced cell proliferation, impaired long-term survival, and inhibited three-dimensional spheroid growth, ultimately leading to the induction of apoptosis. Mechanistically, ganetespib downregulated the expression of the HSP90 client protein cyclin-dependent kinase 1, a key cell cycle regulator controlling G2/M phase transition, which is heavily upregulated in hepatoblastoma. This disruption consequently resulted in cell cycle arrest, further contributing to its anti-tumor effects.

HSP90 inhibition by ganetespib demonstrates significant potential as a novel therapeutic strategy for hepatoblastoma, offering a potential alternative to current cytotoxic treatments with fewer adverse effects.

The stress-associated chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing oncoproteins. Most inhibitors target the key component, heat-shock protein 90 (HSP90). Although HSP90 inhibitors are highly tumor-selective, they fail in clinical trials. These failures are partly due to interference with a negative regulatory feedback loop in the heat-shock response (HSR): in response to HSP90 inhibition, there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock-factor 1 (HSF1). We recently identified that wild-type p53 reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here, we test whether in HSP90-based therapies, simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. We found that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids, and patient-derived organoids (PDOs). Mechanistically, upon combination therapy, CRC cells upregulate p53-associated pathways, apoptosis, and inflammatory pathways. Likewise, in a CRC mouse model, dual HSF1-HSP90 inhibition represses tumor growth and remodels immune cell composition. Importantly, inhibition of the cyclin-dependent kinases 4/6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Moreover, in p53-deficient CRC cells, HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR and reduces cancer growth. Likewise, p53-mutated PDOs respond to dual HSF1-HSP90 inhibition, providing a strategy to target CRC independent of the p53 status. In sum, we provide new options to improve HSP90-based therapies to enhance CRC therapies.

Targeted protein degradation (TPD) technologies, inspired by physiological processes, have recently provided new directions for drug development. Unlike conventional drug development focusing on targeting the active sites of disease-related proteins, TPD can utilize any nook or cranny of a protein to drive degradation through the cell's inherent destruction mechanism. It offers various advantages such as stronger pharmacological effects, an expanded range of drug targets, and higher selectivity. Based on the ubiquitin-proteasome system and the lysosomal degradation pathway, a variety of TPD strategies have been developed including PROTAC, PROTAB, and AUTOTAC. These TPD strategies have continuously enriched the toolbox for targeted protein degradation and expanded the scope of application, providing new ideas for biological research and drug discovery. This review attempts to introduce up-to-date research progress in the TPD strategies, focusing mainly on their design concepts, advantages, potential applications, and challenges, which may provide some inspiration for drug design, drug discovery, and clinical application for biologists and chemists.

Heat shock protein 90 (HSP90) plays a crucial role in the maintenance of protein homeostasis in cancer cells. Inhibition of HSP90 is anticipated to exert anticancer activities by reducing levels of HSP90 client proteins. Pimitespib (TAS-116) has emerged as a potent ATP-competitive inhibitor of both HSP90α and β, demonstrating favorable therapeutic properties in preclinical models. Notably, pimitespib is the first HSP90 inhibitor approved for the treatment of advanced gastrointestinal stromal tumors in Japan. Poly(ADP-ribose) polymerase (PARP) inhibitors target cancers susceptible to the homologous recombination (HR) pathway and are used for treating various types of tumors, particularly those harboring defects in HR repair pathways within DNA damage repair (DDR) such as mutations in breast cancer genes 1 and 2 (BRCA1 and BRCA2, respectively). However, PARP inhibitors have shown limited efficacy in HR-proficient tumors, and the development of resistance to PARP inhibitors via restoration of DDR systems poses a significant challenge. In this study, we explored the potential of pimitespib to enhance PARP inhibitor activity. In PARP inhibitor-insensitive breast cancer cell lines, pimitespib impaired HR pathway function by promoting the proteasome-mediated degradation of proteins involved in HR, such as BRCA1, BRCA2, and Rad51 homologous 1 (RAD51). Consequently, pimitespib enhanced antitumor activity and DNA damage induced by PARP inhibitors in vitro. In human breast cancer xenograft mouse models, pimitespib downregulated RAD51 proteins and augmented the antitumor effects of PARP inhibitors. These findings highlight the potential of pimitespib as a therapeutic agent in combination with PARP inhibitors to treat PARP inhibitor-insensitive cancers.

Glioblastoma (GBM) is the most common primary brain cancer and is resistant to standard-of-care chemoradiation therapy (CRT). Magnetic hyperthermia therapy (MHT) exposes magnetic iron oxide nanoparticles (MIONPs) to an alternating magnetic field (AMF) to generate local hyperthermia. This study evaluated MHT-mediated enhancement of CRT in preclinical GBM models. Cell viability and apoptosis were assessed in GBM cell lines after water bath heating with radiation and/or temozolomide. Heating efficiency of MIONPs after intracranial delivery was measured in healthy mice. MHT with CRT was performed in syngeneic and patient-derived xenograft (PDX) GBM tumors. Tissue sections were analyzed for γ-H2AX, HSP90, CD4 + T cells, and microglial cells. Tumor burden and survival were assessed. Hyperthermia with radiation and temozolomide significantly reduced cell viability and increased apoptosis. Hyperthermia predominantly exhibited additive to synergistic interactions with both treatment modalities and reduced doses needed for tumor cell growth inhibition. In vivo, MHT with CRT decreased tumor burden and increased survival in PDX and syngeneic models. Immunohistochemistry showed increased γ-H2AX, HSP90, microglial activation, and CD4 + T cells after MHT in combination with CRT. Overall, adjuvant hyperthermia enhances CRT efficacy in GBM cells, with MHT improving survival outcomes in rodents. Sufficient intracranial heating and MIONP retention for repeated treatments was achieved, supporting further clinical translation.

Amide synthases catalyze the formation of macrolactam rings from aniline-containing polyketide-derived seco-acids as found in the important class of ansamycin antibiotics. One of these amide synthases is the geldanamycin amide synthase GdmF, which we recombinantly expressed, purified and studied in detail both functionally as well as structurally. Here we show that purified GdmF catalyzes the amide formation using synthetically derived substrates. The atomic structures of the ligand-free enzyme and in complex with simplified substrates reveal distinct structural features of the substrate binding site and a putative role of the flexible interdomain region for the catalysis reaction.

Molecular chaperones, a class of complex client regulatory systems, play significant roles in the prevention of protein misfolding and abnormal aggregation, the modulation of protein homeostasis, and the protection of cells from damage under constantly changing environmental conditions. As the understanding of the biological mechanisms of molecular chaperones has increased, their link with the occurrence and progression of disease has suggested that these proteins are promising targets for therapeutic intervention, drawing intensive interest. Here, we review recent advances in determining the structures of molecular chaperones and heat shock protein 90 (HSP90) chaperone system complexes. We also describe the features of molecular chaperones and shed light on the complicated regulatory mechanism that operates through interactions with various co-chaperones in molecular chaperone cycles. In addition, how molecular chaperones affect diseases by regulating pathogenic proteins has been thoroughly analyzed. Furthermore, we focus on molecular chaperones to systematically discuss recent clinical advances and various drug design strategies in the preclinical stage. Recent studies have identified a variety of novel regulatory strategies targeting molecular chaperone systems with compounds that act through different mechanisms from those of traditional inhibitors. Therefore, as more novel design strategies are developed, targeting molecular chaperones will significantly contribute to the discovery of new potential drugs.

One of the leading causes of mortality for women is gynecologic cancer (GC). Numerous molecules (tumor suppressor genes or oncogenes) are involved in this form of cancer's invasion, metastasis, tumorigenic process, and therapy resistance. Currently, there is a shortage of efficient methods to eliminate these diseases, hence it is crucial to carry out more extensive studies on GCs. Novel pharmaceuticals are required to surmount this predicament. Highly conserved molecular chaperon, heat shock protein (HSP) 90, is essential for the maturation of recently produced polypeptides and offers a refuge for misfolding or denatured proteins to be turned around. In cancer, the client proteins of HSP90 play a role in the entire process of oncogenesis, which is linked to all the characteristic features of cancer. In this study, we explore the various functions of HSPs in GC progression. We also discuss their potential as promising targets for pharmacological therapy.

Heat-shock protein 90 (HSP90) is a highly active molecular chaperone that plays a crucial role in cellular function. It facilitates the folding, assembly and stability of various oncogenic proteins, particularly kinases and transcription factors involved in regulating tumor growth and maintenance signaling pathways. Consequently, HSP90 inhibitors are being explored as drugs for cancer therapy. Crystallographic fragment screening is a novel screening method that has been developed in recent years for fragment-based drug discovery and is known for its high hit rate and its ability to provide direct insights into the complex structures of proteins and compounds. In this paper, high-diffraction-resolution crystals of the N-terminal domain of human HSP90α were employed in crystallographic fragment screening to discover binding fragments and binding sites. A diverse library of 800 structurally distinct fragments was screened, yielding 91 starting points for the fragment-based drug design of new HSP90α N-terminal inhibitors. Nearly a thousand crystals were measured, with 738 being processed and phased using a highly automated data-processing pipeline including data reduction and phasing, refinement and hit identification via PanDDA multi-data-set analysis. The 91 identified compounds bind to eight distinct regions of the HSP90α N-terminus, with 63 fragments located in the ATP-binding pocket and its surroundings, thus demonstrating the potential for the development of HSP90α- and ATP-binding inhibitors. This study emphasizes crystallographic fragment screening as a powerful method that can effectively identify fragment molecules and inhibitors that bind to HSP90α, contributing to ongoing efforts in cancer drug discovery.

Histone deacetylases (HDACs) have become one of the main targets in cancer therapy due to their involvement in various biological processes, including gene regulation, cell proliferation, and differentiation. Microtubules, as key elements of the cell cytoskeleton, also represent important therapeutic targets in anticancer drugs research. These proteins are involved in diverse cellular functions, especially mitosis, cell signaling, and intracellular trafficking. With the emergence of multi-target therapy during the last decades, the combination of HDAC and tubulin inhibitors has been envisioned as a practical approach for optimizing the therapeutic efficacy of antitumor molecules. HDAC/tubulin dual-targeting inhibitors offer the advantages of the synergistic action of both compounds, along with a significant decrease in their respective toxicities and drug resistance. This review will detail the major recent advancements in the development of HDAC/tubulin dual inhibitors over the last decade and their impact on anticancer drugs discovery.

The extracellular localisation of the Heat shock protein 90 (Hsp90) is associated with the diseased state and wound healing and presents a promising opportunity for cancer targeting using Positron Emission Tomography (PET) imaging and molecularly targeted radiotherapy. The aim of this work is to develop a radiotracer with low nanomolar binding affinity to target the extracellular and particularly membrane pool of Hsp90, evaluate it in vitro, and conduct preliminary PET studies in vivo in mouse tumour models. Variable Heavy domain of Heavy chain antibodies, often referred to as Nanobodies, are suitable targeting vectors for the extracellular targets due to their favourable pharmacokinetic properties and low nanomolar target affinities. The main objective of the study is to target tumours expressing extracellular and membrane Hsp90 phenotype with minimal tracer accumulation in the non-target organs, which limited the translation of previously studied small molecule cytosolic Hsp90 tracers suffering from high non-Hsp90 specific background in the abdominal area.

Six nanobodies were obtained after llama immunization with recombinant Hsp90α and ELISA biopanning, produced in E. coli and screened for stability and affinity. We selected one nanobody, 4DAM26, with good thermal stability, no aggregation at elevated temperatures, and low nanomolar affinity towards Hsp90α and Hsp90β isoforms for translation as a PET radiotracer. The nanobody was bioconjugated to p-NCS-NODAGA and radiolabeled with gallium-68 with 75 ± 11% radiochemical yield and > 99% radiochemical purity and remained stable up to 3 h in phosphate buffered saline and mouse serum. Pilot in vivo evaluation using µPET/CT and ex vivo biodistribution demonstrated a favourable pharmacokinetic profile, but the tumour uptake was non-distinguishable from the background tissue.

Compared to the small molecule Hsp90 tracers, the studied Nb-based tracer has improved pharmacokinetics properties including renal clearance and almost no accumulation in the non-target organs. Tumour uptake, on the other hand, was minimal and could not be differentiated from the background in µPET/CT. Our experiments indicate that in the studied models, membrane and extracellular expression of Hsp90 is majorly an artifact of cellular death, as only dead/dying cells had accessible pools of Hsp90 by flow cytometry, a consequence of a leaky membrane. More fundamental research is required to reassess the role of extracellular Hsp90 in cancer, and our future efforts will be focused on improving our inventory of cytosolic Hsp90 tracers with proven Hsp90-specific tumour accumulation.

Despite initial success with FLT3 inhibitors (FLT3is), outcomes for FLT3-ITD acute myeloid leukemia (AML) patients remain unsatisfactory, underscoring the need for more effective treatment options. Epigenetic modifications, such as histone acetylation, contribute to AML's onset and persistence, advocating the potential for epigenetic therapies. However, the poor specificity of pan-histone deacetylase inhibitors (HDACis) leads to undesirable adverse effects, prompting the need for isoform-specific HDACis. This study aims to explore the antileukemic activities and mechanisms of IHCH9033, a novel class I HDACi, alone or combined with FLT3i in FLT3-ITD AML.

The viability of AML cell lines and primary AML cells treated with HDACis alone or in combination with FLT3i was detected by MTT or CCK8 assay. Flow cytometry was utilized to examine cell apoptosis, cell cycle progression and ROS production. RNA sequencing analysis, RT-qPCR, western blotting, and co-immunoprecipitation assays were employed to elucidate the molecule mechanisms. The in vivo anti-leukemia efficacy was tested in xenografted mice models derived from FLT3-ITD cell lines and primary AML patients.

Here, we identified IHCH9033, a novel selective class I HDACi, which exhibited an increased antitumor effect in FLT3-ITD AML through effectively eliminating leukemia burden and overcoming resistance to FLT3i. Mechanically, IHCH9033 selectively inhibited DNA repair in FLT3-ITD AML cells, leading to the accumulation of DNA damage that eventually resulted in cell cycle arrest and apoptosis. Additionally, IHCH9033 induced HSP90 acetylation, FLT3 ubiquitination, and proteasomal degradation of FLT3, thereby inhibiting FLT3 downstream signaling. Notably, IHCH9033 maintained its potency in both FLT3i-resistant AML cell lines and primary-resistant patient samples, and exerted strong synergy with the FLT3i quizartinib, leading to tumor regression in FLT3-ITD/TKD AML xenografts. In patient-derived xenografts, the treatment with IHCH9033, both alone and in combination, led to nearly complete eradication of the AML burden, without significant adverse effects.

Our study shows that IHCH9033, a novel class I HDACi with a desirable pharmacological profile, is a promising drug candidate for FLT3-ITD AML, and suggests a strategy of combining class I HDACis and FLT3is in AML clinical trials to increase efficacy and overcome resistance, thus potentially providing a curative treatment option.

Neurodegenerative diseases (NDs) and Long COVID represent critical and growing global health challenges, characterized by complex pathophysiological mechanisms including neuronal deterioration, protein misfolding, and persistent neuroinflammation. The emergence of innovative therapeutic approaches, such as whole-body hyperthermia (WBH), offers promising potential to modulate underlying pathophysiological mechanisms in NDs and related conditions like Long COVID. WBH, particularly in fever-range, enhances mitochondrial function, induces heat shock proteins (HSPs), and modulates neuroinflammation-benefits that pharmacological treatments often struggle to replicate. HSPs such as HSP70 and HSP90 play pivotal roles in protein folding, aggregation prevention, and cellular protection, directly targeting pathological processes seen in NDs like Alzheimer's, Parkinson's, and Huntington's disease. Preliminary findings also suggest WBH's potential to alleviate neurological symptoms in Long COVID, where persistent neuroinflammation and serotonin dysregulation are prominent. Despite the absence of robust clinical trials, the therapeutic implications of WBH extend to immune modulation and the restoration of disrupted physiological pathways. However, the dual nature of hyperthermia's effects-balancing pro-inflammatory and anti-inflammatory responses-emphasizes the need for dose-controlled applications and stringent patient monitoring to minimize risks in vulnerable populations. While WBH shows potential interest, significant challenges remain. These include individual variability in response, limited accessibility to advanced hyperthermia technologies, and the need for standardized clinical protocols. Future research must focus on targeted clinical trials, biomarker identification, and personalized treatment strategies to optimize WBH's efficacy in NDs and Long COVID. The integration of WBH into therapeutic paradigms could mark a transformative step in addressing these complex conditions.

This study aimed to explore the significance of the expression of pre- and post-treatment eHSP90α in the treatment response evaluation and prognosis of driver-gene-negative non-small cell lung cancer (NSCLC), as well as the significance of eHSP90α expression in the prognosis of immunotherapy.

We collected pre-treatment eHSP90α in 330 driver-gene-negative NSCLC patients and analyzed its relationship with efficacy evaluation and prognosis. Survival curves were used to determine their respective critical values and the relationship between eHSP90α expression and OS and PFS was analyzed. Then, univariable and multivariable Cox regression analyses and LASSO multivariable logistic regression analyses were used to establish the prognostic and efficacy evaluation models.

High expression of pre-treatment eHSP90α in driver-gene-negative NSCLC patients is associated with shorter OS. Pre-treatment eHSP90α, immunotherapy, TTF-1, CA125, and age influence OS in NSCLC patients. In the stratified analysis of immunotherapy, pre-treatment eHSP90α in immunotherapy is an independent influencing factor of OS in NSCLC patients, and pre-treatment eHSP90α, age, CA125, and B lymphocytes in non-immunotherapy are independent influencing factors of OS in NSCLC patients. High expression of pre-treatment eHSP90α in NSCLC patients is associated with shorter PFS. Pre-treatment eHSP90α, M1, Ki-67, and immunotherapy are independent influencing factors of PFS in NSCLC patients. In the stratified analysis of immunotherapy, pre-treatment eHSP90α and CEA in immunotherapy are independent influencing factors of PFS in NSCLC patients, and pre-treatment eHSP90α, M1, Ki-67, CA125, Th/Ts, and age in non-immunotherapy are independent factors affecting PFS in NSCLC patients. Pre-treatment eHSP90α was one of the influencing factors in the efficacy evaluation model.

The expression of pre-treatment eHSP90α has significant significance for the prognosis and efficacy evaluation of driver-gene-negative NSCLC.

The sustained activation of androgen receptor splice variant-7 (AR-V7) is a key factor in the resistance of castration-resistant prostate cancer (CRPC) to second-generation anti-androgens such as enzalutamide (ENZ). The AR/AR-V7 protein is regulated by the E3 ubiquitin ligase STUB1 and a complex involving HSP70, but the precise mechanism remains unclear.

High-throughput RNA sequencing was used to identify differentially expressed circular RNAs (circRNAs) in ENZ-resistant and control CRPC cells. The coding potential of circSRCAP was confirmed by polysome profiling and LC-MS. The function of circSRCAP was validated in vitro and in vivo using gain- and loss-of-function assays. Mechanistic insights were obtained through immunoprecipitation analyses.

A novel ENZ-resistant circRNA, circSRCAP, was identified and shown to be upregulated in ENZ-resistant C4-2B (ENZR-C4-2B) cells, correlating with increased AR-V7 protein levels. circSRCAP is generated via splicing by eIF4A3, forming a loop structure and is exported from the nucleus by the RNA helicase DDX39A. Mechanistically, circSRCAP encodes a 75-amino acid peptide (circSRCAP-75aa) that inhibits the ubiquitination of AR/AR-V7's co-chaperone protein HSP70 by disrupting the interaction with the E3 ligase STUB1. This process results in the upregulation of AR-V7 expression and promotes ENZ resistance in CRPC cells. Xenograft tumor models further confirmed the role of circSRCAP in CRPC progression and its potential as a therapeutic target for ENZ-resistant CRPC.

circSRCAP provides an epigenetic mechanism influencing AR-V7 stability and offers a promising therapeutic target for treating ENZ-resistant CRPC.

Epilepsy is a leading cause of disability and mortality worldwide. However, despite the availability of more than 20 antiseizure medications, more than one-third of patients continue to experience seizures. Given the urgent need to explore new treatment strategies for epilepsy, recent research has highlighted the potential of targeting gliosis, metabolic disturbances, and neural circuit abnormalities as therapeutic strategies. Astrocytes, the largest group of nonneuronal cells in the central nervous system, play several crucial roles in maintaining ionic and energy metabolic homeostasis in neurons, regulating neurotransmitter levels, and modulating synaptic plasticity. This article briefly reviews the critical role of astrocytes in maintaining balance within the central nervous system. Building on previous research, we discuss how astrocyte dysfunction contributes to the onset and progression of epilepsy through four key aspects: the imbalance between excitatory and inhibitory neuronal signaling, dysregulation of metabolic homeostasis in the neuronal microenvironment, neuroinflammation, and the formation of abnormal neural circuits. We summarize relevant basic research conducted over the past 5 years that has focused on modulating astrocytes as a therapeutic approach for epilepsy. We categorize the therapeutic targets proposed by these studies into four areas: restoration of the excitation-inhibition balance, reestablishment of metabolic homeostasis, modulation of immune and inflammatory responses, and reconstruction of abnormal neural circuits. These targets correspond to the pathophysiological mechanisms by which astrocytes contribute to epilepsy. Additionally, we need to consider the potential challenges and limitations of translating these identified therapeutic targets into clinical treatments. These limitations arise from interspecies differences between humans and animal models, as well as the complex comorbidities associated with epilepsy in humans. We also highlight valuable future research directions worth exploring in the treatment of epilepsy and the regulation of astrocytes, such as gene therapy and imaging strategies. The findings presented in this review may help open new therapeutic avenues for patients with drug-resistant epilepsy and for those suffering from other central nervous system disorders associated with astrocytic dysfunction.

Cell division cycle 37 (CDC37) is a member of the molecular chaperone family and acts as a cochaperone of heat shock protein 90 (HSP90), which is overexpressed in many cancer types as a regulator of protein kinase maturation. In this process, CDC37 selectively recognizes and stabilizes protein kinases by forming a HSP90-CDC37-kinase chaperone complex. The protein-protein interactions (PPIs) of HSP90-CDC37 and CDC37-kinase complexes contribute to malignant tumors, as oncogenic kinases in malignant cells depend upon CDC37 expression. Thus, inhibiting CDC37 to disrupt HSP90-CDC37-kinase chaperone complex reveals as a promising way to achieve selective inhibition of oncogenic kinase maturation. Herein, we report a small-molecule CDC37 inhibitor called DDO-6079 that simultaneously inhibits HSP90-CDC37 and CDC37-CDK4/6 chaperone complex by binding to an allosteric site on CDC37. DDO-6079 selectively inhibited the maturation of multiple oncogenic kinases to escape heat shock response (HSR). Furthermore, DDO-6079 decreased the thermostability of CDK6, reversed the resistance of CDK6 to palbociclib (a successful CDK4/6 inhibitor) in colorectal cancer cells and exhibited efficacy in vivo. Together, the results revealed that DDO-6079 is a first-in-class small molecule CDC37 inhibitor that disrupts the HSP90-CDC37-kinase chaperone complex and provides a new way to block kinase maturation.

Hypoxia-inducible factors (HIFs) are essential transcription factors that orchestrate cellular responses to oxygen deprivation. HIF-1α, as an unstable subunit of HIF-1, is usually hydroxylated by prolyl hydroxylase domain enzymes under normoxic conditions, leading to ubiquitination and proteasomal degradation, thereby keeping low levels. Instead of hypoxia, sometimes even in normoxia, HIF-1α translocates into the nucleus, dimerizes with HIF-1β to generate HIF-1, and then activates genes involved in adaptive responses such as angiogenesis, metabolic reprogramming, and cellular survival, which presents new challenges and insights into its role in cellular processes. Thus, the review delves into the mechanisms by which HIF-1 maintains its stability under normoxia including but not limited to giving insights into transcriptional, translational, as well as posttranslational regulation to underscore the pivotal role of HIF-1 in cellular adaptation and malignancy. Moreover, HIF-1 is extensively involved in cancer and cardiovascular diseases and potentially serves as a bridge between them. An overview of HIF-1-related drugs that are approved or in clinical trials is summarized, highlighting their potential capacity for targeting HIF-1 in cancer and cardiovascular toxicity related to cancer treatment. The review provides a comprehensive insight into HIF-1's regulatory mechanism and paves the way for future research and therapeutic development.

Heat shock protein 90 (HSP 90) and its isoforms are a group of homodimeric proteins that regulate several cellular processes, such as the elimination of misfolded proteins, cell development and post-translational modifications of kinase proteins and receptors. Due to its involvement in extracellular matrix (ECM) remodelling, myofibroblast differentiation and apoptosis, HSP 90 has been investigated as a key player in the pathogenesis of lung fibrosis. Idiopathic pulmonary fibrosis (IPF) is the most common and deadly interstitial lung disease, due to the progressive distortion of lung parenchyma related to the overproduction and deposition of altered ECM, driven by transforming growth factor-β (TGF-β) dependent and independent pathways. The inhibition or induction of HSP 90 is associated with a reduced or increased expression of TGF-β receptors, respectively, suggesting a role for HSP 90 as a biomarker and therapeutic target in IPF. Experimental drugs such as geldanamycin and its derivatives 17-AAG (17-N-allylamino-17-demethoxygeldanamicin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxigeldanamycin), along with AUY-922, 1G6-D7, AT-13387, TAS-116 and myricetin, have been found to reduce lung fibrosis in both in vivo and in vitro models, supporting the role of this emerging target. This review aims to illustrate the structure and biological function of HSP 90 in the context of IPF pathobiology, as well as perspective application of this molecule as a biomarker and therapeutic target for IPF.

Excessive lipid accumulation promotes the occurrence and progression of hepatocellular carcinoma (HCC), accompanied by high levels of fatty acid synthetase (FASN) and more active lipogenesis. Heat shock protein 90 (Hsp90) acts as a chaperone to maintain the stability and activity of the client proteins. Studies have revealed that Hsp90 regulates the lipid metabolism of HCC, but the effect of Hsp90 on FASN still remains unknown. This study aims to discover the mechanism of Hsp90 inhibition on lipid accumulation and investigate the different effects of Hsp90 N-terminal domain inhibitor STA9090 and C-terminal domain inhibitor novobiocin on FASN protein stability and transcription pathway in HCC. We found that HCC cells tended to store lipids, which could be disrupted by Hsp90 inhibitors in vivo and in vitro. High levels of Hsp90α and FASN in tumor tissue had correlation with poor prognosis of HCC patients, and Hsp90α interacted with FASN to maintain its protein stability. Furthermore, N-terminal domain of Hsp90α was essential for process of sterol regulatory element binding protein 1 to activate FASN transcription and Hsp90α prevented proteasomal degradation of liver X receptor α to upregulate FASN transcription via liver X receptor α/sterol regulatory element binding protein 1 axis. Our data reveal that Hsp90α promotes lipid accumulation by increasing the protein stability and FASN mRNA transcription, and can be alleviated by Hsp90 inhibitors, which provides a theoretical basis for Hsp90-targeted therapy on lipid metabolism in HCC.

Major depressive disorder (MDD) is characterized by high rates of disability and death and has become a public health problem that threatens human life and health worldwide. HPA axis disorder and neuroinflammation are two common biological abnormalities in MDD patients. Hsp90 is an important molecular chaperone that is widely distributed in the organism. Hsp90 binds to the co-chaperone and goes through a molecular chaperone cycle to complete its regulation of the client protein. Numerous studies have demonstrated that Hsp90 regulates how the HPA axis reacts to stress and how GR, the HPA axis' responsive substrate, matures. In addition, Hsp90 exhibits pro-inflammatory effects that are closely related to neuroinflammation in MDD. Currently, Hsp90 inhibitors have made some progress in the treatment of a variety of human diseases, but they still need to be improved. Further insight into the role of Hsp90 in MDD provides new ideas for the development of new antidepressant drugs targeting Hsp90.

Heat Shock Protein 90 (Hsp90) is responsible for the proper folding and maturation of ~400 client protein substrates, many of which are directly associated with the ten hallmarks of cancer. Hsp90 is a great target for cancer therapy including melanoma, since Hsp90 inhibition can disrupt multiple oncogenic pathways simultaneously. In this study, we report the synthesis and anti-proliferative activity manifested by a series of Hsp90 C-terminal inhibitors against mutant BRAF and wild-type BRAF melanoma cells. Furthermore, we explored structure-activity relationships (SAR) for the amide moiety of 6 (B1), a novel Hsp90C-terminal inhibitor via introduction of amide bioisosteres. Compound 6 displayed an IC50 of 1.01 μM, 0.782 μM, 0.607 μM and 1.413 μM against SKMel173, SKMel103, SKMel19 and A375 cells, respectively.

Due to their impact on several oncogenic client proteins, the Hsp90 family of chaperones has been widely studied for the development of potential anticancer agents. Although several Hsp90 inhibitors have entered clinical trials, most were unsuccessful because they induced a heat shock response (HSR). This issue can be circumvented by using isoform-selective inhibitors, but the high similarity in the ATP-binding sites between the isoforms presents a challenge. Given that Hsp90 shares a conserved Bergerat fold with bacterial DNA gyrase B and human topoisomerase IIα, we repurposed our ATP-competitive inhibitors of these two proteins for Hsp90 inhibition. We virtually screened a library of in-house inhibitors and identified eleven hits for evaluation of Hsp90 binding. Among these, compound 11 displayed low micromolar affinity for Hsp90 and demonstrated a 12-fold selectivity for Hsp90β over its closest isoform, Hsp90α. Out of 29 prepared analogs, 16 showed a preference for Hsp90β over Hsp90α. Furthermore, eleven of these compounds inhibited the growth of several cancer cell lines in vitro. Notably, compound 24e reduced intracellular levels of Hsp90 client proteins in MCF-7 cells, leading to cell cycle arrest in the G0/G1 phase without inducing HSR. This inhibitor exhibited at least a 27-fold preference for Hsp90β and was selective against topoisomerase IIα, a panel of 22 representative protein kinases, and proved to be non-toxic in a zebrafish larvae toxicology model. Finally, molecular modeling, corroborated by STD NMR studies, and the binding of 24e to the S52A mutant of Hsp90α confirmed that the serine to alanine switch drives the selectivity between the two cytoplasmic isoforms.

Hsp90, a crucial molecular chaperone, regulates diverse client proteins, impacting both normal biology and disease. Central to its function is its conformational plasticity, driven by ATPase activity and client interactions. However, comprehensive insights into Hsp90's dynamic molecular transitions remain elusive. Using solution NMR spectroscopy, we reveal how ATP binding, hydrolysis, and client engagement drive conformational and dynamic shifts in E. coli Hsp90, HtpG, through its chaperone cycle. Pronounced conformational fluctuations occur, especially in regions crucial for nucleotide binding and conformational transitions. ATP binding induces slow-exchanging conformations, representing discrete on-path transition states from open to closed forms, while ATP hydrolysis shifts HtpG into a compact conformation. Client binding acts as an allosteric switch, dynamically priming HtpG for elevated chaperone activity and, therefore, its efficient remodeling. Here, we provide atomic-level insights into Hsp90's functional mechanism, highlighting the interplay of conformation, dynamics, nucleotide, and client interactions.