Recent progress in the development of hypoxia-inducible factor 2α (HIF-2α) modulators: Inhibitors, agonists, and degraders (2009-2024).

Hypoxia-inducible factor 2α (HIF-2α) is a critical transcription factor that regulates cellular responses under hypoxic conditions. In situations of insufficient oxygen supply or patients with Von Hippel-Lindau (VHL) mutations, HIF-2α accumulates and forms a heterodimeric complex with aryl hydrocarbon receptor nuclear translocator (ARNT, or HIF-ß). This complex further binds to coactivator p300 and interacts with hypoxia response elements (HREs) on the DNA of downstream target genes, regulating the transcription of a variety of genes (e.g. VEGFA, CCND1, CXCR4, SLC2A1, etc) involved in various processes like angiogenesis, mitochondrial metabolism, cell proliferation, and metastasis. Targeting HIF-2α holds great promise for effectively addressing solid tumors associated with aberrant oxygen-sensing pathways and hypoxia mechanisms, offering broad application prospects. In this review, we provide an overview of recent advancements (2009-2024) in HIF-2α modulators such as inhibitors, agonists, and degraders for cancer therapy. Additionally, we discuss in detail the challenges and future directions regarding HIF-2α modulators.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

HDAC-targeting epigenetic modulators for cancer immunotherapy.

HDAC inhibitors, which can inhibit the activity of HDAC enzymes, have been extensively studied in tumor immunotherapy and have shown potential therapeutic effects in cancer immunotherapy. To date, numerous small molecule HDAC inhibitors have been identified, but many of them suffer from limited clinical efficacy and serious toxicity. Hence, HDAC inhibitor-based combination therapies, and other HDAC modulators (e.g. PROTAC degraders, dual-acting agents) have attracted great attention with significant advancements achieved in the past few years due to their superior efficacy compared to single-target HDAC inhibitors. In this review, we overviewed the recent progress on HDAC-based drug discovery with a focus on HDAC inhibitor-based drug combination therapy and other HDAC-targeting strategies (e.g. selective HDAC inhibitors, HDAC-based dual-target inhibitors, and PROTAC HDAC degraders) for cancer immunotherapy. In addition, we also summarized the reported co-crystal structures of HDAC inhibitors in complex with their target proteins and the binding interactions. Finally, the challenges and future directions for HDAC-based drug discovery in cancer immunotherapy are also discussed in detail.

Quantification of Available Ligand Density on the Surface of Targeted Liposomal Nanomedicines at the Single-Particle Level.

Active targeting has been hailed as one of the most promising strategies to further enhance the therapeutic efficacy of liposomal nanomedicines. Owing to the critical role of ligand density in mediating cellular uptake and the intrinsic heterogeneity of liposomal formulations, precise quantification of the surface ligand density on a single-particle basis is of fundamental importance. In this work, we report a method to simultaneously measure the particle size and the number of ligands on the same liposomal nanoparticles by nanoflow cytometry. Then the ligand density for each individual liposome can be determined. With an analysis rate up to 10 000 particles per minute, a statistically representative distribution of ligand density could be determined in minutes. By utilizing fluorescently labeled recombinant receptors as the detection probe against the conjugated ligands, only those available for cell targeting can be exclusively detected. The influence of ligand input, conjugation strategy, and the polyethylene glycol spacer length on the available ligand density of folate-modified liposomes was investigated. The correlation between the available ligand density and cell targeting capability was assessed in a quantitative perspective for liposomes modified with three different targeting moieties. The optimal ligand density was determined to be 0.5-2.0, 0.7, and 0.2 ligand per 100 nm2 for folate-, transferrin-, and HER2-antibody-conjugated liposomes, respectively. These optimal values agreed well with the spike density of the natural counterparts, viruses. The as-developed approach is generally applicable to a wide range of active-targeting nanocarriers.

High-Throughput Human Telomere Length Analysis at the Single-Chromosome Level by FISH Coupled with Nano-Flow Cytometry.

Telomere length (TL) is a highly relevant biomarker for age-associated diseases and cancer, yet its clinical applications have been hindered by the inability of existing methods to rapidly measure the TL distribution and the percentage of chromosomes with critically short telomeres (CSTs, < 3 kb). Herein, we report the development of a high-throughput method to measure TL at the single-chromosome level. Metaphase chromosomes are isolated, hybridized with the Alexa Fluor 488-labeled telomeric peptide nucleic acid probe, and analyzed using a laboratory-built ultrasensitive nano-flow cytometer. The fluorescence intensity of individual chromosomes is converted to TL in kilobases upon external calibration. With an analysis rate of several thousand chromosomes per minute, a statistically robust TL distribution histogram is acquired in minutes, and the percentage of chromosomes with CSTs can be quickly assessed. By analyzing peripheral blood lymphocytes of 158 healthy donors, TL is found to shorten with age at a rate of 64 ± 3 bp/year and the percentage of chromosomes with CSTs increases with age at a rate of 0.32 ± 0.02%/year. Moreover, the data of 28 patients with chronic myeloid leukemia (CML) indicate that telomeres are significantly shorter at the time of diagnosis and the clinical phases of CML are closely associated with TL and the percentage of chromosomes with CSTs. This powerful tool could greatly deepen our understanding of telomere biology and improve the clinical utility of telomere biomarkers.

Rolling circle amplification integrated with suspension bead array for ultrasensitive multiplex immunodetection of tumor markers.

Multiplex detection of ultra-low abundant tumor markers is extremely important for early diagnosis and therapy evaluation. Herein, an ultrasensitive multiplex immunoassay was developed by combination of rolling circle amplification (RCA) and suspension bead array (SBA) technology. Based on a conventional sandwich-type immunoreaction on beads, the detection antibodies were conjugated with DNA primers, so RCA could be implemented to generate long-stranded DNA with abundant repeated sequences allowing for hybridization with fluorochrome-labeled oligonucleotide probes. Thus the fluorescence signal of immunocomplexes on the encoded beads can be greatly enhanced. Using the as-developed immuno-RCA suspension bead array (iRCA-SBA), simultaneous analysis of multiple tumor markers was achieved with the limits of detection of 3.1 pg/mL (∼0.1 pM) for prostate specific antigen (PSA), 9.1 pg/mL (∼50 fM) for carcinoembryonic antigen (CEA), and 0.66 pg/mL (∼9 fM) for α-fetoprotein (AFP), which are two to three orders of magnitude lower than those obtained by the conventional SBA method. The dynamic range were 4.5, 4.7, and 5.5 orders of magnitude for PSA, CEA, and AFP, respectively. Tests on clinical serum samples demonstrate that the tumor marker concentrations measured by the newly developed iRCA-SBA assay agreed well with those obtained by the conventional SBA method. These results indicate that the iRCA-SBA assay significantly increased the detection sensitivity and dynamic range without sacrificing the reliability and accuracy of conventional SBA. Upon the integration with iRCA, SBA could find more applications in the detection of low abundance protein biomarkers for early diagnosis of cancer and other diseases.

Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry.

A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

High-throughput single-cell analysis of low copy number ß-galactosidase by a laboratory-built high-sensitivity flow cytometer.

Single-cell analysis is vital in providing insights into the heterogeneity in molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. ß-galactosidase (ß-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here we report the development of a high-throughput method for the single-cell analysis of low copy number ß-gal proteins using a laboratory-built high-sensitivity flow cytometer (HSFCM). Upon fluorescence staining with a fluorogenic substrate, quantitative measurements of the basal and near-basal expression of ß-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1-2min, which reveals the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration by HSFCM, the ß-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell. The sensitivity and speed of the HSFCM offers great capability in quantitative analysis of low abundance proteins in single cells, which would help gaining a deeper insight into the heterogeneity and fundamental biological processes in microbial populations.

Detection and quantification of bacterial autofluorescence at the single-cell level by a laboratory-built high-sensitivity flow cytometer.

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.