Karl Ocius

Bioorg Chem. 2025 May 10;162:108577. doi: 10.1016/j.bioorg.2025.108577. Online ahead of print.

ABSTRACT

The mechanisms underlying transcriptional dysregulation in tumorigenesis have received considerable attention as promising therapeutic targets to combat human cancer. Cyclin-dependent kinase 9 (CDK9) and class I histone deacetylases (HDACs) are significant therapeutic targets due to their pivotal roles in the dysregulated transcriptional programs characteristic of many cancers. Furthermore, the combinatorial transcriptional therapy with CDK9 and class I HDAC inhibitors has been shown to have a synergistic anticancer effect. In this study, a series of novel N-(2-amino-phenyl)-5-(4-aryl-pyrimidin-2-yl) amino)-1H-indole-2-carboxamide derivatives were designed and synthesized as novel dual-functional inhibitors targeting CDK9 and HDAC signaling pathways for cancer treatment. Among the synthesized compounds, 13ea demonstrated potent anti-proliferative activities (IC₅₀ < 5.0 μM) in various cancer cell lines (HeLa, MDA-MB-231, HepG2). In addition, 13ea was found to significantly inhibit the phosphorylation function of CDK9 and the deacetylation function of class I HDACs. Furthermore, 13ea was found to inhibit the protein activity of CDK9 (IC₅₀ = 0.17 μM), HDAC1 (IC₅₀ = 1.73 μM), and HDAC3 (IC₅₀ = 1.11 μM). The docking studies predicted the binding patterns of 13ea in the active pockets of CDK9 and HDAC1/3. The cellular assays revealed that 13ea induced mitochondria-related apoptosis and G2/M phase arrest in cancer cells, showing superior activities compared to those of AZD-5438 (a CDK9 inhibitor) and Mocetinostat (an inhibitor of class I HDACs). Notably, the in vivo assay demonstrated that 13ea (30 mg/kg) exhibited significant inhibition on MDA-MB-231 xenograft tumor growth, with a tumor shrinkage rate of 76.83 %. In summary, we have identified 13ea as a novel CDK9/HDAC inhibitor with excellent anticancer activity in vitro and in vivo.

PMID:40383016 | DOI:10.1016/j.bioorg.2025.108577

Cancer Immunol Res. 2025 May 19. doi: 10.1158/2326-6066.CIR-24-0119. Online ahead of print.

ABSTRACT

The efficacy of most immunotherapies for prostate cancer is limited by poor tumor immunogenicity, evidenced by minimal T-cell infiltration. Treatment with T cells engineered to express T-cell receptors (TCR) targeting prostate-specific antigens offers a potential solution by bypassing endogenous T-cell repertoire limitations. Through differential gene expression analysis, we have identified kallikrein-related peptidases 2, 3 and 4 (KLK2, KLK3, KLK4) and homeobox B13 (HOXB13) as strictly prostate lineage-specific genes with high expression in prostate cancer and no expression in healthy tissues of risk. Naturally processed peptides derived from these antigens were identified, enabling T-cell enrichment using peptide-MHC multimers. High-avidity T cells targeting these antigens were isolated from allogeneic HLA-mismatched donors. After screening for on-target tumor specificity and absence of off-target reactivity, TCRs recognizing KLK4 in HLA-A*02:01 and KLK3 in HLA-B*35:01 were sequenced and further tested. TCRs were expressed in T cells through TCR gene transfer and TCRs with best performance were selected. Using combinatorial peptide library scanning, the cross-reactive potential of the KLK4-A2 and KLK3-B35 TCRs was analyzed. The KLK3-B35 TCR exhibited cross-reactivity against two additional peptides derived from LOXHD1 and CDH23, with broad tissue-expression, and was therefore excluded. The KLK4-A2 TCR was highly specific for the KLK4 peptide. Further testing confirmed effective cytotoxic killing potential of KLK4-A2 TCR in vitro and in vivo, underscoring its therapeutic potential. These findings highlight the promise of the KLK4-A2 TCR for prostate cancer immunotherapy and demonstrate that prostate-specific antigens can be effectively targeted using TCR-gene transfer strategies.

PMID:40387827 | DOI:10.1158/2326-6066.CIR-24-0119

ACS Appl Mater Interfaces. 2025 May 21;17(20):29183-29197. doi: 10.1021/acsami.5c01645. Epub 2025 May 7.

ABSTRACT

Macrophage phagocytosis of tumor cells shows significant promise in cancer treatment. However, it faces great challenges due to the upregulation of antiphagocytosis molecules, such as CD47, on the surface of tumor cells. Merely reducing the level of CD47 is insufficient to induce phagocytosis of tumor cells because it lacks enough "eat me" signals. Here, we have developed an engineered bacterial biohybrid system (eVNP@AuNFs) to decrease the expression of CD47 and HSP90 proteins, achieving an enhanced immuno-photothermal combination therapy. The attenuated Salmonella VNP20009, capable of selectively accumulating in hypoxic tumor regions, was intracellularly genetically engineered with CD47 and HSP90 shRNA plasmids and an extracellularly adsorbed flower-like gold nanoparticle (AuNF) photothermal agent, forming an eVNP@AuNF bacterial hybrid. After administration into 4T1 tumor-bearing mice intravenously, the eVNP@AuNF bacterial hybrid could effectively accumulate in tumor tissues and release CD47 and HSP90 shRNA plasmids to reduce the expression of CD47 and HSP90 protein, leading to enhanced macrophage phagocytosis to tumor cells and an improved photothermal effect. Under further NIR-II laser irradiation, extracellular AuNFs of eVNP@AuNFs could photothermally induce immunogenic cell death, including surface calreticulin exposure and high-mobility group box 1 translocation, facilitating the infiltration of the "eat me" signal and multiple immune cells and enhancing tumor immunogenicity. The eVNP@AuNF bacterial hybrid could eradicate the primary tumor and elicit a systemic antitumor immunity response, inhibiting the recurrence of the tumor. This study presents a hybrid system involving bacteria, plasmids, and nanomaterials for tumor therapy, opening an avenue for hierarchical modulation of the tumor immune response.

PMID:40331355 | DOI:10.1021/acsami.5c01645

Sheng Wu Gong Cheng Xue Bao. 2025 Apr 25;41(4):1221-1239. doi: 10.13345/j.cjb.240742.

ABSTRACT

Membrane vesicles (MVs) are non-replicating spherical nanoparticles produced by bacteria. The MVs actively released from Gram-negative bacteria are termed outer membrane vesicles (OMVs). OMVs carry various biomolecules, such as lipopolysaccharides, peptidoglycans, proteins, and nucleic acids for material exchange between cells and perform component-dependent physiological functions. In recent years, OMVs have been developed into various biological products, such as vaccines, adjuvants, drug delivery carriers, and cancer immunotherapy agents because of their unique structures and functions. This review describes the biogenesis, composition, and physiological functions of OMVs of Gram-negative bacteria, summarizes the recent research progress of OMVs in product development and cell modifications or engineering, highlights new methods for OMV preparation and characterization, and provides an outlook on the future research directions, with the aim to provide a good reference for study and development of the application of OMVs in the biomedical field.

PMID:40328692 | DOI:10.13345/j.cjb.240742

Proc Natl Acad Sci U S A. 2025 May 13;122(19):e2422185122. doi: 10.1073/pnas.2422185122. Epub 2025 May 5.

ABSTRACT

Mycobacterium tuberculosis (Mtb) is a leading cause of death worldwide. Mtb cell envelope glycans are potent virulence factors that play key roles in mediating infection of host tissues and modulating the host immune response. However, there are few ways to site-selectively modify and label these or any glycans to study their functions in disease. This gap arises because glycans generally lack functional groups amenable to bioconjugation strategies. Methylthioxylofuranose (MTX), a rare monosaccharide in select pathogenic mycobacteria, is an exception. MTX is appended to mannose-capped lipoarabinomannan (ManLAM), an antigenic glycolipid in the Mtb cell envelope implicated in downregulating the host immune system during infection. MTX is unique not only in its prevalence but also in its functionality-it contains a thioether not present in other glycans. We envisioned exploiting the MTX thioether to selectively label ManLAM with an oxaziridine probe. Here, we show that MTX-containing glycans can be labeled selectively in the test tube and live cells, highlighting the reactivity and accessibility of this motif. Our approach labels ManLAM efficiently despite the presence of protein methionine residues and can distinguish between different mycobacterial species. Using an oxaziridine equipped with a reporter, we could visualize ManLAM localization in live cells and a macrophage infection model, highlighting the stability of the label and the cell envelope in this environment. These studies will enable investigations of dynamic changes in a critical Mtb cell envelope component during infection. Moreover, the selective reactivity of thioethers can be leveraged to expand the repertoire of glycan bioconjugation strategies.

PMID:40324093 | PMC:PMC12088412 | DOI:10.1073/pnas.2422185122

Adv Mater. 2025 May 6:e2503571. doi: 10.1002/adma.202503571. Online ahead of print.

ABSTRACT

Gut-associated lymphoid tissue (GALT) possesses a highly specialized immune system and is rational as a foothold for oral tumor vaccines. Here, a noninvasive oral vaccine (Bif-OVA-Ocur) is designed to engage GALT, inducing both intestinal mucosal and systemic immunity for tumor therapeutics. The vaccine uses Bifidobacterium (Bif) as a delivery vehicle for tumor antigen peptides, which are coated with antigen peptides (OVA) and oxidized curdlan (Ocur) in a layer-by-layer (LBL) manner. Upon oral administration, Bif-OVA-Ocur is efficiently directed to Peyer's patches (PPs) in the intestines and further presented to antigen-presenting cells (APCs), which then migrate to the mesenteric lymph nodes (MLNs) to evoke specific T cell responses. In mouse models, Bif-OVA-Ocur effectively boosts the production of secretory immunoglobin A (SIgA) and promotes a strong mucosal and systemic immune response, leading to significant tumor suppression and resistance to tumor challenges. Importantly, the vaccine shows no systemic toxicity. This approach to harnessing the intestinal mucosal immune system offers valuable insights for the development of other non-invasive oral vaccines and therapeutic agents.

PMID:40326243 | DOI:10.1002/adma.202503571

Sci Rep. 2025 May 5;15(1):15628. doi: 10.1038/s41598-025-99141-2.

ABSTRACT

Peptidoglycan hydrolases are promising alternatives for combating pathogens due to their specificity and potent bacteriolytic activity. In this study, a novel M23 peptidase from Streptococcus thermophilus NCTC10353, designated StM23, was discovered and characterized. It exhibited antibacterial activity against Listeria monocytogenes and other Gram-positive bacteria with meso-DAP-type peptidoglycan, including Bacillus subtilis and Bacillus cereus. To enhance StM23's efficacy and specificity, a chimeric enzyme, StM23_CWT, was engineered by fusing its catalytic domain with a cell wall-targeting domain (CWT) from SpM23B, a peptidoglycan hydrolase found in Staphylococcus pettenkoferi. The engineered chimera demonstrated expanded specificity, showing activity against Staphylococcus aureus and Enterococcus faecium. Its ability to disrupt L. monocytogenes cells was visualized by electron microscopy. The enzyme effectively disrupted biofilm structures and decontaminated surfaces like glass, stainless steel, and silicone, showcasing its industrial potential. Safety evaluations using zebrafish, moth larvae, and human cell models confirmed its non-toxic profile, supporting its broad applicability. Based on these findings, StM23_CWT is a novel and potent antimicrobial agent with significant potential to reduce the risk of listeriosis and control persistent pathogens.

PMID:40325189 | PMC:PMC12052989 | DOI:10.1038/s41598-025-99141-2

J Immunother Cancer. 2025 May 2;13(5):e011074. doi: 10.1136/jitc-2024-011074.

ABSTRACT

BACKGROUND: Human papillomavirus (HPV)-associated malignancies continue to present a major health concern despite the development of prophylactic vaccines. Standard therapies offer limited benefit to patients with advanced-stage disease. Despite improved outcomes with programmed cell death protein-1 (PD-1) targeted therapies, treatment resistance and modest response rates highlight a significant unmet need to develop novel therapies for these patients. PDS0101 (designated HPV vaccine) is a liposomal nanoparticle HPV16-specific therapeutic vaccine that has been shown to generate strong HPV-specific responses in preclinical and clinical studies. Here we assess the efficacy of this HPV vaccine in combination with the tumor-targeting immunocytokine NHS-IL12 (PDS01ADC), plus either αPD-1 or the class I histone deacetylase inhibitor Entinostat.

METHODS: Mice bearing HPV16⁺, αPD-1 refractory TC-1 and mEER tumors were treated with HPV vaccine, NHS-IL12, and either αPD-1 or Entinostat to determine antitumor efficacy and survival benefits. A comprehensive analysis of the tumor microenvironment was performed using flow cytometry, multiplex immunofluorescence, chemokine and cytokine assessment, and single-cell RNA sequencing with T-cell receptor (TCR) enrichment.

RESULTS: Combination of HPV vaccine and NHS-IL12 with either Entinostat or αPD-1 yielded significant antitumor activity and prolonged survival in αPD-1 refractory models of HPV16⁺ cancer, with superior activity employing Entinostat versus αPD-1 combination. Entinostat triple therapy increased overall and HPV16-specific tumor CD8⁺ T-cell infiltration with heightened cytotoxicity. TCR sequencing revealed a CD8⁺ T-cell clone unique to vaccine-treated cohorts, which displayed an enriched cytotoxic transcriptional profile with triple therapy. These effects were paralleled by strong differentiation of tumor-associated macrophages (TAMs) towards pro-inflammatory, antitumor M1-like cell states. Single-cell transcriptomic analysis indicated all three agents were required for highest modulation of both CD8⁺ T cells and TAMs conducive to tumor control. A biomarker signature reflecting the preclinical findings was found to be associated with improved survival in patients with HPV-associated malignancies.

CONCLUSION: Together, these findings provide a rationale for the combination of HPV vaccine, NHS-IL12, and Entinostat in the clinical setting for patients with HPV16-associated malignancies.

PMID:40316302 | PMC:PMC12049950 | DOI:10.1136/jitc-2024-011074

Bioorg Chem. 2025 Apr 28;161:108514. doi: 10.1016/j.bioorg.2025.108514. Online ahead of print.

ABSTRACT

The development of selective histone deacetylase (HDAC) inhibitors represents an encouraging approach for cancer therapy. In this study, we report design, synthesis, and biological evaluation of hydroxamate, amidoxime, and carboxylic acid-based derivatives as novel HDAC inhibitors. The synthesized compounds were assessed for their inhibitory activity against multiple HDAC isoforms, particularly HDAC6, 7, and 8. Compounds 13, 16, 20, and 26 exhibited potent and selective inhibition of HDAC6. Compound 26 exhibited the most potent inhibitory activity against HDAC6, with an IC₅₀ value of 70 nM. Additionally, compounds 17 and 23 demonstrated significant broad-spectrum antiproliferative activity across various cancer cell lines compared to other tested derivatives. Furthermore, compounds 17 and 23 showed promising total pan-HDAC inhibitory activity. Subsequent biological studies revealed that compounds 13, 16, 17, 20, 23, and 26 induced a combination of early and late apoptosis along with necrosis. In silico studies, including molecular docking and ADME predictions, were also conducted. Collectively, these findings highlight the potential of these compounds as promising candidates for the development of a novel class of selective HDAC6 inhibitors in the future.

PMID:40319810 | DOI:10.1016/j.bioorg.2025.108514

Chem Sci. 2025 Apr 29. doi: 10.1039/d4sc08141b. Online ahead of print.

ABSTRACT

The binding of T cell receptors (TCRs) to peptide-MHC I (pMHC) complexes is critical for triggering adaptive immune responses to potential health threats. Developing highly accurate machine learning (ML) models to predict TCR-pMHC binding could significantly accelerate immunotherapy advancements. However, existing ML models for TCR-pMHC binding prediction often underperform with unseen epitopes, severely limiting their applicability. We introduce TRAP, which leverages contrastive learning to enhance model performance by aligning structural and sequence features of pMHC with TCR sequences. TRAP outperforms previous state-of-the-art models in both random and unseen epitope scenarios, achieving an AUPR of 0.84 (a 22% improvement over the second-best model) and an AUC of 0.92 in the random scenario, and an AUC of 0.75 (almost 11% higher than the second-best model) in the unseen epitope scenario. Furthermore, TRAP demonstrates a noteworthy capability to diagnose potential issues of cross-reactivity between TCRs and similar epitopes. This highly robust performance makes it a suitable tool for large-scale predictions in real-world settings. A specific case study confirmed that TRAP can discover hit TCRs with binding free energies comparable to referenced experimental results. These findings highlight TRAP's potential for practical applications and its role as a powerful tool in developing TCR-based immunotherapies.

PMID:40321182 | PMC:PMC12046420 | DOI:10.1039/d4sc08141b

Dev Comp Immunol. 2025 May 2:105384. doi: 10.1016/j.dci.2025.105384. Online ahead of print.

ABSTRACT

Hemorrhagic disease caused by grass carp reovirus (GCRV) poses a significant threat to the health and sustainability of grass carp (Ctenopharyngodon idella) farming. There are no effective measures to control the outbreaks of the disease. While DNA vaccines have proved to be promising to enhance the survival of vaccinated fish to GCRV infection, the protective efficacy is not maximized, and necessitates further improvement. This study explores the immunomodulatory potential of type II interferons (IFNs), including IFN-γ and IFN-γ related molecule (IFN-γrel), as adjuvants for GCRV-VP35 DNA vaccine. Expression plasmids, including pcDNA3.1-VP35, pcDNA3.1-IFN-γ, and/or pcDNA3.1-IFN-γrel, were intramuscularly administered in grass carp, and their effects on the expression of immune genes evaluated. Immunofluorescence microscopy confirmed the localized expression of GCRV-VP35, IFN-γ and IFN-γrel at the injection site, with the persistent expression detected for at least five weeks. Moreover, co-administration of IFN-γ and IFN-γrel plasmids synergistically enhanced the expression of Mx1, Isg15 and Viperin to a greater extent than either plasmid alone. The Igm and Cd8 genes were also upregulated in the spleen and muscle of fish injected with the IFN-γ/IFN-γrel plasmids. Furthermore, our findings reveal that IFN-γ and IFN-γrel robustly upregulated the expression of Mhc I but not Mhc II to promote antigen presentation of VP35 vaccine. The results indicate that type II IFNs have potential as adjuvants to enhance the immunogenicity and efficacy of DNA vaccines in protecting fish against viral infection.

PMID:40320154 | DOI:10.1016/j.dci.2025.105384