Dual-Activity Fluoroquinolone-Transportan 10 Conjugates Offer Alternative Leukemia Therapy during Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is often considered a last resort leukemia treatment, fraught with limited success due to microbial infections, a leading cause of mortality in leukemia patients. To address this critical issue, we explored a novel approach by synthesizing antileukemic agents containing antibacterial substances. This innovative strategy involves conjugating fluoroquinolone antibiotics, such as ciprofloxacin (CIP) or levofloxacin (LVX), with the cell-penetrating peptide transportan 10 (TP10). Here, we demonstrate that the resultant compounds display promising biologic activities in preclinical studies. These novel conjugates not only exhibit potent antimicrobial effects but are also selective against leukemia cells. The cytotoxic mechanism involves rapid disruption of cell membrane asymmetry leading to membrane damage. Importantly, these conjugates penetrated mammalian cells, accumulating within the nuclear membrane without significant effect on cellular architecture or mitochondrial function. Molecular simulations elucidated the aggregation tendencies of TP10 conjugates within lipid bilayers, resulting in membrane disruption and permeabilization. Moreover, mass spectrometry analysis confirmed efficient reduction of disulfide bonds within TP10 conjugates, facilitating release and activation of the fluoroquinolone derivatives. Intriguingly, these compounds inhibited human topoisomerases, setting them apart from traditional fluoroquinolones. Remarkably, TP10 conjugates generated lower intracellular levels of reactive oxygen species compared with CIP and LVX. The combination of antibacterial and antileukemic properties, coupled with selective cytostatic effects and minimal toxicity toward healthy cells, positions TP10 derivatives as promising candidates for innovative therapeutic approaches in the context of antileukemic HCT. This study highlights their potential in search of more effective leukemia treatments. SIGNIFICANCE STATEMENT: Fluoroquinolones are commonly used antibiotics, while transportan 10 (TP10) is a cell-penetrating peptide (CPP) with anticancer properties. In HCT, microbial infections are the primary cause of illness and death. Combining TP10 with fluoroquinolones enhanced their effects on different cell types. The dual pharmacological action of these conjugates offers a promising proof-of-concept solution for leukemic patients undergoing HCT. Strategically designed therapeutics, incorporating CPPs with antibacterial properties, have the potential to reduce microbial infections in the treatment of malignancies.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

Relationship between Telomere Length, TERT Genetic Variability and TERT, TP53, SP1, MYC Gene Co-Expression in the Clinicopathological Profile of Breast Cancer.

The molecular mechanisms of telomerase reverse transcriptase (TERT) upregulation in breast cancer (BC) are complex. We compared genetic variability within TERT and telomere length with the clinical data of patients with BC. Additionally, we assessed the expression of the TERT, MYC, TP53 and SP1 genes in BC patients and in BC organoids (3D cell cultures obtained from breast cancer tissues). We observed the same correlation in the blood of BC patients and in BC organoids between the expression of TERT and TP53. Only in BC patients was a correlation found between the expression of the TERT and MYC genes and between TP53 and MYC. We found associations between TERT genotypes (rs2735940 and rs10069690) and TP53 expression and telomere length. BC patients with the TT genotype rs2735940 have a shorter telomere length, but patients with A allele rs10069690 have a longer telomere length. BC patients with a short allele VNTR-MNS16A showed higher expression of the SP1 and had a longer telomere. Our results bring new insight into the regulation of TERT, MYC, TP53 and SP1 gene expression related to TERT genetic variability and telomere length. Our study also showed for the first time a similar relationship in the expression of the above genes in BC patients and in BC organoids. These findings suggest that TERT genetic variability, expression and telomere length might be useful biomarkers for BC, but their prognostic value may vary depending on the clinical parameters of BC patients and tumor aggressiveness.

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells.

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.

Cancer immune escape: the role of antigen presentation machinery.

The mechanisms of antigen processing and presentation play a crucial role in the recognition and targeting of cancer cells by the immune system. Cancer cells can evade the immune system by downregulating or losing the expression of the proteins recognized by the immune cells as antigens, creating an immunosuppressive microenvironment, and altering their ability to process and present antigens. This review focuses on the mechanisms of cancer immune evasion with a specific emphasis on the role of antigen presentation machinery. The study of the immunopeptidome, or peptidomics, has provided insights into the mechanisms of cancer immune evasion and has potential applications in cancer diagnosis and treatment. Additionally, manipulating the epigenetic landscape of cancer cells plays a critical role in suppressing the immune response against cancer. Targeting these mechanisms through the use of HDACis, DNMTis, and combination therapies has the potential to improve the efficacy of cancer immunotherapy. However, further research is needed to fully understand the mechanisms of action and optimal use of these therapies in the clinical setting.

Development of Safirinium dyes for new applications: fluorescent staining of bacteria, human kidney cells, and the horny layer of the epidermis.

Low-molecular synthetic fluorophores are convenient tools in bioimaging applications. Several derivatives of Safirinium dyes as well as their reactive N-hydroxysuccinimide (NHS) esters bearing diverse substituents were synthesized and evaluated experimentally in terms of their lipophilicity by means of reverse-phase and immobilized artificial membrane high-performance liquid chromatography. Subsequently, the selected compounds were employed as novel cellular imaging agents for staining Gram-positive and Gram-negative bacteria, human kidney cell line, as well as human skin tissue. The analyzed dyes allowed for visualization of cellular structures such as mitochondria, endoplasmic reticulum, and cellular nuclei. They proved to be useful in fluorescent staining of stratum corneum, especially in the aspect of xenobiotic exposure and its penetration into the skin. The best results were obtained with the use of moderately lipophilic NHS esters of Safirinium Q. The development of Safirinium dyes is a promising alternative for commercially available dyes since the reported molecules have low molecular masses and exhibit efficient staining and remarkable water solubility. Moreover, they are relatively simple and low-cost in synthesis.

New Peptide Based Fluconazole Conjugates with Expanded Molecular Targets.

Infections of Candida spp. etiology are frequently treated with azole drugs. Among azoles, the most widely used in the clinical scenario remains fluconazole (FLC). Promising results in treatment of dangerous, systemic Candida infections demonstrate the advantages of combined therapies carried out with combinations of at least two different antifungal agents. Here, we report five conjugates composed of covalently linked FLC and cell penetrating or antimicrobial peptide: TP10-7-NH2, TP10-NH2, LFcinB(2-11)-NH2, LFcinB[Nle1,11]-NH2, and HLopt2-NH2, with aspects of design, chemical synthesis and their biological activities. Two of these compounds, namely FLCpOH-TP10-NH2 and FLCpOH-TP10-7-NH2, exhibit high activity against reference strains and fluconazole-resistant clinical isolates of C. albicans, including strains overproducing drug transporters. Moreover, both of them demonstrate higher fungicidal effects compared to fluconazole. Analysis performed with fluorescence and scanning electron microscopy as well as flow cytometry indicated the cell membrane as a molecular target of synthesized conjugates. An important advantage of FLCpOH-TP10-NH2 and FLCpOH-TP10-7-NH2 is their low cytotoxicity. The IC90 value for the human cells after 72 h treatment was comparable to the MIC50 value after 24 h treatment for most strains of C. albicans. In reported conjugates, FLC was linked to the peptide by its hydroxyl group. It is worth noting that conjugation of FLC by the nitrogen atom of the triazole ring led to practically inactive compounds. Two compounds produced by us and reported herein appear to be potential candidates for novel antifungal agents.