Lactoferricin B Combined with Antibiotics Exhibits Leukemic Selectivity and Antimicrobial Activity.

The fusion of penetrating peptides (PPs), e.g., cell penetration peptides (CPPs) or antimicrobial peptides (AMPs), together with antimicrobial agents is an expanding research field. Specific AMPs, such as lactoferricin B (LfcinB), have demonstrated strong antibacterial, antifungal, and antiparasitic activity, as well as valuable anticancer activity, proving beneficial in the development of anticancer conjugates. The resulting conjugates offer potential dual functionality, acting as both an anticancer and an antimicrobial agent. This is especially necessary in cancer treatment, where microbial infections pose a critical risk. Leukemic cells frequently exhibit altered outer lipid membranes compared to healthy cells, making them more sensitive to compounds that interfere with their membrane. In this study, we revisited and reanalyzed our earlier research on LfcinB and its conjugates. Furthermore, we carried out new experiments with a specific focus on cell proliferation, changes in membrane asymmetric phosphatidylserine location, intracellular reactive oxygen species (ROS) generation, mitochondrial functions, and in vitro bacterial topoisomerase inhibition.

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.

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.

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity.

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25 - 100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10 - 100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable.

Antimicrobial Activity of Chimera Peptides Composed of Human Neutrophil Peptide 1 (HNP-1) Truncated Analogues and Bovine Lactoferrampin.

Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond, whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity toward Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs.

(Very) Small Stem-like Cells in Human Cell Cultures.

Very Small Embryonic-like Stem Cells (VSELSCs) and Very Small Cancer Stem Cells (VSCSCs) are fields of intensive research. Although the presence in vitro of VSELSC and VSCSC cellular stage analogs appear probable, it has yet to be published. Utilizing established human cell cultures with varying populations of primitive cells, stained with CD markers specific to primitive stages, in addition to a fluorescent DNA dye, and following histochemical processing, we have developed a cytological method for detecting Very Small Leukemic Stem-like Cells (VSLSLCs), Very Small Cancer Stem-like Cells (VSCSLCs), and VSELSCs. This detection provides an opportunity to advance research in these areas.

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.

Peptide conjugates of lactoferricin analogues and antimicrobials-Design, chemical synthesis, and evaluation of antimicrobial activity and mammalian cytotoxicity.

Eight new peptide conjugates composed of modified bovine lactoferricin truncated analogues (LFcinB) and one of the three antimicrobials - ciprofloxacin (CIP), levofloxacin (LVX), and fluconazole (FLC) - were synthesized. Four different linkers were applied to connect a peptide and an antimicrobial agent. The FLC-containing peptidic conjugates were synthesized using the "click chemistry" method. This novel approach is reported here for the first time. Unlike their components, CIP- and LVX-based conjugates exerted activity against Candida yeast. Similarly to the constituent peptides, synthesized conjugates showed activity against Gram-positive bacteria, especially S. epidermidis. The most active were the conjugates containing CIP linked to the peptide by the redox-sensitive disulfide bridge. Our results show a significant role of a linker between antimicrobial agent and a peptide. This was also confirmed by the lack of synergistic effects on the antimicrobial activity of the constituent compounds. Moreover, cytotoxicity assays revealed that the proposed conjugates cause a comparatively low cytotoxic effect in reference to antibiotics widely used in therapies. Therefore, they can be deliberated as attractive leading structures for the development of drugs.

Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells.

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7.

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.

Role of heat-shock proteins and cobalamine in maintaining methionine synthase activity.

Atheromatous plaque is one of the most common cardiovascular-related diseases. Reports show a connection between its development and the levels of homocysteine. In pathological states high levels of homocysteine in the organism can be caused by the malfunction of the methionine synthase pathway. Bacterial methionine synthase (MetH) is a homologue of the human methionine syntase (MS). In this study we aimed to investigate the functional relations between MetH and its cofactor--cobalamine--under stress conditions. We have demonstrated that heat shock proteins (Hsp 70/100 system or HtpG) can protect MetH activity under stress conditions. Moreover, in the presence of cobalamine they can restore the activity of partially denatured methionine synthase.