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.

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.

Antibiotic-Based Conjugates Containing Antimicrobial HLopt2 Peptide: Design, Synthesis, Antimicrobial and Cytotoxic Activities.

Recent studies have shown that modified human lactoferrin 20-31 fragment, named HLopt2, possesses antibacterial and antifungal activity. Thus, we decided to synthesize and evaluate the biological activity of a series of conjugates based on this peptide and one of the antimicrobials with proven antibacterial (ciprofloxacin, CIP, and levofloxacin, LVX) or antifungal (fluconazole, FLC) activity. The drugs were covalently connected to the peptide via amide, methylenecarbonyl moieties, or a disulfide bridge. The antibacterial and antifungal activities were evaluated under Clinical and Laboratory Standard Institute (CLSI) recommended conditions or in a low-salt brain-heart infusion diluted medium (BHI1/100). Results showed that conjugation of the peptide with the drug increased its antimicrobial activity up to 4-fold. Under CLSI-recommended conditions, all the compounds revealed rather low efficiency. Among conjugates, the highest antibacterial activity was recorded for the CIP-Cys-S-S-HLopt2-NH2 (III). In BHI1/100, which had lower differentiating properties, all of the conjugates revealed low MIC and MMC (minimum inhibitory and microbicidal concentrations) values. The disulfide bridge used as a linker in the most active conjugate (III) upon incubation with S. aureus cells is reduced, releasing constituent peptide and CIP-Cys. In addition, we showed that its fluorescently labeled analogue and constituent peptide are able to be internalized into both C. albicans and S. aureus cells. Moreover, the invaluable advantage of the presented conjugates was their low toxicity to mammalian cells and very low hemolytic activity. The current research can form a solid basis for further in vivo studies and drug development.

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.

Emerging Anticancer Activity of Candidal Glucoseamine-6-Phosphate Synthase Inhibitors upon Nanoparticle-Mediated Delivery.

Numerous glutamine analogues have been reported as irreversible inhibitors of the glucosamine-6-phosphate (GlcN-6-P) synthase in pathogenic Candida albicans in the last 3.5 decades. Among the reported inhibitors, the most effective N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) has been extensively studied in order to develop its more active analogues. Several peptide-FMDP conjugates were tested to deliver FMDP to its subcellularly located GlcN-6-P synthase target. However, the rapid development of fungal resistance to FMDP-peptides required development of different therapeutic approaches to tackle antifungal resistance. In the current state of the global antifungal resistance, subcellular delivery of FMDP via free diffusion or endocytosis has become crucial. In this study, we report on in vitro nanomedical applications of FMDP and one of its ketoacid analogues, N3- trans-4-oxo-4-phenyl-2-butenoyl-l-2,3-diaminopropanoic acid (BADP). FMDP and BADP covalently attached to polyethylene glycol-coated iron oxide/silica core-shell nanoparticles are tested against intrinsically multidrug-resistant C. albicans. Three different human cancer cell lines potentially overexpressing the GlcN-6-P synthase enzyme are tested to demonstrate the immediate inhibitory effects of nanoparticle conjugates against mammalian cells. It is shown that nanoparticle-mediated delivery transforms FMDP and BADP into strong anticancer agents by inhibiting the growth of the tested cancer cells, whereas their anti-Candidal activity is decreased. This study discusses the emerging inhibitory effect of the FMDP/BADP-nanoparticle conjugates based on their cellular internalization efficiency and biocompatibility.

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.

Molecular Umbrellas Modulate the Selective Toxicity of Polyene Macrolide Antifungals.

Antifungal polyene macrolide antibiotics Amphotericin B (AmB) and Nystatin (NYS) were conjugated through the ω-amino acid linkers with diwalled "molecular umbrellas" composed of spermidine-linked deoxycholic or cholic acids. The presence of "umbrella" substituents modulated biological properties of the antibiotics, especially their selective toxicity. Some of the AmB-umbrella conjugates demonstrated antifungal in vitro activity comparable to that of the mother antibiotic but diminished mammalian toxicity, especially the hemolytic activity. In contrast, antifungal in vitro activity of NYS-umbrella conjugates was strongly reduced and all these conjugates demonstrated poorer than NYS selective toxicity. No correlation between the aggregation state and hemolytic activity of the novel conjugates was found.

Acetate-Induced Disassembly of Spherical Iron Oxide Nanoparticle Clusters into Monodispersed Core-Shell Structures upon Nanoemulsion Fusion.

It has been long known that the physical encapsulation of oleic acid-capped iron oxide nanoparticles (OA-IONPs) with the cetyltrimethylammonium (CTA+) surfactant induces the formation of spherical iron oxide nanoparticle clusters (IONPCs). However, the behavior and functional properties of IONPCs in chemical reactions have been largely neglected and are still not well-understood. Herein, we report an unconventional ligand-exchange function of IONPCs activated when dispersed in an ethyl acetate/acetate buffer system. The ligand exchange can successfully transform hydrophobic OA-IONP building blocks of IONPCs into highly hydrophilic, acetate-capped iron oxide nanoparticles (Ac-IONPs). More importantly, we demonstrate that the addition of silica precursors (tetraethyl orthosilicate and 3-aminopropyltriethoxysilane) to the acetate/oleate ligand-exchange reaction of the IONPs induces the disassembly of the IONPCs into monodispersed iron oxide-acetate-silica core-shell-shell (IONPs@acetate@SiO2) nanoparticles. Our observations evidence that the formation of IONPs@acetate@SiO2 nanoparticles is initiated by a unique micellar fusion mechanism between the Pickering-type emulsions of IONPCs and nanoemulsions of silica precursors formed under ethyl acetate buffered conditions. A dynamic rearrangement of the CTA+-oleate bilayer on the IONPC surfaces is proposed to be responsible for the templating process of the silica shells around the individual IONPs. In comparison to previously reported methods in the literature, our work provides a much more detailed experimental evidence of the silica-coating mechanism in a nanoemulsion system. Overall, ethyl acetate is proven to be a very efficient agent for an effortless preparation of monodispersed IONPs@acetate@SiO2 and hydrophilic Ac-IONPs from IONPCs.

Characterization of recombinant homocitrate synthase from Candida albicans.

LYS21 and LYS22 genes from Candida albicans encoding isoforms of homocitrate synthase (HCS), an enzyme catalyzing the first committed step in the l-lysine biosynthetic pathway, were cloned and expressed as N-oligoHistagged fusion proteins in Escherichia coli. The purified gene products revealed HCS activity, i.e. catalyzed the condensation of α-ketoglutarate with acetyl-coenzyme A to yield homocitrate. The recombinant enzymes were purified to homogeneity and characterized for their physical properties and substrate specificities. As determined by size-exclusion chromatography (SEC) and native page electrophoresis, both isoenzymes adopt multiple quaternary structures, with the homotetrameric one being the most abundant. The KM (acetyl-CoA)=0.8±0.15mM and KM (α-ketoglutarate)=0.113±0.02mM for His6CaLys21p and KM (acetyl-CoA)=0.48±0.09mM and KM (α-ketoglutarate)=0.152±0.03mM values for His6CaLys22p were determined. Both enzyme versions were inhibited by l-Lys, i.e. the end product of the α-aminoadipate pathway but Lys22p was more sensitive than Lys21p, with Ki (L-Lys)=128±8µM for His6CaLys21p and Ki (L-Lys)=4.37±0.68µM for His6CaLys22p. The isoforms of C. albicans HCS exhibited differential sensitivity to several l-Lys analogues. Most notably, dl-α-difluoromethyllysine strongly inhibited His6CaLys22p (IC50 32±3µM) but was not inhibitory at all towards His6CaLys21p. Differential sensitivity of recombinant C. albicans Δlys21/LYS22, LYS21/Δlys22 and Δlys21/Δlys22 mutant strains to lysine analog, 2-aminoethyl-l-cysteine and biochemical properties of homocitrate synthase isoforms suggest different roles of two HCS isoenzymes in α-aminoadipate pathway.

Inactivation of glucosamine-6-phosphate synthase by N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid.

N(3)-Oxoacyl derivatives of L-2,3-diaminopropanoic acid 1-4, containing either an epoxide group or a conjugated double bond system, inactivate Saccharomyces cerevisiae glucosamine-6-phosphate (GlcN-6-P) synthase in a time- and concentration dependent manner. The results of kinetics studies on inactivation suggested a biphasic course, with formation of the enzyme-ligand complex preceding irreversible modification of the enzyme. The examined compounds differed markedly in their affinity to the enzyme active site. Inhibitors containing a phenyl ketone moiety bound much more strongly than their methyl ketone counterparts. The molecular mechanism of enzyme inactivation by phenyl ketone compounds 1 and 3 was elucidated by using a stepwise approach with 2D NMR, MS and UV-visible spectroscopy. A substituted thiazine derivative was identified as the final product of a model reaction between an epoxide compound, 1, and L-cysteine ethyl ester (CEE); and the respective cyclic product, found as a result of reaction between 1 and CGIF tetrapeptide, was identical to the N-terminal fragment of GlcN-6-P synthase. On the other hand, the reaction of a double-bond-containing compound, 3, with CEE, CGIF and GlcN-6-P synthase led to the formation of a C-S bond, without any further conversion or rearrangement. Molecular mechanisms of the reactions studied are proposed.

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase.

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.

Structure-activity relationships for a series of peptidomimetic antimicrobial prodrugs containing glutamine analogues.

Synthetic glutamine analogues such as N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP) inhibit purified glucosamine-6-phosphate synthase, an intracellular enzyme that is essential for microbial cell wall synthesis, but they are inactive against intact organisms because they cannot enter the cell. However, when the analogues are linked to a peptide they can be actively transported, and FMDP peptidomimetics show broad-spectrum antimicrobial activity. To characterize this process in more detail, the antibacterial activities of various synthetic peptidomimetics containing glutamine analogues have been determined against isogenic strains of Escherichia coli in which one or more of its three peptide transporters Dpp, Opp and Tpp have been mutated. In addition, their affinities for DppA and OppA, the binding-protein components of the transporters, have been measured. In general, antibacterial activities against the various transport mutants correlated with binding to DppA and OppA. Xaa-FMDP compounds have greater activities than FMDP-Xaa analogues. To explore structure-activity relationships for the peptidomimetics, molecular modelling was used to determine the conformational forms they adopt in solution. The relative bioactivities of the peptidomimetics correlated with the percentage of conformers that had backbone torsions matching those previously defined for the molecular recognition templates of the peptide transporters. However, the large size of the N-terminal residue in the FMDP-Xaa analogues appears to interfere with transport and thus to limit antibacterial activity. Overall, the results provide the structural rationale for the identification in silico of analogues with optimal bioactivities, which decreases the need for extensive chemical syntheses and testing.

Glucosamine-6-phosphate synthase--the multi-facets enzyme.

L-Glutamine: D-fructose-6-phosphate amidotransferase, known under trivial name of glucosamine-6-phosphate synthase, as the only member of the amidotransferase subfamily of enzymes, does not display any ammonia-dependent activity. This enzyme, catalysing the first committed step in a pathway leading to the eventual formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules. The molecular mechanism of reaction catalysed by GlcN-6-P synthase is complex and involves both amino transfer and sugar isomerisation. Substantial alterations to the enzyme structure and properties have been detected in different neoplastic tissues. GlcN-6-P synthase is inflicted in phenomenon of hexosamine-induced insulin resistance in diabetes. Finally, this enzyme has been proposed as a promising target in antifungal chemotherapy. Most of these issues, especially their molecular aspects, have been extensively studied in recent years. This article provides a comprehensive overview of the present knowledge on this multi-facets enzyme.