(1) Background: Natural peptides supporting the innate immune system studied at the functional and mechanistic level are a rich source of innovative compounds for application in human therapy. Increasing evidence indicates that apart from antimicrobial activity, some of them exhibit selective cytotoxicity towards tumor cells. Their cationic, amphipathic structure enables interactions with the negatively-charged membranes of microbial or malignant cells. It can be modeled in 3D by application of dendrimer chemistry. (2) Methods: Here we presented design principles, synthesis and bioactivity of branched peptides constructed from ornithine (Orn) assembled as proline (Pro)- or histidine (His)-rich dendrons and dendrimers of the bola structure. The impact of the structure and amphipathic properties of dendrons/dendrimers on two glioblastoma cell lines U87 and T98G was studied with the application of proliferation, apoptosis and cell migration assays. Cell morphology/cytoskeleton architecture was visualized by immunofluorescence microscopy. (3) Results: Dimerization of dendrons into bola dendrimers enhanced their bioactivity. Pro- and His-functionalized bola dendrimers displayed cytostatic activity, even though differences in the responsiveness of U87 and T98G cells to these compounds indicate that their bioactivity depends not only on multiple positive charge and amphipathic structure but also on cellular phenotype. (4) Conclusion: Ornithine dendrons/dendrimers represent a group of promising anti-tumor agents and the potential tools to study interrelations between drug bioactivity, its chemical properties and tumor cells’ phenotype.

The compounds 7-ethyl-9-(N-methylamino)methyl-10-hydroxycamptothecin (2) and 7-ethyl-9-(N-morpholino)methyl-10-hydroxycamptothecin (3) are potential topoisomerase I poisons. Moreover, they were shown to have favorable anti-neoplastic effects on several tumor cell lines. Due to these properties, the compounds are being considered for advancement to the preclinical development stage. To gain better insights into the molecular mechanism with the biological target, here, we conducted an investigation into their interactions with model nicked DNA (1) using different techniques. In this work, we observed the complexity of the mechanism of action of the compounds 2 and 3, in addition to their decomposition products: compound 4 and SN38. Using DOSY experiments, evidence of the formation of strongly bonded molecular complexes of SN38 derivatives with DNA duplexes was provided. The molecular modeling based on cross-peaks from the NOESY spectrum also allowed us to assign the geometry of a molecular complex of DNA with compound 2. Confirmation of the alkylation reaction of both compounds was obtained using MALDI–MS. Additionally, in the case of 3, alkylation was confirmed in the recording of cross-peaks in the ¹H/¹³C HSQC spectrum of ¹³C-enriched compound 3. In this work, we showed that the studied compounds—parent compounds 2 and 3, and their potential metabolite 4 and SN38—interact inside the nick of 1, either forming the molecular complex or alkylating the DNA nitrogen bases. In order to confirm the influence of the studied compounds on the topoisomerase I relaxation activity of supercoiled DNA, the test was performed based upon the measurement of the fluorescence of DNA stain which can differentiate between supercoiled and relaxed DNA. The presented results confirmed that studied SN38 derivatives effectively block DNA relaxation mediated by Topo I, which means that they stop the machinery of Topo I activity.

The understanding of the mechanism of Topo I inhibition by organic ligands is a crucial source of information that has led to the design of more effective and safe pharmaceuticals in oncological chemotherapy. The vast number of inhibitors that have been studied in this respect over the last decades have enabled the creation of a concept of an ‘interfacial inhibitor’, thereby describing the machinery of Topo I inhibition. The central module of action of this machinery is the interface of a Topo I/DNA/inhibitor ternary complex. Most of the ‘interfacial inhibitors’ are primarily kinetic inhibitors that form molecular complexes with an “on–off” rate timing; therefore, all of the contacts between the inhibitor and both the enzyme and the DNA are essential to keep the complex stable and reduce the “off rate”. To test this hypothesis, we designed the compound using a C-9-(N-(2′-hydroxyethyl)amino)methyl substituent in an SN38 core, with a view that a flexible substituent may bind inside the nick of a model of the DNA and stabilize the complex, leading to a reduction in the “off rate” of a ligand in a potential ternary complex in vivo. Using docking analysis and molecular dynamics, free energy calculations on the level of the MM-PBSA and MM-GBSA model, here we presented the in silico-calculated structure of a ternary complex involving the studied compound 1. This confirmed our suggestion that compound 1 is situated in a groove of the nicked DNA model in a few conformations. The number of hydrogen bonds between the components of a ternary complex was established, which strengthens the complex and supports our view. The docking analysis and free energy calculations for the receptor structures which were obtained in the MD simulations of the ternary complex 1/DNA/Topo I show that the binding constant is stronger than it was for similar complexes with TPT, CPT, and SN38, which are commonly considered as strong Topo I inhibitors. The binary complex structure 1/DNA was calculated and compared with the experimental results of a complex that was in a solution. The analysis of the cross-peaks in NOESY spectra allowed us to assign the dipolar interactions between the given protons in the calculated structures. A DOSY experiment in the solution confirmed the strong binding of a ligand in a binary complex, having a K_a of 746 mM⁻¹, which was compared with a K_a of 3.78 mM⁻¹ for TPT. The MALDI-ToF MS showed the presence of the biohybrid, thus evidencing the occurrence of DNA alkylation by compound 1. Because of it having a strong molecular complex, alkylation is the most efficient way to reduce the “on–off” timing as it acts as a tool that causes the cog to brake in a working gear, and this is this activity we want to highlight in our contribution. Finally, the Topo I inhibition test showed a lower IC₅₀ of the studied compound than it did for CPT and SN38.