HDAC specificity and kinase off-targeting by purine-benzohydroxamate anti-hematological tumor agents.

A series of hybrid inhibitors, combining pharmacophores of known kinase inhibitors bearing anilino-purines (ruxolitinib, ibrutinib) and benzohydroxamate HDAC inhibitors (nexturastat A), were generated in the present study. The compounds have been synthesized and tested against solid and hematological tumor cell lines. Compounds 4d-f were the most promising in cytotoxicity assays (IC50 ≤ 50 nM) vs. hematological cells and displayed moderate activity in solid tumor models (EC50 = 9.3-21.7 µM). Compound 4d potently inhibited multiple kinase targets of interest for anticancer effects, including JAK2, JAK3, HDAC1, and HDAC6. Molecular dynamics simulations showed that 4d has stable interactions with HDAC and members of the JAK family, with differences in the hinge binding energy conferring selectivity for JAK3 and JAK2 over JAK1. The kinase inhibition profile of compounds 4d-f allows selective cytotoxicity, with minimal effects on non-tumorigenic cells. Moreover, these compounds have favorable pharmacokinetic profiles, with high stability in human liver microsomes (e.g., see t1/2: >120 min for 4f), low intrinsic clearance, and lack of significant inhibition of four major CYP450 isoforms.

Nanostructure Formation and Cell Spheroid Morphogenesis of a Peptide Supramolecular Hydrogel.

Peptide-based hydrogels have attracted much attention due to their extraordinary applications in biomedicine and offer an excellent mimic for the 3D microenvironment of the extracellular matrix. These hydrated matrices comprise fibrous networks held together by a delicate balance of intermolecular forces. Here, we investigate the hydrogelation behavior of a designed decapeptide containing a tetraleucine self-assembling backbone and fibronectin-related tripeptides near both ends of the strand. We have observed that this synthetic peptide can produce hydrogel matrices entrapping >99% wt/vol % water. Ultrastructural analyses combining atomic force microscopy, small-angle neutron scattering, and X-ray diffraction revealed that amyloid-like fibrils form cross-linked networks endowed with remarkable thermal stability, the structure of which is not disrupted up to temperatures >80 °C. We also examined the interaction of peptide hydrogels with either NIH3T3 mouse fibroblasts or HeLa cells and discovered that the matrices sustain cell viability and induce morphogenesis into grape-like cell spheroids. The results presented here show that this decapeptide is a remarkable building block to prepare highly stable scaffolds simultaneously endowed with high water retention capacity and the ability to instruct cell growth into tumor-like spheroids even in noncarcinoma lineages.

Self-assembly and intracellular delivery of DNA by a truncated fragment derived from the Trojan peptide Penetratin.

Penetratin is a short Trojan peptide that attracts great interest in biomedical research for its capacity to translocate biological membranes. Herein, we study in detail both self-assembly and intracellular delivery of DNA by the heptamer KIWFQNR, a truncated peptide derived from Penetratin. This shortened sequence possesses a unique design with bolaamphiphilic characteristics that preserves the longest noncationic amino acid portion found in Penetratin. These features convey amphipathicity to assist self-assembly and make it a suitable model for exploring the role of hydrophobic residues for peptide interaction and cell uptake. We show that the fragment forms peptiplexes (i.e., peptide-DNA complexes), and aggregates into long nanofibers with clear ß-sheet signature. The supramolecular structure of nanofibers is likely composed of DNA cores surrounded by a peptide shell to which the double helix behaves as a template and induces fibrillization. A nucleation and growth mechanism proceeding through liquid-liquid phase separation of coacervates is proposed for describing the self-assembly of peptiplexes. We also demonstrate that peptiplexes deliver double-stranded 200 bp DNA into HeLa cells, indicating its potential for preparing non-viral vectors for oligonucleotides through noncovalent strategies. Since the main structural features of native Penetratin are conserved in this simpler fragment, our findings also highlight the role of uncharged amino acids for structuration, and thus for the ability of Penetratin to cross cell membranes.