We report the synthesis of an original series of oxoazabenzo[de]anthracenes conjugated to an amino acid: Ala, Phe, Pro, Lys, or Gly (4a-e, respectively). The compounds, derived from 1,8-dihydroxyanthracene-9,10-dione, were studied for DNA binding and cytotoxicity. Melting temperature, fluorescence quenching, and surface plasmon resonance methods all indicated that the lysine derivative 4d binds to DNA much more strongly that the Pro, Ala, and Gly conjugates whereas the Phe analogue showed the lowest DNA binding capacity. These compounds form intercalation complexes with DNA, as judged from electric linear dichroism and topoisomerase I-based DNA unwinding experiments. Preferential binding of 4d to defined sequences such as 5'-CTAAAGG and 5'-ATGC was evidenced by DNase I footprinting. This Lys conjugate was found to be over 20 times more cytotoxic to CEM human leukemia cells than the other conjugates, with an IC50 in the submicromolar range. A high antiproliferative activity, likely attributable to the enhanced DNA binding capacity, is maintained despite the incapacity of the compound to stabilize topoisomerase-DNA covalent complexes. The cell cycle effects of 4d consisted in an S phase accumulation of cells coupled with a pro-apoptotic action (appearance of hypodiploid sub-G1 cells) which were confirmed by measuring the inhibition of BrdU incorporation into DNA and labeling of phosphatidylserine residues with annexin V-FITC by means of flow cytometry. Altogether, the work provides interesting structure-activity relationships in the oxoazabenzo[de]anthracene-amino acid conjugate series and identifies the lysine derivative 4d as a promising candidate for further in vivo evaluation and drug design.
The lysosomal endoprotease legumain (asparaginyl endoprotease) has been proposed as a putative biomarker in prostate tumours, in which the enzyme is markedly overexpressed. Overexpression, coupled with highly selective specificity for cleavage of substrates at the C-terminus of asparagine (Asn) residues, make legumain an attractive biochemical target for potential diagnosis, prognosis and treatment. We report the design, synthesis, characterisation and preliminary evaluation of a new rhodamine-B (Rho-B)-labelled legumain peptide substrate probe 5 [Rho-Pro-Ala-Asn-PEG-AQ(4-OH)] and its selective targeting to lysosomes in PC3 prostate cancer cells. Probe 5 was efficiently activated by recombinant human legumain to afford the high quantum yield reporter fluorophore tripeptide 4b (Rho-Pro-Ala-Asn-OH) with concomitant release of intense fluorescence. Furthermore, probe 5 was activated upon incubation with homogenates derived from fresh-frozen tissue material of prostatectomy specimens. Probe 5 represents a new viable biochemical tool for probing the activity of legumain with the potential to be used in ex vivo diagnostics in the cancer pathology laboratory.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.