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Title: Computer-aided search for anthracycline antibiotic reductases inhibitors to optimize anticancer pharmacotherapy


According to World Health Organization forecast, in this century cancers will become the most common cause of death in most of countries, affecting more people than cardiovascular diseases. Effective cancer pharmacotherapy is still one of the most challenging for modern medicine and pharmacy. Despite development and introduction of plenty of innovative anticancer drugs, substances that have been using for many years are still widely used in therapy. Anthracycline antibiotics (ANT) are one of such groups of drugs. They are using in treatment of both solid tumours and haematological malignancies. Unfortunately, their usage is limited due to drug resistance developing during the therapy and increased risk of cardiotoxicity. The reasons behind those effects are still unclear. One of the hypotheses suggests that they are caused by ANT metabolites formed by enzymes called reductases. The main enzymes responsible for this process are carbonyl reductase 1 (CBR1) and aldo-keto reductase 1C3 (AKR1C3). Thus, inhibition of those enzymes should increase drugs activity and limit their cardiotoxicity. The aim of the research presented in this thesis was application of selected computer-aided drug design methods in optimization of selected CBR1 and AKR1C3 three-dimensional crystal structures and then to use the optimized models in searching for new enzymatic inhibito ; rs. The first part of research was focused on searching for new CBR1 inhibitors. Optimization of two crystal structures (PDB codes: 1WMA and 3BHJ) was performed. The structures differed in the presence of glutathione within the active site of CBR1. Using several optimization (Induced-fit docking, flexible docking, molecular dynamics) and evaluation (retrospective virtual screening) methods, two CBR1 models (1WMA_A3 and 3BHJ_N1) were selected. They were then used to dock a library of over 2.3 million compounds in prospective virtual screening. 9 chemotypes of new potential CBR1 inhibitors were selected and then evaluated in CBR1 inhibition assay. Among them the most effective was a derivative of 2-phenyl-3,4-dihydroquinazolin-4-one (compound C2, IC50 = 7,56 μM). Further structure optimization led to discovery of more CBR1 inhibitors, with the most potent one – compound C17 (IC50 = 0,68 μM). In the poses obtained in docking, the compound formed molecular interactions with all crucial CBR1 amino acid residues: Ser139, Tyr 193, Met234 (H-bonds) and Trp229 (π-π hydrophobic interaction). The aim of the second part of the research was to analyse the potential of ASP9521 to inhibit CBR1. The compound is recognised as a potent AKR1C3 inhibitor, with structural features typical for CBR1 inhibitors. Docking studies confirmed that ASP9521 can form the same molec ; ular interactions as the previously described compound C17. Results of molecular dynamics simulation revealed that the compound can keep stable conformation within the CBR1 catalytic site in the whole range of simulation time (20 ns), suggesting that ASP9521 may be a CBR1 ligand. This hypothesis was confirmed in inhibitory enzymatic assay (IC50 = 44,00 μM). Additionally, ASP9521 ameliorated anticancer activity of daunorubicin against pulmonary cancer cells (cell line A549), decreasing by 14,25% drug concentration necessary to inhibit cells growth by a half. Identification of a moderate dual CBR1-AKR1C3 inhibitory activity of ASP9521 led to initiation of searching for new chemotypes of dual inhibitors of main ANT reductases, the combined effect of which may surpass the use of a selective inhibitor. For this purpose, selected CBR1 and AKR1C3 crystal structures were optimized, using computer tools and methods described in the first part of the research. 36 potential CBR1-AKR1C3 inhibitors were selected after prospective virtual screening performed on the best rated models (11E3 and 74H1). Most of the compounds turned out to be moderate or potent AKR1C3 inhibitors (the most potent inhibitor was compound P27, with IC50 AKR1C3 = 2,25 μM), with moderate or weak CBR1 inhibition. Additional analysis of compounds previously pointed as CBR1 inhibitors revealed ; that compound C17 had also potent AKR1C3 inhibitory properties, which made him the second (the first was ASP9521) dual CBR1-AKR1C3 inhibitor described in this thesis (IC50 CBR1 = 0,68 μM; IC50 AKR1C3 = 6,94 μM). Based on combined analysis of both in silico and in vitro research, 1-benzylpiperidine (present in compounds P1-P9) was indicated as a favourable chemotype for further optimization research with intention to discovery of new dual enzymatic inhibitors. In vitro research, performed on A549 cell line, revealed that addition of compounds P18 and P25 increased anticancer activity of daunorubicin – the same effect was observed previously for ASP9521. The research described in this doctoral thesis led to optimized three-dimensional models of selected CBR1 and AKR1C3 crystal structures. The models were applied to identify new inhibitors of main ANT reductases, including compounds with a new, unique dual CBR1-AKR1C3 inhibitory properties. The compound selection was based not only on poses achieved in prospective screenings, but also on evaluation of selected ADMET parameters, predicted by dedicated software, in accordance with rules of modern, rational drug design. Both the best compounds from C series (C2, C17) and P series (P18, P25, P27) did not contain structural features that could enhance a risk of toxicity or cause unfavourable pharmacokinetic ; properties of the described substances.

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2 - studia doktoranckie

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Rada Dyscypliny Nauki farmaceutyczne


Kołaczkowski, Marcin ; Bucki, Adam

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Apr 8, 2024

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Apr 8, 2024

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ZB-139425 Apr 8, 2024


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