Projects / Programmes source: ARIS

Nitroxoline and its derivatives as new antitumour drugs

Research activity

Code Science Field Subfield
4.06.01  Biotechnical sciences  Biotechnology  Recombinant DNA technology 

Code Science Field
T490  Technological sciences  Biotechnology 

Code Science Field
3.04  Medical and Health Sciences  Medical biotechnology 
cathepsin B, protease inhibitor, nitroxoline, cancer
Evaluation (rules)
source: COBISS
Researchers (22)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  26491  PhD Maruška Budič  Biochemistry and molecular biology  Researcher  2014 - 2015  24 
2.  14575  PhD Maja Čemažar  Oncology  Researcher  2013 - 2016  1,408 
3.  15284  PhD Stanislav Gobec  Pharmacy  Researcher  2013 - 2016  827 
4.  20376  Branka Grabovac    Technical associate  2013 - 2016 
5.  12688  PhD Kristina Gruden  Biotechnology  Researcher  2014  983 
6.  04648  PhD Janko Kos  Biotechnical sciences  Head  2013 - 2016  1,154 
7.  32175  PhD Boštjan Markelc  Medical sciences  Researcher  2013 - 2014  212 
8.  19170  PhD Urša Pečar Fonović  Pharmacy  Researcher  2013 - 2016  144 
9.  29966  Dejan Pelko    Technical associate  2015 - 2016 
10.  36596  PhD Milica Perišić Nanut  Biotechnical sciences  Researcher  2014 - 2016  141 
11.  32035  PhD Anja Pišlar  Biotechnology  Researcher  2013 - 2016  162 
12.  26507  PhD Jure Pohleven  Biochemistry and molecular biology  Researcher  2013  75 
13.  15813  PhD Boris Rogelj  Neurobiology  Researcher  2013  410 
14.  23576  PhD Jerica Sabotič  Biochemistry and molecular biology  Researcher  2013 - 2016  315 
15.  08800  PhD Gregor Serša  Oncology  Researcher  2013 - 2016  1,494 
16.  30816  PhD Izidor Sosič  Pharmacy  Researcher  2013 - 2015  247 
17.  36900  Jasna Šlenc  Pharmacy  Researcher  2014 - 2015 
18.  21619  PhD Olga Vasiljeva  Oncology  Researcher  2013 - 2016  183 
19.  35506  PhD Sabina Vatovec  Biology  Researcher  2013  28 
20.  34507  PhD Tjaša Zlobec  Biotechnology  Junior researcher  2013 - 2015  26 
21.  17285  Darja Žunič Kotar    Technical associate  2013 - 2016 
22.  34459  PhD Simon Žurga  Pharmacy  Junior researcher  2013 - 2015  25 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0106  Jožef Stefan Institute  Ljubljana  5051606000  89,990 
2.  0302  Institute of Oncology Ljubljana  Ljubljana  5055733000  15,352 
3.  0787  University of Ljubljana, Faculty of Pharmacy  Ljubljana  1626973  17,522 
Cathepsin B is a lysosomal cysteine protease, ubiquitous in cells and tissues. Its expression, activity and localisation change with various pathological processes the most widely investigated of which is a harmful role in the development and progression of cancer. Cathepsin B is one of the main proteases involved in the degradation of proteins of the extracellular matrix (ECM), a process which promotes invasion and metastasis of tumour cells and tumour angiogenesis. Cathepsin B is unique among cathepsins possessing exo- and endopeptidase activities. The former is important for its physiological role, the latter is involved in the pathological degradation of ECM. The search for new cathepsin B inhibitors as possible anti-tumour drugs is focused on reversible inhibitors which could specifically inhibit harmful endopeptidase activity.   Within our research group we have identified nitroxoline, an established antibiotic used for treating urinary infections, as a potent inhibitor of cathepsin B, using methods of virtual screening, enzyme kinetics and various functional tests. Nitroxoline inhibits cathepsin B endopeptidase activity reversibly in the low micromolar range. It binds S' primed sites of cathepsin B, with aromatic quinolone rings filling the S2' binding site, while the negatively charged nitro group interacts specifically with the positively charged His110 and His111 in the occluding loop. By inhibiting endopeptidase activity, nitroxoline decreased the degradation of protein substrates and tumour cell invasion in vitro. Using the X-ray crystal structure that we determined for a complex of cathepsin B with nitroxoline and the results of enzyme kinetics, we intend to prepare derivatives of nitroxoline with better specificity and inhibition constants in the nanomolar range. To do this we will use computer assisted design and directed systemic synthesis. The latter includes the exchange of the nitro group with bio-isostenic groups known from the literature, the introduction of various substituents at positions 7 and 8 of the quinolone ring and replacement of the heterocycle by the naphthalene ring. Additionally, we will prepare derivatives acting as reversible covalent inhibitors by introducing nitrile groups into the nitroxoline scaffold, thus enabling additional interactions with the active site thiol group.   Nitroxoline together with its derivatives exhibiting the best inhibitory properties, will be tested in vitro using various models of tumour cell adhesion, migration, invasion and angiogenesis. Besides the established ones, new 2D and 3D tumour models will be developed which will better resemble the in vivo tumour status. To test the impact of specific inhibitory properties of nitroxoline and its derivatives on mechanisms of tumour development and progression, various methods of fluorescence microscopy, flow cytometry and real time monitoring of cellular events by electric impedance, will be used. The anti-tumour potential of nitroxoline and its derivatives will be tested in vivo in mouse models of tumour growth and formation of spontaneous metastases. Additionally, nanodelivery systems established by our group, including biodegradable nanoparticles, will be used to improve delivery of the inhibitors to tumour cells, PK/PD properties and biological availability. We expect that the superior inhibitory properties of nitroxoline and its derivatives will result in effective reduction of tumour growth and metastasis formation, enabling their further use in clinical evaluation.
Significance for science
The proposed research is part of the advanced trends in science in the fields of the role of proteolytic enzymes in cancer development and progression. Recent results, obtained by our and other groups, on specific regulation of cathepsin B proteolytic activity and consequently tumorigenic processes, have opened up a new field of research, which has met with a wide response in the scientific community, shown by multiple citations in journals with the highest impact factors, and some groups have continued with the research, based on our results. Knowledge of the mechanisms of regulation of cathepsin B activity enables the understanding of basic processes in tumorigenesis and may explain the events leading to tumour growth and formation of metastasis. This is very important if this protease is to be used as target for the development of new drugs. Only selective inhibition of a particular protease that is truly involved in harmful pathological process can lead to progress in therapy, the use of nonselective general protease inhibitors has been compromised, causing serious side effects. The application in therapy of known irreversible inhibitors, specific for cathepsin B, is also inadequate, first because of their toxicity and low biological availability and secondly, because of their ability to block, sooner or later, off-target proteases. The action of nitroxoline as a cathepsin B reversible inhibitor is an original scientific contribution which served as a platform for further design and synthesis of its derivatives with even better pharmacological properties. Their effectiveness in vitro and in vivo in inhibiting tumour development and metastasis confirm the hypotheses of the crucial role of cathepsin B endopeptidase activity in cancer. Our project group used the most recent techniques and methods, which can be translated to other research fields, the same as the experiences gained through the collaboration with leading researchers in Slovenia and worldwide.
Significance for the country
The results of the proposed project will make possible the development of new technologies and products, which will be economically interesting for pharmaceutical companies in Slovenia. In Slovenia and the European Union, biotechnology and pharmacy constitute a leading scientific and economic priority. The pharmaceutical industry has exhibited outstanding growth in recent years and the implementation of new products, either original or generic, constitutes the main driving force. The scientific knowledge arising from top class research in this field, to which our proposed project contributes, is essential for further development of the pharmaceutical industry. Besides potential new drugs, the result of the project are also the most recent methods and techniques, which can easily be adopted by industrial laboratories. New products which may originate from this project will have high added value and would increase the competitiveness of companies in Slovenia and Europe. The proposed project also contributed to all levels of university education. It enabled students to acquire new knowledge and to the project’s implementation diploma, master and doctoral works were included in fields of pharmaceutical biochemistry, biotechnology and tumour biology. This was possible since the members of the project team are involved in the pedagogical process at public universities in Slovenia and at the Jožef Stefan Postgraduate School. The results of this project also fostered our collaboration with other research groups, increased knowledge exchange and the opportunity to obtain new funds for financing our science.
Most important scientific results Annual report 2013, 2014, 2015, final report
Most important socioeconomically and culturally relevant results Annual report 2013, 2014, 2015, final report
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