Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes found in numerous organisms across the tree of life, encoded by seven genetically distinct CA families: alfa-, beta-, gamma-, delta-, epsilon-, zeta- and teta-CAs . Carbonic anhydrases are metalloenzymes therefore they are catalytically effective only with one metal ion bound within the active site cavity, the apoenzymes being devoid of any catalytic activity.
In the last years, there has been evidence of the role that several CAs isoforms cover in tumorigenesis, as a result of the capability to create an acidic extracellular milieu useful for tumour survival, progression and adoption of metastatic phenotypes. The two transmembrane isoforms CAs IX and XII (tumor-related isoforms) are overexpressed in hypoxic tumors and have been linked to this behaviour, while showing a limited expression in normal tissues.In the light of the roles that hCA IX and XII have in the cancer malignancies, the development of inhibitor which selectively bind and inhibit these two enzymes could be useful in cancer treatment. Up to day, only few hCA IX and XII inhibitors endowed with good therapeutic profiles have been included in clinical trial (as Indisulam and SLC-0111). These outcomes suggest that there is a real possibility, as well as urgencies, to develop compounds able to increase the equipment to fight cancer. Compounds, based on saccharin and acesulfame scaffolds N-/O- substituted with different substituents, are endowed with low nanomolar inhibitory activity against hCA IX and XII, albeit some of them, retained activity against off-targets (hCA I and II). With the aim to ameliorate activity and selectivity towards the two tumor-related hCA isoforms, we will design and study a series of novel derivatives based on these scaffolds introducing various kinds of changes.
The purpose of this project is to develop selective inhibitors of carbonic anhydrase isoforms IX and XII, in the light of the evidences of the overexpression of these enzymes in some tumor types.
Owing to the quite low expression of hCA IX (and in the less extent the hCA XII) in the normal tissues, the design and development of inhibitors directed against these isoforms could be an important breakthrough for therapeutics, because lacking all the drawbacks that classical antitumor drugs have due to their poor selectivity.
Moreover, hCA IX and XII inhibitors can restore the extracellular pH lowered by the activity of these overexpressed enzymes; this extracellular pH (in some cases pH = 6.5) causes the protonation of basic group, that with positive charge cannot cross the cellular membrane, reducing in this way their effectiveness.
The combination of CA IX and XII inhibitors, with classical antitumor drugs can reverse this condition eliciting a synergistic effect, as already observed for other compounds. For example the combination of SLC-0111 (the hCA IX inhibitor actually under clinical trials investigation as monotherapy and in association with other drugs) with drugs as Dacarbazine and Temozolomide (currently used for advanced melanoma), Doxorubicin (used for breast cancer) and 5-Fluorouracil (used for colon cancer) was evaluated [1]. The results showed as SLC-011 potentiates Dacarbazine and Temozolomide cytotoxicity, increased breast cancer cell response to Doxorubicin and enhanced 5-Fluorouracil cytostatic activity on colon cancer cells. Beneficial effects were also obtained by combining selective inhibitor of hCA IX with tumor irradiation, finding enhanced antitumor effects due to the "sensitizing" of the cancer cells to the treatment induced by hCA inhibitor [2].
The saccharin and acesulfame-based compounds designed in this project could represent an important step forward in the development of antitumor drugs. Previously reported compounds whose structure was based on saccharin and acesulfame core, showed the ability to inhibit the tumor-related isoforms in the low nanomolar activity, albeit some of them retained activity against the off-target isoforms hCA I and II. However, these two scaffolds need to be further explored in order to obtain information about the substitutions that they can undergo to improve the activity, investigating also if there are substitutions that are not tolerated by them.
The molecules presented here could fill this void, giving the opportunity to widen the SAR evaluating: (i) the importance of methylene group belonging to the benzyl moiety (usually present as substituent of saccharin-based compounds) both for closed and open form of saccharin core; (ii) the effect that an aromatic ring spacer can elicit when placed between the nitrogen (or oxygen) of saccharin/acesulfame moiety and the (un)substituted phenyl ring; (iii) the synthetic conditions useful to direct the obtaining of N- or O- substituted saccharin/acesulfame derivatives.
All the proposed compounds have not been previously tested on CA IX and XII, therefore the outcomes of this biological investigation could increase the knowledge about these scaffolds.
The study of kinetic and affinity constants of the interaction between the inhibitors considered and the enzyme carbonic anhydrase, through surface plasmon resonance (SPR) will be a good new approach to better understand the interaction enzyme-inhibitor.
References
[1] E. Andreucci, J. Ruzzolini, S. Peppicelli, F. Bianchini, A. Laurenzana, F. Carta, C.T. Supuran, L. Calorini, The carbonic anhydrase IX inhibitor SLC-0111 sensitises cancer cells to conventional chemotherapy, J. Enzyme Inhib. Med. Chem. 34 (2019) 117¿123. doi:10.1080/14756366.2018.1532419.
[2] L. Dubois, S. Peeters, N.G. Lieuwes, N. Geusens, A. Thiry, S. Wigfield, F. Carta, A. McIntyre, A. Scozzafava, J.M. Dogné, C.T. Supuran, A.L. Harris, B. Masereel, P. Lambin, Specific inhibition of carbonic anhydrase IX activity enhances the in vivo therapeutic effect of tumor irradiation, Radiother. Oncol. 99 (2011) 424¿431. doi:10.1016/j.radonc.2011.05.045.