Carbonic Anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in the catalysis of the reversible hydration of carbon dioxide. Up to day, eight genetic CA families alpha, beta, gamma, delta, zeta, eta, theta, and iota-CAs, have been reported in organisms from across the phylogenetic tree. The fifteen human (h) isoforms of carbonic anhydrase (hCAs) belong to the alpha family and are involved in functions such as respiration, transport of carbon dioxide between metabolising tissues and lungs, pH homeostasis, electrolyte secretion in various tissues/ organs as well as biosynthetic reactions covering a pivotal role in a multitude of physiological functions. However, in some cases, these enzymes could contribute to pathological processes taking part in the complex machinery system developed by cells to face the hypoxic conditions of solid tumors and regulate intracellular pH. In particular the two isoforms of hCA IX and XII, called tumor-related isoforms, are overexpressed in different cancers. In this regard, the development of inhibitors that selectively bind and inhibit these two enzymes could be exploited for cancer treatment. The objective of this project is the development of novel and selective inhibitors of human carbonic anhydrase (hCAs) isoforms IX and XII, based on the pyran-2-one core, substituted at the position 5 with carboxamide linker bearing different (hetero)aromatic or aliphatic residues.
The novel compounds will be evaluated in in vitro assay against 4 isoforms of human carbonic anhydrase: the two off-targets hCA I and II and the two targets hCA IX and XII. The derivatives exhibiting the best profiles will be further investigated against proper tumor cell lines. Docking studies and molecular dynamics will be performed in order to assess the putative binding mode of the novel compounds.
The objective of this project is the development of novel and selective inhibitors of hCA IX and XII, whose overexpression has been extensively reported in many tumor types [1-4]. Usually in healthy humans beings, the expression of the CA IX take parts in a limited number of tissues such as stomach and peritoneal lining; on the contrary it is ectopically induced and overexpressed in different tumors including malignancies of the brain, head/neck, breast, lung, bladder, cervix uteri, colon/rectum and kidney; overexpression of CA XII follows similar patterns (this isoform has been showed in renal tumors, metastatic brain tumors and colon) [5-7].
Owing to the quite low expression of hCA IX in the normal tissues (and in a less extent, of the hCA XII), the design and development of inhibitors directed against these isoforms could be an important breakthrough for therapeutics, because lacking the drawbacks that classical antitumor drugs have due to their poor selectivity. The overexpression of these two transmembrane proteins often leads to reduction of extracellular pH, causing the protonation of basic group of antitumoral drugs, that being endowed 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 1-(4-fluorophenyl)-3-(4-sulfamoylphenyl)urea, commonly named SLC-0111 (a hCA IX inhibitor currently 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 [8]. 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 [9]. At present, a phase 1b study involving SLC-0111 (oral) in combination with gemcitabine (IV) for metastatic pancreatic ductal cancer (mPDAC) is ongoing in subjects positive for CAIX, while recently have been published the results of a trial study performed to assess the safety of SLC-0111 after dose escalation administration started at 500 mg oral daily and increased to 1000 and 2000 mg (in 3 cohorts) [10]. The results demonstrated that SLC-0111 was safe in patients and pharmacokinetic data support 1000 mg/d as the dose for further studies involving SLC-0111.
The novel compounds designed in this project could represent an important step forward in the development of antitumor drugs and in the research involving the development of novel inhibitors of human carbonic anhydrases. These compounds have been obtained by the removal of the benzene ring participating in the bicyclic constitution of the coumarin core. This molecular simplification could lead to molecules able to effectively inhibit the two tumor related isoforms. Furthermore, the insertion of the carboxamide group bearing different moieties, could be exploitable in order to tailor the affinity against the enzyme as well as pharmacokinetic studies. This novel scaffold could exhibit a new binding mode inside the active site, paving the way to new and interesting research about the possible hCAs inhibitory mechanisms [11]. 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 this scaffold.
References
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