Chegaev, Konstantin

Fruttero, Roberta

Gasco, Alberto

Gazzano, Elena

Riganti, Chiara

Rolando, Barbara

English

Institutional Research Information System University of Turin

20 November 2023AperTO - Archivio Istituzionale Open Access dell'Università di TorinoOriginal Citation:Multitarget Drugs: No-Donor DoxorubicinsTerms of use:Open Access(Article begins on next page)Anyone can freely access the full text of works made available as "Open Access". Works made availableunder a Creative Commons license can be used according to the terms and conditions of said license. Useof all other works requires consent of the right holder (author or publisher) if not exempted from copyrightprotection by the applicable law.Availability:This is the author's manuscriptThis version is available http://hdl.handle.net/2318/1525583 since 2015-09-25T14:42:06ZHydrolytic stability. One possible explanation for the lack ofcorrelation between intracellular accumulation andcytotoxicity is the different susceptibility to esterase enzymes,which gives the different compounds different hydrolyticstabilities (Table 1).Table 1. Hydrolytic stability of NO-DOXOs in human serum These results could explain the lack of efficacy of compounds6, 7 and 10, and the good efficacy of compounds 8 and 9.However, the highly stable compounds 3-5 were characterizedby the lowest cytotoxic efficacy in the biological assays.MULTITARGET DRUGS: NO-DONOR DOXORUBICINSKonstantin Chegaev,(a) Chiara Riganti,(b) Elena Gazzano,(b) Barbara Rolando,(a)Roberta Fruttero,(a),* Alberto Gasco(a)(a)Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, via P. Giuria, 9, 10125 Torino, Italy;(b)Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, via Santena 5/bis, 10126 Torino, Italy.Figure 1NO-donorIntroduction. Doxorubicin (DOXO) is an antibiotic belonging to the class of anthracyclines, used in treating a wide range of cancers, includinghematological malignancies, many types of carcinoma, and soft tissue sarcomas. However, there are some serious limitations on DOXO’s efficacy incancer therapy mainly due to Multidrug Resistance (MDR) following the overexpression of ATP-Binding Cassette (ABC) transporters, such as P-glycoprotein (Pgp), Multidrug Resistance Related Proteins (MRPs) and Breast-Cancer Resistance Related Protein (BCRP).[1] In previous research ourgroup showed that exogenous nitric oxide (NO) reduced the activity of Pgp and MRPs, by nitrating tyrosine residues crucial for the protein functions,with consequent increase of intracellular DOXO concentration and toxicity in MDR tumor cells.[2] On this basis, new semisynthetic DOXOs werestudied, in which the antibiotic is joined through an ester linkage to NO-donor moieties (Figure 1).[3] These compounds can accumulate indoxorubicin-resistant human colon cancer cells (HT29/dx), inducing cytotoxicity. In order to extend this new class of semisynthetic DOXOs, a smalllibrary of novel chimeras of DOXO were designed, bearing different NO-donor groups at the C-14 position (Scheme 1). A series of experiments werecarried out in order to evaluate their cytotoxicity profile and to explore their different biological behaviour.References.[1]. Takara, K.; Sakaeda, T.; Okumura, K. Curr. Pharm. Des. 2006, 12, 273-286.[2]. Riganti, C.; Miraglia, E.; Viarisio, D. et. al., Cancer Res. 2005, 65(2), 516-525.[3]. Chegaev, K.; Riganti, C.; Lazzarato, L. et. al., ACS Med. Chem. Lett. 2011, 2(7), 494-497.[4]. Fernandez, A.-M.; Dubois, WO 2004/011033, 2004.Synthesis. The designed NO-DOXOs were prepared from a mixture of14-bromo/chloro daunorubicine hydrobromide (1) [4], by the reactionwith carboxylic acid bearing NO-donor in the presence of KF as a weakbase in DMF solution (Scheme 1).Scheme 1. i) RCOOH, KF, DMF dry, rt; ii) HCl in dioxane dry, THF dry.  OOOHONH2OHOOROHOHOOOClHOOO OOOHOHOHONH2OHBr/ClBrHi), ii)1 2-10ONO23 R = OONO2ONO24 R = O ONO25 R = ONO2ONO26 R = ONO27 R = ONO28 R = ON+NSO2PhOO-9 R = OOONN+NO-10 R = ONO2ONO22 R = Intracellular accumulation. All thecompounds except 10 were retained atleast as much as DOXO in HT29 andHT29/dx cells; compounds 2-5, 7, 8accumulated to a greater extent than DOXOin HT29/dx cells (Figure 2). This trend wasin line with the different degree of nitrationof MRP1 and BCRP present on the plasma-membrane (Figure 3).2         3           4          5         6          7         8          9       10Figure 2. Intracellular accumulation of compounds in HT29 and HT29/dx cells2          3            4           5           6            7          8           9         10 Figure 3. Nitration of plasma-membrane associated ABC transporters.Compoundt½ (h) human serumCompoundt½ (h) human serum2 16.0 7 4.93 24.6 8 21.74 34.7 9 >>24 h [a]5 26.7 10 2.86 6.0[a] 73.5% conc. at 24hConclusions. This study shows that the synthetic NO-releasing drugs withphysico-chemical properties and/or conjugated with specific moietiesfavoring intramitochondrial delivery are very effective against DOXO-resistant cells. These features should be considered in the design of futureNO-releasing DOXOs as effective MDR-reversing tools.Cytotoxicity. DOXO is cytotoxic onlyin sensitive HT29 cells, Surprisingly,only compounds 2, 8 and 9 weremore cytotoxic than DOXO in bothsensitive HT29 and resistantHT29/dx cells (Figure 4).Figure 4. Cytotoxicity of compounds in HT29 and HT29/dx cells2       3      4       5      6       7      8       9    10Figure 5. Intramitochondrial accumulation of compounds in HT29 and HT29/dx cells2         3          4         5          8          9       102            8           9        10 2               8            9            10Figure 6. Nitration of mitochondrial ABC transporters in HT29 and HT29/dx cellsIntramitochondrial accumulation. Compound 2, 8 and 9 had more markedintramitochondrial retention than DOXO in both HT29 and HT29/dx cells(Figure 5). These results are in line with mitochondrial ABC transportersnitration (Figure 6). The compounds 3-5 do not nitrate ABC pumps and do notaccumulate in mitochondrion.These results suggest that the cytotoxicity of the NO-releasing DOXOs is closelydependent on their ability to localize within the mitochondria. Indeed,compounds 2, 8 and 9 increased the activation of caspase 9 and 3 while thecompounds that accumulated little within the mitochondria did not activate thecaspase 9/caspase 3 axis. These data may provide a rational explanation for thedifferent cytotoxic efficacy of the NO-releasing DOXOs of the compoundsstudied here.

Multitarget Drugs: No-Donor Doxorubicins

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Multitarget Drugs: No-Donor Doxorubicins

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