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Targeting gliomas: can a new alkylating hybrid compound make a difference? Rui Pinheiro, Cláudia Braga, Gisela Santos, Maria R Bronze, Maria Jesus Perry, Rui Moreira, Dora Brites, and Ana Sofia Falcão ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00169 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on September 28, 2016
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ACS Chemical Neuroscience
Title: Targeting gliomas: can a new alkylating hybrid compound make a difference?
Rui Pinheiro1, Cláudia Braga1,3, Gisela Santos1, Maria R Bronze1,4, Maria J Perry1,3, Rui Moreira1,3, Dora Brites1,2,*, Ana S Falcão1,2,*
1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade
de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal 2
Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade
de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal 3
Department of Pharmaceutical Chemistry and Therapeutics, Faculty of Pharmacy,
Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal 4
Department of Toxicological and Bromatological Sciences, Faculty of Pharmacy,
Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003 Lisbon, Portugal *Corresponding authors Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa Avenida Professor Gama Pinto 1649-003 Lisbon Portugal Phone: +3512179464900; Fax: +351217946491 E-mail:
[email protected];
[email protected] 1 ACS Paragon Plus Environment
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Abstract Glioblastoma (GBM) is the most common and aggressive type of brain tumor in adults. The
triazene
temozolomide
(TMZ),
an
alkylating
drug,
is
the
classical
chemotherapeutic agent for gliomas, but has been disappointing against the highly invasive and resistant nature of GBM. Hybrid compounds may open new horizons within this challenge. The multicomponent therapeutic strategy here used resides on a combination of two repurposing drugs acting by different but potentially synergistic mechanisms, improved efficacy and lower resistance effects. We synthesized a new hybrid compound (HYBCOM) by covalently binding a TMZ analogue to valproic acid, a histone deacetylase inhibitor drug that was shown to sensitize TMZ-resistant glioma cells. Advantages of this new molecule as compared to TMZ, in terms of chemotherapeutic efficacy, were investigated. Our results evidenced that HYBCOM more efficiently decreased the viability and proliferation of the GL261 glioma cells, while showing to better target the tumor cells than the functionally normal astrocytes. Increased cytotoxicity by HYBCOM may be a consequence of the improved autophagic process observed. Additionally, HYBCOM changed the morphology of GL261 cells into a non-polar, more rounded shape, impairing cell migration ability. Most interesting, and in opposite to TMZ, cells exposed to HYBCOM did not enhance the expression of drug resistance proteins, a major issue in the treatment of GBM. Overall, our studies indicate that HYBCOM has promising chemotherapeutic benefits over the classical TMZ, and future studies should further assess if the treatment translates into efficacy in glioblastoma experimental models and reveal clinical benefits in GBM patients.
Keywords: Autophagy; Glioblastoma; Glioma cell migration; Hybrid compound; Multidrug resistance; Temozolomide
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INTRODUCTION
Malignant gliomas are heterogeneous, highly invasive brain tumors. Glioblastoma multiforme (GBM), classified by World Health Organization as a grade IV astrocytoma is particularly aggressive. Most patients diagnosed with this tumor die within one year from the diagnosis and only 5% survive more than 5 years despite aggressive therapies.1 Thus, regardless the international scientific community efforts, treatment of GBM remain one of the most challenging tasks in clinical oncology. Surgical resection followed by radiotherapy with concomitant and adjuvant chemotherapy is the standard treatment in patients with GBM.2 Temozolomide (TMZ) is the classical anticancer drug for gliomas, a triazene alkylating prodrug that can be taken orally. Due to its small size and lipophilic features, the drug is able to cross the blood-brain barrier.3 Once in the central nervous system (CNS), TMZ is spontaneously converted into the active metabolite 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) through a basecatalyzed nucleophilic attack by water. The unstable carbamic acid decarboxylates at pH 7.4 and further hydrolyzes into 4-amino-5-imidazole-carboxamide (AIC) and methydiazonium ion. This cation is highly reactive and alkylates DNA species, particularly purine bases (O6-guanine, N7-guanine and N3-adenine)4-6, inducing carcinogenic, mutagenic and toxic lesions.7 However, the therapeutic effectiveness of TMZ is unsatisfactory in GBM, largely in consequence of the lack of selectivity for tumor cells and insufficient drug concentration in the intracellular target site due to 9
cancer drug resistance.8 In fact, in addition to an increased mechanism of DNA repair , there are other defense mechanisms, such as high expression of drug efflux transporters10, 11, which can lead to multidrug resistance (MDR) and to the consequent failure of chemotherapy in GBM. Thus, most patients will have a recurrence of GBM, raising the need to urgently develop alternative treatments able at improving survival. On this regard, histone deacetylases (HDACs) have been identified as potential targets in GBM. In fact, altered expression and mutations of genes that encode HDACs have 3 ACS Paragon Plus Environment
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been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis.12 Recently, it has been proposed the combination of valproic acid (VPA) administration with chemotherapy and radiotherapy in GBM. VPA is an antiepileptic drug and HDAC inhibitor used to control seizures in patients with brain tumors.13 Epigenetic modifiers, such as HDAC inhibitors, have shown to sensitize cancer cells to ionizing radiation, while protecting normal cells and tissues from deleterious effects of radiotherapy.14 Interestingly, exposure of glioma cell lines to VPA led to histone hyperacetylation, increased cellular radiosensitivity, and synergistic tumor-growth delay in animals treated with a combination of VPA and radiotherapy.15 There is also evidence that the combination of VPA and TMZ significantly increased antitumor effects in TMZ-resistant glioma cells due to VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, a DNA repair mechanism with an important role in cellular resistance to alkylating agents.9 In a phase II study using concurrent radiotherapy, TMZ and VPA in patients with newly diagnosed GBM showed that VPA may result in improved outcomes compared to historical data and merits further investigation in a phase III study.16 The majority of these studies were performed using combined therapies, but an emerging promising research line consists in the development of the so-called hybrid molecules, which may have increased efficacy.17 Hybrid drugs are defined as the combination of two different and independently acting molecules linked through a covalent bond. The presence of two drug pharmacophores in a single molecule may produce a synergistic effect, leading to a pharmacological potency greater than the sum of each individual moiety’s potencies.
18
Also, one of the hybrid
parts may be incorporated to counterbalance the known side effects associated with the other hybrid part. Additionally, combined drugs that impact multiple targets are less prone to drug resistance.19 Thus, hybrid anticancer drugs present emerging interest in the discovery of highly potent and selective molecules that triggers two or more 4 ACS Paragon Plus Environment
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pharmacological mechanisms of action working in synergy to inhibit cancer tumor growth. Taking advantage of such findings we synthesized a hybrid compound (HYBCOM) with a triazene linked to a HDAC inhibitor (from short-chain fatty acid class), both with recognized anti-tumor roles, and investigated the potential therapeutic benefits of this molecule in comparison to the classical TMZ. We demonstrated that in the GL261 mouse glioma cell line, HYBCOM more efficiently decreased glioma cell viability and proliferation as compared to TMZ. The induction of cytotoxicity by our hybrid molecule indicates to be a consequence of the improved autophagic process. Treated GL261 cells switched from a polar to a non-polar morphology, which revealed to implicate lower migration ability and a consequent decrease on their invasive properties. Finally, cells exposed to HYBCOM showed maintained drug resistance levels, in contrast with TMZ effects, where levels were significantly enhanced when compared to those of control
conditions.
Overall,
HYBCOM
revealed
promising
chemotherapeutic
advantages over TMZ and should additionally be explored as a valuable tool in glioma therapy.
RESULTS AND DISCUSSION An aryltriazene was linked to VPA by an amide linkage. The synthesis of
HYBCOM (6) comprised the steps described in Scheme 1. As previously reported, diazotation of the commercially available 4-aminobenzonitrile 1 with nitrous acid led to the formation of the diazonium salt 2 and the subsequent reaction with HCHO/MeNH2 provided the hydroxymethyltriazene 3.20 In the presence of a three-fold excess of MeNH2,
the
hydroxymethyltriazene
monomethyltriazene
4,
with
the
3
decomposed
elimination
of
into
the
corresponding
formaldehyde.21 In
situ,
N-
methylmorpholine (NMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) formed the
coupling
reagent
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium 5 ACS Paragon Plus Environment
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chloride (DMTMM), responsible for the activation of VPA 5. Monomethyltriazene 4 was activated with NaH to enhance its reactivity as nucleophile and its later reaction with the activated ester intermediate yielded the desired product 6.
Scheme 1. Synthetic route to HYBCOM (6)
HYBCOM induces higher decrease in glioma cell viability and proliferation than TMZ. First, we evaluated the time- and dose-dependent viability of glioma cells exposed to TMZ, the standard molecule to be compared with the newly synthetized HYBCOM. After incubation with TMZ at concentrations from 50 to 250 µM for 24, 48 and 72 h, cell viability was assessed by the MTS reduction test. The dose–response curves (Figure 1) showed that up to a 50 µM TMZ exposure, glioma cells remain insensitive. Upon a 100 µM TMZ incubation, viability of GL261 cells showed a significant reduction only after 72 h (22%, p
