Structure-based identification of a NEDD8-activating enzyme inhibitor via drug repurposing.

NEDD8-activating enzyme (NAE) is an essential player of the NEDD8 conjugation pathway that regulates protein degradation. Meanwhile, drug repurposing is a cost-efficient strategy to identify new therapeutic uses for existing scaffolds. In this report, mitoxantrone (1) was repurposed as an inhibitor of NAE by virtual screening of an FDA-approved drug database. Compound 1 inhibited NAE activity in cell-free and cell-based systems with high selectivity and was competitive with ATP. Furthermore, compound 1 induced apoptosis of colorectal adenocarcinoma cancer cells through inhibiting the degradation of the neddylation substrate p53.

Oestrone-targeted liposomes for mitoxantrone delivery via oestrogen receptor - synthesis, physicochemical characterization and in-vitro evaluation.

OBJECTIVES: Targeted delivery of mitoxantrone (MTO, an anthraquinone drug with high antitumour effect) may be achieved using a novel nanoparticulate delivery system via binding the oestrogen receptor (ER, highly expressed in a variety of human tumours). METHODS: A novel liposomal nanoparticle (NP) was developed using a conjugate derived from 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG2000 -NH2 ) and oestrone (ES, is known to bind the ER) to produce an ES-targeted PEGylated liposome (ES-SSL). The resulting targeted NP was loaded with MTO to produce a targeted liposome-MTO formulation (ES-SSL-MTO). KEY FINDINGS: The targeted formulation (~140 nm, 1.5 mV) achieved over 95% drug encapsulation efficiency and a favourable stability at 4, 25 and 37 °C up to 48 h. The flow cytometric data indicated that cellular uptake of ES-SSL into human leukaemia HL-60 cells was mediated via binding the oestrogen receptor. In addition, the ES-SSL-MTO significantly reduced the growth of HL-60 cells. CONCLUSIONS: Our results provide a proof of principle that ES-modified PEGylated liposomes can target the ER, thereby potentially improving the therapeutic benefits in ER-overexpressed tumours.

Synthesis and tau RNA binding evaluation of ametantrone-containing ligands.

We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules.

Novel ametantrone-amsacrine related hybrids as topoisomerase IIß poisons and cytotoxic agents.

The precise definition of the structural requirements for effective topoisomerase II poisoning by drug molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we prepared several conjugates combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human topoisomerase IIß isoform, and cytotoxic activity resembling that of the parent compounds. In addition, the properties of the lateral groups linked to the anthracenedione group play an important role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19 appear to be promising for further development.

Cationic drug-derived nanoparticles for multifunctional delivery of anticancer siRNA.

Combined treatment of anticancer drugs and small interfering RNAs (siRNAs) have emerged as a new modality of anticancer therapy. Here, we describe a co-delivery system of anticancer drugs and siRNA in which anticancer drug-derived lipids form cationic nanoparticles for siRNA complexation. The anticancer drug mitoxantrone (MTO) was conjugated to palmitoleic acid, generating two types of palmitoleyl MTO (Pal-MTO) lipids: monopalmitoleyl MTO (mono-Pal-MTO) and dipalmitoleyl MTO (di-Pal-MTO). Among various lipid compositions of MTO, nanoparticles containing mono-Pal-MTO and di-Pal-MTO at a molar ratio of 1:1 (md11-Pal-MTO nanoparticles) showed the most efficient cellular delivery of siRNA, higher than that of Lipofectamine 2000. Delivery of red fluorescence protein-specific siRNA into B16F10-RFP cells using md11-Pal-MTO nanoparticles reduced the expression of RFP at both mRNA and protein levels, demonstrating silencing of the siRNA target gene. Moreover, delivery of Mcl-1-specific anticancer siRNA (siMcl-1) using md11-Pal-MTO enhanced antitumor activity in vitro, reducing tumor cell viability by 81% compared to a reduction of 68% following Lipofectamine 2000-mediated transfection of siMcl-1. Intratumoral administration of siMcl-1 using md11-Pal-MTO nanoparticles significantly inhibited tumor growth, reducing tumor size by 83% compared to untreated controls. Our results suggest the potential of md11-Pal-MTO multifunctional nanoparticles for co-delivery of anticancer siRNAs for effective combination therapy.

Asymmetric enzymatic glycosylation of mitoxantrone.

Using a uniquely promiscuous engineered glycosyltransferase (GT) derived from the macrolide-inactivating GT OleD, a single-step asymmetric glucosylation of one 'arm' of the drug mitoxantrone was efficiently achieved in high stereo- and regiospecificity. The synthesis, structural elucidation, and anticancer activity of the corresponding mitoxantrone 4'-ß-D-glucoside are described.

New anthracenedione derivatives with improved biological activity by virtue of stable drug-DNA adduct formation.

Mitoxantrone is an anticancer agent that acts as a topoisomerase II poison, however, it can also be activated by formaldehyde to form DNA adducts. Pixantrone, a 2-aza-anthracenedione with terminal primary amino groups in its side chains, forms formaldehyde-mediated adducts with DNA more efficiently than mitoxantrone. Molecular modeling studies indicated that extension of the "linker" region of anthracenedione side arms would allow the terminal primary amino greater flexibility and thus access to the guanine residues on the opposite DNA strand. New derivatives based on the pixantrone and mitoxantrone backbones were synthesized, and these incorporated primary amino groups as well as extended side chains. The stability of DNA adducts increased with increasing side chain length of the derivatives. A mitoxantrone derivative bearing extended side chains (7) formed the most stable adducts with ∼100-fold enhanced stability compared to mitoxantrone. This finding is of great interest because long-lived drug-DNA adducts are expected to perturb DNA-dependent functions at all stages of the cell cycle.

Rational design, synthesis, and DNA binding properties of novel sequence-selective peptidyl congeners of ametantrone.

Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases.

Mitoxantrone analogues as ligands for a stem-loop structure of tau pre-mRNA.

A series of mitoxantrone (MTX) analogues have been designed, synthesized, and evaluated for binding to and stabilizing a stem-loop structure that serves as a splicing regulatory element in the pre-mRNA of tau, which is involved in Alzheimer's and other neurodegenerative diseases. Several compounds showed significantly improved binding activity relative to the original screening hit mitoxantrone. These findings establish essential structure-activity relationships to further optimize the activity of this promising class of compounds.

Synthesis and cytotoxic properties of 4,11-bis[(aminoethyl)amino]anthra[2,3-b]thiophene-5,10-diones, novel analogues of antitumor anthracene-9,10-diones.

We developed the synthesis of a series of thiophene-fused tetracyclic analogues of the antitumor drug ametantrone. The reactions included nucleophilic substitution of methoxy groups in 4,11-dimethoxyanthra[2,3-b]thiophene-5,10-diones with ethylenediamines, producing the derivatives of 4,11-diaminoanthra[2,3-b]thiophene-5,10-dione in good yields. Several compounds showed marked antiproliferative potency against doxorubicin-selected, P-glycoprotein-expressing tumor cells and p53(-/-) cells. The cytotoxicity of some novel compounds for P-glycoprotein-positive cells is highly dependent on N-substituent at the terminal amino group of ethylenediamine moiety. The cytotoxic potency of selected compounds correlated with their ability to attenuate the functions of topoisomerase I and telomerase, strongly suggesting that these enzymes are the major targets of antitumor activity of anthra[2,3-b]thiophene-5,10-dione derivatives.

Synthesis and biological evaluation of novel cytotoxic azanaphthoquinone annelated pyrrolo oximes.

Two series of azanaphthoquinone annelated pyrrolo oximes have been synthesized. The antiproliferative activities of 10 compounds were evaluated on at least four different cell lines. One series of pyrrolo derivatives showed high cytotoxic activity. The effects on cell cycle and caspase activity were investigated. Compounds 9a and 9b showed an accumulation of cells in G2/M phase. Substantial and dose-dependent caspase activity was found after treatment of cells with 9a and 9b. This indicates an apoptosis inducing property of these compounds.

[Selective tumor-accumulation of HPMA copolymer-mitoxantrone conjugates].

AIM: To increase the accumulation of mitoxantrone in solid tumor by synthesis and characterization of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-mitoxantrone conjugate (p-DHAQ). METHODS: HPMA copolymer-mitoxantrone conjugate was prepared by free radical precipitation copolymerization method. The in vitro stability of conjugate was investigated under different conditions. Biodistribution was examined in mice bearing Ehrlich solid tumor. RESULTS: The p-DHAQ conjugate was characterized by UV, HPLC and size exclusion chromatography. The conjugate was stable in buffers of different pH and in mice plasma while the rate of drug liberation was faster in tumor. It appeared that the circulation lifetime of HPMA copolymer-bound mitoxantrone were three times more than that of the drug in free form. The AUC of p-DHAQ was three times more than the AUC of free drug. The p-DHAQ level in heart was five times lower than free drug. This reduces the possibility of toxicity to the heart. CONCLUSION: HPMA copolymer-mitoxantrone conjugate was successfully synthesized and characterized. The biodistribution results showed the possibility of targeting anticancer drug-mitoxantrone with secondary amino residue to the tumor tissue by HPMA copolymer as carrier.

[Biodistribution of N-(2-hydroxypropyl) methacrylamide copolymer-bound mitoxantrone in mice bearing Ehrlich solid tumor].

N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-Mitoxantrone conjugates was successfully synthesized and characterized as an anti-cancer drug for solid tumors. The biodistribution and pharmacokinetics of the conjugates were examined in mice bearing Ehrlich solid tumor. The biodistribution of the HPMA copolymer-bound Mitoxantrone in tumor-bearing mice was significantly different from that of the free drug. It appeared that the circulation life times of the conjugates were three times more than those of the drugs in the free form. The concentrations of HPMA copolymer-bound Mitoxantrone in tumor reached maximum levels 24 h post injection. AUC is three times higher than that of free drug. The P-Mitoxantrone level in heart was five times lower than that of free drug. This reduces the possibility of toxicity to heart. In particular, HPMA copolymer-bound Mitoxantrone accumulated at higher levels in tumor tissues. This may be explained by "Enhanced Permeability and Retention effect" (EPR effect). These results show the possibility of targeting anticancer drug-Mitoxantrone with secondary amino residue to the tumor tissue by HPMA copolymer as carrier.

Synthesis and characterization of the tumor targeting mitoxantrone-insulin conjugate.

Anticancer drugs have serious side effects arising from their poor malignant cells selectivity. Since insulin receptors highly express on the cytomembrane of some kind of tumor cells, using insulin as the vector was expected to reduce serious side effects of the drugs. The objective of this study was to evaluate the tumor targeting effect of the newly synthesized mitoxantrone-insulin conjugate (MIT-INS) with the drug loading of 11.68%. In vitro stability trials showed MIT-INS were stable in buffers with different pH (2-8) at 37 degrees C within 120 h (less than 3% of free MIT released), and were also stable in mouse plasma within 48 h (less than 1% of free MIT released). In vivo study on tumor-bearing mice showed that, compared with MIT [75.92 microg x h/g of the area under the concentration-time curve (AUC) and 86.85 h of mean residence time (MRT)], the conjugates had better tumor-targeting efficiency with enhanced tumor AUC of 126.53 microg x h/g and MTR of 151.95 h. The conjugate had much lower toxicity to most other tissues with targeting indexes (TIC) no larger than 0.3 besides good tumor targeting efficiency with TIC of 1.67. The results suggest the feasibility to promote the curative effect in cancer chemotherapy by using insulin as the vector of anti-cancer drugs.

Aza and diaza bioisosteric anthracene-9,10-diones as antitumor agents.

Synthetic routes to aza and diaza bioisosteres related to the anthracene-9,10-dione, mitoxantrone, have been developed. The antitumor properties of these chemotypes are compared with those exhibited by the corresponding carbocyclic analogues. The sensitivity of the expressed cytotoxicities on the position(s) of the nitrogen atom(s) are discussed in terms of potential cellular targets. Several analogues show potential for clinical evaluations.

Biological evaluation of mitoxantrone dihydrochloride synthesized by a new method. I. Acute toxicity and antitumor activity in mouse transplantable tumor systems.

A high antitumor activity of mitoxantrone dihydrochloride, synthesized according to a new method, was demonstrated in mice with i.p. growing tumors: P388 leukemia in CD2F1 and B16 melanoma in B6D2F1 hybrid strains. The preparation was ineffective when administered to mice with subcutaneously implanted solid tumors: Lewis Lung carcinoma, 16/C mammary adenocarcinoma or B16 melanoma. This finding is consistent with data reported by others for mitoxantrone produced by American Cyanamid Co. Acute toxicity of the tested compound was evaluated after single i.p. or i.v. administration to male and female CD2F1 and B6D2F1 mice.

Biological evaluation of mitoxantrone dihydrochloride synthesized by a new method. II. Comparison of Now 85/34 and Novantrone (Lederle) acute toxicity and antitumor activity.

A basic biological similarity of the newly synthesized mitoxantrone dihydrochloride preparation Now 85 with the Lederle's Novantrone was tested in simultaneously carried out comparative evaluations of acute toxicity and antitumor activity.

Anthrapyrazole anticancer agents. Synthesis and structure-activity relationships against murine leukemias.

Chromophore modification of the anthracenediones related to mitoxantrone in an attempt to provide agents with diminished or no cardiotoxicity has resulted in a novel class of DNA binders, the anthrapyrazoles. Their synthesis was carried out by a two-stage condensation sequence starting from requisite 1,4- or 1,5-dichloro-9,10-anthracenedione precursors. Reaction with a monoalkylhydrazine gave a chloroanthrapyrazole intermediate whose subsequent condensation with primary or secondary alkylamines provided the target "two-armed" anthrapyrazoles. A-ring 7,10-dihydroxy anthrapyrazoles were derived from amine condensation with intermediate 5-chloro-7,10-dihydroxyanthrapyrazoles or, alternatively, from intermediate 5-chloro-7,10-bis(benzyloxy)anthrapyrazoles followed by hydrogenolysis of the benzyl protecting groups to provide the target compounds. Potent in vitro activity was demonstrated against murine L1210 leukemia in vitro (IC50 = 10(-7)-10(-8) M) as well as against P388 leukemia in vivo over a wide range of structural variants. In general, activity against the P388 line was maximized by basic side chains at N-2 and C-5, two to three carbon spacers between proximal and distal nitrogens of the side chain, and A-ring hydroxylation. Besides having curative activity against the P388 line, the more active compounds were curative against murine B-16 melanoma in vivo. On the basis of their exceptional in vivo anticancer activity, A-ring dihydroxy compounds 71 and 74 reported in this study have been selected for development toward clinical trials.