Edinburgh Napier University

  The development of drug resistance, notably multidrug resistance (MDR) and adverse side effects due to treatment with current anticancer drugs are considerable obstacles in cancer therapy and together with dose-limiting toxicity lead to therapeutic failure in the cancer clinic. The aim of this study is to design and deliver new anticancer agents selectively to the tumour site to increase efficacy and reduce toxic side effects to normal cells, thereby increasing the therapeutic index; this would afford significant improvement for patients. The P-glycoprotein (P-gp; p-170) efflux pump is the most notable factor contributing to MDR. In one approach, a series of novel ester-linked aminoanthraquinone-triphenylphosphonium (AQ-TPP) conjugates, structurally related to the DNA-binding, Pgp- substrate anticancer drug mitoxantrone, was, synthesised and characterized by mass spectrometry, 1H and 13C NMR spectroscopy. AQ-conjugates of the lipophilic cation TPP+ were designed and shown to utilize the large mitochondrial membrane potential (ΔΨm) in cancer cells to drive drug accumulation inside the mitochondria and concomitantly circumvent Pgp-mediated resistance. Correspondingly, enhanced DNA-binding affinity and cytotoxicity were achieved for the AQ-TPP conjugate series compared with unconjugated precursors, exemplified by conversion of the non-DNA binding, non-cytotoxic hydroxyalkylanthraquinone NU:UB 238 to its TPP conjugate SH1; that bound strongly to DNA (Kapp 0.1×107M-1) and was equitoxic in the highly resistant Pg-p expressing HCT 15 colon carcinoma and sensitive MCF7 breast carcinoma cell lines. [IC50 (µM) 9.48 ± 0.32 and 9.69 ± 3.35, respectively, by MTT assay]. Confocal microscopy live cell imaging in HCT116 colon cells, using MitoTrackerFM tracer dye showed significant colocalisation of cationic and lipophilic SH1 (Log D: 2.68 ± 0.04) to mitochondria and not to lysosomes, whereas precursor NU:UB 238 (Log D:1.97 ± 0.12) was not taken up by cells, establishing proof of principle.

In another approach to achieve selective targeting of anthraquinone-based cytotoxics to cancer cells, a novel theranostic (therapeutic and diagnostic) anticancer prodrug Rho-Pro-Ala-Asn-Gly-APA-AQ(4,8-di-OH) (OM50) and FRET substrate of the endoprotease legumain, highly overexpressed in cancer, was designed and synthesized, using solution phase and solid phase peptide synthesis, to incorporate experimental anticancer drug NU:UB 51, a cytotoxic bis-hydroxylated, propyl-spaced, aminoanthraquinone glycine conjugate with in vivo activity to confer cancer cell selectivity by legumain-mediated prodrug OM50 activation in the tumour microenvironment. The active drug was attached to the C-terminal asparagine (Asn) residue of the peptide substrate to exploit the unique proteolytic cleavage specificity of the enzyme for the acid terminus of Asn. Incubation of OM50 (10 μM) with rh-legumain (0.40 ng/μL) at 37 °C for 2 h in MES assay buffer (pH 5.0) afforded a time-dependent increase in relative fluorescence intensity, monitored at the emission wavelength (λem 583 nm) of rhodamine-labelled peptide, confirming proof of principle for activation of the prodrug and direct release of the active anthraquinone NU:UB 51.

The above approaches offer the prospect of improved methods for selectively targeting the anthraquinone class of cytotoxics to cancer cells with therapeutic potential.