PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity

Abstract

Background and purpose: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity.

Experimental approach: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models.

Key results: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals.

Conclusions and implications: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.

Figure 1

Chemical structure and DNA binding properties of PM01183. (A) Structure of PM01183; (B) binding to naked DNA. Drugs were incubated with a 250 bp PCR product at 25°C during 1 h and the electrophoresis was run in 2% agarose-TAE; (C) relative binding affinities for different DNA triplet sequences. PM01183 was incubated with the labelled double-strand oligodeoxynucleotides at 25°C during 1 h; then the melting assay was started in a 7500 Fast Real-Time PCR System by increasing the temperature up to 95°C in small steps of 1°C·min⁻¹. Analysis of the 1/C₅₀ parameter (drug affinity for specific DNA sequences) and ΔT_m(max) (stability of the drug–DNA complex) were analysed with an in-house developed Visual Basic Application (VBA) running on Microsoft Excel. The values correspond to the mean and standard deviation from at least two independent experiments for each sequence.

Figure 2

Modelling of DNA–PM01183 complexes and induction of DNA double-strand breaks following exposure to PM01183. (A) Refined and equilibrated molecular models of the pre-covalent (left) and covalent (right) complexes between PM01183 (C atoms in cyan) and the CGG-containing oligonucleotide (C atoms in green). The dotted lines represent hydrogen bonds. (B) A549 cells were treated with either PM01183 or trabectedin at the indicated concentrations during 6 h, followed by additional 18 h of incubation without the drug. After fixation, cells were immunostained for γ-H2AX and nuclei were visualized with Hoechst 33342.

Figure 3

PM01183 induces apoptosis in living cells. (A) Flow cytometric DNA histograms showing sub-G1 peak induced by PM01183 and trabectedin (both drugs at 150 nM after 24 h exposure) in A549 cells. (B) Representative microphotographs of chromatin condensation of A549 cells without (Control) and after 24 h exposure to 150 nM of PM01183 or trabectedin. (C) Western blots of PARP in A549 cells after exposure to 150 nM of PM01183 and trabectedin at different time intervals. (D) Detection of cleaved cytokeratin-18 using the M30-Apoptosense kit in A549 cells after 24-hour exposure to PM01183 (150 nM) or not (NT).

Figure 4

In vivo anti-tumour activity of PM01183. Treatment with PM01183 results in tumour growth inhibition in NCI-H460 lung (A), A2780 ovarian (B), HT29 colon (C) and HGC-27 gastric (D) xenograft models compared with vehicle-treated animals. Treatments started at a tumour volume size of c. 150 mm³ and were intravenously administered in three cycles of three consecutive weekly doses at 0.18 mg·kg⁻¹. Each point represents the median value (n = 10); *P < 0.05.