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


METHODS
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.


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
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.
Cell Biology Department, PharmaMar SA, Spain Department of Biochemistry and Molecular Biology, University of Alcalá, Spain.
J F M
JF
Leal
M
M
Martínez-Díez
V
V
García-Hernández
V
V
Moneo
A
A
Domingo
J A
JA
Bueren-Calabuig
A
A
Negri
F
F
Gago
M J
MJ
Guillén-Navarro
P
P
Avilés
C
C
Cuevas
L F
LF
García-Fernández
C M
CM
Galmarini
D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D009369:Neoplasms / Q000188:drug therapy* / Q000473:pathology; D044005:Tetrahydroisoquinolines / Q000008:administration & dosage / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D018736:DNA Adducts; D044005:Tetrahydroisoquinolines; D004247:DNA