1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M): I. Direct inhibition of O6-alkylguanine-DNA alkyltransferase (AGT) by electrophilic species generated by decomposition.
OBJECTIVE
To investigate the interaction of the electrophilic species generated by the decomposition of the antineoplastic prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (VNP40101M) on the ability of O(6)-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine DNA lesions.


METHODS
The contributions of inhibitory electrophilic species generated from VNP40101M towards AGT was assessed using analogues that selectively generated either the chloroethylating or the carbamoylating components of VNP40101M. The activity of AGT was determined from the inhibition of crosslink formation from O(6)-chloroethylguanine and/or N(1),O(6)-ethanoguanine lesions. The half-lives of sulfonylhydrazine derivatives and isocyanates were measured using an acidification assay which gives a change in absorbance proportional to the release or consumption of small quantities of protons.


RESULTS
Both of the reactive components produced by VNP40101M directly inactivated cloned human AGT; the carbamoylating moiety (IC(50) about 13 micro M) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC(50) about 100 micro M). These inhibitory actions were moderated by the addition of naked T5 bacteriophage DNA. Thus, AGT bound to DNA was markedly more resistant than free AGT to these electrophilic species. DNA also blocked the spontaneous loss of AGT activity which occurred upon incubation of this protein under mild conditions.


CONCLUSIONS
The reaction of AGT with the methyl isocyanate generated from the decomposition of VNP40101M increased the net number of crosslinks generated by VNP40101M compared to a sulfonylhydrazine prodrug that formed the equivalent alkylating species in the absence of the cogeneration of methyl isocyanate. These actions may be of significance to the antineoplastic activity of VNP40101M.
Penketh
P G
PG
Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
Shyam
K
K
Baumann
R P
RP
Remack
J S
JS
Brent
T P
TP
Sartorelli
A C
AC
D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000494:pharmacology*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage; D004260:DNA Repair; D004279:DNA, Viral / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006834:Hydrazines / Q000737:chemistry / Q000494:pharmacology*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors*; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D017906:Siphoviridae
C424963:1,2-bis(methylsulfonyl),1-(2-chloroethyl)-2-(methylamino)carbonylhydrazine; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D006834:Hydrazines; D011355:Prodrugs; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase