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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

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Abstract

Purpose

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 O6-alkylguanine-DNA alkyltransferase (AGT) to repair alkylated O6-chloroethylguanine and/or N1,O6-ethanoguanine DNA lesions.

Materials and 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 O6-chloroethylguanine and/or N1,O6-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 (IC50 about 13 μM) was approximately seven- to eight-fold more potent than the alkylating component(s) (IC50 about 100 μM). 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.

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Fig. 1
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Fig. 3A, B
Fig. 4A–D

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Notes

  1. Ten μl of 100 μg/ml of T5 DNA = 1 μg DNA; T5 DNA Mr = 68,000,000 = 15 fmol DNA; 30–40% crosslinking = 4.5–6.0 fmol of crosslinks generated by 2 nmol 90CE (10 μl of 0.2 mM). Moles of crosslinks/mole alkylator = crosslink molar yield = 0.025%

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Authors and Affiliations

  1. Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA

    P. G. Penketh, K. Shyam, R. P. Baumann & A. C. Sartorelli

  2. Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA

    J. S. Remack & T. P. Brent

Authors
  1. P. G. Penketh
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  2. K. Shyam
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  3. R. P. Baumann
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  4. J. S. Remack
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  5. T. P. Brent
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  6. A. C. Sartorelli
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Corresponding author

Correspondence to A. C. Sartorelli.

Additional information

This work was supported in part by U.S. Public Health Service Grants CA-90671 (A.C.S.) and CA-14799 (T.P.B.) from the National Cancer Institute.

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Penketh, P.G., Shyam, K., Baumann, R.P. et al. 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. Cancer Chemother Pharmacol 53, 279–287 (2004). https://doi.org/10.1007/s00280-003-0740-7

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