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Research Articles: Therapeutics, Targets, and Development

In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase–mediated cancer diagnosis and therapy

¹ Department of Radiology, Harvard Medical School, Boston, Massachusetts and ² Bauer Center for Genomics Research, Harvard University, Cambridge, Massachusetts

Requests for reprints: Amin I. Kassis, Department of Radiology, Harvard Medical School, Armenise Building, Room 137, 200 Longwood Avenue, Boston, MA 02115. Phone: 617-432-7777; Fax: 617-432-2419. E-mail: amin_kassis{at}hms.harvard.edu

Abstract

As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap water-insoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2'-phosphoryloxyphenyl)-6-[¹²⁵I]iodo-4-(3H)-quinazolinone (¹²⁵IQ_2-P), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2'-hydroxyphenyl)-6-[¹²⁵I]iodo-4-(3H)-quinazolinone (¹²⁵IQ_2-OH). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ_2-P(I) and IQ_2-P), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ_2-P, but not IQ_2-P(I), fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser⁹², which plays an important role in the hydrolytic process. The binding free energies (G_binding) of the isoforms to PLAP predict that IQ_2-P will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ_2-P only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ_2-P with mouse and human tumor cells causes the extracellular, alkaline phosphatase–mediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ_2-OH. No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of ¹²⁵IQ_2-P into alkaline phosphatase–expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of 70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection (¹²³I/¹²⁴I) and therapy (¹³¹I) of solid tumors. [Mol Cancer Ther 2006;5(12):3001–13]

Grant support: U.S. Department of Defense grants W81XWH-04-1-0499-Radiodetection and Radiotherapy of Breast Cancer (A.I. Kassis) and W81XWH-06-1-0204-Radiodiagnosis and Radiotherapy of Ovarian Cancer (A.I. Kassis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

³ K. Wang et al. Evaluation of chemical, physical and biologic properties of a potential anti-tumor radioiodinated quinazolinone derivative. Bioconjugate Chem, submitted for publication.

⁴ http://www.expasy.org/spdbv/.

⁵ Pospisil P, Wang K, Al Aoward AF, Iyer LK, Adelstein SJ, Kassis AI. Computational modeling, synthesis, and evaluation of a novel prodrug for targeting the extracellular space of prostate tumors. Cancer Res, submitted for publication.

⁶ Unpublished results.

Received 8/ 7/06; revised 9/23/06; accepted 10/26/06.

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