Enzyme-Catalyzed Activation of Anticancer Prodrugs

Rooseboom, Martijn; Commandeur, Jan N. M.; Vermeulen, Nico

doi:10.1124/pr.56.1.3

Cited by 441 publications

(308 citation statements)

References 257 publications

(455 reference statements)

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“…We applied the method to altering the specificity of human guanine deaminase (hGDA) with the long-term goal of introducing cytosine deaminase activity into a human protein scaffold. A designed cytosine deaminase with a sequence close to that of a human protein would solve an important problem in suicide gene therapy (16)(17)(18) by providing prodrug-activating ability (19)(20)(21)(22) while retaining low immunogenicity, as described in the SI Text. We consider hGDA to be the best starting point for such an effort based on the complement of deaminases that exist in the human genome (23,24).…”

Section: Resultsmentioning

confidence: 99%

Alteration of enzyme specificity by computational loop remodeling and design

Murphy

Bolduc

Gallaher

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-Å C␣ RMSD of the computational model. computational protein design ͉ loop modeling C omputational protein design methodology has been used to optimize properties such as protein stability (1, 2) and to introduce functions such as binding of small molecules (3), proteins (4), and nucleic acids (5), as well as enzymatic activity (6, 7). In most of these studies, the implicit assumption that the structure of the polypeptide backbone would remain largely fixed despite mutations of amino acid side chains was made for the sake of computational tractability.Explicit remodeling of the polypeptide backbone makes possible further optimization of these and other structural or functional properties. The set of combinations of protein sequences and structures is considerably larger when backbone flexibility is allowed and is likely to contain conformations that optimize a desired property to a greater degree than the original scaffold. This is illustrated by the backbone shifts that accompany functional divergence in natural protein evolution. Previous studies have achieved functional changes by backbone alteration, but relied on grafting methods that are restricted to sequences of known structure and function (8-10), which may be suboptimal with respect to the desired property.De novo protein structure prediction methods are well suited for sampling novel backbone conformations (11). These methods have recently been extended to focus sampling on conformations that satisfy specific positional constraints (12, 13). Computational design algorithms that iterate between sequence design and backbone optimization using structure-prediction methods have been used to design previously unobserved protein fold and loop conformations (2, 14), but have not yet been applied to achieving functional changes such as alteration of an enzyme's substrate specificity.We have developed a computational design algorithm that uses constr...

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Section: Resultsmentioning

confidence: 99%

Alteration of enzyme specificity by computational loop remodeling and design

Murphy

Bolduc

Gallaher

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

113

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“…Lack of selectivity for malignant cells leading to dose-limiting toxicity is one of the major obstacles for the success of cancer chemotherapy. Gene-directed enzyme prodrug therapy (GDEPT) is potentially an elegant way to improve the therapeutic index of active anticancer drugs, but one of the major limitations for the clinical application of this approach is the lack of strong cancerspecific promoters (Aghi et al, 2000;Rooseboom et al, 2004).Carcinoembryonic antigen (CEA) is not expressed in normal and overexpressed in epithelial cancer cells (Shively and Beatty, 1985). Four cis-acting DNA-binding elements mapped on the CEA promoter region within 300 bp upstream of CEA translation starting point, especially, the first three elements are essential for specific CEA transcription (Hauck and Stanners, 1995;Richards et al, 1995a).…”

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confidence: 99%

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confidence: 99%

In vitro evaluation of cancer-specific NF-κB-CEA enhancer–promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines

et al. 2007

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Nuclear factor-kappa B (NF-kB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer -promoter system, kB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kB DNA-binding sites (kB4). In combination with a kB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three-to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kB4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kB4, kB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kB4-CEA205-and CMV-driven TP cDNAtransfected cancer cell lines (H630 and RKO). The kB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV-and kB4-CEA205-driven TP cDNA transiently transfected cells were 8-to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5 0 -deoxy-5-fluorouradine (5 0 -DFUR), in contrast to only 1.5-to 2-fold sensitised by the kB4-and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV-and kB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy. Lack of selectivity for malignant cells leading to dose-limiting toxicity is one of the major obstacles for the success of cancer chemotherapy. Gene-directed enzyme prodrug therapy (GDEPT) is potentially an elegant way to improve the therapeutic index of active anticancer drugs, but one of the major limitations for the clinical application of this approach is the lack of strong cancerspecific promoters (Aghi et al, 2000;Rooseboom et al, 2004).Carcinoembryonic antigen (CEA) is not expressed in normal and overexpressed in epithelial cancer cells (Shively and Beatty, 1985). Four cis-acting DNA-binding elements mapped on the CEA promoter region within 300 bp upstream of CEA translation starting point, especially, the first three elements are essential for specific CEA transcription (Hauck and Stanners, 1995;Richards et al, 1995a). Utilisation of the CEA promoter in an adenovirus vector for cytosine deaminase (CD) gene expression improves the selectivity of 5 0 -deoxy-5-fluorocytidine (5-DFCR)/5-fluorouracil (5-FU) conversion in CEA-expressing tumours (Richards et al, 1995a;Lan et al, 1996a). However, the transcriptional activity of CEA in most cancer cell lines is 10-to 300-fold lower than that of CMV and RSV viral promoters (Lan et al, 1996b;Ueda et al, 2001).Even when using adenovirus to deliver CEA promoter-driven suicide gene in vivo, few reports have demonstrated sufficient antitumou...

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“…In principle, GSTs may have two opposite effects on drugs, namely by inactivating drugs or by activating ineffective pro-drugs. The latter effects have been employed as an anticancer drug strategy (Rooseboom et al, 2004) and may be further developed as an anti-Echinococcus strategy.…”

Section: (2) Studies On Antiproliferative Drugsmentioning

confidence: 99%

Alveolar and cystic echinococcosis: towards novel chemotherapeutical treatment options

Hemphill

Müller

2009

J. Helminthol.

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Echinococcus granulosus and Echinococcus multilocularis are cestode parasites, of which the metacestode (larval) stages cause the neglected diseases cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. The benzimidazoles albendazole and mebendazole are presently used for the chemotherapeutical treatment, alone or prior to and after surgery. However, in AE these benzimidazoles do not appear to be parasiticidal in vivo. In addition, failures in drug treatments as well as the occurrence of side-effects have been reported, leading to discontinuation of treatment or to progressive disease. Therefore, new drugs are needed to cure AE and CE. Strategies that are currently employed in order to identify novel chemotherapeutical treatment options include in vitro and in vivo testing of broad-spectrum anti-infective drugs or drugs that interfere with unlimited proliferation of cancer cells. The fact that the genome of E. multilocularis has recently been sequenced has opened other avenues, such as the selection of novel drugs that interfere with the parasite signalling machinery, and the application of in silico approaches by employing the Echinococcus genome information to search for suitable targets for compounds of known mode of action.

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Enzyme-Catalyzed Activation of Anticancer Prodrugs

Cited by 441 publications

References 257 publications

Alteration of enzyme specificity by computational loop remodeling and design

Alteration of enzyme specificity by computational loop remodeling and design

In vitro evaluation of cancer-specific NF-κB-CEA enhancer–promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines

Alveolar and cystic echinococcosis: towards novel chemotherapeutical treatment options

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