Part:BBa_K2688003

cpg2_tu

Executive summary : This BioBrick is a full translation unit encoding CPG2, an enzyme that converts the anticancer drug methotrexate (MTX) into less toxic compounds. When expressed in E. coli, we have shown that it can rapidly remove MTX from the culture medium. This is proven conclusively by HPLC, and a bioassay that measures residual toxicity.

The Pseudomonas carboxypeptidase G2 (CPG2 or ‘glucarpidase’) is an hydrolase that cleaves the (poly)glutamate tail off folates and analogs, leaving behind a pteroate ring system^[1]). There is broad substrate specificity, including endogenous folates, and notably the chemotherapeutic drug MTX (MTX). Its metabolite DAMPA (2,4-diamino-N10-methylpteroic acid) (Figure 1) has little antifolate activity in vitro, and norelevant clinical effects compared to MTX^[2].

This has led to its use as both an antidote in case of MTX poisoning^[3], and as a general purpose platform for novel drug delivery methods, where CPG22 would activate polyglutamated, soluble prodrugs in situ^[4].

Figure 1 : The MTX biotransformation by Pseudomonas CPG2

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 132
Illegal NgoMIV site found at 820
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 919

↑ Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., & Brick, P. (1997). Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure (London, England : 1993), 5(3), 337–347. https://doi.org/10.1016/S0969-2126(97)00191-3
↑ Widemann, B. C., Sung, E., Anderson, L., Salzer, W. L., Balis, F. M., Monitjo, K. S., … Adamson, P. C. (2000). Pharmacokinetics and metabolism of the methotrexate metabolite 2, 4-diamino-N(10)-methylpteroic acid. The Journal of Pharmacology and Experimental Therapeutics, 294(3), 894–901.
↑ Widemann, B. C., Balis, F. M., Kim, A. R., Boron, M., Jayaprakash, N., Shalabi, A., … Adamson, P. C. (2010). Glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction: Clinical and pharmacologic factors affecting outcome. Journal of Clinical Oncology, 28(25), 3979–3986. https://doi.org/10.1200/JCO.2009.25.4540
↑ Masterson, L. A., Spanswick, V. J., Hartley, J. A., Begent, R. H., Howard, P. W., & Thurston, D. E. (2006). Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy. Bioorganic & Medicinal Chemistry Letters, 16(2), 252–256. https://doi.org/10.1016/J.BMCL.2005.10.017

[Roswell1997-1] Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., & Brick, P. (1997). Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure (London, England : 1993), 5(3), 337–347. https://doi.org/10.1016/S0969-2126(97)00191-3

[Widemann2000-2] Widemann, B. C., Sung, E., Anderson, L., Salzer, W. L., Balis, F. M., Monitjo, K. S., … Adamson, P. C. (2000). Pharmacokinetics and metabolism of the methotrexate metabolite 2, 4-diamino-N(10)-methylpteroic acid. The Journal of Pharmacology and Experimental Therapeutics, 294(3), 894–901.

[Widemann2010-3] Widemann, B. C., Balis, F. M., Kim, A. R., Boron, M., Jayaprakash, N., Shalabi, A., … Adamson, P. C. (2010). Glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction: Clinical and pharmacologic factors affecting outcome. Journal of Clinical Oncology, 28(25), 3979–3986. https://doi.org/10.1200/JCO.2009.25.4540

[Masterson2006-4] Masterson, L. A., Spanswick, V. J., Hartley, J. A., Begent, R. H., Howard, P. W., & Thurston, D. E. (2006). Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy. Bioorganic & Medicinal Chemistry Letters, 16(2), 252–256. https://doi.org/10.1016/J.BMCL.2005.10.017

[1]

[2]

[3]

[4]

ec_num	3.4.17.11
ligands	folates, folate analogues, various polyglutamated small molecules, various short peptides
uniprot	P06621