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AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase

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Summary

The glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoyl-L-glutamic acid (AG2034), was designed from the X-ray structure of the GARFT domain of the human tri functional enzyme. AG2034 inhibits human GARFT (Ki = 28 nM), has a high affinity for the folate receptor (K d = 0.0042 nM), and is a substrate for rat liver folylpolyglutamate synthetase (K m = 6.4 μM, V max = 0.48 nmole/hr/mg). The IC50 for growth inhibition was 4 nM against L1210 cells and 2.9 nM for CCRF-CEM cells in culture. In vitro growth inhibition can be reversed by addition of either hypoxanthine or AICA (5-aminoimidazole-4-carboxamide) to the culture medium.

A cell line with impaired transport of reduced folates, L1210/CI920 [1], was resistant to AG2034 indicating that this compound can enter cells by utilizing the reduced folate carrier. AG2034 showed in vivo antitumor activity against the 6C3HED, C3HBA, and B-16 murine tumors and in the HxGC3, KM20L2, LX-1, and H460 human xenograft models, and has been selected for preclinical development towards clinical trials.

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Abbreviations

GARFT:

glycinamide ribonucleotide formyltransferase

FBP:

human folate-binding protein

FPGS:

folylpolyglutamate synthetase

BSA:

bovine serum albumin

FDDF:

N10-formyl-5,8-dideazafolate

HEP-ES:

N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid

Tris:

tris(hydroxymethyl)-aminomethane

EDTA:

Ethylene diamine tetraacetic acid

PMSF:

phenylmethylsulfonyl fluoride

e294 :

extinction coefficient at 294 nm

K i :

inhibition constant

K i, app :

apparent inhibition constant

K d :

equilibrium dissociation constant

K m :

Michaelis constant

V max :

maximal velocity

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Authors and Affiliations

  1. Agouron Pharmaceuticals Inc., 92121, San Diego, CA, USA

    Theodore J. Boritzki, Charlotte A. Barlett, Cathy Zhang, Eleanor F. Howland, Stephen A. Margosiak, Cindy L. Palmer, William H. Romines & Robert C. Jackson

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  1. Theodore J. Boritzki
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  2. Charlotte A. Barlett
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  3. Cathy Zhang
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  4. Eleanor F. Howland
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  5. Stephen A. Margosiak
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  6. Cindy L. Palmer
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  7. William H. Romines
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  8. Robert C. Jackson
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Boritzki, T.J., Barlett, C.A., Zhang, C. et al. AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase. Invest New Drugs 14, 295–303 (1996). https://doi.org/10.1007/BF00194533

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