Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1997-01-02
1998-07-28
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 23, 435 24, 435 4, 435 692, 435 681, 536 221, 536 276, 536 2626, 504116, 935 64, 935 67, 800200, C12Q 134, C12Q 137, C12Q 100, C12N 1500
Patent
active
057861650
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method for identifying potential herbicides and the use of inhibitors, or pro-inhibitors, of AMP deaminase as herbicides.
PRIOR ART
Coformycin and carbocyclic coformycin have previously been reported to have herbicidal activity (Isaac et al (1991), J. Antibiotics 44, 729 and Bush et al (1993), Phytochem. 32, 737 respectively).
Coformycin is known to be a potent inhibitor of the enzyme adenosine deaminase, EC 3.5.4.4 (Nakamura et al 1974, J. Am. Chem. Soc. 96, 4327). Coformycin is also an inhibitor of the related enzyme AMP deaminase, EC 3.5.4.6, although phosphorylation at position 5' of the ribose is required to achieve very potent inhibition (Frieden et al, 1980, Biochem. 19, 5303; Merkler et al, 1990, Biochem., 29, 8358).
Inhibitors of adenosine deaminase are of interest for cancer chemotherapy, as immunosuppressive agents for autoimmune disease, and for their ability to enhance the activity of adenine nucleoside analogues for the treatment of viral diseases and cancer that could be rendered inactive through the Medicinal Chemistry (Eds Hansch C., Sammes P. G. and Taylor J. B., Vol 2, pp 449)!
It is generally accepted that plants do not contain adenosine deaminase al (1985), Physiol. Plant Pathol., 27, 65; Brady and Hegarty (1966), Nature, 209, 1027; Staub et al (1985), J. Am. Soc. Hort. Sci., 110, 426; Le Floch et al (1982), Plant Sci. Lett., 27, 309!. Consequently, the herbicidal activity of coformycin cannot be due to its known ability to inhibit this enzyme. While the related enzyme AMP deaminase has been proposed to have a role in maintaining the intracellular adenylate energy charge in plants (Raymond et al, 1987, in The Biochemistry of Plants, Vol II, Chapter 5, pp 129, Ed: Davis D. D.), no direct experimental evidence has been published, to our knowledge, to support this theory in plants. Although coformycin has been reported to perturb adenine nucleotide levels in a suspension culture of Catharanthus roseus, inhibition of AMP deaminase was not directly demonstrated (Yabuki and Ashihara, 1991, Biochim. Biophys. Acta, 1073, 474) and coformycin is known to be a relatively weak inhibitor of this enzyme when compared both to its effects on adenosine deaminase and the potency of its 5'-phosphate derivative against AMP deaminase.
We have discovered that the potent herbicidal activity of carbocyclic coformycin is due to phosphorylation of the compound in planta and subsequent inhibition of the enzyme AMP deaminase (EC 3.5.4.6). This mode of action has not previously been described for herbicides.
DISCLOSURE OF THE INVENTION
We have shown carbocyclic coformycin to be a potent inhibitor of mammalian adenosine deaminase (Test Example 1).
Investigations of the metabolism of .sup.14 C-adenosine by crude, desalted homogenates from pea seedlings confirmed the literature data that adenosine deaminase was not present. The related enzyme, AMP deaminase was, however, clearly present (Test Example 2).
Application of carbocyclic coformycin to pea seedlings via the transpiration stream resulted in a rapid decrease in the extractable levels of AMP deaminase (Test Example 3).
Although carbocyclic coformycin was only a relatively poor inhibitor of plant AMP deaminase, its 5'-phosphate derivative was a potent inhibitor (Test Example 4).
The above observations suggested that carbocyclic coformycin was phosphorylated in planta and that this phosphorylated derivative caused the observed reduction in the levels of the enzyme activity. Further evidence that phosphorylation does occur in vivo was provided by the application of .sup.3 H-carbocyclic coformycin to pea seedlings via the transpiration stream and monitoring the metabolites formed. A compound that co-chromatographed with authentic carbocyclic coformycin-5'-phosphate was shown to be formed and, moreover, this compound could be isolated bound to a soluble protein fraction containing AMP deaminase, providing strong evidence that the 5'-phosphate derivative does inhibit the enzyme in planta (Test Example 5).
AMP
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Dancer et al; "Bioorg. Med. Chem. Lett.", vol. 6(17), pp. 2131-2136, 1996. Month Not Available.
Dancer Jane Elizabeth
Lindell Stephen David
Agrevo UK Limited
Leary Louise N.
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