Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-06-02
1999-11-09
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 18, 4353201, 4352523, 43525233, 435 732, 435 737, 536 232, 536 237, C12Q 168, C12N 1555, C12N 1563
Patent
active
059811846
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The invention relates to a screening model for the determination of the action of substances inhibiting the P-ATPase activity of Helicobacter.
PRIOR ART
Helicobacter pylori (H. pylori) is a human-pathogenic, gastric bacterium whose eradication from the stomach is today considered an obligatory requirement for the lasting cure of H. pylori-associated diseases. The microorganism is found in the stomach, preferably in and under the mucous membrane, occasionally also between the epithelial cells. Owing to the uniqueness of the medium in which the microorganism is found, H. pylori must have developed metabolic/physiological strategies which make possible its survival in the gastrointestinal region of man.
It is known today that the occurrence of the microorganism often is causally associated with the development of gastritis, ulcer and certain forms of stomach cancer. The forms of therapy customary today are based on the administration of substances which inhibit the activity of the gastric proton pump, H.sup.+ /K.sup.+ -ATPase, together with substances having antibiotic activity, which on the one hand are not always understood as regards mechanism of action and on the other hand comprise the risk of the formation of resistance. On account of the wide distribution of the microorganism and of the unsatisfactory therapeutic strategies to date, the need for new therapeutic strategies is therefore great.
Primary screening for helicobactericidal substances is based today, on the one hand, largely on in vitro methods such as the agar dilution test, using which the minimum inhibitory concentration with respect to H. pylori growth can be determined. In addition, Helicobacter-infected animals are often employed as an in vivo model for the screening of helicobactericidal substances. In both cases this necessitates the relatively complicated handling of the microorganism, which on the one hand is distinguished by relatively slow growth, which is why relatively long breeding or culturing times are needed, and on the other hand by demanding media conditions (microaerophilia, serum addition is necessary). Moreover, H. pylori is a human pathogen (classified as S2 according to the German State Contagious Diseases Act) and handling is therefore associated with appropriate safety measures and restrictions. In the development of helicobactericidal medicaments, the need for simple in vitro primary screening models, with the aid of which the use of experimental animals in the primary screening phase can also be reduced, is great. Such screening models can be the basis for the development of suitable, safe and efficient forms of therapy for the eradication of this human-pathogenic bacterium. In order to guarantee a safe and, if possible, Helicobacter-specific action, it should be possible to describe the targets of future eradication strategies, if possible, in their molecular structure and biochemical-physiological significance for the microorganism. On the basis of recombinant screening models, in which the action of drugs on cloned Helicobacter functions can be investigated, the development and improvement of drugs taking into account drug-target interactions into the molecular region is possible.
DESCRIPTION OF THE INVENTION
One object of the invention is to be seen in making available a screening model which, without specific safety restrictions and without the use of experimental animals, allows the primary screening of substances having Helicobacter-inhibiting action.
It has now been found that the measurement of the metabolic activity of a recombinant organism which is transformed using at least one Helicobacter-specific P-ATPase gene controllable via a promoter in the presence of cations which inhibit the metabolic activity of the recombinant organism only together with Helicobacter P-ATPase can be used for the effective and highly selective determination of the Helicobacter-inhibiting action of substances to be investigated.
One subject of the invention is therefore a screening model for the determin
REFERENCES:
Ge et al. "Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport". Mol. Microbiol. 15 (1): 97-106, Jan. 1995.
Harman et al. "Characterization of a P-type ATPase of Helicobacter pylori." Am. J. Gastroenterology 89(8): 1288, Aug. 1994.
Stokes et al. "Structures of P-type and F-type ion pumps." Curr. Opin. Struct. Biol. 4(2):197-203, 1994.
Lutsenko et al. " Organization of P-Type ATPases: Significance of Structural Diversity". Biochemistry 34(48):15607-15613, Dec. 5, 1995.
Pesci et al. Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Camoylobacter jejuni. Infection & Immunity 62(7): 2687-2694, Jul. 1994.
Chemical Abstracts, 119 (1993); Beil et al., Pharmacology 1993, 47(2), 135-140.
Bugaisky Gabriele
ByK Gulden Lomberg Chemische Fabrik GmbH
Wax Robert A.
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