Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2001-03-27
2003-11-11
Swartz, Rodney P (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S009100, C424S009200, C424S184100, C424S185100, C424S190100, C424S200100, C530S300000, C530S350000, C536S023100, C536S023700
Reexamination Certificate
active
06645505
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the development of a reporter gene based drug screening system against tuberculosis by using essential regulatory genes of Mycobacterium tuberculosis H37Rv as a target. Such regulatory genes are more particularly, the whiB genes of mycobacteria whose functions are essential for the survival and normal growth of mycobacteria.
BACKGROUND OF THE INVENTION
The Streptomycetes are dimorphic organisms. After reaching the late log phase of growth, the substrate mycelia differentiate into the aerial mycelia. The tip of the aerial mycelium then differentiates into a chain of spores. Each spore represents a single cell and is separated by a septum. In 1992, Davis and Chater (Davis, N. K. and Chater, K. F. 1992. Mol. Gen. Genet: 232: 352-358) reported that any mutation in the whiB gene of
Streptomyces coelicolor
A3(2) results into a non sporulating organism. These mutants were also white in colour since they had lost capability-to, produce deep reddish blue pigment which is a characteristic of the wild type strain of
Streptomyces coelicolor
A3(2). It was further confirmed that a fully functional whiB gene is essential for the sporulation of the
Streptomyces coelicolor
A3(2) and whiB gene may be a transcription activator. A whiB homologue was also reported from Streptoverticillium sps,
Streptomyces aurofaciens
and Rhodococcus opacus (Kormanec and Homerova, 1993 Nucl. Acid Res. 21 :2512; Seibert,V., Kourbatova, E. M., Golowela, L. M. and M. Schlomann. 1998. 180:3503-3508; Soliveri, J. E., Vijg nboom, E., Granozzi, C., Plaskitt, K. A. and K. F. Chater. 1993. J.Gen.Microbiol. 139:2569-2578). However, unexpectedly, the genome sequence of
Mycobacterium tuberculosis
H37Rv showed the presence of four genes that is whiBI/Rv3219-254 bp, whiB2/Rv3260c-269 bp, whiB3/Rv 3416-308 bp and whiB4/Rv368l c-302 bp whose deduced amino acid products were similar to the whiB gene of
Streptomyces coelicolor
A3(2). The amino acid sequences of whiB genes of
M. tuberculosis
H37Rv show 32-35 percent homology to the amino acid sequences of Streptomyces whiB genes. Although the homology is relatively low, the general property of the predicted protein remains conserved. General morphology of mycobacteria is bacillus, unlike the species of streptomycetes, which are filamentous. So far, sporulation of mycobacteria has not been reported. Therefore, the presence of whiB like genes, which controls the sporulation, in a non-sporulating organism is highly intriguing. The predicted amino acid sequence of whiB gene suggests that the whiB gene may code for a transcription activator. If that were so, then whiB genes would be a regulatory gene. However, so far it has not been reported that the whiB genes indeed code for a transcription activator, If the whiB genes are indeed a set of regulatory genes then the question is what kind of genes do they control? Recently, Gomez and Bishai (Gomez, J. E. and Bishai, W. R., 2000. Proc. Nati. Acad. Sci. 97: 8554-8559) have shown that a whiB2 homologue of
Mycobacterium tuberculosis
H37Rv is present in
Mycobacterium smegmatis
and is essential for its survival.
Mycobacterium smegmatis
is a fast growing and non-pathogenic organism. However no report or patent could be found related to the invention described in this application.
The present invention describes that out of the four whiB genes originally described in the
Mycobacterium tuberculosis
H37Rv genome sequence, the whiB1/Rv 3219 is essential to the survival of the
Mycobacterium bovis
BCG and whiB3/Rv 3416 appears to control septa formation during cell division. The properties of these genes were not reported in the genome sequence of
Mycobacterium tuberculosis
H37Rv nor have they been published in the literature (Cole et al. Nature 1998. 393: 537-544.).
Tuberculosis is still one of the major killers of human lives in India and in most of the developing countries. The spread of HIV has compounded the problems of tuberculosis because several species of mycobacteria, which were otherwise not known to infect humans, have also been found to be associated with the HIV infected patients. More than often these new pathogens are not sensitive to the conventional therapy of tuberculosis. Further the incidence of the tuberculosis caused by the drug resistant mycobacteria are also increasing at an alarming rate. These resistant bacteria do not respond to conventional therapy. Thus there is need to find either new drugs or first to find new drug targets that have not yet developed the capability to modify themselves and then search for a drug, which would attack at a particular target. The search for a new drug without any specific target usually leads to the rediscovery of known drugs. Secondly, the mode of action of the new drug discovered by random search is usually not known, thus the study of pharmaco-kinetics can be a laborious process. Further, if the organism develops resistance to the new drug then modification of these drugs which, would suit to the target of resistant organism would be very difficult. With the advancement in computer simulation studies and modeling software, it is possible to design a drug if the target is known.
The present invention uses the DNA binding property of the whiB genes of
Mycobacterium tuberculosis
H37Rv that has been demonstrated for the first time in this invention. The invention is based on the activation of the reporter gene lacZ, which produces the enzyme &bgr;-galactosidase. In a normal circumstance the &bgr;-galactosidase activity remain repressed because the whiB genes, which code for a DNA binding protein, can bind to the lexA operators present in certain strains of
Saccharomyces cerevisiae
as well as in reporter plasmids. Binding of whiB gene products with the operator's sequence results in the repression of the transcription of the lacZ gene, which produces &bgr;-galactosidase enzyme. However if a drug binds to the whiB genes then its products will not be available for binding to the operators and the lacZ transcription will continue. Activation or repression of &bgr;-galactosidase can be monitored either by adding 5-Bromo-4-chloro-3-indolyl &bgr;-D-galactopyranoside (X-gal) into the growth medium, which produces blue color or by adding o-nitrophenol &bgr;-D-galactopyranoside (ONPG) which produces yellow color in presence of &bgr;-galactosidase. Intensity of the color production is directly related to the activation of &bgr;-galactosidase enzyme and therefore would be directly related to the binding capability of a drug. In other words, a strong binder to the target will allow strong activation of &bgr;-galactosidase, however, a poor binder to the target would permit low activation of &bgr;-galactosidase enzyme. The method in this invention is fully compatible to any automated High-Through-Put screening system or any other automated or non-automated screening system.
The present invention is thus based upon the need to find a new drug target in
Mycobacterium tuberculosis
H37Rv and develop a drug screening system based upon the identified target. To suit the fast developing technology and the urgent need to find a better cure, it is desired that the screening system is compatible to any automated High-Through-Put screening or any other mechanised screening procedure.
OBJECTS OF THE INVENTION
The main objective of the present invention is to provide a new drug target, which would facilitate target specific screening of anti-tuberculosis drugs.
Another objective of the present invention is to develop a drug screening system, which is fast and uses the properties of the target gene.
SUMMARY OF THE INVENTION
The present invention relates to a drug screening method taking advantage of the yeast two-hybrid system, known in the literature. The invention also describes that the whiB genes are essential regulatory genes of
Mycobacterium tuberculosis
H37Rv and appear to be conserved amongst the pathogenic and slow growing mycobacteria. Thus the novelty of the method of the invention is to use the whiB genes which are
Agrawal Pushpa
Khandrika Lakshami Pathi
Soni Vishal
Council of Scientific and Industrial Research
Ladas & Parry
Swartz Rodney P
LandOfFree
Reporter gene based method for the screening of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Reporter gene based method for the screening of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Reporter gene based method for the screening of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3178298