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GSJ: Received Apr. 16, 2007:
http://wbabin.net/saba/saba73.htm
Discovery of Protease Modulators via Pyrophosphate Signaling
James Saba
Proteases, particularly viral proteases are emerging as viable drug targets (1).
A new an apparently significant advance in DNA sequencing is pyrosequencing (2,3,4), and it has been recognized, and claimed herein, that the this technology is nicely suited to identify ligands which alter the functioning of proteases.
As Figure 1 delineates, the process for identifying protease inhibitors involves the release of pyrophosphate during amino acylation (5)
We start with a polypeptide substrate which is chemically modified (capped) at one or both termini, such that termini are no longer substrates for an exopeptidase (6).
Within this polypeptide substrate there are one or more amino acid sequences recognized by the target protease whose function we are attempting to modulate.
Of important, only after the capped polypeptide is cleaved by the target protease, can there be release of individual amino acids by the exopeptidase.
These free amino acids then are substrates for amino acyl transferases which release pyrophosphate upon activation of the amino acids.
Finally the pyrophosphate is the source of our signal. For example if we use the technology of pyrosequencing, ATP sulfurylase converts PPi to ATP in the presence of AMP, and this ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin and photon.
Notice that the reagents after the potential modulator optionally can be added together (at the same time).
As not to limit the scope of the invention, the precise form of the assay used will not be specified. Numerous possibilities are apparent to those skilled in the art of assay design. Of course the microsphere-fiber optic based technology (1,2,3) is a prime candidate.
Notice that activators as well as inhibitors could be identified, as could allosteric modulators of known activators or inhibitors.
If it should be that the above invention is indeed novel any patentable rights I may have, I freely give away.
It is hoped that others will honor the invention as delineated above and by the following claims.
Claims
1) Materials and methods of monitoring a protease activity which involves the use of pyrophosphate as a signaling molecule.
2) The materials and methods of claim 1 which also involves the use of one or more amino acyl transferases.
3) The process of claim 1 or 2 which also involves the use of a bead-bases library of potential protease modulating molecules.
4) The process of any of the above, which also utilizes a a fiber-optic picotiter plate.
5) The process of any of the above where the protease is of viral, fungal, or bacterial origin.
6) The process of claim 5 where the protease is from a virus.
2) Pyrosequencing using fiber-optic picotiter plates.
3)
Sequencing by synthesis based ordered restriction mapping.
Fuerst,
et al US Patent Appl 20070082358 April 12, 2007
4)
Nucleic acid sequencing using microsphere arrays.
Chee, et al US Patent Appl 20050191698 September 1, 2005
5)
The Enzymic Synthesis of Amino Acyl Derivatives of Ribonucleic Acid.
Berg, et al J. Biol. Chem. June 1961
Of course this techique could also be applied in screening for unknown proteases and/or their encoding nucleic acids.
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