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GSJ: Received June 1, 2005:
http://wbabin.net/saba/saba47.htm
Microarrays for Discovering Allosteric Ligands of Enzymes
James Saba
In a prior report (1) the use of microarrays or encoded particles for discovering allosteric ligands for receptors was detailed.
Herein the use of a similar material and methods are utilized to discover ligands for enzymes, such as proteases, nucleases, kinases, or polymerases. The materials and methods are especially useful for finding allosteric ligands which modulate the substrate utilization capacity of wild-type (wt) or mutant enzymes.
For example in Figure 1, we are seeking ligands which allosterically can restore the normal enzymatic function of a mutant enzyme.
This microarray is quite similar to that previously described (1) except that adjacent to each arrayed library of ligands, is a common enzyme substrate probe.
These acceptor probes are often proteins or nucleic acids. For example they could be a protein which is cleaved by a protease or phosphorylated by a kinase, or a nucleic acid which is extended by a polymerase as previously described (2). Substrate probes may be precisely positioned adjacent to each ligand, or randomly placed yet so prevalent so as to be adjacent to each ligand. Conceivably they could be directly conjugated to the ligands.
As shown in Figure 2, microarrays as in Figure 1 can also be utilized to find ligands which function allosterically towards a wt enzyme such that it now can utilize a substrate is would not normally utilize.
A similar processes could be utilized to find ligands which enhance or diminish substrate utility of a wt enzyme, perhaps reducing host toxicity.
Figure 3 depicts a process for finding ligands which are able to allosterically restore the functioning of a protease or nuclease.
Therein the substrate probe is a peptide or oligonucleotide comprising a quenched fluorescent dye. Cleavage of the probe terminates quenching. Numerous other labeling schemes for this process and those in Figures 1 and 2 could be devised.
Particles, preferably encoded, could replace the array support.
The array could comprise partitions, such as microwells, such that the ligands and substrate probes could if desired be free in solution.
If the conditions of an enzyme's modification of the substrate probe could be made dependent on the proximity of these reagents, then non-allosteric ligands could also be discovered.
This invention is considered valuable and a US patent application is anticipated to be filed. However, it is hoped that others with laboratory facilities will investigate its full potential.
The following provisional claims are an attempt to encompass important aspects of this invention.
Claims
2) A process of discovering allosteric modulators of enzymes utilizing an library of potential ligands, each thereof affixed to an optionally encoded particle.
3) A microarray wherein at each locus is positioned a different ligand and a common enzyme substrate probe.
4) Any of the processes above comprising the use of a known drug.
5) A molecule an allosteric ligand identified utilizing any of the processes above.
6) A molecule which while never being previously found to function as a ligand for a particular drug target, is found to do so via any of the processes above.
2)
Allosteric Restoration or Expansion of Mutator
Phenotypes of Microbial Polymerases.
Saba, JA Gen
Sci J 2005 May 31
From what I gather from this abstract, the enzyme does not efficiently label the bead-affixed substrate unless brought into proximity by ligand.
I had considered such assays with the new kind of microarray as described above, but was concerned that the enzyme would label the array support-affixed substrate regardless of binding to an adjacent array support-affixed ligand. However, from the above reference it appears possible to find conditions which this does not appreciable occur.
Therefore, not only are arrays in the figures above useful for finding allosteric ligands, they are useful for finding ligands in general.
Addendum 6/6/05 II
Previously the use of soluble or support-affixed multiply conjugated polymers, particularly PEG was described (1). It has been recognized that the processes of the present article could be utilized in conjunction with such soluble or support-affixed polymers, as exemplified in the following figure.
Herein only two members of a library are shown, and while in this example they are soluble, they could be affixed to a support.
Herein the decoding is done by addressing the oligo encoded tails to complementary arrayed oligos, yet other decoding schemes are possible.
As just eluded to in the prior addendum, this process while shown in the identification of allosteric ligands, could potentially be used for identification of non-allosteric ligands.