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GSJ: Received June 1, 2005:
http://wbabin.net/saba/saba45.htm
Allosteric Drug Discovery Utilizing Microarrayed Ligands
James Saba
Recently interesting means of utilizing encoded libraries for discovering allosteric modulators of target-drug interactions were described (1,2). Therein encoded libraries were soluble and the drug or target were immobilized.
Herein the library of ligands are support-affixed on beads, or to form a microarray as exemplified in self explanatory Figures 1.
In this figure we start with an array of potential allosteric ligands, perhaps photolithographically produced. We contact this array with a mutant target and a drug which binds wild-type (wt) but not the mutant target. In essence, only when mutant target is induced by the a ligand to bind the drug, is a signal produced. In this example scintillation proximity via a radioactive drug and modified array support it utilized. Other detection schemes are possible, such as using a fluorescent dye-conjugated or biotin-conjugated drug; ELISA; or immono-RCA.
Figure 2 is a derivation of Figure 1 useful in drug discovery.
Particles, preferably encoded, could replace the array support.
The array could comprise partitions, such as microwells, such that the ligands could be free in solution, perhaps as a consequence of photo liberation from a support. Using partitions would allow functional screening for allosteric modulators (2).
Previously described were methods of making exceptionally dense protein arrays utilizing random arraying of cells, viruses, or nucleic acids to partitioned loci, followed by their clonal expansion (3). It has now been recognized that similar methods could be utilized to distribute libraries of particle-conjugated molecules to partitioned loci. One preferred method of distributing the particles to the partitioned loci is spraying (Figure 3).
Subsequent to assaying, which perhaps comprises photoliberation of conjugated molecules from the particles, those particles with active conjugated molecules are characterized, perhaps subsequent to being micromanipulated out.
In addition to utilizing liquid aerosol for spraying, interesting alternatives would include an electostatic-based spraying in a vacuum onto an empty partitioned microarray; or a particle shooting gun (4)
Figures 4 depicts another possible means of randomly distributing cells, viruses, or particles to form highly dense microarrays.
As previously mentioned (1) the 'ligand' as well as the target may be a relatively large molecule, such as a high molecular weight protein, and the processes above could be utilized to identify and analyze ligands which induce or disrupt their interaction
Also as previously mentioned (2) a drug's toxicity may be reduced by allosterically enhancing its binding to a target. Likewise, a drug's side effect could be reduced by allosterically reducing it binding by host non-targets.
Finally, notice that the previously described ligand dimers comprising hybridized ligand-conjugated polynucleotides (7) could be utilized.
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 target-ligand interaction wherein a library of potential allosteric modulators are affixed to particles.
3) A microarray detection scheme utilizing scintillation proximity.
4) A process comprising of randomly distributing a library of particles, each of which is conjugated to different molecule, and is optionally encoded.
5) The process of claim 4 which involves spraying.
6) Any of the processes above which utilizes a known drug.
7) A molecule which is isolated utilizing any of the processes above.
8) A molecule which while never being previously found to function as a ligand or substrate for a particular drug target, is found to do so do to allosteric alteration of the drug target, via the ligand or claim 7.
9) A process comprising contacting a support-affixed library of ligand candidates with a target protein and drug (or anticipated drug).
2) A method of detecting the covalent or noncovalent binding of a radioactive molecule to a support-affixed polymer, which utilizes a photographic emulsion to record the radioactive molecule's disintegrations.
3) The method of claim 2 wherein support-affixed polymer is a member of a library of different support-affixed polymers.
4) The method of claim 1, 2 or 3 wherein the support is a microarray support or particle.
5) Any method above wherein the polymer is a protein.
6) Any method above useful in analysis of ligands which allosterically modulate receptor binding or enzyme catalysis.
7) Any method above useful in the isolation of enantiomer-selective polymers.
2)
Allosteric Restoration or Expansion of Mutator
Phenotypes of Microbial Polymerases.
Saba, JA Gen
Sci J 2005 May 31
3)
Functional Screening Utilizing an Arrayed Library
of Clonally Expanded Cells, Viruses, or Nucleic Acids.
Saba, JA Gen Sci J 2005 Feb 24
4)
DNA vaccines: protective immunizations by
parenteral, mucosal, and gene-gun inoculations.
Fynan, et al Proc Natl Acad Sci U S A. 1993 Dec
15;90(24):11478-82
5)
Ligand-Conjugated Polynucleotides and Microarrays
of Combinatorial Libraries Thereof - Update (contains
serveral important references, particularly concerning
library synthesis).
Saba, JA Gen Sci J 2004 June 22
6)
Going to the well no more: lawn format assays for
ultra-high-throughput screening.
Marron, et al Curr
Opin Chem Biol. 2003 Jun;7(3):395-401
7)Ligand-Conjugated Polynucleotides and Microarrays
of Combinatorial Libraries Thereof -
If binding of radioactive target molecule by a protein is sufficiently strong such the unbound radioactive molecules can be washed away, then alternatively to scintillation proximity, a radiation-sensitive photographic emulsion could be layered over the array (1). If beads was used, subsequent to washing unbound radioactive molecules, the beads could be distributed within a thin layer of the emulsion. Since use of an emulsion gives a cumulative record via 'grains', it could be more sensitive than scintillation proximity.
2) Measurement of radioligand binding by scintillation proximity assay. Berry, et al Methods Mol Biol. 2005;306:121-37
Addendum 6/6/05
A distinguished G-protein coupled receptor (GPCR) pharmacologist writes:
"The development of novel allosteric interactions with G-protein coupled receptors and ion channels is a very important contemporary challenge. I wonder about tethering membrane bound G-protein coupled receptors and/or ion channels to solid phase linkages by interactions with "other proteins" in the membrane (perhaps a common protein that is transfected into the membrane). In this approach, one would bind different sets of membrane bound GPCRs or ion channels to compartmentalized solid phase supports (through the unrelated transmembrane protein) and then add the allosteric compound + fluorescently-tagged substrate of interest (e.g. ED50 concentration of agonist). The model would test for enhanced or reduced agonist binding. One could evaluate multiple membrane bound GPCRs per well - perhaps a family of GPCRs or ion channels in each well, such as opioid receptors, NMDA receptors or AMPA receptors."
This is very fine feedback and brings out the important of utilizing cells in membrane receptor assay.
Of course the target in the previously described process (1) could reside in a cell membrane. However, it was not previously made clear that cell-activation could be a valuable means of assaying the modulatory capacity of a ligand, such as depicted in the following figure.
The assay could also identify allosteric ligands which inhibit activation as well as enhance activation. Further, the assay could examine for new primary receptor site binding ligands, via the use of a known soluble allosteric regulator. The professors concept of tethering ligands to help secure cells to support may find utility utilize here.
The kind professor also correctly points out:
"I wonder about the value of locating the allosteric ligands on the solid phase. Would this approach spatially restrict the alignments necessary for strong interactions with proteins? In addition, I would be concerned about the chemistry necessary to bind a wide range of allosteric ligands to the same solid phase tethers. The chemistry may be inappropriate for may small molecule allosteric ligands and/or the chemistry may bias the interaction of the allosteric ligand with the added protein."
One interesting solution to this problem has involved dispersing a library of bead-affixed molecules in a gel covering a layer of cells (1, 2).
Perhaps known, but worthy of mention is a similar process can be envisioned wherein the library of molecules are arrayed via photocleavable linkers, and the cells layered over to this array prior to photoliberation, as depicted in the figure below.
2)
Going to the well no more: lawn format assays for
ultra-high-throughput screening.
Marron, et al Curr
Opin Chem Biol. 2003 Jun;7(3):395-401
Mass spectrometry could be utilized to identify those array loci wherein noncovalent binding of a drug or ligand to a protein has occurred.
2) Evaluation of automated nano-electrospray mass
spectrometry in the determination of non-covalent
protein-ligand complexes.
De Vriendt, et al Rapid
Commun Mass Spectrom. 2004;18(24):3061-7.