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GSJ: Received May 31, 2005: http://wbabin.net/saba/saba44.htm

Allosteric Restoration or Expansion of Mutator Phenotypes of Microbial Polymerases.

James Saba

In the prior disclosure (1) interesting methods of utilizing target-based selection of encoded library members to restore the binding of drug resistant mutant to the native drugs, and similar means of expanding the susceptibility of drug targets, was described.

Herein is described methods useful in functional assays, rather than binding assays. Particularly we are concerned with microbial enzymes, such as polymerases and proteases.

Concerning viral polymerases, it has been recognized that there is a fine line between a viral's need to mutate as a means of evasion, and its need to keep a relatively intact genome (2).

Figure 1 describes a scintillation proximity assay useful in screening preselected library members of a phage display library.

More specifically, individual isolated members of a phage library preselected using wild type (wt) or mutant RT are screened for their ability to bind and alter the conformation of a mutant RT sufficiently such that it can incorporate radioactive AZT. The figure illustrates the case wherein the preselected and isolated phage is indeed able to restore the wt phenotype of the RT.

Figure 2 describes a similar assay using wt RT, with the objective of inducing it to accept a previously unacceptable nucleotide derivative as substrate.

Figures 3 and 4 describe analogous processes of restoring or expanding a viral polymerases substrate specificity.

Herein the nucleotide derivatives are not chain terminators (e.g. ribavirin). Note other labeling processes, such as the use of fluorescent NTPs could be utilized.

Of course other functional assays using other enzymes, for example HSV thymidine kinase, HIV or HCV protease could be imagined. For example, one could use a fluorogenic peptide substrate in the case of the proteases.

Also, modifications discussed in (1) such as using other encoded libraries are applicable.

Finally, notice that a drug's toxicity may be reduced by allosterically enhancing its enzymatic utilization or binding to a target. Likewise, a drug's side effect could be reduced by allosterically reducing it binding or utilization by host non-targets, via molecules discovered as described above and in (1).

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.

1) A process of restoring the substrate capacity of a mutant enzyme, or expanding the substrate capacity of a wt enzyme, comprising
i) screening a library (preferably encoded) for members which bind the wt and/or mutant enzyme,
ii) assaying (preferably individually) the capacity of selected library members to restore or expand the substrate capacity of the mutant or wt enzyme.

 2) The process of claim 1 wherein the enzyme in a microbial enzyme.

3) The process of claim 2 wherein the enzyme is a viral enzyme.

4) The process of any of the above wherein the enzyme is a polymerase.

5) The process of any of the above wherein the enzyme is a protease.

6) The process of any of the above wherein the enzyme is a nucleotide kinase.

7) The process of any of the above wherein the process utilizes scintillation proximity.

8) The process of any of the above wherein the process utilizes a fluorogenic protease substrate.

9) The process of any of the above wherein the encoded library is phage display library.

10) The process of any of the above wherein the encoded library is cell display library.

11) The process of any of the above wherein the encoded library is bead-based encoded library.

12) The process of any of the above wherein the encoded library is in vitro synthesized relatively small organic library.

13) The process of any of the above wherein the enzymes substrate is support-affixed.

14) A molecule which is isolated by any of the processes above.

15) A molecule which while never being previously found to function as a substrate for a particular enzyme, is found to do so do to allosteric alteration of the enzyme by the molecule of claim 14.

 New Provisional Claims

1) A method of screening a support-affixed polymer library which comprises the use of a radioactive molecule and photographic emulsion.

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.

 References

1) Phage Display Libraries in Affinity-based Screenings for Molecules which Induce or Disrupt Molecular Associations.
Saba, JA Gen Sci J May 29, 2005

2) Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications.
Vignuzzi, et al Virus Res. 2005 Feb;107(2):173-81

3) A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity.
Pfeiffer, et al Proc Natl Acad Sci U S A. 2003 Jun 10;100(12):7289-94

4) Measurement of radioligand binding by scintillation proximity assay.
Berry, et al Methods Mol Biol. 2005;306:121-37

5) Preparation of encoded combinatorial libraries for drug discovery.
Guo, et al Methods Mol Biol. 2002;201:23-39

6) Methods of screening combinatorial libraries using immobilized or restrained receptors.
Woodbury, et al J Chromatogr B Biomed Sci Appl. 1999 Apr 2;725(1):113-37

 References added 6/1/05

7) Site-specific incorporation of nucleoside analogs by HIV-1 reverse transcriptase and the template grip mutant P157S. Template interactions influence substrate recognition at the polymerase active site.
Klarmann, et al J Biol Chem. 2000 Jan 7;275(1):359-66

8) A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase.
Meyer, et al Mol Cell. 1999 Jul;4(1):35-43.

9) Overexpression of DNA polymerase beta sensitizes mammalian cells to 2',3'-deoxycytidine and 3'-azido-3'-deoxythymidine.
Bouayadi, et al Cancer Res. 1997 Jan 1;57(1):110-6

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Addendum 6/3/05

Claim: A process for discovering allosteric modulators of proteins or polynucleotide aptamers which utilizes scintillation proximity.

Addendum 6/4/05

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.

1) Radioactive labels: autoradiography and choice of emulsions for in situ hybridization. Brady, et al (1990) In: In Situ Hybridization: Principle and Practice (J. M. Polak & J. O'D. McGee eds.) Oxford University Press.

2) Measurement of radioligand binding by scintillation proximity assay. Berry, et al Methods Mol Biol. 2005;306:121-37