Molecular Pathways, Inc.Molecular Pathways, Inc.

The goal: analyze The goal: analyze complex enzymatic complex enzymatic pathwayspathways

How do you increase the specificity of enzyme detection?

Molecular Pathways, Inc.Molecular Pathways, Inc.

The specificity problemThe specificity problem• In order to be useful for global enzymatic

analysis, each microarray spot should be as specific as possible to just one unique enzyme

• Short peptides, commonly used for enzymatic analysis, have a lot of cross-talk between different enzymes

• Need general methods to improve the specificity of enzymatic targets

Molecular Pathways, Inc.Molecular Pathways, Inc.

How to increase the How to increase the specificity of synthetic specificity of synthetic enzyme targets?enzyme targets?

• Problem is that short peptide substrates don’t have enough binding specificity

• Binding specificity determined by more than just enzyme’s active site

• Need to make substrates that are sensitive to these other “steric” factors

Molecular Pathways, Inc.Molecular Pathways, Inc.

Thrombin substrate Thrombin substrate insightsinsights

• Previous workers studied a number of different thrombin protease substrates, coupled to different fluorescent groups

• The peptide “Tos-Gly-Pro-Arg” has a different enzymatic specificity depending upon which fluorescent group was used

• Fluorescent group is acting as a “steric restrictor” helping to confer additional specificity onto the substrate peptide.

• Generalize this finding to “steric restrictor” concept

Molecular Pathways, Inc.Molecular Pathways, Inc.

Conventional enzyme Conventional enzyme substratessubstrates

Enzyme 1

Substrate 1

Enzyme 2

Substrate 2

Cross-reaction

• Conventional substrates are too small to fully distinguish between enzyme types

Molecular Pathways, Inc.Molecular Pathways, Inc.

Steric restrictor Steric restrictor concept:concept:

• Start with a substrate peptide, and modify it to increase specificity

• Specificity is increased by the addition of two or more carefully optimized “steric restrictor groups”.

• D-amino acids are good for this: good variation & protease resistant Enzyme

Substrate

Molecular Pathways, Inc.Molecular Pathways, Inc.

MP’s high specificity MP’s high specificity substratessubstrates

• Additional “steric restrictor” groups minimize cross-reactions

Molecular Pathways, Inc.Molecular Pathways, Inc.

Selecting for high Selecting for high specificityspecificity

Candidate substrates are bound to a solid phase support, and screened for (-) reaction for non-target enzymes, and (+) reaction with target enzymes.

Molecular Pathways, Inc.Molecular Pathways, Inc.

High specificity substrate High specificity substrate application 20030113768 application 20030113768 A1A1

• Use of “steric restrictor groups” to enhance enzymatic specificity for all peptide enzymatic substrates

• Specific claims on types of steric restrictor groups

• Specific claims on methods to generate steric restrictor groups