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Probes

Serine Hydrolase Probes

Comprised of over 200 members including proteases, lipases, esterases, and amidases the serine hydrolase enzyme super-family is an attractive target for pharmaceutical exploration. Several known serine hydrolases are validated drug targets, with successfully marketed products treating major disease categories. Many serine hydrolases are also being developed as diagnostic markers. However, less than 5% of the serine hydrolase super-family members have been studied in any detail.

To date, ActivX has identified a large fraction of the known serine hydrolase enzymes, as well as several novel members of the serine hydrolase family.  All ActivX serine hydrolase probes use the fluorophosphonate reactive group (Figure 1).  While the fluorophosphonate (FP) reactive group in general is capable of reacting with the majority of serine hydrolases, ActivX has developed several different versions of FP probes for different applications.  For example cell permeable probes have been developed that allow for profiling of serine hydrolases directly in living cells.  Cell impermeable probes as well as probes with differing kinetic profiles are also available.  The serine hydrolase probes can be analyzed using all three ActivX technology platforms (Xhibit, Xceed, and XsiteTM).

Protein Kinase/ATPase Probes

Protein kinases represent the largest family of mammalian enzymes with more than 500 human members.  Through post-translational phosphorylation of their substrates, protein kinases are able to quickly alter the activity or function of target proteins without the need for changes in mRNA or protein synthesis.  Because of their rapid action, protein kinases play central roles in nearly all signal transduction pathways, controlling processes such as cell division, inflammation, angiogenesis, and metabolism.  The success of several recent protein kinase directed drugs such as Iressa, and Glivec, have prompted tremendous pharmaceutical interest in this enzyme family.

All protein kinases share a common structural motif that directs the transfer of the g-phosphate of ATP to a protein or peptide substrate.  Thus all protein kinases have affinity for ATP and contain conserved amino acids to facilitate the phosphate transfer.  Specifically, protein kinases all contain at least one conserved lysine residue in their active site.ActivX has taken advantage of these structural and mechanistic commonalities to develop two probes that in principle can label all protein kinases in their ATP binding sites.  The probes are acylphosphates of ATP (AX7809) and ADP (AX7844), with a biotin tag (Figure 2).  Upon binding to the ATP site on a target kinase, the acylphosphate group reacts with a conserved lysine e-amino group which generates a stable amide bond between the kinase and the biotin tag (Figure 3).  To date ActivX has already identified more than 80% of the known human protein kinases, more than 400 distinct enzymes (Figure 4).

Because of the ATP binding motif on these probes, numerous ATP and nucleotide co-factor binding enzymes are labeled in addition to the protein kinases (Figure 5).  Metabolic kinases, chaperones/HSPs, sulfotransferases, and many oxidoreductase are examples of different enzyme classes labeled by the probes in their co-factor binding sites.  The wide spectrum of labeling by these probes leads to a very large number of targets in proteomic samples (often >300 enzymes) and thus these samples can only be analyzed by the highest resolution XsiteTM platform.

The protein kinase/ATPase profiling methods at ActivX are particularly valuable for profiling candidate kinase (or other ATPase) directed therapeutic agents for selectivity (Figure 6).  Because most kinase inhibitors are directed against the ATP binding sites of the target kinase, there is a high propensity for off-target activity against other protein kinases and other ATPases in general.  Many kinases, and an even larger fraction of other ATPases do not have screening assays available and thus cannot currently be counter-screened by any other method.  Additionally, protein kinases are subjected to a variety of post-translation modifications and activity altering protein-protein interactions.  These factors are largely lost in recombinant enzyme based assays.  The ActivX method ensures that compounds are profiled against the potential target enzymes in their native state. 

Other Activity Based Probes

Cathepsin Probes

An acyloxymethyl ketone based probe is available that broadly targets cysteine proteases of the Cathepsin class.  The probe is fluorescent and can be used on all three technology platforms.  The probe is also cell permeable, and therefore can be used to profile Cathepsins in live cells.  Cathepsins B, C, L, H, K, S, and Z are all robustly labeled by this probe.  Other Cathepsins have not been evaluated for labeling. 

Caspase Probes

An acyloxymethyl ketone based probe is available that broadly targets cysteine proteases of the caspase class.  The probe is fluorescent and can be used on all three technology platforms.  The probe is also cell permeable, and therefore can be used to profile caspases in live cells.  Caspases 1-10 are all robustly labeled by this probe.  Other Caspases have not been evaluated for labeling.

Other Nucleotide Probes

A series of probes similar to the ATP/ADP based Kinase probes are available with other nucleotides as the binding group.  GTP, TTP, CTP, and UTP based probes have all been synthesized and tested.  Generally these probes have partially overlapping labeling profiles with several distinct targets consistent with their nucleotide binding group.  GTP based probes, for example, label GTPases including the small GTPase family much more efficiently than other nucleotide probes. 

Motif-Based and Non-Directed Probes

A number of probes have been developed and characterized at ActivX that label numerous enzymes from diverse families.  These include probes with sulfonate(references?), a-chloroacetamide, and fluorosulfonyl benzoyl reactive groups.  A wide range of enzymes including aldehyde dehydrogenases, alcohol dehydrogenases, glutathione-s-transferases, sulfotransferases, and cysteine hydrolases are labeled by these probes.

    

© 2010 ActivX Biosciences, Inc.
A wholly owned subsidiary of Kyorin Pharmaceutical Co, Ltd.		 envelopeinfo@activx.com