Glutamate Carboxypeptidase II as a Therapeutic Target - Proteinases as Drug Targets - page 81

cleft is defined only by three amino acid residues (Trp541, Arg511, and Arg463),
it drastically (4100-fold) enhances the small-molecule binding anity. Inter-
estingly, the arene-binding cleft is also implicated in the binding of natural GCPII
substrates, namely the pteroatemoiety of folate-g-glutamates 3-5 (Navratil et al.,
unpublished). Consequently, the arene-binding cleft (and possibly other RBSs)
might play an active role in physiological processes Figure 3.4.
3.7.2 Macromolecules
In addition to low-molecular-weight GCPII inhibitors/ligands, a variety of
macromolecular entities have been developed to interact specifically with GCPII.
Anti-GCPII aptamers, nuclease-stabilized RNA molecules specifically targeting
humanGCPII, were isolated using the SELEX (systematic evolution of ligands by
exponential enrichment) methodology by Lupold and co-workers in 2002. 109 The
aptamers have a low nanomolar anity for human GCPII. The aptamers and
their derivatives were used by several laboratories in a variety of experimental
setups for targeting GCPII-expressing cells in vitro and in vivo, including the
formation of nanoparticles for toxin delivery or imaging, delivery of therapeutic
siRNAs, and construction of quantum dot-aptamer conjugates. 110-113 At present,
there is no structural information regarding the aptamer-GCPII interaction.
Several groups have reported isolation and characterization of monoclonal
antibodies and fragments thereof targeting GCPII (Table 3.1). At present,
7E11-C5 and J591, two monoclonal antibodies recognizing the intracellular
and extracellular part of GCPII, respectively, are relevant to clinical practice.
3.8 Applications
3.8.1 Experimental Anti-Neurodegeneration Therapy
NAAG is the most prevalent peptide neurotransmitter in the human CNS. 8 By
cleaving NAAG, GCPII contributes to the elevation of extracellular glutamate.
Excessive glutamatergic transmission can be neurotoxic, most significantly by
overactivating NMDA receptors. While no dramatic physiologic deficit seems
to accompany the loss of GCPII activity under normal conditions, the effects of
NAAG hydrolysis can be profound under stressed conditions. 53
Figure 3.3 A 'generalized' NAAG-based inhibitor. (A) Schematic representation in
the middle. The NAAG-based inhibitor can be viewed as a composite of
several interconnected parts: the P1 0 moiety (blue), the non-hydrolyzable
peptide bond surrogate (gray), P1 linker (green), and the distal part (red).
The introduction of one or two carboxylic groups (magenta) interacting
with positively charged amino acids of GCPII substantially increases
inhibitor anity. Specific examples of the individual parts together with
interacting residues are shown surrounding the generalized inhibitor. (B)
Examples of GCPII-specific NAAG-based inhibitors.