Glutamate Carboxypeptidase II as a Therapeutic Target - Proteinases as Drug Targets - page 77

intricate network of enzyme-inhibitor interactions that are described below in
detail and illustrated in Figure 3.2.
The a-carboxylate of a C-terminal glutamate is engaged by the Arg210
guanidinium group. Not only does this interaction ensure GCPII selectivity as a
carboxypeptidase, but mutagenesis, SAR, and structural studies suggest that
the Arg210-inhibitor interactions are one of the major determinants of inhi-
bitor anity for GCPII.
The side chains of Phe209 and Leu428 define opposite walls of the S1 0 pocket
with an internal spacing of approximately 8.0 A ˚ , imposing limits on the size
and placement of a potential inhibitor molecule. Phe209 and Leu428 also con-
tribute prominently to the non-polar interactions involved in inhibitor binding. 78,82
The bottom of the S1 0 pocket is closed by the glutarate sensor (residues 692-
704). Both Lys699 and Tyr700 contribute to the specificity and anity of
inhibitor binding by engaging the a-carboxylate (Tyr700 OH) and g-carboxy-
late (Lys699 Nz) groups of S1 0 -bound glutamate (or glutamate-like moiety).
Although shielded from direct interactions with the S1 0 -bound glutamate or
glutamate-like residues by an array of ordered water molecules, the amino acid
segment Leu259-Asn262 serves as an example of S1 0 pocket plasticity. In sev-
eral structures of GCPII complexes with compounds featuring non-glutamate
P1 0 residues, this loop reorients itself to better accommodate such functional-
ities (Plechanovova et al., unpublished). 83

A B

Figure 3.2 Glutamate-based GCPII inhibitors. (A) Inhibitors derived from the
glutamate (the reaction product; middle) scaffold by substituting the
amino group with a zinc binding functionality (blue squares; from
the top—hydroxamate, phosphinate, thiol, phosphonate). The attach-
ment of the Zn 21 binding group increases the inhibitor binding anity
up to 10 6 -fold. Concurrently, the glutamate side chain (red circle) can be
modified to adjust the desired inhibitor properties, such as lipophilicity.
The GCPII substrate/inhibitor binding cavity is represented by a sur-
rounding irregular shape. GCPII amino acids interacting with the inhi-
bitor are shown. (B) Examples of GCPII-specific inhibitors based on
glutamate. 2-MPPA—first orally available GCPII inhibitor. 2-PMPA—
also known as GPI5000, the most widely used GCPII-specific inhibitor in
proof-of-principle studies. CMBA—a derivative of 2-MPPA with more
lipophilic properties.