Glutamate Carboxypeptidase II as a Therapeutic Target - Proteinases as Drug Targets - page 76

(though energetically unfavorable) 'all-gauche' conformation. In this con-
formation, the Arg534 guanidinium group is well placed to engage an acidic
functionality of a substrate/inhibitor. At the same time, the positional
invariability of Arg534 contrasts with the conformational flexibility of the
side chains of Arg536 and, to a lesser extent, Arg463.

3.7 GCPII Inhibitors and Ligands
Early efforts to identify low-molecular-weight GCPII inhibitors relied mainly
on the knowledge of GCPII substrate specificity. As a result, two major cate-
gories of GCPII-specific inhibitors that exist at present are analogs of NAAG
(GCPII substrate) and derivatives of glutamic acid (reaction product). A wealth
of structural data later allowed for a more rational approach to inhibitor
design, yet the basic distinction, glutamate-like vs. NAAG-like inhibitors, is still
valid and will be used in the rest of the chapter. In addition to low-molecular
weight inhibitors/ligands, several macromolecular entities (antibodies, apta-
mers) have been identified as GCPII-specific ligands and are considered in more
detail in the following sections.

3.7.1 Low-Molecular-Weight Ligands

3.7.1.1 Derivatives of Glutamate
The architecture of the S1 0 pocket of GCPII is 'optimized' for glutamate
binding, although glutamate itself is a fairly weak inhibitor with an IC 50 of
30-500 mM. 2,78 Several glutamate isosteres have higher anities, including
quisqualate (IC 50 o 10 mM) and willardine (IC 50 B

67 mM), but truly potent
GCPII-specific inhibitors were obtained only upon the addition of a zinc-
binding group to the glutamate scaffold. 2-(Phosphonomethyl)pentanedioic
acid (2-PMPA), synthesized by the Jackson laboratory in 1996, was the first
competitive GCPII inhibitor with a high selectivity for GCPII (over various
other glutamate-associated receptors, transporters, and metabolizing enzymes)
and a picomolar inhibition constant (200 pM) 89 (see Figure 3.2).
For the design of a series of subsequent glutamate(like)-based inhibitors, a
similar overall strategy was exploited—the glutarate part of the inhibitor
ensured the specificity, while a zinc-binding group was used to impart a high
anity for GCPII. Phosphoramidates, hydroxamates, and thiols have been
used as alternative zinc-binding groups, and the decreased polarity of thiol-
based compounds led to the discovery of 2-MPPA (K i ¼ 90 nM), the first orally
bioavailable GCPII inhibitor. 88,90,91 Later, more potent and more lipophilic
thiol-based inhibitors, such as 3-(2-carboxy-mercaptopentyl)-benzoic acid
(CMBA; K i ¼ 15 nM), were synthesized via modifications of the glutarate side
chain of 2-MPPA. 91

3.7.1.1.1 GCPII-Inhibitor Interactions Within the S1 0 Pocket. The selectiv-
ity of glutamate and glutamate-like compounds for GCPII is achieved via an