Glutamate-based functionalities are instrumental for selective targeting of human GCPII in applications ranging from prostate cancer (PCa) imaging to the experimental treatment of neurodegenerative conditions.8
Since the GCPII pharmacophore (S1′) pocket is “optimized” for glutamate-like scaffolds, the presence of these functionalities assures both high affinity and specificity of corresponding inhibitors.31, 32
Several groups reported structure-activity relationship (SAR) studies focusing on substituting the P1′ glutamate in GCPII inhibitors. Majer et al33
designed and tested a series of thiol-based inhibitors containing a benzyl moiety at the P1′ position to increase lipophilicity of 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA), the first orally available GCPII inhibitor. In addition to higher lipophilicity, the best candidates were found to be more potent than the parent molecule and showed effectiveness in rat chronic constriction injury model of neuropathic pain. The Kozikowski group34
applied the SAR approach using N
)-1-Carboxy-3-methylbutyl]amino]carbonyl]-L-glutamic acid (ZJ-43, a urea-based NAAG analogue as a lead compound) to decrease polarity for more efficient targeting of GCPII in the nervous system, especially in the PNS. We evaluated a series of DCIBzL-based isosteres and identified several non-glutamate inhibitors with Ki
values below 20nM that exhibited selective binding to GCPII-expressing tumors by single photon emission computed tomography (SPECT-CT) imaging in mice.32
This report uses the rational design to extend and complement the above-mentioned SAR studies with the objective of preparing potent GCPII inhibitors with enhanced lipophilicity. Based on our earlier kinetic data we first designed and characterized a set of novel dipeptidic GCPII substrates and provided the structural evidence for recognition of such dipeptides by GCPII. Next, we designed a series of inhibitors, where the P1′ moiety is derived from the dipeptidic substrates and the P1 part features 4-iodo-benzoyl-ε-lysine, the functionality shown by us previously to augment interactions with GCPII24
; the P1 and P1′ parts are connected via
a urea linker (). The most potent molecule (compound 8I
) has Ki
= 29 nM and ClogD = -0.23. Although the binding affinity of 8I
is markedly lower compared to the parent glutamate-based compound (29 nM vs 10 pM, respectively), its affinity is sufficient for imaging PCa.32
Furthermore, substantially increased lipophilicity (- 0.23 vs -5.16) can be translated into a better pharmacokinetic profile in the periphery, with increased likelihood of the penetration into the CNS. Last but not least, in the phase I human clinical trial using N
one of the glutamate-based PET agents targeting GCPII that was developed for prostate cancer imaging, we observed somewhat increased signal from the blood pool in human subjects, suggesting potential binding of the compound to an unidentified plasma protein. The prime suspect in the case is plasma glutamate carboxypeptidase, a circulating plasma protein with 27% overall sequence identity and overlapping substrate specificity to GCPII.36, 37
Given the substitution of glutamate by non-natural amino acids in novel inhibitors presented here, the likelihood of off-target interactions with endogenous proteins might be less pronounced in the latter. To prove these assumptions, however, additional in vivo
studies are needed.
For both substrate and inhibitor synthesis, 2-amino acids with pentyl to heptyl side chains were used as a racemic mixture for both substrate and inhibitor synthesis. The corresponding products (substrates and inhibitors) are therefore equimolar mixtures of two diastereomers. In the case inhibitors, the individual diastereomers were separated by HPLC and their inhibition potency assayed. As expected, only compounds with the (S) stereochemistry at the C-terminus were inhibitory, while their (R) counterparts turned out to be inactive (data not shown). In the case of substrates, a mixture of diastereomers was used for kinetic studies. Following the substrate incubation with rhGCPII for 24 hours at 37°C, we analyzed the reaction mixture using HPLC with UV detection after pre-column derivatization of released C-terminal amino acids with the Marfey's reagent (1-fluoro-2-4-dinitrophenyl-5-L-alanine amide), a chiral reagent used for distinguishing (S)- and (R)-amino acids. In all cases, we observed peaks corresponding to only a single, presumably (S), enantiomer (data not shown). Since previously reported data suggested that GCPII is inactive towards (R)-amino acids at the P1′ position, we concluded that only (S)-amino acid-containing dipeptides serve as efficient substrates of rhGCPII.
The SAR studies suggest that the non-prime GCPII specificity pocket(s) are rather insensitive to structural changes of GCPII inhibitors and can accommodate (or at least tolerate) surprising diversity of functional groups of inhibitors.4, 7, 24, 26, 28, 34
On the contrary, the S1′ (or pharmacophore) pocket in GCPII is highly selective for glutamate and glutamate-like moieties. The selectivity is achieved via
an intricate network of mostly polar interactions between GCPII and an inhibitor, with the most prominent being the ion pairing between Arg210-α-carboxylate and Lys699-γ-carboxylate.22, 31
Structural data that characterize the S1′ pocket as fairly compact, small-sized and unyielding, in contrast to the much larger and quite flexible non-prime site (the funnel emanating from the active site zinc to the surface of the protein), are in agreement with these observations.
This report expands the above concept in two ways: (i) it documents for the first time substantial plasticity of the GCPII pharmacophore pocket achieved by the relocation of the Lys699 side chain leading to the considerable enlargement (by 3.9 Å) of the S1′ site. Furthermore, this work directly demonstrates the importance of non-polar interactions, mediated by the side chains of Phe209, Asn257, Leu428, and Lys699 for GCPII affinity towards small-molecule compounds featuring hydrophobic moieties in the P1′ position. Although ionic interactions between the Arg210 guanidinum group and the α-carboxylate group of the C-terminal (P1′ position) residue are common to all dipeptidic substrates tested in this study, these are obviously not sufficient with respect to efficient substrate positioning and subsequent hydrolysis, as the dipeptide with glycine in the P1′ position is not cleaved. Additionally, the dipeptide with a C-terminal alanine, the amino acid with the shortest side-chain, in the P1′ position is the least efficient GCPII substrate (see ).
In summary, the new findings presented here expand the chemical space that can be explored during the rational design of GCPII inhibitors with increased lipophilicity. By linking a lipophilic “non-glutamate” C-terminal moiety to a non-polar P1 functionality one can design inhibitors with increased lipophilicity that are more likely to penetrate the blood-brain barrier. It should be noted, however, that lipophilicity is only one of physicochemical parameters related to the drug-like molecular properties (others being e.g. molecular weight, polar surface area, number of hydrogen bond donors/acceptors, number of rotatable bonds). In this regard, compounds presented here can be viewed as a precedent for the development of GCPII-specific inhibitor analogs, with the ultimate goal of designing the truly BBB-permeable compounds.