SERINE RACEMASE INHIBITORS
SR is a rational target for
the treatment of diseases described in section 7. However, in spite of its relevance,
investigations carried out towards the development of potent inhibitors or
effectors are surprisingly rather limited. This fact might be dependent on at
least two factors: i) the yield and stability of the enzyme are low, making inhibition
studies difficult, and ii) inhibitors should be very specific and cross the
blood-brain barrier. In order to generate specific inhibitors, the structural
and functional features of SR active site have been exploited as well as the
well known reactivity of the coenzyme. SR inhibitors have been developed to
reduce the excitoxic effects of high levels of D-serine. Specifically, these
compounds are aimed at reducing SR activity, thus decreasing D-serine
concentration, and/or at increasing the b-elimination operating on D-serine, thus
speeding up the depletion of the neurotransmitter.
SR effectors that are under development can be
grouped based on their mechanism of action:
1. PLP targeting with
reversible modifications;
2. PLP targeting with
irreversible modifications;
3. Active site targeting;
4. Allosteric effectors;
5. Modulators of SR-protein
interactions.
To our knowledge, inhibitors so far identified
belong exclusively to the first three groups. In Table 2 the structure and
binding affinities of the most potent inhibitors are reported.
Table Structure and activity of the most potent
inhibitors of SR identified to date
Inhibitors targeting PLP
|
Structure
|
Ki (mM)
|
Species
|
Reference
|
|
Reversible
|
Glycine
|
![]() |
0.36
1.64
|
hSR
mSR
|
(51)
(22)
|
L-erythro-3-hydroxyaspartate
|
![]() |
0.01
0.04
|
hSR
mSR
|
(51)
(22)
|
|
Irreversible
|
Succinodihydroxamic
acid
|
![]() |
0.003
|
mSR
|
(109)
|
L-aspartic
acid b-hydroxamate
|
![]() |
0.10
|
mSR
|
(109)
|
|
Reversible Competitive
|
Malonate
|
![]() |
0.03-0.06
0.07
0.06
|
hSR
mSR
rSR
|
(17, 51)
(22)
(17)
|
PLP targeting with reversible modifications
L- and D-amino acids were assayed as substrate
analogs and inhibitors (20, 50, 56).
Among them the stronger inhibitor was the NMDAR ligand glycine (56) that
exhibits a Ki of 366 and 1640 mM for hSR and mSR, respectively (51).
Asparagine, aspartate and oxolacetate also bind to the enzyme indicating that
neither an amine group nor a negative charge in the lateral chain are essential
for inhibition, and that compounds with bulkier groups can enter in SR active site
(56). Sulfhydryl containing amino acids, capable of reacting with PLP to form thiazolidine derivatives, are also SR
inhibitors (50, 56). Furthermore,
L-serine derivatives, such as L-serine O-sulfate,
were found to be effective uncompetitive SR inhibitors in the absence of
ATP (50). L-serine O-sulfate undergoes a b-elimination
reaction to sulfate, ammonia and pyruvate. This finding indicates that an a-aminoacrylate species is first formed, and successively
hydrolyzed. However, there is not yet an explanation for the observed
uncompetitive inhibition.
A
comprehensive analysis of compounds derived from serine and L-Ser-O-sulfate
led to the identification of the so far strongest mSR competitive inhibitor,
(2S,3R) L-erythro 3-hydroxyaspartate,
exhibiting a Ki of 43 mM
(22) (Table 2). Interestingly, the Ki for hSR is
almost four fold lower than for mSR, 11 mM
(51). This
compound is a four carbon amino acid analog that reacts with PLP, forming a
stable external aldimine. Accordingly to the docked conformation proposed by
Jiraskova-Vanickova et al. (12), one carboxylic group interacts with the
backbone nitrogen of Ser84 and His87, the other with Arg135 and Asn174, while
the hydroxyl moiety contacts the side chain of Ser84. SR is a close relative of
bacterial L-threo 3-hydroxyaspartate
dehydratases and L-threo-3-hydroxyaspartate
(2S,3S) is a substrate of the murine (22) and human enzymes (12)
that degrade it to oxaloacetate and ammonia. In contrast, D-threo-3-hydroxyaspartate
(2R,3R) is only a weak inhibitor (22).
Further
studies identified b-haloalanines as SR inhibitors (56), similarly to that observed for
other PLP-dependent enzymes belonging to fold type II, as tryptophan synthase
(14, 107) and O-acetylserine
sulfhydrylase (15, 108). This class of compounds reacts with PLP forming an
external aldimine that, given the favourable b-leaving group, undergoes a b-elimination reaction with
formation of an a-aminoacrylate derivative.
PLP targeting with irreversible modifications
The
first reported SR inhibitor of this class was aminooxoacetic acid, an aspecific
PLP-dependent enzyme inhibitor that reacts with the
Schiff base linkage between PLP and the enzyme, forming an aldoxime (50). A series of small
aliphatic hydroxamic and dihydroxamic acids were assayed as potential mSR
inhibitors (109). It was found that the
most effective were malonodihydroxamic
acid, succinodihydroxamic acid,
glutarodihydroxamic acid and L-aspartic acid β-hydroxamate. The Ki value calculated
for succinodihydroxamic acid was about 3 mM, the most potent SR inhibitor so far
identified (Table 2), whereas L-aspartic acid β-hydroxamate exhibited a Ki of about
100 μM. A clear disadvantage of
hydroxamic acid derivatives as SR inhibitors and potential drugs is their lack
of specificity, as they react with several PLP-dependent enzymes (109).
Cell migration assays
were used for the identification of SR inhibitors. It was found that phenazine,
phenazine methosulfate, and phenazine ethosulfate are effective inhibitors of
SR with IC50 of 3 and 5 μM for the methosulfate and ethosulfate
derivatives, respectively (66). However, no information on the assay conditions
were reported. Furthermore, much higher IC50 values for these
compounds were reported by Kovalinka and coworkers (12).
Active site targeting
This approach is aimed at
identifying compounds that do not react with PLP and exploit for binding their
tight functional and geometric complementarity with the active site. A recent
example is the interaction between OASS and pentapeptides mimicking the
C-terminal of serine acetyltransferase
(110, 111).
Several dicarboxylic acids
were tested as SR active site inhibitors.
It was found that malonate is the best binder with a Kdiss of
33 mM for
hSR (22) (Table 2). A slightly different value, 59 mM, was reported by Smith et
al. (17), for hSR, and 111 mM for a mutant hSR in which Cys2 and Cys6 were substituted with Asp residues. A larger discrepancy for the affinity of
malonate to mSR was found with a Ki of 71 mM (12, 22, 51), and a Ki of 568 mM to rSR and 1599 mM to
the mutant (17).
Malonate is a dicarboxylic acid that is orthosteric with L-serine. Its ability
to interact with SR is due to the small dimension and the carboxylate groups
that make strong ionic bonds with active site residues. As reported in Figure
2, one carboxylate of malonate interacts with the nitrogen backbone of Ser84,
Asn86 and His87, with the side chain of Ser83 and with one water molecule,
while the other forms a salt bridge type ionic interactions with Arg135, and
also contacts Ser84 and Ser242, in addition to a pair of water molecules. As
suggested, Arg135 might play a similar role to the arginine finger identified
in the active site of G-proteins, involved in the stabilization of the reaction
transition state and in the enhancement of the reaction rate (112, 113). On the
other side, the N-terminal loop, containing Ser83, Ser84, Gly85, Asn86 and
His87, is recognized as the asparagine loop, located in the PLP O3’ side, and
acting as the recognition site for the ligand in the closed enzyme conformation
(25). Interestingly, Arg135, which appears fundamental for the stabilization of
the enzyme-inhibitor complex is not conserved in other PLP-dependent enzymes
belonging to the fold-type II (18). A series of malonate derivatives were
synthesized and tested (12). No significant improvement with respect to the
parent molecule was observed. Among a series of dicarboxylic and
tricarboxylicacids meso-diaminosuccinic as well as meso-tartaric
exhibits a degree of rSR inhibition comparable with malonate.
A different approach towards the identification
of SR inhibitors was followed by Dixon et al (114). A 74088 tripeptide library
bound to individual beads was screened with a fluorescent-labelled human
SR. Sixty peptide-beads were
found to be positive and twenty five of them were randomly selected,
identified, synthesized and assayed for SR inhibition. It was found that most
of the identified peptides contained either histidine or phenylpropionic
moieties. The best binders exhibit Ki
values between 300 and 600 mM
with the inhibition mechanism involving a fast interaction with the enzyme
followed by a slow binding.
A still different method is pursued by Spyrakis et al.
(quoted in (1)). In silico screening
using both the open and the closed conformational states of SR, docking,
scoring and best hits experimental evaluation was aimed at identifying active
site SR binders. Studies are still ongoing (Spyrakis et al., unpublished
results).
Allosteric effectors and modulators of SR-protein interactions
No investigation has been so far carried out aimed at
developing compounds that target the multiple sites that allosterically
modulate SR activity, i.e. the ATP binding site with the reactive Cys113, the
cation binding site and the sites of the interaction between SR and effector
proteins, GRIP, PICK1, and GOLGA3. This is a fascinating and challenging
approach that might allow to generate effectors that either activate or inhibit
SR activity.
Post Comment
No comments