The Development of a Regulator of Human Serine Racemase for N-Methyl-D-aspartate Function

Abstract

It is crucial to regulate N-methyl-D-aspartate (NMDA) function bivalently depending on the central nervous system (CNS) conditions. CNS disorders with NMDA hyperfunction are involved in the pathogenesis of neurotoxic and/or neurodegenerative disorders with elevated D-serine, one of the NMDA receptor co-agonists. On the contrary, NMDA-enhancing agents have been demonstrated to improve psychotic symptoms and cognition in CNS disorders with NMDA hypofunction. Serine racemase (SR), the enzyme regulating both D- and L-serine levels through both racemization (catalysis from L-serine to D-serine) and β-elimination (degradation of both D- and L-serine), emerges as a promising target for bidirectional regulation of NMDA function. In this study, we explored using dimethyl malonate (DMM), a pro-drug of the SR inhibitor malonate, to modulate NMDA activity in C57BL/6J male mice via intravenous administration. Unexpectedly, 400 mg/kg DMM significantly elevated, rather than decreased (as a racemization inhibitor), D-serine levels in the cerebral cortex and plasma. This outcome prompted us to investigate the regulatory effects of dodecagalloyl-α-D-xylose (α12G), a synthesized tannic acid analog, on SR activity. Our findings showed that α12G enhanced the racemization activity of human SR by about 8-fold. The simulated and fluorescent assay of binding affinity suggested a noncooperative binding close to the catalytic residues, Lys56 and Ser84. Moreover, α12G treatment can improve behaviors associated with major CNS disorders with NMDA hypofunction including hyperactivity, prepulse inhibition deficit, and memory impairment in animal models of positive symptoms and cognitive impairment of psychosis. In sum, our findings suggested α12G is a potential therapeutic for treating CNS disorders with NMDA hypofunction.

Keywords: NMDA; enzyme activator; malonate; racemization; serine racemase; tannic acid; β-elimination.

Scheme 1

Synthesis steps of α12G ((2R,3R,4S,5R)-tetrahydro-2H-pyran-2,3,4,5-tetrayl tetrakis(3-((3,4-dihydroxy-5-((3,4,5-trihydroxybenzoyl)oxy)benzoyl)ox)-4,5-dihydroxybenzoate)).

Figure 1

In vivo regulation of SR in C57BL/6J mice by dimethyl malonate (DMM) administration. The levels of D-serine (A) and L-serine (B) in the cerebral cortex and plasma 30 min after a single intravenous administration of DMM. Data are presented as mean ± SEM and were analyzed with Student’s t-test in comparison with the vehicle group. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Purification and identification of recombinant human serine racemase (hSR). Size-exclusion chromatography of hSR showing a symmetric peak corresponding to a theoretical molecular mass of ~74 kDa, which was about double its calculated molecular mass of 37 kDa. It suggested that hSR was a homodimer in solution in 20 mM Tris-HCl, 100 mM NaCl, 2 mM MgCl_2, 50 µM PLP, and 5 mM DTT at pH 8.0. SDS-PAGE gel analysis of purified hSR also showed that hSR has a molecular mass of 37 kDa.

Figure 3

Enzymatic characterization of hSR and the regulatory/inhibitory effects of α12G on hSR and D-amino acid oxidase (DAAO). (A) Within the physiological range of ATP concentration, 0.25 mM ATP promotes racemization of hSR the best. (B) The IC₅₀ of α12G on DAAO was determined to be 0.16 µM. (C) The EC₁₀₀ of α12G on hSR was 31.25 µM, and the EC₅₀ of α12G on hSR was determined to be 8.82 µM (favoring racemization) and 220.80 µM (progressively inhibiting racemization). The dotted line indicates the basal level of hSR racemization (without compound) The black line indicates the half-maximal effective concentration of α12G on hSR.

Figure 4

The binding site and affinity of α12G on hSR. (A) Interactions between active site residues of hSR with α12G. Active residues Lys 56 + pyridoxal 5′-phosphate (PLP) are shown as orange sticks, and Ser 84 is shown as yellow sticks. The H-bonds between molecules are presented as green lines. Distances of hydrogen bonds are as follows: Asn86 (2.400 Å), Lys114 (2.647 Å), Pro233 (2.694 Å), Lys241 (2.019 Å), and Lys241 (2.639 Å). Grid score =−164.83 kcal/mol. (B) The binding affinity of α12G on hSR. The dissociation constant (Kd) = 0.24 µM, the Hill coefficient (n) = 1.28, and the Bmax = 96.86 fmol/mg based on the Hill slope model. R² = 0.99.

Figure 5

Pharmacodynamic findings of α12G treatment in MK-801-treated mice. (A) In open field test, (B) prepulse inhibition (PPI) test, and (C) novel object recognition test, α12G could alleviate the MK-801-induced hyperactivity, PPI deficit, and memory impairment with optimal doses at 1 or 10 mg. Data are presented as mean ± SEM and were analyzed with Student’s t-test and/or Dunnett’s multiple comparisons test. * denotes a significant difference compared with the MK-801 group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. # denotes a significant difference compared with dosing groups. ^#, p < 0.05.

Figure 5

Pharmacodynamic findings of α12G treatment in MK-801-treated mice. (A) In open field test, (B) prepulse inhibition (PPI) test, and (C) novel object recognition test, α12G could alleviate the MK-801-induced hyperactivity, PPI deficit, and memory impairment with optimal doses at 1 or 10 mg. Data are presented as mean ± SEM and were analyzed with Student’s t-test and/or Dunnett’s multiple comparisons test. * denotes a significant difference compared with the MK-801 group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. # denotes a significant difference compared with dosing groups. ^#, p < 0.05.