Accelerated identification of serine racemase inhibitor from Centella asiatica

Abstract

Serine racemase (SR) converts the free form of L-serine into D-serine (DS) in the mammalian brain. The DS functions as a co-agonist of N-methyl D-aspartate (NMDA) receptor. The over-activation of NMDA receptor leads to many neurological disorders like stroke, amyotrophic lateral sclerosis, Alzheimer's disease and an effective inhibitor of SR could be a corrective method for the receptor over-activation. We report for the first time here a rapid way of purifying and identifying an inhibitor from medicinal plants known to have the neuro-protective effect. We have purified SR inhibitor from the methanolic extract of Centella asiatica by affinity method. High resolution mass spectrometry and infrared spectroscopy were used to identify the ligand to be madecassoside. We have shown the madecassoside binding in silico and its inhibition of recombinant human serine racemase in vitro and ex vivo. D-serine (DS), an endogenous D-amino acid, is produced by serine racemase (SR) in glial cells and neurons 1 . SR belongs to the class of pyridoxal-5-phosphate (PLP)-dependent enzymes and it converts the free form of L-serine into DS. The free DS functions as a co-agonist of N-methyl-D aspartate (NMDA) receptor in the brain 1 , retina 2 , keratinocytes 3 and in chondrocytes 4 . Incorporation of D-serine causes significant changes in the functional properties of many proteins that are detrimental to humans. Racemization of serine residues is known to occur in beta-amyloid senile plaques of Alzheimer's disease (AD) 5 , human lens protein αcrystallin 6 , human myelin basic protein 7 , osteoarthritic articular and meniscal cartilages 8 , the funnel-web spider toxin 9 , egg ovalbumin 10 and in skin 3 . The conversion of L-serine to D-serine was conclusively established by the discovery of SR in the rat brain by Wolosker et al. in 1999. Active SR is a dimer and needs divalent cations to become functionally active 11 . In addition to serine isomerization, SR also performs α, β-elimination reaction with both L-serine and D-serine to produce pyruvate and ammonia 12 . D-serine is also catabolized by the D-amino acid oxidase (DAAO) 13 . Thus, the D-serine levels are controlled by two enzymes: SR and DAAO.The NMDA receptors are the major ionotropic glutamate receptors in the central nervous system. They control the flow of Ca 2+ ions into the cell, which is essential for the synaptic plasticity, synapse formation, rhythmogenesis, and nerve excitation 14 . One of the main features of the NMDA receptor is that its activation requires simultaneous binding of its agonist glutamate and co-agonist glycine. However, DS also binds to NMDA receptor at the glycine site with three-fold higher affinity than glycine 15 . The efficacy of DS to activate the NMDA receptor is 10 times higher in comparison to glycine 16 . Hyper activation of the NMDA receptor takes place in excess of DS 17 . The hyperactivity of the NMDA receptor is involved in many neurological diseases such as Alzheimer's disease 18,19 amyotrophic lateral sclerosis 20 , epilepsy 21 , neuropa...

Tyrosine 121 moves revealing a ligandable pocket that couples catalysis to ATP-binding in serine racemase

“…It would be interesting to determine whether such compounds utilize the Tyr 121 pocket. In 2020, it was reported that the natural product madecassoside is an inhibitor of SR 49 , although a supporting co-crystal structure has not yet been solved. While malonate and related acids (Supplementary Table 6 ) remain useful tool compounds, they are likely too polar to be developed as drugs.…”

“…A 2017 paper published an inhibitor of mouse SR, 13 J, that could suppress neuronal overactivation in vivo 39 and produced better IC 50 values (140 µM) than malonate (770 µM). In 2020, a novel plant derivative SR inhibitor, madecassoside (IC 50 26 µM), was reported and described as a far better inhibitor than malonate (IC 50 449 µM) 49 . IC 50 values are known to be dependent on conditions under which they are measured.…”

Tyrosine 121 moves revealing a druggable pocket that couples catalysis to ATP-binding in serine racemase

“…It would be interesting to determine whether such compounds utilize the Tyr 121 pocket. In 2020, it was reported that the natural product madecassoside is an inhibitor of SR 55 , although a supporting co-crystal structure has not yet been solved. While malonate and related acids (Supplementary Table 6) remain useful tool compounds, they are likely too polar to be developed as drugs.…”

“…A 2017 paper published an inhibitor of mouse SR, 13J, that could suppress neuronal overactivation in vivo 38 and produced better IC 50 values (140 μM) than malonate (770 μM). In 2020, a novel plant derivative SR inhibitor, madecassoside (IC 50 26 μM), was reported and described as a far better inhibitor than malonate (IC 50 449 μM) 48 . IC 50 values are known to be dependent on conditions under which they are measured.…”

Commentary on Urinary l-erythro-β-hydroxyasparagine: a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase

“…Taken together the degradation of l -β-EHAsn by Dsd1p and its inhibitory effects on serine racemase, results suggest the involvement of l -β-EHAsn in metabolic regulation of pathways related d -ser. Unfortunately, it has not been shown that l -β-EHAsn inhibits human serine racemase, a more relevant model; if this inhibition is proven true, modified versions of l -β-EHAsn could serve as new start points for drug discovery programs targeting human serine racemase [ 7 ].…”