Proteins of glutamatergic NMDA receptor signaling pathways have been studied as targets for intervention in a variety of neuropathological conditions, including neurodegenerations, epilepsy, neuropathic pain, drug addiction, and schizophrenia. High activity NMDA-blocking agents have been designed to treat some of these disorders; however, their effect is often compromised by undesirable side effects. Therefore, alternative ways of modulating NMDA receptor function need to be sought after. The opening of the NMDA receptor ion channel requires occupation of two distinct binding sites, the glutamate site and the glycine site. It has been shown that D-serine, rather than glycine, can trigger the physiological NMDA receptor function. D-serine is a product of the activity of a specific enzyme, serine racemase (SR), which was identified a decade ago. SR has therefore emerged as a new potential target for the NMDA-receptor-based diseases. There is evidence linking increased levels of D-Ser to amyotrophic lateral sclerosis and Alzheimer's disease and decreased concentrations of D-serine to schizophrenia. SR is a pyridoxal-5'-phosphate dependent enzyme found in the cytosol of glial and neuronal cells. It is activated by ATP, divalent cations like Mg(2+) or Ca(2+), and reducing agents. This paper reviews the present literature on the activity and inhibition of mammalian SRs. It summarizes approaches that have been applied to design SR inhibitors and lists the known active compounds. Based on biochemical and docking analyses, i) we delineate for the first time the ATP binding site of human SR, and ii) we suggest possible mechanisms of action for the active compounds. In the end, we discuss the SR features that make the discovery of its inhibitors a challenging, yet very important, task of medicinal chemistry.
- MeSH
- adenosintrifosfát metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- lidé MeSH
- nemoci nervového systému farmakoterapie patofyziologie MeSH
- racemasy a epimerasy antagonisté a inhibitory metabolismus MeSH
- racionální návrh léčiv MeSH
- receptory N-methyl-D-aspartátu účinky léků metabolismus MeSH
- serin metabolismus MeSH
- systémy cílené aplikace léků MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme responsible for production of D-serine in the central nervous system. D-Serine acts as an endogenous coagonist of N-methyl-D-aspartate receptor ion channels. Increased levels of D-serine have been linked to amyotrophic lateral sclerosis and Alzheimer's disease, indicating that SR inhibitors might be useful tools for investigation or treatment of neurodegenerative diseases. However, despite hSR's promise as a therapeutic target, relatively few specific inhibitors have been identified, which is due in part to the lack of a three-dimensional structure of the enzyme. Here, we present a strategy for the generation and screening of random hSR mutants. From a library of randomly mutated hSR variants, twenty-seven soluble mutants were selected, expressed, and evaluated for enzymatic activity. Taking three carefully characterized mutants as an example, we show how this strategy can be used to pinpoint structurally and functionally important residues. In particular, we identify S84 and P111 as residues crucial for hSR activity and C217 and K221 as residues important for binding of the Mg2+ cofactor as well as for overall stability of the enzyme.
D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research.
- MeSH
- chromatografie afinitní MeSH
- cirkulární dichroismus MeSH
- financování organizované MeSH
- klonování DNA MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- racemasy a epimerasy MeSH
- rekombinantní proteiny MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- síran amonný MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
Serine racemase (SR) is an enzyme responsible for the biosynthesis of D-serine, the coagonist of the N-methyl-D-aspartate receptor, in the brain. Therefore, it has been suggested as a possible therapeutic target for the treatment of various neurodegenerative diseases. To develop a potent inhibitor of SR, a simple, sensitive, fast, and robust assay is needed. In this paper, a new CE method for the determination of D-serine is described. Serine enantiomers are resolved in the form of o-phthaldialdehyde (OPA)/2-mercaptoethanol (2-ME) derivatives in an alkaline BGE composed of 50 mM sodium tetraborate, pH 9.7, and containing 40 mM 2-hydroxypropyl-gamma-CD as a chiral selector. The problem of time-limited stability of OPA/2-ME derivatives has been overcome by employing in-capillary derivatization of the sample, i.e., the derivatization reaction was carried out directly in the separation capillary in the first phase of the CE run. UV-absorption detection at 230 nm allowed concentration detection limit of 3 microM. Baseline resolution of D- and L-serine derivatives was achieved in less than 10 min. This fact, together with the simple sample pretreatment, allowed application of the method to medium-throughput monitoring of SR activity, such as the screening of potential SR inhibitors. A good agreement was achieved between the developed CE method and the previously established HPLC method for determination of the inhibition constant, K(i), of a new SR inhibitor, L-erythro-3-hydroxyaspartate.
- MeSH
- elektroforéza kapilární metody MeSH
- financování organizované MeSH
- myši MeSH
- racemasy a epimerasy metabolismus MeSH
- serin analogy a deriváty analýza MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- validační studie MeSH
NMDA receptory jsou široce rozšířené, Ugandy otevírané a napěťově závislé iontové kanály, citlivé současně na L-glutamát a glycin. Tyto receptory jsou nezbytné pro normální funkci excitační glutamátergní neurotransmise. Komplexy receptor/iontový kanál jsou vysoce propustné pro Ca2+ a podléhají regulaci prostřednictvím řady faktorů. NMDA receptory charakterizuje tetramerní podjednotková stavba, na které se podílejí 3 skupiny podjednotek označované NRl, NR2 aNR3. Všechny funkční receptorové komplexy musí obsahovat dvojí zastoupení NRl podjednotky. Současná přítomnost NR2 a NR3 podjednotek nebo jejich sestřižených forem zásadním způsobem ovlivňuje funkční charakteristiky NMDA receptoru, jeho vazebné afinity pro oba agonisty (L-glutamát a glycin) a další modulátory. Změněná funkce NMDA receptoru je dávána do souvislosti s etiologií některých duševních nemocí, především jako součást „glutamátergní hypotézy schizofrenie". Změny ve funkčních vlastnostech NMDA receptoru jsou alespoň částečně podmíněny abnormální expresí genů pro jednotlivé proteinové podjednotky. Navíc se NMDA receptory významně podílejí na perinatálním neurotoxickém poškození mozku, které je integrální součástí „neurovývojové hypotézy schizofrenie". Přitom nedávné nálezy ukázaly, že D-serin jako další endogenní koagonista NMDA receptoru se v případe narušení své homeostázy významně spolupodílí na patofyziologii schizofrenie. Předpokládaná úloha NMDA receptoru v etiopatogenezi schizofrenie, vycházející z jejich zásadního významu pro vývoj mozku a rozvoj jeho funkcí, činí z těchto receptoru cílovou strukturu pro působení farmak. Proto pokračování studií za použití animálních modelů a kombinace genetických studií a zobrazovacích metod u pacientů trpících schizofrenií může významně přispět při vyhledávání a ověřování nových skupin neuroleptik.
NMDA receptors are widely distributed, rapidly responding ligand-gated and voltage-dependent cationic channels sensitive to L-g lutamate and glycine. The NMDA receptors are essential for the normal function of the excitatory glutamatergic neurotransmission. The re ceptor/channel complexes are highly permeable to Ca 2+ and are subject to regulation by a variety of factors. Usually present as tetramers of protein subunits, NMDA receptors are formed by NR1, NR2 and/or NR3 subunits; however, all functional complexes must include NR1 subunits. The pre sence of NR2 and NR3 subunits and/or the nature of their splice variants may influence a range of NMDA receptor characteristics such as binding affinities for glutamate and glycine. Altered function of NMDA receptors has been linked to the etiology of mental disease, par ticularly as a part of the “glutamatergic hypothesis of schizophrenia“. The functional changes in NMDA receptors could result from genetical ly determined variations in individual protein subunits or from their abnormal expression. In addition, NMDA receptors can mediate perinatal neurotoxic insults such as those postulated in the “developmental hypothesis of schizophrenia“. D-Serine is another endogenous co-agonist at NMDA receptors and recent evidence suggests that disturbances in D-serine homeostasis may also contribute to mechanisms of schizophr enia. Putative involvement of NMDA receptors in schizophrenia, their crucial role in the normal brain function and the complex but ex - tensively studied nature of their regulation makes NMDA receptors promising targets for drugs. Additional investigations using either animal models or combination of genetic studies and imaging techniques in human patients in vivo would greatly aid the developm ent of novel classes of neuroleptics.
- MeSH
- antipsychotika terapeutické užití MeSH
- finanční podpora výzkumu jako téma MeSH
- glutamáty biosyntéza farmakokinetika MeSH
- glycin farmakokinetika MeSH
- lidé MeSH
- modely u zvířat MeSH
- receptory N-methyl-D-aspartátu fyziologie terapeutické užití MeSH
- schizofrenie farmakoterapie MeSH
- serin agonisté MeSH
- Check Tag
- lidé MeSH
Mouse serine racemase (mSR) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the biosynthesis of the N-methyl-d-aspartate receptor coagonist d-serine in the brain. Furthermore, mSR catalyzes beta-elimination of serine and l-serine-O-sulfate into pyruvate. The biological significance of this beta-elimination activity and the factors influencing mSR substrate and reaction specificity, which are crucial for prospective inhibitor design, are poorly understood. Using a bacterial expression system and ATP-agarose affinity chromatography, we have generated a pure and active recombinant mSR and investigated its substrate and reaction specificity in vitro by analyzing a systematic series of compounds derived from l-Ser and l-serine-O-sulfate. The analysis revealed several competitive inhibitors of serine racemization including glycine (K(I) = 1.63 mM), several dicarboxylic acids including malonate (K(I) = 0.077 mM), and l-erythro-3-hydroxyaspartate (K(I) = 0.049 mM). The latter compound represents the most effective inhibitor of SR reported to date. A simple inversion of the beta-carbon configuration of the compound yields an excellent beta-elimination substrate l-threo-3-hydroxyaspartate. Inhibition analysis indicates that racemization and beta-elimination activities of mSR reside at the same active site. While the racemization activity is specific to serine, the beta-elimination activity has a broader specificity for l-amino acids with a suitable leaving group at the beta-carbon and optimal spatial orientation of the alpha-carboxyl and leaving groups. The possible implications of our observations for inhibitor design, regulation of activity, and function of mSR are discussed.
- MeSH
- aminokyseliny metabolismus MeSH
- financování organizované MeSH
- inhibitory enzymů MeSH
- kinetika MeSH
- klonování DNA MeSH
- kyseliny dikarboxylové farmakologie MeSH
- lyasy metabolismus MeSH
- myši MeSH
- racemasy a epimerasy antagonisté a inhibitory genetika metabolismus MeSH
- serin metabolismus MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- biopotraviny MeSH
- fyziologie výživy MeSH
- kardiovaskulární nemoci mortalita MeSH
- lidé MeSH
- lipidy krev MeSH
- mastné kyseliny krev MeSH
- západní svět MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- směrnice MeSH
