Identification and characterisation of a novel inhibitor of serine racemase through a fragment-based drug discovery strategy

Koulouris, Chloe Rose (2018) Identification and characterisation of a novel inhibitor of serine racemase through a fragment-based drug discovery strategy. Doctoral thesis (PhD), University of Sussex.

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The N-methyl-D-aspartate (NMDA) receptors (NMDAR) are a subtype of ionotropic glutamate receptors that are highly expressed in the central nervous system (CNS) and are involved in the excitatory synaptic transmission and synaptic plasticity that underpin many critical CNS functions. NMDAR dysfunction has been implicated in Alzheimer’s disease, neuropathic pain, schizophrenia, and depression, among others. Most non-selective NMDAR antagonists (e.g. ketamine) have undesirable side-effects that restrict their clinical utility for relieving symptoms of neuropathic pain and treatment-resistant depression, but indirect modulators of NMDAR function offer the potential to have reduced side-effects relative to non-selective antagonists. One approach is to inhibit the production of the NMDAR co-agonist, D-serine, which is produced endogenously by conversion of L-serine to D-serine by the enzyme serine racemase (SR). Inhibitors of SR that reduce the production of D-serine may therefore have therapeutic benefits in disorders associated with NMDAR hyperfunction. In this thesis, human SR(hSR) was expressed in a bacterial host and isolated to a high degree of purity (>90%). The crystal structure of SR was solved in-house using the purified protein, to a resolution of 2.2Å (in complex with malonate) and 1.9Å (holoenzyme). To measure SR activity, a coupled biochemical assay was optimised, in which produced D-serine is degraded by D-amino acid oxidase (DAO) and the resulting H2O2 is quantified by a chemiluminescence reaction with HRP and luminol to produce light. Assay parameters such as Kmand IC50 were determined, and following further optimisation to enable medium-through put screening, a single-point screen of 3000 fragments was performed to identify novel inhibitory hit matter. A final list of 61 specific hits was collated of fragments that did not exhibit nonspecific inhibition in a DAO counter screen, and demonstrated IC50 values against SR approximately 1mM or below. These specific hits were characterised with biophysical methods adapted for SR (thermal shift, microscale thermophoresis) to determine binding affinities and structure stabilisation. Finally, via crystal soaking, the crystal structure of SR bound to an inhibitory fragment (KD=960μM) was revealed to a resolution of 1.8Å.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry > QD0431 Proteins, peptides, amino acids, etc.
R Medicine > RM Therapeutics. Pharmacology > RM0300 Drugs and their actions
Depositing User: Library Cataloguing
Date Deposited: 26 Apr 2018 09:15
Last Modified: 28 May 2021 09:10

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