Programme Contents

Discovery and characterization of potent biphasic α5GABA_A receptor modulators

Therapeutic drugs targeting GABA type-A chloride channel receptors (GABA_ARs) are used as anxiolytics, sedatives, and antiepileptics. These drugs are allosteric positive modulators of GABA_ARs that enhance GABA activity. As these drugs non-selectively target many GABA_AR subtypes causing unwanted side effects, subunit-specific drugs are now desirable. Positive modulators selective for α5-containing GABA_ARs (α5GABA_ARs) have shown promising results in treating cognitive symptoms of schizophrenia and age-related dementia, whereas α5GABA_AR negative modulators have proven worth as cognition enhancers, in post-stroke recovery and prevention of general anesthetic-induced amnesia, without causing sedation and anxiety. The aim of this project is to discover and characterize novel α5GABA_AR-selective compounds.

This was accomplished by screening a library of synthetic compounds on HEK293 cells, transfected with α5β3γ2L GABA_ARs and an anion-quenchable yellow fluorescent protein (YFP), using a high-throughput fluorescence-based anion influx assay. Modulation on other GABA_AR subtypes (α1β2γ2L, α5β2γ2L) and drug binding sites were explored via functional studies using two-electrode voltage clamp electrophysiology (TEVC). To prepare for TEVC, Xenopus borealis frogs were anesthetized with MS-222 and surgically incised to obtain the oocytes, which were digested and injected with GABA_AR mRNAs.

TEVC recordings revealed that isomeric compounds RM 68, RM 69 and RM 70 are potent GABA_AR modulators. These isomers, except RM 69, demonstrated biphasic modulation, selectively potentiating α5GABA_ARs in the nanomolar (sometimes picomolar) range, but inhibiting GABA_ARs non-selectively at higher micromolar concentrations. Removing mercaptoacetaldehyde from RM 68 to give RM 96 eliminated its α5GAB_AAR selectivity. Flumazenil managed to neutralize the potentiation of GABA responses by the 4 compounds, suggesting that the potentiating effect of these compounds was modulated via the benzodiazepine site.

RM compounds can be potential leads to design novel subtype specific drugs for α5-related disorders and to study the pharmacology of α5GABA_ARs.