SummaryType 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

No abstract

GABA A receptors composed of ␣ 1 , ␤ 2 , ␥ 1 subunits are expressed in only a few areas of the brain and thus represent interesting drug targets. The pharmacological properties of this receptor subtype, however, are largely unknown. In the present study, we expressed ␣ 1 ␤ 2 ␥ 1 -GABA A receptors in Xenopus laevis oocytes and analyzed their modulation by 21 ligands from 12 structural classes making use of the two-microelectrode voltage-clamp method and a fast perfusion system. Modulation of GABA-induced chloride currents (I GABA ) was studied at GABA concentrations eliciting 5 to 10% of the maximal response. Triazolam, clotiazepam, midazolam, 2-(4-methoxyphenyl)-2, 3,5,6,7,8,9,10-octahydro-cyclohepta-(b) (CGS 9896), diazepam, zolpidem, and bretazenil at 1 M concentrations were able to significantly (Ͼ20%) enhance I GABA in ␣ 1 ␤ 2 ␥ 1 receptors. Methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate, 3-methyl-6-[3-trifluoromethyl-phenyl]-1,2,4-triazolo [4,3-b]pyridazine (Cl 218,872), clobazam, flumazenil,, and 6-ethyl-7-methoxy-5-methylimidazo[1,2-a]pyrimidin-2-yl)phenylmethanone (Ru 32698) (1 M each) had no significant effect, and flunitrazepam and 2-phenyl-4-(4-ethyl-piperidinyl)-quinoline (PK 8165) inhibited I GABA . The most potent compounds triazolam, clotiazepam, midazolam, and CGS 20625 were investigated in more detail on ␣ 1 ␤ 2 ␥ 1 and ␣ 1 ␤ 2 ␥ 2S receptors. The potency and efficiency of these compounds for modulating I GABA was smaller for ␣ 1 ␤ 2 ␥ 1 than for ␣ 1 ␤ 2 ␥ 2S receptors, and their effects on ␣ 1 ␤ 2 ␥ 1 could not be blocked by flumazenil. CGS 20625 displayed the highest efficiency by enhancing at 100 M I GABA (␣ 1 ␤ 2 ␥ 2 ) by 775 Ϯ 17% versus 526 Ϯ 14% I GABA (␣ 1 ␤ 2 ␥ 1 ) and 157 Ϯ 17% I GABA (␣ 1 ␤ 2 ) (p Ͻ 0.05). These data provide new insight into the pharmacological properties of GABA A receptors containing ␥ 1 subunits and may aid in the design of specific ligands for this receptor subtype.GABA is the principal inhibitory neurotransmitter in the mammalian brain. It mediates fast synaptic inhibition by interaction with the GABA A receptor. GABA A receptors are ligandgated ion channels that are modulated by a large number of clinically relevant drugs such as benzodiazepines (BZs), barbiturates, neurosteroids, and anesthetics (Sieghart, 1995). They are assembled from individual subunits forming a pentameric structure. Nineteen isoforms of mammalian GABA A receptor subunits have been cloned: ␣ 1-6 , ␤ 1-3 , ␥ 1-3 , ␦, ⑀, p, 1-3 , and (Barnard et al., 1998;Simon et al., 2004). The major receptor subtype of the GABA A receptor in adults consists of ␣ 1 , ␤ 2 , and ␥ 2 subunits, and the most likely stoichiometry is two ␣ subunits, two ␤ subunits, and one ␥ subunit (Sieghart and Sperk, 2002). CGS 20625, 2-(4-methoxyphenyl)-2,3,5,6,7,8,9,10-octahydro-cyclohepta-(b) ABBREVIATIONS:

A plant extract library was screened for GABA(A) receptor activity making use of a two-microelectrode voltage clamp assay on Xenopus laevis oocytes. An ethyl acetate extract of black pepper fruits [Piper nigrum L. (Piperaceae) 100 microg/mL] potentiated GABA-induced chloride currents through GABA(A) receptors (composed of alpha(1), beta(2), and gamma(2S) subunits) by 169.1 +/- 2.4%. With the aid of an HPLC-based activity profiling approach, piperine (5) was identified as the main active compound, together with 12 structurally related less active or inactive piperamides (1-4, 6-13). Identification was achieved by on-line high-resolution mass spectrometry and off-line microprobe 1D and 2D NMR spectroscopy, using only milligram amounts of extract. Compound 5 induced a maximum potentiation of the chloride currents by 301.9 +/- 26.5% with an EC(50) of 52.4 +/- 9.4 microM. A comparison of the modulatory activity of 5 and other naturally occurring piperamides enabled insights into structural features critical for GABA(A) receptor modulation. The stimulation of chloride currents through GABA(A) receptors by compound 5 was not antagonized by flumazenil (10 microM). These data show that piperine (5) represents a new scaffold of positive allosteric GABA(A) receptor modulators targeting a benzodiazepine-independent binding site.