S29434, a Quinone Reductase 2 Inhibitor: Main Biochemical and Cellular Characterization.

Quinone reductase 2 (QR2, E.C. 1.10.5.1) is an enzyme with a feature that has attracted attention for several decades: in standard conditions, instead of recognizing NAD(P)H as an electron donor, it recognizes putative metabolites of NADH, such as N-methyl- and N-ribosyl-dihydronicotinamide. QR2 has been particularly associated with reactive oxygen species and memory, strongly suggesting a link among QR2 (as a possible key element in pro-oxidation), autophagy, and neurodegeneration. In molecular and cellular pharmacology, understanding physiopathological associations can be difficult because of a lack of specific and powerful tools. Here, we present a thorough description of the potent, nanomolar inhibitor [2-(2-methoxy-5H-1,4b,9-triaza(indeno[2,1-a]inden-10-yl)ethyl]-2-furamide (S29434 or NMDPEF; IC50 = 5-16 nM) of QR2 at different organizational levels. We provide full detailed syntheses, describe its cocrystallization with and behavior at QR2 on a millisecond timeline, show that it penetrates cell membranes and inhibits QR2-mediated reactive oxygen species (ROS) production within the 100 nM range, and describe its actions in several in vivo models and lack of actions in various ROS-producing systems. The inhibitor is fairly stable in vivo, penetrates cells, specifically inhibits QR2, and shows activities that suggest a key role for this enzyme in different pathologic conditions, including neurodegenerative diseases.

A novel family of potent negative allosteric modulators of group II metabotropic glutamate receptors.

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.