Thomas Johannes Woltering

Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimers disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Characterization of [3H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using Semliki Forest virus vectors

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

β-Secretase (BACE1) Inhibitors with High in Vivo Efficacy Suitable for Clinical Evaluation in Alzheimer’s Disease

An extensive fluorine scan of 1,3-oxazines revealed the power of fluorine(s) to lower the pKa and thereby dramatically change the pharmacological profile of this class of BACE1 inhibitors. The CF3 substituted oxazine 89, a potent and highly brain penetrant BACE1 inhibitor, was able to reduce significantly CSF Aβ40 and 42 in rats at oral doses as low as 1 mg/kg. The effect was long lasting, showing a significant reduction of Aβ40 and 42 even after 24 h. In contrast to 89, compound 1b lacking the CF3 group was virtually inactive in vivo.

Structure-activity relationships of substituted 5H-thiazolo[3,2-a]pyrimidines as group 2 metabotropic glutamate receptor antagonists.

A series of 5H-thiazolo[3,2-a]pyrimidine derivatives 1 was studied with respect to the inhibition of 1S,3R-ACPD (10 microM)-stimulated GTP gamma35S binding on rat mGlu2 receptor transfected cell membranes. The influence of substituents at position 6 and 7 as well as the substitution pattern of the two phenyl-rings in position 2 and 5 on the activity is discussed.

Characterization of (2S,2′R,3′R)‐2‐(2′,3′‐[3H]‐Dicarboxycyclopropyl)glycine Binding in Rat Brain

Abstract: [(2S,2′R,3′R)‐2‐(2′,3′‐[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a KD value of 180 ± 33 nM and a Bmax of 780 ± 70 fmol/mg of protein. The nonspecific binding, measured using 100 µM LY354740, was <30%. NMDA, AMPA, kainate, l(−)‐threo‐3‐hydroxyaspartic acid, and (S)‐3,5‐dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration‐dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1′S,2′S)‐2‐(2‐carboxycyclopropyl)glycine > (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid > (2S,1′S,2′S)‐2‐methyl‐2‐(2‐carboxycyclopropyl)glycine > l‐glutamate = ibotenate > quisqualate > (RS)‐α‐methyl‐4‐phosphonophenylglycine = l(+)‐2‐amino‐3‐phosphonopropionic acid > (S)‐α‐methyl‐4‐carboxyphenylglycine > (2S)‐α‐ethylglutamic acid > l(+)‐2‐amino‐4‐phosphonobutyric acid. N‐Acetyl‐l‐aspartyl‐l‐glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 µM for the high‐affinity component. The binding was also affected by GTPγS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre‐ and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPγS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.

Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators

BACKGROUND AND PURPOSE Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu2) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu2/3 receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu2/3 NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu2 is characterized.

A Real-World Perspective on Molecular Design.

We present a series of small molecule drug discovery case studies where computational methods were prospectively employed to impact Roche research projects, with the aim of highlighting those methods that provide real added value. Our brief accounts encompass a broad range of methods and techniques applied to a variety of enzymes and receptors. Most of these are based on judicious application of knowledge about molecular conformations and interactions: filling of lipophilic pockets to gain affinity or selectivity, addition of polar substituents, scaffold hopping, transfer of SAR, conformation analysis, and molecular overlays. A case study of sequence-driven focused screening is presented to illustrate how appropriate preprocessing of information enables effective exploitation of prior knowledge. We conclude that qualitative statements enabling chemists to focus on promising regions of chemical space are often more impactful than quantitative prediction.

Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives : Part 4. In vivo active potent and selective non-competitive metabotropic glutamate receptor 2/3 antagonists

This study completes a series of papers devoted to the characterization of the non-competitive mGluR2/3 antagonist properties of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives with particular emphasis on derivatizations compatible with brain penetration and in vivo activity. Especially the compounds bearing a para-pyridine consistently showed in vivo activity in rat behavioral models after oral administration, for example, blockade of the mGluR2/3 agonist LY354740-induced hypoactivity and improvement of a working memory deficit induced either by LY354740 or scopolamine in the delayed match to position task (DMTP). Moreover, combination studies with a cholinesterase inhibitor show apparent synergistic effects on working memory impairment induced by scopolamine.

BACE1 inhibitors: a head group scan on a series of amides.

A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimers disease treatment.

Alkyl diphenylacetyl, 9H-xanthene- and 9H-thioxanthene-carbonyl carbamates as positive allosteric modulators of mGlu1 receptors.

Starting from the random-screening hit 1a, a series of alkyl diphenylacetyl, 9H-xanthene- and 9H-thioxanthene-carbonyl carbamates 1 has been prepared. These derivatives turned out to be selective positive allosteric modulators of mGlu1 receptors. These compounds do not directly activate mGlu1 receptors but markedly potentiate agonist stimulated responses, increasing potency and maximum efficacy.