Rene Imhof

From moclobemide to Ro 19-6327 and Ro 41-1049: the development of a new class of reversible, selective MAO-A and MAO-B inhibitors

This study describes the serendipitous discovery of moclobemide, a short-acting MAO-A inhibitor which is in an advanced stage of clinical development as an antidepressant. The short duration of action of this MAO inhibitor containing a morpholine ring moiety is due to the complete reversibility (probably by metabolism of the inhibitory molecular species) of MAO-A inhibition. Since moclobemide is much more effective in vivo than expected from its in vitro activity, investigations to identify a possible metabolite(s) more active as MAO-A inhibitor than the parent compound were carried out. The study of the MAO inhibitory characteristics of several known and putative moclobemide metabolites did not allow the identification of a potent MAO-A inhibitor but led to the discovery of Ro 16-6491, a potent MAO-B inhibitor of novel chemical structure. Systematic chemical modification of the aromatic ring system of Ro 16-6491 finally provided Ro 19-6327 and Ro 41-1049 which are highly selective and reversible inhibitors of MAO-B and MAO-A, respectively. Tritiated derivatives of Ro 19-6327 and Ro 41-1049 were used in binding studies to elucidate their mechanisms of action and to study their cellular distribution by quantitative enzyme radioautography.

[3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

This study demonstrated the existence of specific binding sites for [3H]Ro 19-6327 in human platelet membranes. This compound is a novel, time-dependent inhibitor of monoamine oxidase type B (MAO-B) and is structurally closely related to [3H]Ro 16-6491. The density of the sites labelled with high affinity by [3H]Ro 19-6327 was similar to that observed in previous studies with [3H]Ro 16-6491 as ligand. Binding was reversible at 20 degrees C and showed a relatively slow dissociation (t1/2 = 220 min). The dissociation rate was markedly decreased (t1/2 = greater than 24h) at 0 degrees C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [3H]Ro 19-6327. Like [3H]Ro 16-6491, [3H]Ro 19-6327 is recognized as a substrate by MAO-B, being eventually deaminated by the enzyme. Since the deaminated aldehyde derivative of Ro 19-6327 did not inhibit MAO-B, a still unidentified reversible adduct, formed at the MAO-B active site, might explain the high potency and selectivity of [3H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH3CN and acetic acid (to pH 4.5) caused the irreversible incorporation of the radioactivity into a single polypeptide as shown by SDS-PAGE analysis. This polypeptide had a molecular weight identical to that of the MAO-B subunit, i.e. 58,000. The presence of unlabelled MAO-B inhibitors in the incubation mixture prevented the covalent incorporation of [3H]Ro 19-6327. The irreversible MAO-B inhibitor, [3H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [3H]Ro 19-6327.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of [3H]Ro 16-6491, a Reversible Inhibitor of Monoamine Oxidase Type B, to Human Brain Mitochondria and Platelet Membranes

Abstract: The reversible inhibitor of monoamine oxidase type B (MAO‐B) [3H]Ro 16‐6491 binds specifically and with high affinity to a single population of binding sites in human frontal cortex crude mitochondria and platelet membranes. In both tissues binding equilibrium was reached after l h incubation at 20°C. Dissociation of bound radioactivity was relatively fast at 20°C (t1/2= 90–120 min) whereas at 0°C [3H]Ro 16–6491 showed the characteristics of a slowly dissociating ligand. Inhibitors and substrates of MAO‐B inhibited binding of [3H]Ro 16‐6491, whereas MAO‐A blockers were much less potent. Ro 16‐6491 was also a substrate for MAO‐B and a stable unidentified intermediate of the oxidation of Ro 16‐6491 possessing high affinity for the enzyme may account for the marked MAO‐B inhibitory effect of the drug. According to this hypothesis Ro 16‐6491 would behave as a mechanism‐based reversible inhibitor. In conclusion, [3H]Ro 16‐6491 binds selectively to MAO‐B and represents an excellent new radioligand probe for studying the regional tissue distribution of this enzyme in normal and pathological conditions.

[3H]Ro 16–6491, a Selective Probe for Affinity Labelling of Monoamine Oxidase Type B in Human Brain and Platelet Membranes

Abstract: [3H]Ro 16–6491 [N‐(2‐aminoethyl)‐p‐chloroben‐zamide HCl], a reversible “mechanism‐based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO‐B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO‐B inhibitor /‐deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO‐B, e.g., by /‐deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO‐A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO‐B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO‐B activity whereas MAO‐A activity was unaffected. Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO‐B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO‐B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491‐MAO‐B complex results in the formation of a stable adduct with the amino acid chain of the MAO‐B or with its prosthetic group, FAD, remains to be elucidated.

An efficient resolution of 3-PPP and assignment of the absolute configuration.

Abstract ( + )-3-PPP has been resolved by means of (+)- and (-)- 2,2′-(1,1′-binaphthyl)phosphoric acid (BNPPA) in high optical purity; (+)-3-PPP has been assigned the R configuraiton on the basis of a single crystal X-ray analysis.

Mode of action and characteristics of monoamine oxidase-A inhibition by moclobemide

The mode of interaction of the reversible monoamine oxidase-A (MAO-A) inhibitor moclobemide with the enzyme was investigated. The inhibition of rat brain or human placenta MAO-A by moclobemide showed an initial competitive phase, with a relatively low affinity (KI=0.2–0.4 mM). However, the potency of the inhibitor was increased with incubation time. The time-dependent component of the association of moclobemide with MAO-A followed pseudo-first order kinetics. In contrast to mechanism-based inhibitors, no indication for adduct or product formation was detected after incubation of moclobemide with the enzyme. Even though some aspects of the moclobemide interaction with MAO-A are still not completely elucidated, this compound seems to have the characteristics of a slow-binding inhibitor.

Characterization of [3H]Ro 16-6491 binding to digitonin solubilized monoamine oxidase-B and purification of the enzyme from human platelets by affinity chromatography

In the present paper we describe the use of digitonin as a suitable detergent for the effective solubilization of MAO-B from human platelets and frontal cortex membrane preparations and the characteristics of the solubilized enzyme using the selective MAO-B inhibitor [ 3 H] Ro 16-6491 [N-(2-aminoethyl)-p-chlorobenzamide HCl] as binding probe. In addition, we describe an affinity chromatography method for human platelet MAO-B, using as affinity ligand a derivative of Ro 19-6327 [N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide HCl], a compound in clinical trial belonging to a novel class of reversible and selective MAO-B inhibitors