Kaspar Zimmermann

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE, THE PUTATIVE TARGET OF THE ANTIAPOPTOTIC COMPOUNDS CGP 3466 AND R-(-)-DEPRENYL

R-(−)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson’s disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog ofR-(−)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore® technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.

Cyclophilin D as a Drug Target

The mitochondrial permeability transition (MPT) plays an important role in damage-induced cell death, and agents inhibiting the MPT may have a therapeutic potential for treating human conditions such as ischemia/reperfusion injury, trauma, and neurodegenerative diseases. The mitochondrial matrix protein, cyclophilin D (CYP D), a member of a family of highly homologous peptidylprolyl cis-trans isomerases (PPIases), plays a decisive role in MPT, being an integral constituent of the MPT pore. Other putative MPT pore proteins include the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC). In an alternative model, the MPT pore is formed by clusters of misfolded membrane proteins outlining aqueous channels that are regulated by CYP D and other chaperone-like proteins. Like cyclophilin A (CYP A) and other cyclophilin family members, CYP D is targeted by the immunosuppressant cyclosporin A (CsA). CsA is cytoprotective in many cellular and animal models, but protection may result from either inhibition of the MPT through an interaction with CYP D or inhibition of calcineurin-mediated dephosphorylation of BAD through an interaction with CYP A. The relevance of MPT inhibition by CsA for its cytoprotective effects is well documented in many cellular models. Mechanisms of action in vivo are more difficult to define, and accordingly the evidence is as yet less compelling in in vivo animal models of ischemia/reperfusion injury, trauma and neurodegenerative diseases. Notwithstanding, CYP D is a drug target of high interest. Structural considerations suggest feasibility of designing CYP D ligands without immunosuppressant properties. This is highly desirable, since they have the potential of being useful therapeutic agents in a variety of disease states. It might be a tougher challenge to obtain compounds specific for CYP D vs. other cyclophilins, and/or of small molecular weight, allowing brain penetration to make them suitable for treating neurodegenerative diseases.

Blocking Metabotropic Glutamate Receptor Subtype 7 (mGlu7) via the Venus Flytrap Domain (VFTD) Inhibits Amygdala Plasticity, Stress, and Anxiety-related Behavior

Background: Behavioral genetics identified mGlu7 as a key regulator of brain emotion circuits. Results: An mGlu7-selective, Venus flytrap domain (VFTD)-directed antagonist inhibits fear, synaptic plasticity, stress, and anxiety in rodents. Conclusion: Pharmacological blockers of mGlu7 may represent promising future anxiolytics and antidepressants in man. Significance: The VFTD region of class C GPCRs provides a promising target for computer-assisted drug design. The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7s extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7s Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Discovery of novel indolinone-based, potent, selective and brain penetrant inhibitors of LRRK2

Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common genetic cause of Parkinsons disease (PD). The most frequent kinase-enhancing mutation is the G2019S residing in the kinase activation domain. This opens up a promising therapeutic avenue for drug discovery targeting the kinase activity of LRRK2 in PD. Several LRRK2 inhibitors have been reported to date. Here, we report a selective, brain penetrant LRRK2 inhibitor and demonstrate by a competition pulldown assay in vivo target engagement in mice.

Dibenzoxepines as treatments for neurodegenerative diseases

In recent years, apoptotic cell death has been implicated with different progressive neurodegenerative diseases such as Parkinsons disease, Huntingtons disease, Amyotrophic Lateral Sclerosis or Alzheimers disease. The hypothesis emerged, that a drug preventing apoptosis may slow or even halt the disease progression. (±)-Deprenyl was reported to rescue neurons from cell death in different in vitro and in vivo systems. However, deprenyl suffers the antagonizing actions of its major metabolites. We set up a screening for compounds with neurorescuing properties, lacking deprenyls metabolic problems. 10-Aminomethyldibenzo[b,f]oxepin derivatives were identi®ed to show marked effects in a survival assay of trophically-withdrawn PC12 cells. Dibenzo[b,f]oxepines bearing different aminomethyl sidechains and aromatic substituents were prepared in a multistep synthesis, and a structureactivity relationship was established. In particular the N-methyl-N-propargylaminomethyl derivative, CGP 3466, shows neurorescuing properties at concentrations as low as 10 M in different in vitro test systems. In vivo, CGP 3466 prevents the death of dopaminergic cells in the mouse substantia nigra after MPTP-lesion. It also rescues mouse facial motor neurons after axotomy and increases the life-span of mice with progressive motor neuronopathy. Glyceraldehyde-3-phosphate dehydrogenase was identi®ed as the putative molecular target of CGP 3466-derivatives by means of af®nity binding and photoaf®nity labeling.

Synthesis of tools for target identification of the anti-apoptotic compound CGP 3466; Part I

Immobilized compounds for BIAcore studies and affinity precipitation as well as a fluorescent-labeled compound were prepared in order to identify the molecular target of the anti-apoptotic, neurorescuing compound CGP 3466 (N-methyl-N-propargyl-10-aminomethyl-dibenzo[b,f]oxepin).

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) ∼2 μM) and its C(18)-analogues (IC(50) <10 μM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) ∼8 μM) growth of JURKAT cells.

Electrophysiological characterization of CGP6873OA a N-methyl-D-aspartate antagonist acting at the strychnine-insensitive glycine site

1. Electrophysiological experiments were performed in vitro and in vivo. Voltage clamp recordings were done in Xenopus oocytes. Extracellular recordings were done in vitro in the neocortical slice and in the CA1 region of the hippocampal slice and in vivo in the CA1 region of the hippocampus of the anaesthetized rat. 2. In oocytes expressing either the human NMDAR1A/2A or 1A/2B subunit combinations, CGP68730A [sodium (-)-9-bromo-2,3,6,7-tetrahydro-5,6-dioxo-5H-pyrazino[1,2,3-de]-1,4-benzo thiazine-3-acetic acid] antagonized L-glutamate / glycine induced currents with calculated IC50s of 20.5 and 81.6 nM, respectively. 3. In vitro, CGP68730A was tested on NMDA induced depolarizations in the neocortical slice preparation and on epileptiform activity in hippocampal slices bathed in Mg2+-free-medium, which is known to be NMDA mediated. In both in vitro models CGP68730A exhibited antagonistic effects on the NMDA receptor mediated responses. 4. In vivo CGP68730A was tested on NMDA induced excitations in the CA1 region. CGP68730A abolished NMDA induced excitations when applied microiontophoretically. However, only weak effects on NMDA induced excitation were observed after systemic administration at 100 mg/kg i.v.. These results indicate that CGP68730A has poor central nervous system bioavailability. 5. In oocytes, an increase in the glycine concentration from the EC80 to the EC95.99 shifted the inhibition curves for CGP68730A to the right. Furthermore, in neocortical slices and in anaesthetized rats CGP68730A inhibited NMDA mediated depolarizations, and this effect could be reversed by the addition of the glycine mimetic D-serine. This indicates that these effects of CGP68730A are mediated by an action on the strychnine-insensitive glycine site. 6. Selectivity tests in oocytes and in the neocortical slice preparation, using NMDA, kainate and AMPA showed that CGP68730A was selective in antagonizing NMDA receptor mediated responses. In oocytes, the compound was about 1000 times less potent on the rat GluR3 and the human GluR6 receptors than on the human NMDAR1A/2A subunit combination. In the neocortical slice preparationCGP68730A had no effects on AMPA or kainate induced depolarizations at concentrations of 3 and 10 microM. At 30 microM CGP68730A reduced the effects of each of the three agonists tested. 7. Thus, CGP68730A seems to be a selective antagonist at the strychnine-insensitive glycine coagonist site of the NMDA receptor. However, the compound showed no obvious central NMDA antagonistic effects following intravenous application.

Design and synthesis of a biotinylated dopamine transporter ligand for the purification and labeling of dopaminergic neurons

The design and synthesis of a new tool for labeling and purification of dopaminergic neurons is described.