Target-Mediated Brain Tissue Binding for Small Molecule Inhibitors of Heat Shock Protein 90.

Drug distribution in the brain is generally associated with an affinity for fatty brain tissues and therefore known to be species- and concentration-independent. We report here the effect of target affinity on brain tissue binding for 10 small molecules designed to inhibit brain heat shock protein 90 (HSP90), a widespread protein whose expression is 1-2% of total cytosolic proteins in eucaryotes. Our results show that increasing the test item concentrations from 0.3 to 100 µM increased the unbound fraction 32-fold for the most potent molecules, with no change for the inactive one (1.1 fold change). Saturation of HSP90 led to normal concentration-independent brain tissue binding. In vivo pharmacokinetics performed in rats showed that the overall volume of distribution of compounds is correlated with their affinity for HSP90. The in vitro binding and in vivo pharmacokinetics (PK) performed in rats showed that small molecule HSP90 inhibitors followed the principle of target-mediated drug disposition. We demonstrate that assessing unbound fractions in brain homogenate was subject to HSP90 target interference; this may challenge the process of linking systemic-free drug concentrations to central nervous system unbound concentrations necessary to establish the proper pharmacokinetics/pharmacodynamics (PK/PD) relation needed for human dose prediction.

Linking HSP90 target occupancy to HSP70 induction and efficacy in mouse brain.

HSP90 (Heat shock protein 90) is a molecular chaperone protein ubiquitously expressed throughout all tissues in the body. HSP90 has been proposed as a target to increase turnover of pathological proteins leading to neurodegeneration in Huntington's disease, Parkinson's disease and Alzheimer's disease. The mechanism of how HSP90 inhibition leads to clearance of misfolded proteins is not fully understood. It may involve direct effects of inhibiting ATPase function, indirect effects by inducing the heat-shock-response resulting in upregulation of other chaperone proteins like HSP70 or a combination of both. In the current work we established a methodology to investigate the relationship between HSP90 target occupancy and HSP70 induction in vivo. We also characterized the acute effect of two different HSP90 inhibitors in the rTg4510 transgenic mouse model of Alzheimer's disease which displays a tau-mediated synaptic dysfunction. We show that reversal of synaptic impairments in this model can be obtained with a compound which has a high HSP70 induction capacity. The current developed assay methodologies may thus be of significant use in the further elucidation of the mechanism involved in the in vivo effect of HSP90 inhibition in models of neurodegeneration. Further on, the ability of HSP90 inhibitors to normalize synaptic dysfunction in an in vivo disease model of Alzheimer's disease could have therapeutic relevance and further strengthens the usefulness of this animal model to establish pharmacodynamic effect of HSP90 inhibition.

The identification of GPR3 inverse agonist AF64394; the first small molecule inhibitor of GPR3 receptor function.

The identification of the novel and selective GPR3 inverse agonist AF64394, the first small molecule inhibitor of GPR3 receptor function, is described. Structure activity relationships and syntheses based around AF64394 are reported.

Characterization of [³H]Lu AE60157 ([³H]8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) binding to 5-hydroxytryptamine6 (5-HT6) receptors in vivo.

The serotonin6 (5-HT(6)) receptor has received attention for its proposed role in cognitive impairments associated with schizophrenia and Alzheimer's disease. This has lead to a search for selective 5-HT(6) receptor ligands useful for in vivo imaging in animals and humans. The novel 5-HT(6) receptor antagonist Lu AE60157 (8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) displays high affinity for the human (h) 5-HT(6) receptor (K(d) 0.2nM), and broad profiling in 60 additional binding and enzyme assays showed that Lu AE60157 displays 16-fold selectivity to the h5-HT(2A) receptor (K(i) 3.2nM) and >100-fold selectivity to all other evaluated targets. Lu AE60157 was labeled with tritium in the N-methyl group and evaluated as a radioligand in vitro as well as in vivo in rats and mice. Autoradiography experiments showed that [(3)H]Lu AE60157 bound preferentially to rat brain regions with expected high 5-HT(6) receptor density. Furthermore, [(3)H]Lu AE60157 showed good brain penetration after systemic administration and high (about 75%) specific in vivo binding to the striatal 5-HT(6) receptor in rats. The striatal binding of [(3)H]Lu AE60157 was fully displaced by selective 5-HT(6) receptor antagonists (SB-742457; Lu AE58054) and antipsychotics known to inhibit the binding of 5-HT(6) receptors in vitro (clozapine; olanzapine; sertindole), but was not displaced by antipsychotics lacking high 5-HT(6) receptor affinities (risperidone; haloperidol; quetiapine). No specific binding to mouse brain tissue in vivo could be obtained. In conclusion, [(3)H]Lu AE60157 is suitable for measuring in vivo occupancies of 5-HT(6) receptor ligands in rat brain regions in which 5-HT(2A) receptors do not interfere.

Discovery of 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine (Lu AA21004): a novel multimodal compound for the treatment of major depressive disorder.

The synthesis and structure-activity relationship of a novel series of compounds with combined effects on 5-HT(3A) and 5-HT(1A) receptors and on the serotonin (5-HT) transporter (SERT) are described. Compound 5m (Lu AA21004) was the lead compound, displaying high affinity for recombinant human 5-HT(1A) (K(i) = 15 nM), 5-HT(1B) (K(i) = 33 nM), 5-HT(3A) (K(i) = 3.7 nM), 5-HT(7) (K(i) = 19 nM), and noradrenergic ß(1) (K(i) = 46 nM) receptors, and SERT (K(i) = 1.6 nM). Compound 5m displayed antagonistic properties at 5-HT(3A) and 5-HT(7) receptors, partial agonist properties at 5-HT(1B) receptors, agonistic properties at 5-HT(1A) receptors, and potent inhibition of SERT. In conscious rats, 5m significantly increased extracellular 5-HT levels in the brain after acute and 3 days of treatment. Following the 3-day treatment (5 or 10 (mg/kg)/day) SERT occupancies were only 43% and 57%, respectively. These characteristics indicate that 5m is a novel multimodal serotonergic compound, and 5m is currently in clinical development for major depressive disorder.

Novel NK(3) receptor antagonists for the treatment of schizophrenia and other CNS indications.

The cloning of the three tachykinin receptors in the late 1980s formed the basis of intense preclinical research efforts into the systems relating to the tachykinin receptors, as well as compound screening campaigns. Remarkably, orally active non-peptide antagonists were successfully identified for all three of the tachykinin receptors, providing tools for further evaluation of the pharmacology of these receptor systems. The NK3 receptor (mammalian tachykinin receptor 3), which exhibited a discrete expression pattern and the modulatory regulation of various transmitter systems in the CNS, has attracted significant interest. Preclinical studies demonstrated that the NK3 receptor might be a promising target for CNS disorders, and clinical trials with non-peptide NK3 receptor antagonists have been performed for indications such as schizophrenia, major depressive disorder, panic attacks and Parkinson's disease. In particular, the positive results of the schizophrenia meta-trial with osanetant increased the focus on the NK3 receptor system and its clinical potential. Consequently, a significant number of patents covering non-peptide antagonists for the NK3 receptor have been published during the past decade. This review describes the most recent NK3 receptor antagonists (published from 2004 to 2009), which are classified into seven unique templates.