Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

Presenilin (PS1 or PS2) is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing

Conditioned media collected from the cell lines that express wt PS1, PS1-D385TG or PS1-M146V and were treated with either DMSO or 100 μM Sulindac Sulfide

Equal amount of the media was immunoprecipitated with monoclonal antibody 4G8 and the extensively washed immunoprecipitates were resolved on a Tricine-Urea gel. The blot was reacted with antibody 6E10 for detection.<p><b>Copyright information:</b></p><p>Taken from "Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1"</p><p>http://www.molecularneurodegeneration.com/content/3/1/6</p><p>Molecular Neurodegeneration 2008;3():6-6.</p><p>Published online 12 May 2008</p><p>PMCID:PMC2405781.</p><p></p

Dopamine D3/D2 Receptor Antagonist PF-4363467 Attenuates Opioid Drug-Seeking Behavior without Concomitant D2 Side Effects

Dopamine receptor antagonism is a compelling molecular target for the treatment of a range of psychiatric disorders, including substance use disorders. From our corporate compound file, we identified a structurally unique D3 receptor (D3R) antagonist scaffold, <b>1</b>. Through a hybrid approach, we merged key pharmacophore elements from <b>1</b> and D3 agonist <b>2</b> to yield the novel D3R/D2R antagonist PF-4363467 (<b>3</b>). Compound <b>3</b> was designed to possess CNS drug-like properties as defined by its CNS MPO desirability score (≥4/6). In addition to good physicochemical properties, <b>3</b> exhibited low nanomolar affinity for the D3R (D3 <i>K</i>_i = 3.1 nM), good subtype selectivity over D2R (D2 <i>K</i>_i = 692 nM), and high selectivity for D3R versus other biogenic amine receptors. In vivo, <b>3</b> dose-dependently attenuated opioid self-administration and opioid drug-seeking behavior in a rat operant reinstatement model using animals trained to self-administer fentanyl. Further, traditional extrapyramidal symptoms (EPS), adverse side effects arising from D2R antagonism, were not observed despite high D2 receptor occupancy (RO) in rodents, suggesting that compound <b>3</b> has a unique in vivo profile. Collectively, our data support further investigation of dual D3R and D2R antagonists for the treatment of drug addiction