Amyloid modifier SERF1a interacts with polyQ-expanded huntingtin-exon 1 via helical interactions and exacerbates polyQ-induced toxicity.

Abnormal polyglutamine (polyQ) expansion and fibrillization occur in Huntington's disease (HD). Amyloid modifier SERF enhances amyloid formation, but the underlying mechanism is not revealed. Here, the fibrillization and toxicity effect of SERF1a on Htt-exon1 are examined. SERF1a enhances the fibrillization of and interacts with mutant thioredoxin (Trx)-fused Httex1. NMR studies with Htt peptides show that TrxHttex1-39Q interacts with the helical regions in SERF1a and SERF1a preferentially interacts with the N-terminal 17 residues of Htt. Time-course analysis shows that SERF1a induces mutant TrxHttex1 to a single conformation enriched of ß-sheet. Co-expression of SERF1a and Httex1-polyQ in neuroblastoma and lentiviral infection of SERF1a in HD-induced polypotent stem cell (iPSC)-derived neurons demonstrates the detrimental effect of SERF1a in HD. Higher level of SERF1a transcript or protein is detected in HD iPSC, transgenic mice, and HD plasma. Overall, this study provides molecular mechanism for SERF1a and mutant Httex1 to facilitate therapeutic development for HD.

Discovery of small molecular (D)-leucinamides as potent, Notch-sparing γ-secretase modulators.

Structural optimization of the prior lead 3 led to the small molecular (D)-leucinamides with potent modulating activity and Notch-sparing selectivity on the proteolytic processing of amyloid-ß precursor proteins. The N-(R)-epoxypropyl analog 10c exhibited potent γ-secretase modulation compared to DAPT and showed substantial substrate selection for APP cleavage over Notch cleavage, while N-(2-fluoro)benzyl analog 10e showed the most potent γ-secretase inhibition with dull selectivity. The exceptional suppression of ERK-mediated activation suggested that these potent γ-secretase modulators may adapt an alternative pathway to prominently induce the differential inhibition of C99 cleavage by γ-secretase.

Difluorophenylglycinols as new modulators of proteolytic processing of amyloid precursor proteins.

Synthesis and evaluation of difluorophenylglycinols as new modulators of proteolytic processing of the amyloid-ß precursor proteins for Alzheimer's therapies were described. A range of N-substituted (R)- and (S)-difluorophenylglycinols, structured on the amino alcohol framework, were explored by incorporating the arylsulfonyl moieties and various N-substituents. Evans' chiral auxiliary strategy was employed for the asymmetric synthesis of these enantiomeric difluorophenylglycinols. Compounds with effects on the γ-secretase inhibition and ERK-mediated signaling pathways were evaluated on cell-based assays. Among them, N-cyclopropylmethyl derivatives R-12c and R-13c showed modest γ-secretase inhibition as well as ERK-dependent activation.

Retinoic acid-elicited RARα/RXRα signaling attenuates Aß production by directly inhibiting γ-secretase-mediated cleavage of amyloid precursor protein.

Retinoic acid (RA)-elicited signaling has been shown to play critical roles in development, organogenesis, and the immune response. RA regulates expression of Alzheimer's disease (AD)-related genes and attenuates amyloid pathology in a transgenic mouse model. In this study, we investigated whether RA can suppress the production of amyloid-ß (Aß) through direct inhibition of γ-secretase activity. We report that RA treatment of cells results in significant inhibition of γ-secretase-mediated processing of the amyloid precursor protein C-terminal fragment APP-C99, compared with DMSO-treated controls. RA-elicited signaling was found to significantly increase accumulation of APP-C99 and decrease production of secreted Aß40. In addition, RA-induced inhibition of γ-secretase activity was found to be mediated through significant activation of extracellular signal-regulated kinases (ERK1/2). Treatment of cells with the specific ERK inhibitor PD98059 completely abolished RA-mediated inhibition of γ-secretase. Consistent with these findings, RA was observed to inhibit secretase-mediated proteolysis of full-length APP. Finally, we have established that RA inhibits γ-secretase through nuclear retinoic acid receptor-α (RARα) and retinoid X receptor-α (RXRα). Our findings provide a new mechanistic explanation for the neuroprotective role of RA in AD pathology and add to the previous data showing the importance of RA signaling as a target for AD therapy.

Enzymatic and nonenzymatic synthesis of glutathione conjugates: application to the understanding of a parasite's defense system and alternative to the discovery of potent glutathione S-transferase inhibitors.

A primary pathway for metabolism of electrophilic compounds in Schistosoma japonicum involves glutathione S-transferase (SjGST)-catalyzed formation of glutathione (GSH) conjugates. As part of a program aimed at gaining a better understanding of the defense system of parasites, a series of aromatic halides (1-8), aliphatic halides (9, 10), epoxides (11-20), alpha,beta-unsaturated esters (21, 22), and alpha,beta-unsaturated amides (23, 24) were prepared, and their participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymatic reactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS) analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed. Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3, 4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold, respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to other electrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a process that forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways are generated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymatic GSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymatic GSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5, 0.96, 19.0, 152, and 0.36 microM, and they display moderate inhibitory activities against human GSTA2. Direct evidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than that of its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-based technique that can be used to characterize glutathione conjugates derived from small molecule libraries as part of efforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applications to the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.