Alan Nadin

Selected Non-steroidal Anti-inflammatory Drugs and Their Derivatives Target γ-Secretase at a Novel Site EVIDENCE FOR AN ALLOSTERIC MECHANISM

γ-Secretase is a multi-component enzyme complex that performs an intramembranous cleavage, releasing amyloid-β (Aβ) peptides from processing intermediates of the β-amyloid precursor protein. Because Aβ peptides are thought to be causative for Alzheimers disease, inhibiting γ-secretase represents a potential treatment for this neurodegenerative condition. Whereas inhibitors directed at the active center of γ-secretase inhibit the cleavage of all its substrates, certain non-steroidal antiinflammatory drugs (NSAIDs) have been shown to selectively reduce the production of the more amyloidogenic Aβ(1–42) peptide without inhibiting alternative cleavages. In contrast to the majority of previous studies, however, we demonstrate that in cell-free systems the mode of action of selected NSAIDs and their derivatives, depending on the concentrations used, can either be classified as modulatory or inhibitory. At modulatory concentrations, a selective and, with respect to the substrate, noncompetitive inhibition of Aβ(1–42) production was observed. At inhibitory concentrations, on the other hand, biochemical readouts reminiscent of a nonselective γ-secretase inhibition were obtained. When these compounds were analyzed for their ability to displace a radiolabeled, transition-state analog inhibitor from solubilized enzyme, noncompetitive antagonism was observed. The allosteric nature of radioligand displacement suggests that NSAID-like inhibitors change the conformation of the γ-secretase enzyme complex by binding to a novel site, which is discrete from the binding site for transition-state analogs and therefore distinct from the catalytic center. Consequently, drug discovery efforts aimed at this site may identify novel allosteric inhibitors that could benefit from a wider window for inhibition of γ (42)-cleavage over alternative cleavages in the β-amyloid precursor protein and, more importantly, alternative substrates.

Synthesis of tricyclic pyridones by radical cyclization

Abstract A general and novel route for the synthesis of tricyclic pyridones by 5-, 6- and 7- exo-trig radical cyclization is described. The use of Pd-catalyzed cross-coupling reactions to introduce functionality at the 5-position of a pyridone is also presented.

γ-Secretase: characterization and implication for Alzheimer disease therapy

gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimers disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

Saturated nitrogen heterocycles

Reviewing the literature published in 1999. Continuing the coverage in J. Chem. Soc., Perkin Trans. 1, 1999, 2553.

Potent and selective inhibitors of PI3Kδ: obtaining isoform selectivity from the affinity pocket and tryptophan shelf.

A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

γ‐Secretase performs the final processing step in the generation of amyloid‐β (Aβ) peptides, which are believed to be causative for Alzheimers disease. Presenilins (PS) are required for γ‐secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen‐bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue γ‐secretase inhibitor. Here we report that replacement of either tyrosine residue alters γ‐secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Aβ(42) peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.

Synthesis and γ-secretase activity of APP substrate-based hydroxyethylene dipeptide isosteres

Two new APP substrate-based hydroxyethylene isosteres (AT and VI) were prepared and their dipeptide conjugates shown not to inhibit the γ-secretase-mediated formation of either Aβ1-40 or Aβ1-42. The FG isostere and a des-hydroxy hydroxyethylene isostere also gave inactive compounds. Conversely, a number of compounds containing the intact substrate-unrelated Phe-Phe (FF) hydroxyethylene isostere were shown to be potent inhibitors (ED50=14–732 nM). These results show that the factors governing the substrate-based design of γ-secretase inhibitors are more complicated than first thought.

Synthesis of optically active α-phenylselenyl carbonyl derivatives

Abstract Homochiral silyl enol ethers derived from N -acyl oxazolidinones can be phenyselenylated in high diastereoselectivity and good yield with phenylselenyl chloride. Removal of the chiral auxiliary is accompanied by some epimerisation, leading to isolation of the corresponding α-phenylselenyl methyl esters in moderate enantiomeric excess. A slightly improved enantiomeric excess is achieved if 4( S )-benzyl-2-oxazolidinethione 17 is used as a chiral auxiliary.

New methodology for the synthesis of unsaturated 8-, 9- and 10-membered lactams

Unsaturated 8-, 9- and 10-membered medium ring lactams 1 (n= 1,2,3) have been prepared in good yield by the Claisen rearrangement of the vinyl-substituted precursors 3 in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

Medium ring lactams in molecular recognition

Abstract β-Turns are ubiquitous in bioactive conformations of important peptides. We have designed a family of seven-membered lactam dipeptide mimics which are expected to act as conformational constraints in enforcing type VIa β-turns in peptide chains. Molecular modelling using Macromodel predicts that both 3(S)-acetylamino-7(S)-carboxy-azepan-2-one N-methyl amide (1, the cis-disubstituted lactam, mm2 ) and 3(S)-acetylamino-7(R)-carboxy-azepan-2-one N-methyl amide (2a, the trans-disubstituted lactam, mm2 and amber ) should exhibit features of type VI β-turns. The synthesis and structural analysis of the cis-(1) and trans-disubstituted (2) seven-membered lactam constraints are reported. X-Ray crystallographic analysis and solution NMR measurements offer insight into the preferred conformations of these molecules. Variable temperature NMR data and vapour pressure osmometry of 1 in solution indicate that it exhibits a strong cooperative recognition phenomenon to form a novel dimer, a feature not demonstrated by the trans-isomer 2a, which has a type VIa β-turn-like conformation in the solid state. These phenomena can be realistically modelled in molecular dynamics simulations, and demonstrate potential for the design of new materials by self-assembly.