Frédéric Bihel

New protease inhibitors prevent γ-secretase-mediated production of Aβ40/42 without affecting Notch cleavage

We have designed new non-peptidic potential inhibitors of γ-secretase and examined their ability to prevent production of amyloid-β 40 (Aβ40) and Aβ42 by human cells expressing wild-type and Swedish-mutant β-amyloid precursor protein (βAPP). Here we identify three such agents that markedly reduce recovery of both Aβ40 and Aβ42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of βAPP that are derived from β-and α-secretase, respectively. Furthermore, we show that these inhibitors do not affect endoproteolysis of endogenous or overexpressed presenilins. These inhibitors are totally unable to affect the mΔEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent γ-secretase cleavage of βAPP without affecting processing of mΔEnotch-1 or endoproteolysis of presenilins. The distinction between these two proteolytic events, which are both prevented by disruption of presenilin genes, indicates that although they are intimately linked with βAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave γ-secretase and Notch.

Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new β-amyloid peptide production inhibitors and their activities on various classes of protease

A series of new 7-substituted-4-chloro-3-alkoxy isocoumarin derivatives were synthesized and evaluated as inhibitors of representative classes of proteases: serine protease (alpha-chymotrypsin, trypsin), cysteine protease (Caspase-3), and aspartyl protease (HIV-protease), 20S proteasome and also as inhibitors of amyloid peptide gamma-secretase-mediated production. Protease inhibition selectivity is directly related to the structure of the substituent at the 7-position of the isocoumarin nucleus. 7-Nitro-isocoumarin derivatives (4c, 4d, 4f) are potent alpha-chymotrypsin inhibitors but slightly active or inactive on HIV-protease, as well as on cysteine protease. In contrast, only derivatives bearing a free amino (5d, 5f) or a substituted amino group (6f) at the 7-position of the isocoumarin nucleus, were found weakly active or inactive on alpha-chymotrypsin, trypsin, Caspase-3 and HIV-protease, but prevent gamma-secretase-mediated production of Abeta 40/42 amyloid peptides, which is known to be involved in Alzheimers disease. Moreover, the most active compounds on beta-amyloid peptide production [JLK6 (5d), JLK2 (5f) and JLK7 (6f)] show only weak or moderate inhibitory activity on the 20S proteasome. The obtained results suggest that the described new isocoumarin analogues could be of interest, since compounds like JLK6 (5d), JLK2 (5f) and JLK7 (6f) can be considered as possible hits for the development of new agents directed towards Alzheimers disease.

Novel synthesis of 3,4-dihydro-5-bromo[1,4]oxazin-2-one derivatives, new protease inhibitor scaffold

We designed and synthesized a new class of serine protease inhibitors based on the oxazinone core. To this end, we first developed a short and efficient route to synthesize a new 3,4-dihydro[1,4]oxazin-2-one ring. Then we successfully synthesised the corresponding 5-bromo derivatives which have never been reported before, and demonstrated their inhibitory activities on alpha-chymotrypsin.

New β-strand macrocyclic peptidomimetic analogues containing α-(O-, S- or NH-)aryl substituted glycine residues: synthesis, chemical and enzymatic properties

In so much as bis-macrocyclic peptidomimetics have been recognized as high affinity substrates for HIV-1 protease, we were interested in the design and synthesis of new bis-macrocyclic bioisosteric analogues whose general structure is displayed on Fig. 2. The structures of these new analogues are characterized by the specific replacement of the methylene of the benzyl group directly attached to the N-acyl glycine residue in the original molecule 1, by its main bioisosteres, i.e. O-, S- and NH-aryl groups. Knowing that an intermediate in which an heteroatomic aryl group is directly linked to a free amine glycine residue is not stable, we developed an original synthetic pathway which involved the coupling of a specific side chain to the exocyclic carboxylic acid function, followed by an elegant oxidation–nucleophilic substitution Steglich-type reaction. Analogues 2a–d were then submitted to chemical and enzymatic hydrolysis. We demonstrated that, as expected, the specific cleavage of the exocyclic N-acyl bond led to the release of aryl moieties (phenol, thiophenol and aniline species). These chemical and enzymatic stability studies brought to light the biological potential of such macrocyclic analogues in infected cells.

Design of potential new HIV protease inhibitors: enantioconvergent synthesis of new pyrrolidin-3-ol, and pyrrolidin-3-one peptide conjugates

Novel potential HIV protease inhibitors are obtained by an enantioconvergent synthesis of mimicking Phe-Pro dipeptides, achieved through the coupling between Boc(L)Phe or Boc(L)Tyr and both enantiomers of syn-2-benzylpyrrolidin-3-ol and their corresponding pyrrolidin-3-one analogs. The stereochemistry and enantiopurity of intermediate 3-hydroxypyrrolidines 5a and 5b are determined through 1H NMR analysis, and through the synthesis and 19F NMR assignments of the corresponding Mosher’s esters 13a and 13b. The enantiopure compounds 5a and 5b are obtained with 100% diastereoselectivity using specific experimental reductive conditions upon Meldrum’s acid derivatives of activated aromatic amino acids.

Reactivity studies of 3-alkoxy-7-amino-4-chloroisocoumarins (β-amyloid peptide inhibitors)versus different classes of amines

3-Alkoxy-7-amino-4-chloroisocoumarins have been shown to lower the beta-amyloid secretion (a major component of the senile plaques involved in Alzheimers disease). This paper reports the characterization of new adducts resulting from the reaction between the isocoumarin synthon and different classes of amines. This study allows on the one hand, a better understanding of the biological molecular processes by which such isocoumarin derivatives may interact with enzyme nucleophiles and, on the other hand, brings out the chemical potential of these synthons to generate new polycyclic derivatives.

New Non-Peptidic Inhibitors of γ-Secretase Abolish Aβ Production Without Modifying Notch Cleavage

The amyloidogenic cascade hypothesis to explain Alzheimer’s disease pathology implies a key strategic role of β and γ-secretases, which are the proteolytic activities responsible for the release of the N- and C-termini of Aβ peptides, respectively (for review, see Checler, 1995). A consensus now exists concerning β-secretase, a novel type of membrane-bound aspartyl protease recently discovered by several teams and referred to as BACE1 (β-site APP-cleaving enzyme), memapsin2 or Asp2 (for review, see Vassar and Citron 2000). By contrast, the nature of γ-secretase is still a matter of intense discussion (Checler 2001; Wolfe 2001).