Flap I

Fig. 3.5.1. BACE complexed to Glu-Val-Asn-C(Leu-Ala)-Ala-Glu-Phe (3, OM99-2) (1FKN).

ment, although their normal regulatory function in healthy tissue is not fully understood (Ab or peptide models thereof are also dealt with in Chapters 2.3 and 2.4).

Although several reviews on secretase inhibition have been published [10-12], the rapid progress in the field demands continuous survey. And despite this significant progress potent non-peptidic inhibitors of b-secretase are still unknown. Several peptide-based inhibitors were patented or reported immediately after J. Tang's disclosure of the BACE-inhibitor complex X-ray structure in 2000. Figures 3.5.1 and 3.5.2 show fragments of the homodimeric structures, which were reviewed recently [13]. Non-peptidic inhibitors of presenilin are known from patents

Fig. 3.5.2. BACE complexed to Glu-Val-Asn-C(Leu-Ala)-Ala-Glu-Phe (3, OM99-2) (1FKN).

by Elan/Eli Lilly, Bristol Myers Squibb, and DuPont. A single original publication appeared for DAPT (16, difluorophenylacetylaminopropionylaminophenylacetic acid tert-butyl ester) and its drug-metabolism and pharmacokinetics (DMPK) outside that literature, but thanks to the commercialization of DAPT, which is a phase II candidate by Eli Lilly, it will turn into the standard for other compounds to come. Peptidic presenilin inhibitors [14, 15], for example Merck's L-685 458, which is still the most potent inhibitor, were patented prior to publication in scientific journals [16].

The well known, beneficial influence of non-steroidal anti-inflammatory drugs (NSAID) on the progress of Alzheimer's disease has been confirmed for some NSAID subtypes. The work by Weggen et al. indicates the potential of COX 1 inhibitors (e.g. Diclofenac, Sulindac, Indomethacin, Ibuprofen, but not the most prominent, Aspirin) in PS inhibition [17].

The reports of Nicastrin, which is a protein linked to familial dementia in the Italian town Nicastro, and its co-precipitation with presenilin by presenilin-specific antibodies [18] stimulated the ongoing debate about the identity of y-secretase/PS. C. Haass has suggested that mature nicastrin plays a crucial role in PS1 trafficking from the endoplasmic reticulum to the plasma membrane [19].

^-Secretase (BACE) was established as an aspartic protease (Box 13) by molecular biology despite the initial lack of selective inhibitors. It bears all the hallmarks of a typical aspartic protease including the flexible flap region, which is crucial for substrate docking. The two states, open and closed, contribute to the selectivity and activity of the enzyme [20]. BACE1 is anchored to the membrane via its transmembrane domain (455-480); the catalytic domain is stabilized by three cystines in analogy to other aspartic proteases. The fully active BACE1 used by Tang for co-crystallization lacks the transmembrane and intracellular domains and some flexible N-terminal regions were not resolved by X-ray structure determination. The inhibitor is placed in the active site as intended by design (Figure 3.5.2): the transition state analog hydroxyethylene is coordinated through four hydrogen bonds to the two catalytic aspartic acids. Another ten hydrogen bonds are established between inhibitor, binding pocket, and flap region. Despite analogies with other as-partic proteases, there are significant differences in side-chain preferences. S4, S3' are hydrophilic and readily accessible by water, the hydrophilic S4', which holds the phenylalanine, is located at the surface and contributes less to binding. Therefore shortened peptidomimetics 4-6 retain activity. The S1' position has space for more than just an alanine, as in the co-crystallized inhibitor 3. This was realized by ethyl substitution of the hydroxyurea 5. The importance of the flap region for structural reorganization and activity modulation was concluded from kinetics of statine-based peptides (hydroxyethylene) [21]. A detailed analysis of BACE distribution, structure, species variation, and properties was published recently [13]. BACE2, which is very similar, leads to additional hydrolysis close to Phe20 (Scheme 3.5.3).

Aspartic proteases hydrolyze the amide bond as a result of concerted effort by an aspartic acid and an aspartate (Box 13). The aspartic acid protonates and activates the peptide A (Scheme 3.5.2) towards nucleophilic attack, and the aspartate is re-

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