. Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting. Science. 2008 Apr 25;320(5875):520-3. PubMed.

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  1. This work described an interesting concept for targeting β-secretase to endosomes. The results show some promise. However, the improvement in the inhibition of Aβ production by sterol-modified inhibitors over the unmodified inhibitor was accomplished with an early-stage inhibitor. Although the inhibitor structure was not revealed in the paper and supplements, the fact that the inhibitor was synthesized by automated peptide synthesizer suggests that it may be similar to the first inhibitor OM99-2 we described in 2000 (Hong et al., 2000; Lin et al., 2000).

    The first-generation peptidic inhibitors have poor ability to penetrate membranes. Thus, the sterol modification would help their activity in cells and in vivo. However, the late-generation inhibitors are no longer peptidic and are relatively small. Their ability to penetrate membranes and inhibit Aβ production is well demonstrated now in transgenic mice (both i.v. and oral) and in human clinical trials (by CoMentis).

    It is not clear that the membrane-anchoring modification would help develop better β-secretase inhibitor drugs. Of special concern is that the conjugation makes inhibitors considerable larger. Since the blood-brain barrier has a cutoff size of about 500 Da, people designing drugs are strained to put selectivity, blood-brain-barrier penetration, and potency all in a small package. The sterol conjugation does not seem to help blood-brain barrier penetration, as the transgenic experiment was done with direct injection of the conjugated inhibitor into the brain, thus bypassing the blood-brain barrier.

    On the scientific side, the results do demonstrate that a membrane-anchored inhibitor can inhibit β-secretase, which suggests that the protease has considerable freedom of gyration in endosomes. This is good to know.

    References:

    . Structure of the protease domain of memapsin 2 (beta-secretase) complexed with inhibitor. Science. 2000 Oct 6;290(5489):150-3. PubMed.

    . Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1456-60. PubMed.

    View all comments by Jordan Tang
  2. This work clearly demonstrates the importance of targeting to the right subcellular localization in the inhibition of BACE.

    To test if a BACE inhibitor can reduce Aβ production in vivo, the authors used the APPswe/PS mutant transgenic mouse model. It is unclear exactly to me which mouse model was used in the current study. Although it was stated in the text that APPswe/PS1delta9 mice were used, an article describing APPswe/PS1L166P transgenic mice was cited.

    If APPswe/PS1Δ9 mice were used, it might be still reasonable to test the effect of the BACE inhibitor after four hours of injection, given the very low plaque load at four to five months of age in APPswe/PS1Δ9 mice.

    However, if APPswe/PS1L166P mice were used, their huge plaque load at four to five months of age might confound the interpretation, even with PBS extraction and a bead grinder homogenizer.

    View all comments by Jungsu Kim
  3. I have to thank Alzforum's new reporter, Esther Landhuis, for the coverage on our recent study. Great work!

    The comment by Drs. Tang and Augelli-Szafran on the non-drug-likeness of our inhibitor is extremely important. Membrane anchoring of an inhibitor requires the presence of a lipid-anchor and also a linker molecule, and hence, considerably increases the size of the inhibitor. Size definitely matters when it comes to crossing the blood-brain barrier. We also share the same concern these commentators have as to whether our drug can pass the BBB. We are currently performing mice experiments in collaboration with Mikael Simons from the Max Planck Institute in Goettingen to see if the inhibitor, when administered through several different routes, can pass the BBB.

    Having said that, I want to emphasize here that we do not claim that we have a “drug” against AD. Our work is more a proof for the principle of subcellular targeting: since much of the active β-secretase is found only in the endosomal compartment, it is important that transition-state inhibitors against β-secretase reach this compartment. We hypothesized that membrane anchoring would render the anchored inhibitor endocytosis-competent and thereby traffic the inhibitor to the endosomes. As a proof-of-principle, we membrane-anchored a non-permeable inhibitor and compared it with the unanchored version of the inhibitor (all structures—parent inhibitor and the modifications—are shown in Supplementary Figure 3). The anchored version was more effective than the free inhibitor in inhibiting β-secretase suggesting that the inhibitors should reach this endosomal compartment in order to effectively inhibit β-secretase. Inhibitors that accomplish this task by permeating through the membrane, thereby gaining access to the endosomal β-secretase, also support the hypothesis.

    We are also trying to vary the pharmacophore (currently of peptidic nature) to small-molecule inhibitors of β-secretase along with variations in the linker lengths. This would enable us to formulate a considerably smaller, yet membrane-anchored version of the inhibitor.

    Inferring from our Drosophila experiments, one could assume that the sterol-linked inhibitor that was mixed with the feed crossed the gut epithelium via transcytosis to reach other tissues. Since transcytosis is a mechanism by which several drugs cross the blood-brain barrier, it is not impossible that such an inhibitor could also be transported across the blood-brain barrier and be delivered to the brain. Lipoprotein particles could aid in the delivery. These are, of course, assumptions that have to be tested by further work.

    One aspect that we couldn’t emphasize much in the paper is that membrane anchoring reduced the dimensionality of the otherwise soluble inhibitor. By membrane anchoring, the inhibitor partitions readily into the membrane plane, thereby increasing the concentration of the inhibitor in the membrane plane. We will now test the reduction in dimensionality principle by membrane anchoring a membrane-permeable inhibitor.

    Thanks to Jungsu for pointing out the mistake regarding the nomenclature of the mice—we have written an erratum. Thanks once again to all commentators.

    View all comments by Lawrence Rajendran