At the 8th annual Eibsee Meeting on Cellular Mechanisms of Alzheimer Disease, the theme of hitting two secretases at once to deal a blow to AD came up twice. Phil Wong of Johns Hopkins University, Baltimore, explored it with genetic experiments, and Stefan Lichtenthaler at the Ludwig-Maximilians University of Munich followed up with a small-molecule approach. For his part, Michael Willem, who is in Haass’s group at LMU Munich, added new data to the understanding of BACE’s physiological role, which is still in its infancy.
Wong got things going by arguing that while total inhibition of either APP-processing enzyme would lead to untoward side effects, moderately inhibiting both might be safe. Research by several groups has enlisted normal functions and substrates for BACE that make its wholesale block unsafe, Wong said. His own group used BACE knockout mice to show that this enzyme’s participation in neuregulin processing (Willem et al. 2006; Yan et al., 2006) partly explains why its complete loss leads to behaviors resembling certain endophenotypes of schizophrenia (Savonenko et al., 2008). Moreover, total BACE inhibition subtly impairs synaptic function, as well as BACE’s role in adult myelination of peripheral nerves (see ARF related news story). In contrast, paring back BACE by half reduced amyloid deposition while avoiding those side effects, Wong said.
On γ-secretase, the danger of inhibiting notch cleavage is well known. Wong’s group has used various different strains of heterozygous and homozygous knockout mice that ranged from 25 to 75 percent of normal γ-secretase activity to show that γ-secretase serves as a tumor suppressor in the skin and, at least in mice, can be safely inhibited globally by no more than a third. That was enough to reduce amyloid deposition (Li et al., 2007).
New at the Eibsee conference, Wong presented data on combining moderate inhibition of both BACE and γ-secretase. The idea behind this approach is that because both enzymes act on APP but the majority of their other substrates do not overlap, hitting both might enhance the desired effect and dilute the unwanted effects. To see whether this pans out, Wong’s group crossed mice heterozygous for both BACE and the γ-secretase component Aph1a with a mutant PS/APP strain and found that this moderate genetic reduction of both secretases achieved a greater attenuation of amyloidosis in the brain as compared to either of the single heterozygotes. The side effect profile was encouraging: myelination, a series of schizophrenia endophenotype tests used on prior knockout strains, were all normal, as was the skin and spleen, which are commonly affected in γ-secretase knockouts. These new mice are still aging but appear to have a normal lifespan. “ The therapeutic effect was additive, the side effects were not. This might be an attractive therapeutic strategy for AD, particularly for prevention, because these moderate reductions are not associated with toxic side effects,” Wong said.
Others at the conference agreed with this concept but pointed out that titrating the precise extent of the inhibition may be less practical in elderly humans than in genetic models. For example, some γ-secretase inhibitors have behaved paradoxically where low doses of inhibitor unexpectedly increased Aβ and only higher drug doses decreased the peptide (Lanz et al., 2006; Burton et al., 2008).
In his talk, Michael Willem of the University of Munich, noted that the potential risks of BACE inhibition as a drug target are only beginning to come to light. One controversy at this point is whether BACE participates in myelination both in the brain and peripheral nervous system, or only the latter. Willem’s ongoing research reinforces his and Christian Haass’s initial finding that BACE’s role in myelination plays out primarily in Schwann cells, not oligodendrocytes, hence is not needed for myelination in the brain (for a paper suggesting the opposite, see Hu et al., 2006). Recent crosses of BACE knockout mice to neuregulin heterozygote knockout mice showed profound hypomyelination, Willem reported, but again only of peripheral nerves.
But even if BACE1’s supporting role in myelination is predominantly peripheral, what that means for BACE inhibition in people with AD remains to be understood. Willem addressed this issue with LY-2434074, a BACE inhibitor made by Eli Lilly and company that will remain an experimental tool because the P glycoprotein transporter pumps it out of the brain too fast for it to be a serious drug candidate. When given to mice, this inhibitor effectively reduced Aβ levels in the brain and plasma of mice; however, the BACE substrate neuregulin accumulated along with APP, Willem noted. “Every substrate will accumulate to some extent if BACE is blocked,” he said. To what extent this will happen in the human brain is far from clear, in part because other proteases, including perhaps ADAM-17, appear to compensate partially for the loss of BACE1, Willem added (see also Sankaranarayanan et al., 2008). Neuregulin has many different functions in the nervous system. Many of them occur during development, but particularly BACE1s possible role in synaptic plasticity should be carefully studied in the course of pursuing BACE inhibition, Willem urged (see also Wang et al., 2008).
On the pharmaceutical front, new BACE inhibitors keep appearing in the literature (e.g., Chirapu et al., 2008; Cole et al., 2008; Silvestri, 2008) and known inhibitors are becoming better understood preclinically (Sankaranarayanan et al., 2008; Meredith et al., 2008). But to date, no Phase 2 trials of BACE inhibitors have yet been reported, even though several large companies are said to have begun human testing (see ARF related Keystone story for a Phase 1 example).
One-Two Punch: Can a Single Drug Block Two Enzymes?
Stefan Lichtenthaler picked up Wong’s concept of putting a mild damper on both β-secretase and γ-secretase in the hope of reducing amyloidosis safely. This would seem to require two drugs, as these two enzymes differ in all sorts of regards—where they are in the cell, how they cut, and what level pH they prefer, for example. But surprisingly, Lichtenthaler reported that he has come across compounds that he believes might eventually be able to pull off such a twofer. Lichtenthaler’s group ran a chemical genetics screen to look for small molecules that tweak APP shedding. Besides some known activators and inhibitors, the screen turned up bepridil, an old drug that is still being used in France and Japan to treat heart disease, particular chronic angina. This clinical record established that bepridil is safe for chronic use at plasma concentrations of some 3 to 10 micromolar. Bepridil has a role in blocking calcium channels and calmodulin. Curiously, closer examination of this drug showed that it is a dual modulator: it inhibits β-secretase and, separately, also tweaks γ-secretase, Lichtenthaler said.
The scientists first examined bepridil’s effect on BACE. In cell culture, bepridil blocks the enzyme with an IC50 of 6 micromolar. That is in the range achievable in human plasma in clinical practice, and bepridil is known to cross the blood-brain barrier. Whether this IC50 is potent enough to do much to BACE in the brain is unclear at this point; animal studies measuring Aβ levels in brain will address this question. (Again in cell culture, bepridil also blocked cleavage of neuregulin, Lichtenthaler noted.) To get a sense of how the drug might interfere with BACE1, the scientists first looked to its known role antagonizing calmodulin, but soon ruled that out. Instead, the group is pursuing a working hypothesis whereby bepridil affects BACE indirectly by raising the pH near the membrane of endosomes, where BACE is thought to work, upward from BACE’s low acidic comfort level.
Regarding bepridil’s effect on γ-secretase, the scientists found that it behaves like a typical “inverse” GSM. That is, in cells expressing the γ-secretase APP substrate C99, it boosted Aβ42, lowered Aβ38, and left unchanged Aβ40 and total Aβ. This, clearly, is the opposite of what AD scientists want from a γ-secretase modulator. Intriguingly, though, previous research by conference attendee Boris Schmidt of the Technical University of Darmstadt has shown that such inverse modulators can be flipped chemically to do the right thing. Turn a certain ester moiety into a carboxylic acid and, voila, you’ve got yourself an Aβ42-lowering GSM (Narlawar et al., 2007). Lichtenthaler is collaborating with Schmidt’s group to synthesize derivatives of bepridil that behave like a “straight” (i.e., Aβ42-lowering) GSM yet maintain their penchant for blocking BACE, as well. On his wish list of chemical characteristics for a perfect dual modulator are an aromatic core that lets the compound sink into the membrane to affect γ-secretase there, a COOH group to make sure the compound modulates the enzyme complex in the desired direction and, finally, an amine group necessary for upping the endosomal pH. At this stage, these studies are primarily geared to understanding the dual mechanism of bepridil and similar compounds. If this line research ever comes to pass as a dual-mechanism drug, one advantage would be that the patient would have to take one pill, not two, Lichtenthaler noted. —Gabrielle Strobel.
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