Nonpeptidic lysosomal modulators derived from Z-Phe-Ala-diazomethylketone for treating protein accumulation diseases

Article abstract of DOI:10.1021/ml300197h

Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak cathepsin B and L

Full text of DOI:10.1021/ml300197h

Letter
pubs.acs.org/acsmedchemlett
Nonpeptidic Lysosomal Modulators Derived from Z‑Phe-Ala-
Diazomethylketone for Treating Protein Accumulation Diseases
Kishore Viswanathan,† Dennis J. Hoover,‡ Jeannie Hwang,†,§ Meagan L. Wisniewski,§ Uzoma S. Ikonne,§
Ben A. Bahr,†,§ and Dennis L. Wright*,†,‡
†Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Connecticut 06269, United States
‡Synaptic Dynamics Inc., Farmington, Connecticut 06032, United States
§William C. Friday Laboratory, Biotechnology Research and Training Center, University of North CarolinaPembroke, Pembroke,
North Carolina 28372, United States
S
* Supporting Information
ABSTRACT: Lysosomes are involved in protein turnover and removing misfolded species, and their enzymes have the potential
to offset the defect in proteolytic clearance that contributes to the age-related dementia Alzheimer's disease (AD). The weak
cathepsin B and L inhibitor Z-Phe-Ala-diazomethylketone (PADK) enhances lysosomal cathepsin levels at low concentrations,
thereby eliciting protective clearance of PHF-τ and Aβ42 in the hippocampus and other brain regions. Here, a class of positive
modulators is established with compounds decoupled from the cathepsin inhibitory properties. We utilized PADK as a departure
point to develop nonpeptidic structures with the hydroxyethyl isostere. The first-in-class modulators SD1002 and SD1003 exhibit
improved levels of cathepsin up-regulation but almost complete removal of cathepsin inhibitory properties as compared to
PADK. Isomers of the lead compound SD1002 were synthesized, and the modulatory activity was determined to be
stereoselective. In addition, the lead compound was tested in transgenic mice with results indicating protection against AD-type
protein accumulation pathology.
KEYWORDS: Alzheimer's disease, cathepsin B, lysosomal modulator, PADK, nonpeptidic inhibitors
educing protein accumulation events is essential for
slowing the progression of Alzheimer's disease (AD), the
Lysosomes play an important role in normal protein turnover
and the clearance of misfolded or aggregated protein species.
The cathepsin family of lysosomal proteases appears to be
particularly responsive to accumulating proteins in neurons.
Protein accumulation stress up-regulates the message, protein,
and activity levels of cathepsins,11−13 including the stress
produced by Aβ1−42. Such responses may keep protein
accumulation partially in check and account for the gradual
nature of AD type pathology that can extend over many years
in patients. Enhancement of the lysosomal system has been
suggested as a plausible strategy to reduce aberrant protein
accumulation in age-related neurodegenerative disorders.11,13,14
Shown in Figure 1, the positive lysosomal modulators Z-Phe-
Ala-diazomethylketone (PADK; 1A) and Z-Phe-Phe-diazome-
thylketone (PPDK; 1B) are small molecules found in many
R
most frequent form of dementia. Accumulations of Aβ peptides,
PHF-τ, and related paired helical filaments are implicated as
contributing to the disease.1 There are no current treatments
that reduce the levels of protein accumulation, although several
different strategies are currently under development. There are
two classes of drugs that are approved for the treatment of AD.
They are (i) inhibitors of acetylcholinesterase that only affect
the symptoms of AD and do little to slow or reverse the course
of disease progression and (ii) an antagonist of the N-methyl-D-
aspartate (NMDA) receptor that is no better than placebo in
treating mild AD patients, and there is only meager evidence
for a benefit in a moderate form of the condition.2 New
treatments that directly affect the underlying pathology of the
disease are desperately needed.
The events associated with protein accumulation pathology
point to protein clearance as a treatment avenue to offset AD
type synaptic pathology, which is the best neurobiological
correlate of cognitive decline.3,4 In response to protein
accumulation, the lysosomal system exhibits regulatory events
as found in AD5−8 as well as in areas of the aged brain.9,10
Special Issue: Alzheimer's Disease
Received: July 13, 2012
Accepted: September 9, 2012
Published: September 9, 2012
© 2012 American Chemical Society
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Letter
a
Scheme 1. Synthesis of lead series of peptidomimetics
Figure 1. Structure of PADK and PPDK.
studies to enhance lysosomal enzyme levels and elicit protective
clearance of PHF-τ and Aβ42 in the brain.11,12,14−16
Recently, Butler et al.17 demonstrated that the pharmaco-
logically controlled modulation of the lysosomal system
promotes clearance of Aβ species in two mouse models of
AD. The findings also indicate that reducing intracellular
protein accumulation stress protects against AD type synaptic
decline. These observations suggested that PADK could serve
as an important lead in the development of a new class of
agents with the potential to treat protein accumulation
pathology. Although PADK has good therapeutic potential in
treating neurodegenerative disorders, there are obvious
concerns about its drug-likeness, namely, a protein reactive
diazomethylketone and a metabolically active peptide bond.
Herein, we report the enantioselective synthesis of peptidomi-
metics of PADK and PPDK with improved druglike properties,
enhanced up-regulation of cathepsin B, and neuroprotective
properties in a transgenic mouse model of AD.
a
Reagents and conditions. (a)TsCl, pyridine; (b) dihydropyran, PPTS,
CH2Cl2; (c) benzylamine, THF, reflux; (d) H2, Pd/C, EtOH; (e)
PhCH2OC(O)Cl, NaHCO3; (f) PPTS, EtOH, 60 °C; (g) Ac2O,
Et3N,DMAP, THF; (h) diketene, EtOH; (i) TsN3, DBU, AcCN; (j)
LiOH/H2O; (k) PPTS, EtOH, 40 °C.
Peptides generally suffer from poor in vivo stability and
bioavailability. A peptidomimetic strategy based on a
hydroxylethyl isostere was designed that would allow retention
of the pendant functionality found in PADK and PPDK. We
also chose to explore if the electrophilic diazoamide could be
replaced with a nonreactive capping group such that the
modulators would be expected to bind in a reversible manner
to their targets.
The synthesis of nonpeptidic lysosomal modulators (Scheme
1) started with the enantiometrically pure azidodiol 2, which
was prepared according to literature procedures.18 The
enantiomeric purity of the azidodiol was confirmed by optical
rotation (+31.2), which compared favorably with reported
values for this compound.19,20 Tosylation of the primary
alcohol followed by protection of the secondary alcohol as the
corresponding THP ether gave 3 in good overall yield.
Displacement of the tosyl group proceeded smoothly with
benzylamine to give the intermediate 4. Catalytic hydro-
genation of the azide followed by incorporation of a
carbobenzyloxy group on to the primary amine yielded the
common intermediate 5. Acylation of 5 followed by
deprotection of the THP group under mild acidic conditions
yielded analogue SD1002. Alternatively, with the intermediate
5, acetoacetylation was achieved with diketene, and subsequent
diazo transfer proceeded smoothly with tosylazide. Hydrolysis
under mild basic conditions effected deacetylation to yield the
desired compound SD1003, which retains the electrophilic end
group.
Figure 2. Cathepsin B inhibition by peptidomimetics. PADK and
nonpeptidyl compounds SD1002 and SD1003 were assessed for their
effects on cathepsin B activity. The cathepsin B assay used the
fluorogenic substrate Z-Arg-Arg AMC, and mean fluorescence units
SEMs were normalized to percent control.
a
Table 1. Extent of Cathepsin B Inhibition and Potency
compd
extent of inhibition (%)
IC50 (μM)
PADK
100 (P < 0.0001)
8−11 (NS)
13−17 (P < 0.01)
100 (P < 0.0001)
7−10
>100
SD1002
SD1003
CA074me
2−5
0.08−0.11
a
The cathepsin B assay used the fluorogenic substrate Z-Arg-Arg AMC
to titrate the effects of different compounds. Nonlinear regression
analyses used one-site competition equations to determine percent
inhibition and range of IC50 values. Unpaired t tests compared
untreated control levels of activity to data from the highest compound
concentrations used.
The nonpeptidic compounds SD1002 and SD1003 were
evaluated for cathepsin B inhibition since the parent PADK was
originally classified as an irreversible cathepsin B inhibitor
(Figure 2 and Table 1). PADK weakly inhibited cathepsin B
activity (IC50 = 9.4 2.4 μM); however, in hippocampal slices,
PADK at 3−10 μM up-regulated multiple cathepsin isoforms
without affecting the levels of actin and other proteins (Figure
3a). Note that PADK was unable to enhance cathepsin levels in
the presence of brefeldin A (Figure 3b), which impairs
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irreversible inhibition is not a prerequisite for lysosomal
modulation.
On the basis of the profile of compound SD1002, it is the
best suited to serve as a lead compound in future development.
To establish additional structure−activity relationships for this
new lead modulator, the role of configuration was probed
through the synthesis of two diastereoisomers of SD1002
(Scheme 2). The commercially available amino acid derivative
a
Scheme 2. Synthesis of Diastereomers of Lead Compound
Figure 3. Positive lysosomal modulation in hippocampal slice cultures.
Organotypic cultures prepared from rat hippocampi were treated daily
with vehicle, PADK, SD1002, or SD1003 (10 μM; n = 4−5 groups of
eight slices each), CA074me (0.3−1 μM; n = 6 groups), or PADK in
the presence of 1 μg/mL brefeldin A (BFA). (a) After 4 days, slice
groups were gently harvested and homogenized, and equal protein
aliquots were analyzed by immunoblot for active cathepsin isoforms
and actin. (b) The cathepsin immunoreactivity was determined by
image analysis, and mean IODs SEMs are shown. Representative
immunoblots are also shown for a separate experiment in which slice
cultures were treated with the enantiomers SD1006 and SD1007 daily
at 20 μM (c), twice the parent SD1002's modulatory concentration, to
illustrate the stereoselective lysosomal enhancement. Unpaired t tests
compared to control slices: *P < 0.01, **P < 0.001, and ***P <
a
Enantioselective synthesis of diastereomers. (a) MeI, NaHCO3,
DMF. (b) DIBAL, MeOH. (c) NaHSO3, KCN. (d) TBSCl, imidazole,
DMF. (e) H2, 5% Rh/C, EtOH/NH3. (f) Benzaldehyde, NaCNBH3,
MeOH. (g) AcCl, Et3N, CH2Cl2. (h) HCl, dioxane. (i) Cbz-Cl, Et3N,
CH2Cl2. (j) HF/pyridine, acetonitrile.
Boc-L-Phe 7 was converted into the known protected
aminoalcohol through the intermediacy of the cyanohydrins,
and reductive amination with benzaldehyde gave 8.23,24
Acylation of the amine gave acetamide 10 and the removal of
the Boc group under acidic conditions yielded the free amine.
Reacylation with a carbobenzyloxy group was followed by a
final desilylation to yield analogue SD1006. Following the same
synthetic route, the enantiomer of SD1006 (SD1007) was
synthesized from Boc-D-Phe 11.
The two enantiomers were tested for their ability to up-
regulate cathepsins B and D in cultured hippocampal slices. In
the right immunoblots of Figure 3a, only the SD1006 treatment
caused the positive modulation of active cathepsin isoforms.
Similar to previous reports on PADK,11,17 SD1006 has a bigger
modulatory effect on cathepsin B than cathepsin D. It is clear
from the quantitative results that cathepsin up-regulation is
stereospecific, as the SD1007 diastereoisomer shows negligible
modulation, whereas the enantiomer SD1006 produces an
effect comparable to that of SD1002 (Figure 3c). Interestingly,
the active enantiomer SD1006 showed no significant inhibition
of cathepsin B activity, indicating that the lysosomal
modulation is independent of enzyme inhibition.
SD1002 was used in a proof-of-concept study for protective
enhancement of lysosomal capacity by a nonreactive
peptidomimetic in an animal model. We had previously
employed this model to study the in vivo lysosomal modulation
by PADK. The APPSwInd mice were previously found to
exhibit synaptic decline and associated behavioral deficits,17 and
here, their hippocampal samples had a 35% reduction in GluR1
as compared to age-matched wild-type mice (P < 0.01; n = 10).
The lysosomal modulator SD1002 was administered daily into
the APPSwInd mice that express mutant human amyloid
precursor protein (hAPP), modified from the B6.Cg-Tg-
#
0.0001; test compared to PADK: P = 0.001.
endosome to lysosome traffic,21 further indicating as in
previous reports22 that the lysosomal enhancement involves
modulated trafficking/maturation of cathepsins. Interestingly,
cathepsin up-regulation was not observed upon treatment with
the cathepsin B inhibitor CA074me that is nearly 100 times
more potent than PADK (Table 1). Thus, more potent
cathepsin inhibition does not correspond with improved
lysosomal modulation. This led us to regard the weak inhibitor
PADK more properly as a lysosomal modulator and to expect
that similar phenotypic responses could be seen with analogous
compounds. In fact, the nonpeptidic analogues possess very
weak (SD1003) or negligible (SD1002) cathepsin B inhibition
as compared to PADK (Figure 2) but maintain the ability to
positively modulate cathepsin levels (Figure 3b).
The synthetic derivative SD1003 produced significant
lysosomal modulation in hippocampal slice cultures, as much
as twice the level produced by PADK (P = 0.01). Although
SD1003 could be a good point to do lead modification, the
presence of the reactive diazo group would produce significant
concern for a drug lead. The reactive group likely explains
SD1003's modest inhibitory activity with a similar IC50 as
PADK (see Table 1), while SD1002 in the absence of the diazo
group does not exhibit appreciable cathepsin B inhibition,
especially not in the 1−10 μM range. Even without the reactive
diazo moiety, SD1002 enhanced cathepsin levels to an extent
that was also significantly more than that produced by PADK,
and no statistical difference was found between the modulatory
effects of SD1002 and SD1003 (Figure 3b). Importantly, the
effects of the nonprotein reactive SD1002 suggest that the
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(PDGFB-APPSwInd)20Lms/2J strain (genotype confirmed by
PCR on tail DNAs). Loss of the postsynaptic GluR1 protein
and the more modest reduction in the presynaptic marker
amphiphysin I were both attenuated by SD1002 (Figure 4a).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 860-486-9451. Fax: 860-486-6857. E-mail: dennis.
wright@uconn.edu.
Funding
This work was supported in part by University of Connecticut's
Center for Science and Technology Commercialization, by the
Oliver Smithies Grant from the North Carolina Biotechnology
Center (Research Triangle Park, NC), and the Alzheimer's
Drug Discovery Foundation and the Association for Fronto-
temporal Dementias. The funding agencies had no role in study
design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Notes
The authors declare the following competing financial
interest(s): Prof. Ben Bahr and Prof. Dennis Wright are co-
founders of Synaptic Dynamics Inc..
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and characterization data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
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