Development of noviomimetics that modulate molecular chaperones and manifest neuroprotective effects

Article abstract of DOI:10.1016/j.ejmech.2017.10.038

Heat shock protein 90 (Hsp90) is a chaperone under investigation for the treatment of cancer and neurodegenerative diseases. Neuroprotective Hsp90 C-terminal inhibitors derived from novobiocin (novologues) include KU-32 and KU-596. These novologues modulate molecular chaperones and result in an induction of Heat Shock Protein 70 (Hsp70). “Noviomimetics” replace the synthetically complex noviose sugar with a simple cyclohexyl moiety to maintain biological efficacy as compared to novologues KU-596 and KU-32. In this study, we further explore the development of noviomimetics and evaluate their efficacy using a luciferase refolding assay, immunoblot analysis, a c-jun assay, and an assay measuring mitochondrial bioenergetics. These new noviomimetics were designed and synthesized and found to induce Hsp70 and improve biological activity. Noviomimetics 39e and 40a were found to induce Hsp70 and exhibit promising effects in cellular assays.

Full text of DOI:10.1016/j.ejmech.2017.10.038

Accepted Manuscript
Development of noviomimetics that modulate molecular chaperones and manifest
neuroprotective effects
Leah K. Forsberg, Mercy Anyika, Zhenyuan You, Sean Emery, Mason McMullen, Rick
T. Dobrowsky, Brian S.J. Blagg
PII:
S0223-5234(17)30838-3
10.1016/j.ejmech.2017.10.038
EJMECH 9830
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 August 2017
Revised Date: 9 October 2017
Accepted Date: 14 October 2017
Please cite this article as: L.K. Forsberg, M. Anyika, Z. You, S. Emery, M. McMullen, R.T.
Dobrowsky, B.S.J. Blagg, Development of noviomimetics that modulate molecular chaperones and
manifest neuroprotective effects, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.038.
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ACCEPTED MANUSCRIPT
Development of Noviomimetics that Modulate Molecular
Chaperones and Manifest Neuroprotective Effects
Leah K. Forsberg, † Mercy Anyika,† Zhenyuan You, ‡ Sean Emery, ‡ Mason McMullen,‡ Rick
T. Dobrowsky‡ and Brian S. J. Blagg*,$
†Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The University
of Kansas, Lawrence, Kansas 66045-7563
‡Department of Pharmacology and Toxicology Department, The University of Kansas,
Lawrence, Kansas 66045, United States
$Department of Chemistry and Biochemistry, The University of Notre Dame, Notre Dame,
Indiana 46556, United States
*Author to whom correspondence should be addressed. Phone: (574) 631-6877. Email:
bblagg@nd.edu
TOC Abstract:
H
R
N
O
O
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MeO
H
HO
F
R
OH
N
H
Additional Interactions
with the binding pocket
O
N
O
X
O
O
Dually Improved
Noviomimetics
F
H
N
O
MeO
KU-596
HO
F
OH
O
O
X
Simplified
Synthesis
F
Abstract
Heat shock protein 90 (Hsp90) is a chaperone under investigation for the treatment of cancer
and neurodegenerative diseases. Neuroprotective Hsp90 C-terminal inhibitors derived from
1

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novobiocin (novologues) include KU-32 and KU-596. These novologues modulate
molecular chaperones and result in an induction of Heat Shock Protein 70 (Hsp70).
“Noviomimetics” replace the synthetically complex noviose sugar with a simple cyclohexyl
moiety to maintain biological efficacy as compared to novologues KU-596 and KU-32. In
this study, we further explore the development of noviomimetics and evaluate their efficacy
using a luciferase refolding assay, immunoblot analysis, a c-jun assay, and an assay
measuring mitochondrial bioenergetics. These new noviomimetics were designed and
synthesized and were found to induce Hsp70 and result in improved biological activity.
Noviomimetics 39e and 40a were found to potently induce Hsp70 and in very promising
effects in cellular assays.
Keywords
Molecular Chaperones, Heat Shock Protein 90, Heat Shock Protein 70, Novologues,
Noviomimetics, Neurodegeneration
Introduction
Molecular chaperones represent promising therapeutic targets for both cancer and
neurodegenerative diseases.1-4 The 90kDa Heat Shock Protein 90 (Hsp90) is a molecular
chaperone that is responsible for the folding and maturation of more than 300 client protein
substrates.5 Hsp90 modulates client protein substrates associated with all 10 hallmarks of
cancer, supporting that inhibition of this chaperone may be useful for the treatment of
cancer.6-8 However, Hsp90 is also a target for neurodegenerative diseases since molecular
chaperones can refold denatured proteins and resolubilize protein aggregates.3, 9-14 The N-
terminus of Hsp90 contains an ATP-binding site, and ATP hydrolysis provides the requisite
energy needed to fold client protein substrates.15-16 Currently, there are six N-terminal
inhibitors undergoing clinical evaluation for the potential treatment of cancer.17-18
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Unfortunately, inhibition of the N-terminus and subsequent client protein degradation also
leads to induction of the heat shock response (HSR), which results in the overexpression of
19-21
the heat shock proteins, Hsp27, Hsp40, Hsp70 and Hsp90.
In contrast to N-terminal
inhibitors, C-terminal inhibitors can segregate client protein degradation from induction of
the pro-survival heat shock response.22
The DNA gyrase inhibitor, novobiocin (1), was the first Hsp90 C-terminal inhibitor
discovered.23 Through extensive structure-activity relationships, the natural product was
modified to exhibit anti-proliferative activity.24-26 Continued development of the potential
cancer therapeutics led to the identification of cytotoxic replacements of the noviose sugar,27-
29 which improved inhibitor synthesis, as well as identification of new cores that target Hsp90,
which could be used instead of the coumarin core.30-33 These cytotoxic compounds provide
an excellent platform for the treatment of cancer, as these analogs induce the degradation of
Hsp90-client proteins, which in cancer cells leads to the disruption of multiple oncogenic
pathways.2
Structure-activity relationship studies previously identified KU-32 (2) as an
efficacious neuroprotective agent in cellular models of Alzheimer’s disease.34-35 Modulation
of Hsp90 by 2 results in the induction of Hsp70 levels, which can refold denatured proteins,
decrease the levels of abnormal proteins, and prevent protein aggregation, which can
ultimately alleviate the phenotypes associated with many neurodegenerative diseases.36
Subsequent studies demonstrated that 2 reversed clinical symptoms of Diabetic Peripheral
Neuropathy (DPN) in vivo and protected against neuronal glucotoxicity.37-39 A homology
model of the Hsp90 C-terminus was then utilized to design a second generation of analogues,
termed novologues (novobiocin analogues), which exchanged the coumarin core of 2 with a
biphenyl ring system, and ultimately produced 3 (Figure 1A: Previous studies).40-41 Although
3 exhibits promising activity and is undergoing pre-clinical evaluation, modeling studies with
3 identified unexplored regions within the C-terminal binding site that may improve its
3

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activity, which ultimately led to the identification of noviomimetics (a non-noviosylated
compound) 4.42 Preliminary studies with 4 confirmed that these noviomimetics maintained
the desired biological activity of 3, but are more synthetically desirable due to removal of the
complex noviose sugar.42-46 As a result, a new library of novologues and noviomimetics were
designed in which modifications were incorporated to gain additional interactions with the
binding pocket (Figure 1A: Current Study). Several compounds obtained from this third
generation library exhibit improved activity when compared to 3 and 4 as described herein.
A.
Current Study:
Previous Studies:
H
H
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O
MeO
MeO
OH
KU-596
3
O
MeO
KU-32
2
O
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OH
F
HO
F
HO
OH
OH
Additional interactions within
the binding pocket
F
Dually improved
noviomimetics
H
N
OH
H
N
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N
O
O
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MeO
KU-1202
4
Novobiocin
1
Simplified synthesis
O
F
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OH
H2N
O
X
B.
OMe
H
H
H
N
N
N
O
O
O
O
O
O
29
4
3
O
O
MeO
MeO
HO
F
F
HO
F
OH
OH
O
Figure 1. A). A summary of prior and current studies presented herein. B). Compounds 3
(KU-596), 4, and 29 docked to Hsp90 C-terminal homology model.
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Results and Discussion
A homology model of the Hsp90 C-terminus was utilized for the design of new
novologues, and demonstrated the potential for a binding pocket adjacent to the noviose
sugar in 3 as illustrated in Figure 1B. During prior studies wherein replacements of the
noviose sugar were explored, 4 was identified as a simplified novologue that exhibited
similar biological activity to 3.42 As shown in Figure 1B, the noviose surrogate projects into
this previously unexplored binding pocket and therefore modifications to the benzyl ring on 4
were explored to identify improved modulators. Modifications at the ortho- position were
investigated to determine whether hydrogen bonding interactions could be established with
GLU537, GLN682, or nearby amino acids (Figure 1B). Substituents at the meta- and para-
positions were explored to identify potential hydrophobic interactions at this location. In
addition, modification to the central ring of 3 was sought to determine whether hydrogen
bond donors or acceptors could interact with the peptide backbone (Figure 1B). The
substituents chosen for these studies included both electronic and steric functionalities to
establish preliminary structure-activity relationships.
Synthesis of phenol 941 (Scheme 1A) commenced by benzyl protection of 2,4-
dihydroxybenaldehyde, 5. The resulting benzyl ether was converted to
trifluoromethanesulfonate 6, in the presence of N-Phenyl bis-trifluoromethane sulfonimide
and potassium carbonate using microwave conditions.47 A subsequent Suzuki coupling
reaction with commercially available 3-fluorophenyl boronic acid was employed to generate
the biaryl ring system found in 7, which was then subjected to a Henry reaction, followed by
simultaneous reduction of both the nitro and olefin functionalities with lithium aluminum
hydride to yield amine 8. Acylation of 8 was followed by cleavage of the benzyl ether under
hydrogenolysis conditions to afford the phenol, 9.
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Scheme 1. A). Synthesis of phenol 9. Reaction conditions to make phenol 9: a. BnBr,
NaHCO3, CH3CN, 75%; b.(CF3CO)2O, Et3N, DCM, 60%; c.3-fluoro-phenylboronic acid,
Pd(PPh3)4, K2CO3, DMF, 90%; d.CH3NO2, NH4OAc, 98%; e.LiAlH4, THF, 30 min, f.
Ac2O, Et3N, 72% over 2 steps, g.H2, Pd/C, MeOH, 90%. B). Synthesis of Noviomimetics
14b-u: h. TsCl, Pyridine, 0 oC-rt, on CHCl3; i. NaH, Acetonitrile, rt, 16hr; j. K2CO3, DMF,
80oC, 2d, 55-80%; b: x = o-OCH3; c: x = o-CH3; d: x = p-Br; e: x = p-Cl; f: x = o-F; g: x =
o-Br; h: x = o-Cl; i: x = m-Br; j: x = m-Cl; k: x = m-OCH3, l: x = p-F; m: x = p-OCH3; n: x
= p-CH3; o: x = p-CF3; p: x = p-tBu; q: x = 2, 6-dichloro; r: x = 2-OCH3, 4-Cl; s: x = 2-Br,
4-Cl; t: x = 2-naphthalene; u: x = 2-OCH3, 4-CH3. C). Synthesis of compounds 18 and 19:
k. NaH, DMF, rt, on, 60%; l. PPh3, DIAD, THF, rt, 3h, 58%; m. OsO4, NMO, THF/H2O,
on, 83%.
Compounds 14b-u were obtained via an SN2 substitution reaction between phenol 9
and the toluenesulfonates of the corresponding sugar surrogates (Scheme 1B).48 Compound
18 was obtained by a Mitsunobu reaction between phenol 9 and the corresponding sugar
mimic. Novologue 18 was then subjected to an osmium tetroxide catalyzed dihydroxylation
to obtain the corresponding diol, 19 (Scheme 1C).
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Scheme 2. A). Synthesis of 26. a. (CF3CO)2O, Et3N, DCM, 60%; b.
3-fluoro-phenylboronic acid, Pd(PPh3)4, K2CO3, DMF, 90%;
c.CH3NO2, NH4OAc, 98%; d. LiAlH4, THF, 30 min; e. Ac2O, Et3N,
72% over 2 steps; f. H2, Pd/C, MeOH, 90%; g. BF3OEt, DCM,
40%; h. Et3N, MeOH, 85%. B). Synthesis of 29: i. Me2SO4, K2CO3,
H2O/Acetone (1/1), 55%; C). Synthesis 31: j. MOMCl, DIPA,
DCM, 65%; D). Synthesis of 34: k. NaHCO3, BnBr, MeCN, 45%; l.
MOMCl, DIPEA, DCM, 70%; E). Synthesis of 37: m.
Hexamethylenetetramine, Trifluoroacetic Acid, 25%; n. NaHCO3,
BnBr, KI, MeCN, 75%.
Synthesis of each derivative that contained a modified core began via different
starting materials. Synthesis of compound 26 commenced by reduction of commercially
available 2,4-dihydroxybenzaldehyde with sodium cyanoborohydride in the presence of HCl
(Scheme 2A).49 The resulting methylbenzene was formylated to obtain 2,4-dihydroxy-5-
methylbenzaldehyde, which was then benzyl protected to produce 20.50 Compound 20 was
then subjected to the same conditions utilized to produce phenol 9, and ultimately yielded
phenol 25 (Scheme 2A). The noviose sugar was synthesized using a previously reported
7

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procedure.43-46 The protected sugar was activated (24)51 and then coupled with 25 using
Schmidt conditions.52 Solvolysis of the carbonate with triethylamine and methanol provided
the final product, 26. Synthesis of 29 and 31 began by formylation of phloroglucinol,
followed by benzyl protection of 2,4,6-trihydroxybenzaldehyde using potassium carbonate
and benzyl bromide to yield 27. To achieve the final product 29, 27 was subjected to
dimethyl sulfide in water and acetone to yield the methyl ether, 28 (Scheme 2B).53 This key
intermediate was then subjected to the conditions previously described in Scheme 2B, to
obtain 29.
Compound 31 was obtained by the treatment of 27 with chloromethyl methyl ether
chloride to produce the mono-protected phenol 30 (Scheme 2C),54-55 which was then
subjected to the conditions previously described to give 31. Synthesis of compound 34 began
by selective benzylation of 2,3,4-trihydroxybenzyaldehyde, which was then subjected to
chloromethyl methyl ether formation to produce intermediate 33. Similarly, compound 33
was subjected to conditions previously described to achieve the free phenol, which was then
coupled with the trichloroacetamide of noviose carbonate and upon solvolysis, gave 34.
Synthesis of compound 37 was initiated by subjecting 2-nitrobenzene 1,3-diol to a Duff
reaction.56 The resulting product was then benzyl protected to yield compound 36, which was
modified according to the conditions highlighted in Scheme 2E to obtain the final product, 37.
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We have previously shown that the cytoprotective activity manifested by KU-32 and
KU-596 requires Hsp70 induction. Therefore, upon construction of the library of
noviomimetics, we evaluated Hsp70 induction via a luciferase reporter assay (Figure 2A).
Figure 2. A). Evaluation of Hsp70 induction via noviomimetics in a Luciferase
Reporter Assay. Results are mean ±SEM. (n = 3-9). c: x = o-CH3; d: x = p-Br; e: x
= p-Cl; f: x = o-F; g: x = o-Br; h: x = o-Cl; j: x = m-Cl; k: x = m-OCH3, l: x = p-F;
m: x = p-OCH3; n: x = p-CH3; o: x = p-CF3; p: x = p-tBu; q: x = 2, 6-dichloro; r: x
= 2-OCH3, 4-Cl; s: x = 2-Br, 4-Cl; t: x = 2-naphthalene; u: x = 2-OCH3, 4-CH3. B).
Induction of Hsp70 protein expression by select noviomimetics. 50B11 cells were
treated for 24hr with 1 µM of the indicated Novologues or DMSO, the negative
control. Immunoblot analysis was performed and densitometry was used to report
fold induction of Hsp70. A representative blot is located in Supplemental Figure 2.
As shown in Figure 2A, 14e induced the most robust Hsp70 levels for the new
noviomimetics. Generally, the noviomimetics with substitutions at the para- and ortho-
positions appear to manifest increased Hsp70 levels. Whereas, substitutions at the meta-
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position appear detrimental, as less Hsp70 induction occurred, 14i-k. The para-substituted
noviomimetics showed greater induction of Hsp70 than the corresponding ortho-
noviomimetics (14c, f-h). Generally, electron-withdrawing groups at the para- position
produced the most robust Hsp70 induction, followed by electron donating groups at the
ortho- position. Similarly, 19 is a para-substituted noviomimetic that contains a cyclohexyl
cis-diol and also yielded good activation of the luciferase promoter. The cis-diol of 19,
mimics the noviose sugar more than the unsubstituted cyclohexane ring, which may explain
its enhanced activity.
Immunoblot analyses were then performed to further examine the effect of select
noviomimetics on Hsp70 protein expression. Compounds 4, 14b, 14g, and 14h all resulted in
induction of Hsp70. Consistent with the induction of luciferase activity via the Hsp70
promoter; ortho- and para-substituted noviomimetics induced Hsp70 protein expression as
evidenced by 14b, 14g, 14h, 18, and 19 (Figure 2B). Additionally meta-substituted
noviomimetics resulted in a decreased induction of Hsp70 when compared to similarly
substituted ortho- and para-noviomimetics, as evidenced by 14q and 14o. Dually modified,
14r, demonstrated the least amount of Hsp70 induction, suggesting that the binding pocket
does not tolerate modifications at both the ortho- and para-position. Surprisingly, 18 and 19
were less effective in the immunoblot assay, despite a robust induction of luciferase activity.
The remaining para- and ortho- substituted compounds were similarly effective when
comparing activity between the luciferase assay and the immunoblot assay. Due to the
promising biological activity in both assays, 4 noviomimetic side chains (14b-e) were
selected for further investigation.
Comparison studies between the noviomimetic side chains and the original KU-32
coumarin scaffold were carried out to further elucidate positive neuroprotective effects.
Additionally, comparison studies were conducted to verify which core could provide the
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greatest Hsp70 induction. The coumarin containing noviomimetics (46-49, supporting
information, Figure SI) were synthesized with selected noviomimetic side chains. Using the
luciferase reporter assay, the phenol for each scaffold and noviomimetics 46-49, were
evaluated (Supporting Information, Figure S1). Scaffolds 9 and 45 did not contain any sugar
moiety/surrogate and were found to be inactive. Noviomimetics containing the KU-32
coumarin scaffold exhibited less activity than their biaryl counterparts (Figure SI).
The decrease in Hsp70 induction by 46-49 could result from the rigid coumarin core.
The biphenyl core offers enhanced flexibility that is not available with the coumarin core,
which may allow for improved interactions within the binding pocket. Increasing flexibility
may also be important due to the increase in length of the noviomimetic side chain compared
to noviose sugar. Substitutions on the central ring were evaluated using immunoblot analysis
to observe Hsp70 level and to support improved interactions with the binding pocket. The
novologues were analyzed at varying concentrations (30, 100, 300, 1000, and 3000nM) as
shown in Figure 3A.
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Figure 3. A). Immunoblot analysis of Hsp70 induction of novologues in
nontransfected 50B11 cells. 50B11 cells were treated for 24hr with the indicated
Novologues or DMSO as the negative control. Immunoblot analysis was performed
and densitometry was used to report fold induction of Hsp70. Results are mean ±SEM.
(n >2). B). Immunoblot analysis of Hsp70 induction of dually modified noviomimetics
in nontransfected 50B11 cells. 50B11 cells were treated for 24hr with the indicated
Noviomimetic or DMSO as the negative control. Immunoblot analysis was performed
and densitometry was used to report fold induction of Hsp70. A representative
immunoblot can be found in supplemental figure 3. Results are mean ±SEM. (n >2).
Novologues containing substituted central rings produced improved Hsp70 induction
as compared to 3 (Figure 3A). Novologues 26 and 34 resulted in the largest induction of
Hsp70. It is possible that the methyl substituent present in 26, overlays in three-dimensional
space with 2, and reinstates interactions that are lost when moving from the coumarin core to
the biphenyl ring system. Furthermore, the data supports the importance of having a
hydrophobic substitution on the central core, as 26 resulted in the greatest induction of Hsp70.
In continuation, the novologues containing central core substituents were coupled with
selected noviomimetics (13a-e). These noviomimetics were synthesized as described in
Scheme 2 and then analyzed via immunoblot analysis to evaluate Hsp70 induction.
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As shown in Figure 3B, noviomimetics 39a, 39d, 40a, and 41d resulted in the greatest
Hsp70 induction. Collectively, all noviomimetics with a methoxy group at the 3-position
(39a-e) resulted in increased Hsp70 levels compared to noviomimetics with other central ring
modifications. Interestingly, none of the methyl containing noviomimetics (38a-d) resulted in
improved Hsp70 induction, which was unexpected since 26 resulted in the greatest induction
of all of the central ring modifications. It is likely that switching from the noviose sugar to a
noviomimetic alters binding, and ultimately leads to a decrease in Hsp70 levels.
Noviomimetics containing a phenol on the central ring were similarly active when located at
the 3- or 6- positions (40a-e and 41a-e), and only the noviomimetic side chain seemed to
impact induction of Hsp70. Noviomimetic side chains a and d appear to produce the most
robust induction of Hsp70, regardless of the central ring modification, and therefore provide
desired neuroprotective activity.
Validation of Hsp90 Inhibition via the c-Jun Assay and Mitochondrial Bioenergetic
(mtBE) Assessment
Further assessment of the neuroprotective activity manifested by select noviomimetics
was carried out after confirmation of the desired cytoprotective response and induction of
Hsp70 levels. As such, noviomimetics 29, 31, 39d, 39e, 40a, 40b, and 41d were selected for
further biological evaluation in an assay to evaluate c-jun levels.
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Figure 4. c-Jun levels in 50B11 cells upon treatment with
1.0 µM of noviomimetic for 24 hours. C-Jun was
upregulated by placing the cells in 10% serum containing
media for 2 hours. The cell lysates were then collected for
immunoblot analysis.
A). c-Jun levels in response to
Noviomimetic treatment, normalized to SF Serum. B).
Representative immunoblot of c-Jun and Actin (control) for
the tested noviomimetics. Immunoblot analysis was
performed and densitometry was used to report fold
induction of c-Jun. Results are mean ±SEM. (n >2).
Hsp70 regulates c-jun levels by increasing its degradation via the proteasome (Figure
4).57 Therefore, induction of Hsp70 via noviomimetics results in the degradation of c-jun.
The assay was carried out by treating 50B11 cells with serum free media for 24 hours,
resulting in cell cycle arrest and a decrease in the expression of c-jun. The cells were then
treated with vehicle or 1.0 µM of the indicated noviomimetic, and c-jun was induced by
placing the cells in medium containing 10% FBS for 2 hours. The protein was harvested and
c-Jun levels were evaluated.
All of the selected noviomimetics decreased c-Jun levels when compared to the
untreated control (Figure 4). Importantly, most noviomimetics exhibited improved activity
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when compared to 3, and a few had improved activity as compared to 4. The general trend of
the data illustrates that noviomimetics with a free phenol at the 3-position were more
effective than noviomimetics with a methoxy at the 3-position. Excitingly, noviomimetic
40a resulted in the greatest reduction in c-Jun levels. Reduction of c-Jun levels by all
noviomimetics suggests that the mechanism of action occurs via induction of Hsp70.
Prior work indicated that the neuroprotective efficacy manifested by 2 to improve the
symptoms of diabetic peripheral neuropathy was associated with an increase in mitochondrial
respiration. To determine whether the increase in Hsp70 expression correlated with
mitochondrial respiration, 50B11 cells were treated with various noviomimetics for 24 hours
(Figure 5). Mitochondrial respiration was then measured in intact cells using an XF96
extracellular flux analyzer, which provides concurrent measures of mitochondrial oxygen
consumption rates (OCR) and extracellular acidification rates (ECAR) as an indication of
glycolytic activity. A variety of noviomimetics were selected to examine this correlation
between Hsp70 expression and mitochondrial respiration effects on mitochondrial respiration.
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Figure 5. Compound 19 enhances mitochondrial function. A). 50B11 cells were seeded
into the 96 well plates, maintained in high glucose DMEM supplemented with 10% fetal
bovine serum. After 12 hours, 50B11 cells were changed into high glucose DMEM
medium with 1% FBS, treated together with 1.0 µM 3, 4, 39d, 39e, 40a, 31. After 24 hr,
the cells were stressed with 0.5mM H2O2 for 2hr. H2O2 can largely inhibit 50B11
mitochondrial bioenergetics, indicated by largely decreased response to FCCP. 1µM 3,4
and 39e can significantly increase cellular Maximal Respiratory Capacity (MRC) of
oxidative stress. 50B11 cells were treated for 16 hr with DMSO, 3, 4, 31, 39e, 39d, 40a.
B). Bar graph representation of data acquired in A, * p<0.05 vs H2O2.
Hydrogenperoxide can induce cellular apoptosis through mitochondrial dysfunction.
0.5mM hydrogen peroxide can cause mitochondrial bioenergetics (mtBE) loss, indicated by
largely decreased response to FCCP. 3 and 4 effectively improved mtBE, however the dually
modified noviomimetics resulted in decreased maximal respiratory capacity (Figure 5).
Surprisingly, noviomimetic 39e was the most effective at increasing the MRC, even though it
was not as efficacious in the c-jun assay as the other noviomimetics. The different results
obtained from these two assays are due to each assay relying upon a different cellular
mechanism for the analyzed biological result.
Conclusion
In conclusion, a library of novologues was designed with the goal of replacing the
synthetically complex noviose sugar with a simplified sugar surrogates. The current work
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supports that noviomimetics can successfully retain the ability to increase Hsp70 expression
and mitochondrial function, two mechanistic features that are associated with the
neuroprotective efficacy of both biaryl-based novologues such as 4 and novobiocin-based
compounds such as 3. Noviomimetic 40a exhibited promising effects in the c-Jun assay,
while 39e was effective at increasing the MRC. Thus, noviomimetics may serve as a new
series of synthetically simplistic, neuroprotective compounds for treating various
neurodegenerative diseases.
Acknowledgments
This work was supported by grants [CA109265] to BSJB; [DK095911] to RTD and
[NS075311] to BSJB and RTD from The National Institutes of Health.
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Highlights
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•
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Treatment of 50B11 cells with Noviomimetics induced Hsp70 levels
Noviomimetics decreased c-JUN levels in 50B11 cells
Noviomimetic treatment improved mitochondrial bioenergetics