A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity

Article abstract of DOI:10.1021/acschemneuro.7b00350

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aβ and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is rep

Full text of DOI:10.1021/acschemneuro.7b00350

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Letter
A Small Molecule Mimetic of the Humanin Peptide as a
Candidate for Modulating NMDA-Induced Neurotoxicity
Mohammad Parvez Alam, Tina Bilousova, Patricia Spilman, Kanagasabai
Vadivel, Dongsheng Bai, Denis Evseenko, and Varghese John
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00350 • Publication Date (Web): 21 Nov 2017
Downloaded from http://pubs.acs.org on November 23, 2017
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A Small Molecule Mimetic of the Humanin Peptide as a Candidate for
Modulating NMDA-Induced Neurotoxicity
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Mohammad Parvez Alam , Tina Bilousova , Patricia Spilman , Kanagasabai Vadivel , Dongsheng Bai ,
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Chris J. Elias , Denis Evseenko , and Varghese John
Affiliations:
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Drug Discovery Laboratory, Department of Neurology, UCLA, Los Angeles, CA, 90095, USA
Department of Orthopedic Surgery, DGSOM, UCLA, Los Angeles, CA, 90095, USA
Department of Orthopedic Surgery, University of Southern California (USC), Los Angeles, CA, 90033,
‡
§
USA
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ABSTRACT: Humanin (HN), a 24ꢀamino acid bioactive peptide, has been shown to increase cell
survival of neurons after exposure to Aβ and NMDAꢀinduced toxicity and thus could be beneficial in the
treatment of Alzheimer’s disease (AD). The neuroprotection by HN is reported to be primarily through
its agonist binding properties to the gp130 receptor. However, peptidic nature of HN presents challenges
in its development as a therapeutic for AD. We report here for the first time the elucidation of the
binding site of Humanin (HN) peptide to gp130 receptor extracellular domain through modeling and the
synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and
provide protection against NMDAꢀinduced neurotoxicity in primary hippocampal neurons. A brain
permeable small molecule mimetic was identified through exploratory medicinal chemistry using
microfluidic flow chemistry to facilitate the synthesis of new analogs for screening and SAR
optimization.
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KEYWORDS:
Humanin, gp130 receptor, NMDA, excitotoxicity, primary neurons, microfluidics
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Alzheimer’s disease (AD) is the most prevalent ageꢀrelated dementia and currently approved
therapeutics provide only temporary symptomatic relief. Therefore new approaches to therapeutic
development are urgently needed. Neuronal cell death is a key feature of AD pathology and factors
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leading to neuronal loss are many and not completely elucidated.
In 2001, Hashimoto et al. identified Humanin (HN) a 24ꢀamino acid peptide from occipital lobe of
postmortem AD patient brain tissue and showed that it protected neurons from amyloidꢀbeta (Aβ)ꢀ
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related toxicity. Muzumdar and coworkers reported a decrease in the endogenous HN plasma level with
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age, suggesting that decreased HN levels may be linked to cognitive decline during aging. The potent
HN analog HNG, with a substitution of serineꢀ14 to glycine, showed neuroprotection against Aβ1ꢀ42ꢀ
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induced toxicity in primary neurons at nanomolar levels. HN exerts its function through binding to both
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extracellular receptors and intracellular binding partners. The proposed extracellular binding partners
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include Gꢀprotein coupled receptors, formyl peptide receptor like (FPRL)ꢀ1 and 2;
and a trimeric
receptor complex of gp130, ciliary neurotrophic factor receptor α (CNTFR), and the ILꢀ27 receptor
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subunit, WSXꢀ1.
Hashimoto and coworkers reported that HN confers its protection against Aβ1ꢀ42 neurotoxicity through
agonism of gp130 and that is required for HNꢀmediated protection against Aβ1ꢀ42ꢀinduced death. In F11
neurohybrid cells transfected with amyloid precursor protein (APP) with the familial AD mutant V642Iꢀ
APP, there is significant neuronal cell death in vitro. Treatment with HN protects against this cell death,
but this protection is eradicated if the F11 cells are also treated with a gp130 neutralizing antibody. In
addition, the study shows that activation of STAT3 is essential for the HN activity, thus treatment of the
F11 cells with HN resulted in upꢀregulation of the tyrosine phosphorylation of STAT3 (pTyr705ꢀ
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STAT3). Immunofluorescence (IF) labeling in aged Tg2576 AD model mice showed that pSTAT3 levels
were significantly reduced in the dentate gyrus (DG) and CA1 brain regions of vehicleꢀtreated mice
while treatment intranasally with the HN derivative peptide Colivelin (CLN) restored pSTAT3 levels
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completely up to levels seen in nonꢀtransgenic mice. Furthermore, HNG has been shown to act through
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the gp130/IL6ST receptor complex to activate STAT3, AKT and ERK1/2 signaling pathways. These
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results support the reports, which suggest that an increase in gp130 signaling may be of benefit in AD.
Others have suggested that the antiꢀAD therapeutic benefits of HN could be through interaction with
residues (17–28) of monomeric Aβ that results in prevention of Aβ oligomerization and provides
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neuroprotection.
Recent studies have shown that HN can rescue neurons from NMDAꢀmediated toxicity in a manner
similar to the NMDA receptor antagonist MK801, but the mechanism of the rescue by HN is not through
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antagonism of the NMDA receptor. Excitotoxicity through NMDA receptor overꢀactivation is
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associated with disease progression in AD, therefore over the years there has been significant drug
discovery efforts to identify new NMDA receptor antagonists for treatment of AD and other neurological
disorders such as Parkinson’s disease, cerebral ischemia, and stroke wherein excitotoxicity is thought to
play a role. However, such antagonists have been associated with significant adverse effects and
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neurotoxicity. A partial antagonist of the NMDA receptor called memantine (Namenda ) was approved
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for AD as a symptomatic therapy over a decade ago but provides only limited benefit.
We show here ꢀ for the first time ꢀ that HN acts to rescue the NMDAꢀmediated toxicity in primary
hippocampal neurons through its interaction with the domainsꢀ4 and ꢀ5 (D4ꢀD5) of the gp130 receptor.
Our discovery was based on computational modeling of HN peptide and the extracellular domains of
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gp130. Interestingly, through screening and exploratory medicinal chemistry approaches we identified a
small molecule series that also binds to gp130 receptor in the same binding site between domains D4ꢀD5
as HN. The small molecule mimetics, like HN, rescue neurons from NMDAꢀmediated toxicity.
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Furthermore, our modeling analysis shows that a known antagonist, SC144, of gp130 also binds to the
same HN binding site and reverses the neuroprotection conferred by these mimetics providing
experimental evidence of their direct interaction with gp130 pathway. Thus, these small molecules are
HN mimetics revealing a new mechanism for suppressing NMDA receptor overꢀactivation and
excitotoxicity for new therapeutic development in neurodegenerative disorders such as AD.
RESULTS AND DISCUSSION
Our discovery efforts for small molecule HN mimetic began with a screening hit (RCGD)ꢀ423
(compound 1), which was found to be a regulator of cartilage growth and differentiation. This compound
was identified by our collaborator at UCLA through an HTS effort using the 200,000 UCLA screening
library, to find compounds that increase articular chondrocyte activation and facilitate joint repair.
Compound 1 was shown to bind gp130 initiating a signaling cascade comprising activation of the JAKꢀ
STAT3 pathway, phosphorylation of STAT3 (pSTAT3), and ultimately regulation of gene expression in
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chondrocytes (Shkhyan et al. submitted manuscript) and hair follicle stem cells. Given that HN has
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been shown to elicit its neuroprotective effects through gp130 receptor agonism, we wanted to use
compound 1 as a starting lead to conduct exploratory medicinal chemistry to find gp130 interacting HN
mimetics. We rationally designed and synthesized compounds 2–8 and evaluated their protective effects
against NMDAꢀinduced neurotoxicity.
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The ClusPro docking server was used to model the interactions between HN and the extracellular
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domains of gp130; this also revealed the HN binding domains D4ꢀD5 as a potential highꢀaffinity
binding sites for the HN peptide in the gp130 extracellular region (Figure 1). In addition, for compounds
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–8 we performed modeling studies using the Swissdock program; these again revealed domains D4ꢀD5
as a potential highꢀaffinity binding site in the gp130 extracellular region (Figure 1, Supporting
information, Figures S1–S7). The binding free energy of HN and compound 2 to gp130 D4D6 were
determined to be ꢀ46.99 (± 9.65) and ꢀ18.61 (± 2.30) kcal/mol, respectively using the MM/PBSA method
implemented in AMBER (see Supporting Information). The compound 2 remained bound to the gp130
D4D6 domain during the 50 ns molecular dynamic simulations similar to that seen with the larger 24
amino acid peptide HN and which has more interactions to the receptor due to its relatively larger size
than 2. For the small molecule mimetic 2 this implies good affinity of compound 2 for gp130 receptor at
the D4D6 domain, the calculated binding energy for compound 2 is ~2.5 fold lower than that of HN. The
known gp130 antagonist, SC144, was also modeled in parallel to investigate its binding specificity
(Supporting information, Figure S8). SC144 was found to have highꢀaffinity binding for the same site in
the D4ꢀD5 domains. Thus, modeling reveals that the compounds 1–8, the gp130 antagonist SC144 and
HN all bound to domains (D4ꢀD5) of gp130 receptor.
Compound 1 and analogs belong to the 2ꢀaminothiazoles family of compounds, and while many
methods have been reported to synthesize 2ꢀaminothiazoles, including solution, solid phase synthesis, a
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tandem oneꢀpot aqueous phase synthesis and microwave promoted synthesis. Our method for 2ꢀ
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aminothiazoles preparation involves Hantzsch condensation using 2ꢀbromoꢀ1ꢀphenylethanꢀ1ꢀone (and
its derivatives) and 1ꢀ(4ꢀbromophenyl)thiourea (and its derivatives) in a microfluidic reactor without the
use of a catalyst which is unprecedented (Table 1).
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We use a flowꢀchemistry approach that we have previously described, for our synthesis. Microfluidic
flow chemistry facilitated rapid syntheses of the new analogs. This is a green chemistry approach with a
cleaner reaction profile, smaller carbon footprint, higher yields, and quick scalability with minimum
reaction optimization enabling us to quickly gain SAR insights into the optimal gp130 binding
molecules for neuroprotection. The reactions are rapid, proceeding to completion in a few minutes and
products are obtained in good yields, in milligram quantities. As a result of these efforts, we report the
synthesis of new chemical entities (NCEs) such as Nꢀ(2,4ꢀdifluorophenyl)ꢀ4ꢀ(pyridinꢀ3ꢀyl)thiazolꢀ2ꢀ
amine (compound 6). With a number of analogs in hand, we next determined the ability of the small
molecules to confer neuroprotection against NMDAꢀinduced toxicity (Table 1).
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In this study, the cytotoxicity of NMDA was quantified by measuring the activity of lactate
dehydrogenase (LDH) released from primary cultured hippocampal neurons into the media and
confirmed by living cell density analysis using calcein staining. Our results showed that NMDA
treatment (100 ꢀM) for 2.5 h caused cytotoxicity in the primary neurons in vitro and triggered release of
LDH (Table 1 and Figure 2A). LDH level in the NMDA treatment group was about 26% higher than in
the DMSO control. MK801, a noncompetitive antagonist of NMDA receptor, was used as a positive
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control in these studies. As shown in Table 1 and Figure 2A, the addition of MK801 significantly
reduced, by 43 %, the release of LDH from NMDAꢀtreated neurons. We evaluated different batches of
HN and HNG from different vendors in our assay and only one of them (Tocris Biosciences, cat# 5154,
SI) was active and exhibited protection against NMDAꢀinduced neurotoxicity (Supplementary Figure
S9). Neither HN nor the compounds by itself had any effect on LDH levels from neurons which were
not treated with NMDA. However, the release of LDH was significantly inhibited in NMDAꢀtreated
neurons when treated with the HN mimetics. Neurons were pretreated with compounds for 16 h before
exposure to NMDA.
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The 2ꢀaminothiazole derivatives showed varied neuroprotective activity depending on the aryl
substitution on either side of the central aminothiazole core. As shown in Table 1 the levels of NMDA
induced LDH release was inhibited by 20% on treatment with our initial screening hit 1 while greater
inhibition of the LDH release was seen with the HN mimetic analogs 2–8 at 10 ꢀM. The levels of
NMDA induced LDH release was inhibited by 21% on treatment with compound 3, the pyridine analog
of compound 2. While compound 4, decrease the LDH levels by 32% compound 5 with two fluoro
groups at the ortho and para ꢀ position inhibited LDH release by 43%, similar to the activity of MK801.
Compound 6 is the pyridine analog of compound 5, and it inhibited NMDA induced LDH release
similar to what observed for compound 3. Compound 7 with the phenethyl group showed similar
inhibition as compound 3 and compound 6. Introduction of fluoro group at para position in both the
rings, compound 8, showed slightly worse inhibition of LDH release as compared to when the fluoro
group was just present only in one ring. Compound 2 was one of the best and its neuroprotective activity
was similar to MK801. The known gp130 antagonist, SC144, treatment reversed the neuroprotection
provided by compounds 2 or 8 against NMDAꢀinduced neurotoxicity (Figure 2A).
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Compound 2 was assessed for its downstream signaling based on its effects on STAT3, AKT and ERK
activation as previously reported upon HNG treatment in HEK293 cells, SHꢀSY5Y cells and primary
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hippocampal neurons. For our experiments we used the potent HN analog HNG17 in SHꢀSY5Y cells
and primary hippocampal neurons. Treatment of the cells with HNG17 and compound 2 (10µM) in
serum free media were performed for varying time periods as indicated in supplementary Figure S11. In
SHꢀSY5Y cells, compound 2 treatment showed a 2ꢀfold increase (as measured using the ratio of
densitometry values for the pꢀSTA3 and STAT3 bands) in phosphorylation of STAT3 within 10 minutes
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at its regulatory Tyr site (Supplementary Figure S11A), this is similar to that seen with HNG17 at 5
minutes. However, in primary hippocampal neuronal cultures the pꢀSTAT3 levels were below levels of
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detection both for HNG17 and compound 2 at all time points (Supplementary Figure S11B), this could
be due lower cross reactivity of the pꢀSTAT3 antibody for the mouse protein. Compound 2 treatment
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showed 9ꢀfold increase in phosphorylation of AKT at its regulatory Thr site in the serum free media
condition in SHꢀSY5Y cells (Supplementary Figure S11A) after 15 minutes incubation and 4ꢀfold
increase in primary cortical neurons for the same time (Supplementary Figure S11B). These increases in
pꢀAKT were greater than that seen with HNG17 in these cells. Compound 2 treatment caused a rapid 2ꢀ
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fold increase in phosphorylation of ERK1/2 at its regulatory Thr /Tyr site in the serum free media
condition in SHꢀSY5Y cell (Supplementary Figure S11A) and 1.2 fold in primary cortical neurons
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Supplementary Figure S11B), similar increase were seen with HNG17 in the SHꢀSY5Y cells and less so
in the primary neuronal cells. In SHꢀSY5Y cells, coꢀtreatment of compound 2 with gp130 antagonist,
SC144, inhibited phosphorylation of STAT3, AKT and ERK1/2, this effect is similar to what was
reported on treatment with the HN analog HNG treatment and with the antiꢀgp130 antibody or the
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gp130 antagonist SC144. This data supports the downstream signaling effects of compound 2 primarily
through its interaction with the gp130 receptor. Furthermore, coꢀtreatment of compound 2 with AG490,
a JAK inhibitor, greatly reduced the AKT phosphorylation indicating compound 2 acts upstream to JAK.
In contrast, ERK1/2 phosphorylation did not change much, as there are alternate pathways involved
besides through the trimeric gp130 complex involving ciliary neurotrophic factor receptor α (CNTFR),
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and the ILꢀ27 receptor subunit, WSXꢀ1, for activation of the ERK pathway. These data support
compound 2 being a HN mimetic at the gp130 receptor.
Calcein (calceinꢀacetoxymethyl ester; calceinꢀAM), a highly lipophilic nonꢀfluorescent and cell
permeable compound, staining was used to evaluate the density of living primary neurons. CalceinꢀAM
can be converted by intracellular esterases into calcein, an anionic fluorescent form, only labeling viable
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cells, and thus provides both morphological and functional information about living neuronal cells.
NMDA (100 ꢀM, 2.5 h) alone induced a significant decrease in neuronal survival ─ more than 50% cell
death observed. With MK801 pretreatment, the density of NMDAꢀtreated neurons was rescued to levels
better than DMSOꢀonly control, this indicates that the toxicity was induced by NMDA receptor
activation (Figure 2Bꢀ2D, Supplementary Figure S10). Furthermore, there was no difference in density
of living neurons between the compound 2 group and the DMSOꢀonly control group, suggesting that
compound 2 could also reduce NMDAꢀinduced toxicity similar to the direct antagonist, MK801.
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The neuroprotective effects of the compound 2 make it a promising candidate for further testing in AD
models. Since any potential therapeutic for AD needs to cross the bloodꢀbrain barrier (BBB), we further
evaluated the brain permeability of compound 2, in mice. Mice were dosed orally at 10 or 30 mg/kg, or
injected subcutaneously (SQ) at 10 mg/kg, and euthanized after 1, 2, 4, 6, and 8 hours postꢀdose. We
found that at 2 h after SQ delivery at 10 mg/kg the brain Cmax was 161 ng/g while dosing at 30 mg/kg
orally, however, resulted in the brain Cmax of 156 ng/g (0.57 ꢀM) as shown in Figure 3. The brain to
plasma ratio for 2 was ~4:1 for oral 30 mg/kg; and ~7.5:1 for 10 mg/kg SQ injection. Based on the good
brain permeability of compound 2 it is a promising lead candidate to conduct proofꢀofꢀconcept testing in
a mouse model of NMDAꢀinduced toxicity in future studies.
In the present study, we elucidate using modeling, the binding site of HN in the extracellular domains of
the receptor gp130. Our results show that HN and small molecule mimetics binds to the same site at the
interface of the D4ꢀD5 domain and provide neuroprotection from the NMDA neurotoxicity in primary
hippocampal neurons. The small molecule HN mimetics hold great promise towards developing an
orally available bioactive small molecule that confers the benefits of the humanin peptide. HN has
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shown to have multiple beneficial effects in neurodegeneration, diabetes, and artherosclerosis, but due to
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its peptidic structure, presents challenges in its development as a therapeutic.
Small molecule
mimetics of HN are attractive agents for therapeutic development both in AD and other neurological
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disorders where HN has shown beneficial effects such as in traumatic brain injury (TBI), stroke, Aβꢀ
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induced cerebrovascular dementia, and amyotrophic lateral sclerosis (ALS). Humanin has a number
of different cytoprotective and metaboloprotective effects such as cardioprotection against cellular
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apoptosis during myocardial injury, along with that HN modulate systemic insulin sensitivity and
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glucose homeostasis. Given the good oral brain permeability of the HN mimetic compound 2, it
represents a promising molecular tool for in vivo proofꢀofꢀconcept testing as a neuroprotective agent in
NMDA and Aβꢀinduced toxicity models. Further efforts will be made to explore SAR and expand our
library of small molecule HN mimetics, while evaluating the underlying gp130 signaling mechanism.
These agents could lead to a new pharmacological class of therapeutic agents for AD.
METHODS
General Procedure for Synthesis. The chemical synthesis of compound 2 began from commercially
available 2ꢀbromoꢀ1ꢀphenylethanꢀ1ꢀone and 1ꢀ(4ꢀfluorophenyl)thiourea. They were premixed in a
separate vial and were pumped through a preheated glass microfluidic reactor maintained at 80°C and 2
bar pressure (Syrris Asia Flow Chemistry Module) at a 500 ꢀL/min flow rate with a one minute
residence time in the reactor using Syrris Asia pumps to afford compound 2 from the output stream.
Other compounds were synthesized (Table 1) by similarly pumping corresponding starting material
through the preheated glass microfluidic reactor. The general procedure for cell testing and
pharmacokinetic analysis is provided in Supporting Information.
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Statistical Analysis. All the data were expressed as the mean ± SEM. Significant differences were
determined by Student’s tꢀtests, and oneꢀway ANOVA followed by Dunnett's multiple comparisons test
using GraphPad Prism 7 software. Values of * < 0.05, ** < 0.01, *** < 0.001 were considered
statistically significant.
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ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge on the ACS Publication website at DOI:
Supplementary data including biological methods, experimental procedures, western blot, additional in
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silico modeling figures, H NMR and C NMR of the synthesized compounds are provided (PDF).
AUTHOR INFORMATION
Corresponding Author
*
Varghese John, PhD. Associate Professor, Drug Discovery Laboratory, Department of Neurology,
UCLA, 3131 Reed, 710 Westwood Plaza, Los Angeles, CA, 90095, USA. Eꢀmail:
VJohn@mednet.ucla.edu.
Author Contributions
M.P.A., V.J., and P.S. has wrote the manuscript. V.J. contributed to the design and interpretation of the
experiments. M.P.A. synthesized the compounds, designed the experiments, helped T.B. with the
biochemical experiments, and analyzed the data. T.B. has designed and performed primary neuronal
culture, cell culture, biochemical experiments, and analyzed the data. K.V. has performed docking and
molecular dynamics studies. D.B. has performed PK study. C.J.E. has contributed by performing
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western blot. D.E. has provided the structure of initial hit. All authors have given approval to the final
version of the manuscript.
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Funding Sources
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This research was supported by the funding provided to V.J. by the Mary S. Easton Center for
Alzheimer’s disease Research
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
Mass Spectrometry Instrumentation was made available through the support of Dr. Greg Khitrov at the
University of California, Los Angeles Molecular Instrumentation Center – Mass Spectrometry Facility
in the Department of Chemistry.
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Table 1. Schematic for the microfluidic reactor for syntheses of compounds 1–8 and effect of small
molecule HN mimetics on NMDAꢀtriggered LDH release in primary hippocampal neurons.
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Figures
Figure 1. The predicted binding site for HN, compound 2 and SC144 in the extracellular domains D4ꢀ
D6 of gp130. (A) HN, compound 2 and SC144 are predicted to bind to the interface region
between extracellular domains 4 and 5 of gp130. The extracellular domains D4ꢀD6 of gp130 and HN
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(green) are shown in the model. Compound 2 (magenta) and SC144 (cyan) are shown in stick
representation. (B) Molecular surface representation of the gp130 extracellular domains 4ꢀ6 are shown
with their electrostatic potential. (C) and (D) The gp130 residues involved in interaction with the
compound 2 (magenta) and HN (green) are shown, respectively. Carbon atoms in the D4 domain are
colored light brown while the carbon atoms in the D5 domain are shown in cyan. The residues within 5
Å radius of compound 2 or HN are shown in the models.
Figure 2. (A) Effect of coꢀtreatment of gp130 antagonist, SC144, MK801 and compounds 2 and 8 on
NMDAꢀtriggered LDH release in cultured hippocampal neurons. NMDA increased LDH release (bar 2),
and both MK801 and compound 2 rescued the LDH increase (bars 3 and 4, respectively). Antagonist
SC144 prevented compound 2 rescue (bar 5). Compound 8 also rescued LDH and SC144 prevented this
rescue (bars 6 and 7, respectively). Each bar represents mean ± SEM of four independent observations.
Statistical significance was at P < 0.05. Effect of compound 2 on density of hippocampal neurons under
NMDAꢀinduced toxicity. Fluorescent microscopy was used to show representative calcein staining, and
cellular density of each group. (B) Control (DMSOꢀonly) density is shown. (C) A clear reduction of
neuronal density is seen as a result of NMDAꢀinduced toxicity. (D) With MK801 pretreatment, the
density of NMDAꢀtreated neurons was similar to the DMSOꢀonly control. (E) Pretreatment with
compound 2 also prevented NMDAꢀinduced toxicity and the density of living neurons was similar to
DMSOꢀonly control.
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Figure 3. Compound 2 levels in mice brain and plasma. Mice were dosed orally at 10 or 30 mg/kg, or
injected subcutaneously (SQ) at 10 mg/kg. Compound 2 is orally bioavailable at a dose of 30 mg/kg
with a Cmax ~0.57 µM level in the brain at 1 h post dose.
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