Neurotropic activity and safety of methylene-cycloalkylacetate (MCA) derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid

Article abstract of DOI:10.1021/acschemneuro.0c00255

Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model. In the present study we extended the characterizations of the in vitro neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro “off-target” pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic pharmacological characterizations including therapeutic efficacy in in vivo animal models of polyneuropathies, toward development of a clinically relevant neurotropic drug.

Full text of DOI:10.1021/acschemneuro.0c00255

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Article
Neurotropic activity and safety of methylene-cycloalkylacetate
(MCA) derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid
Adi Lahiani, Dikla Haham-Yaako, David Lankri, Susan Cornell-
Kennon, Erik M Schaefer, Dmitry Tsvelikhovsky, and Philip Lazarovici
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.0c00255 • Publication Date (Web): 15 Jul 2020
Downloaded from pubs.acs.org on July 16, 2020
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Neurotropic activity and safety of methylene-cycloalkylacetate (MCA) derivative
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-(3-allyl-2-methylenecyclohexyl) propanoic acid
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Adi Lahiani , Dikla Haham-Yaako
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Dmitry Tsvelikhovsky
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and Philip Lazarovici
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†The Institute for Drug Research, Division of Medicinal Chemistry, School of Pharmacy, Faculty of Medicine,
The Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
‡The Institute for Drug Research, Division of Pharmacology, School of Pharmacy, Faculty of Medicine, The
Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
+AssayQuant Technologies Inc. 260 Cedar Hill Street, Marlboro, MA 01752, USA
Corresponding Authors
*
E-mail: dmitryt@ekmd.huji.ac.il. Phone: (+972)2-675-7032. Fax: (+972)2-675-7076.
*E-mail: philipl@ekmd.huji.ac.il. Phone: (+972)2-675-8729. Fax: (+972)2-675-7490.
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Abstract
Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the
major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery
and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a
recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore
as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the
PC12 neuronal model. In the present study we extended the characterizations of the in vitro
neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on
dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood
biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro “off-target”
pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic
pharmacological characterizations including therapeutic efficacy in in vivo animal models of
polyneuropathies, towards development of a clinically relevant neurotropic drug.
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Keywords: Methylene-cycloalkylacetate, neurotropic activity, safety, off-target, GPCR, transporter,
enzyme, PGE
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, kinome, PC12, DRG, spinal cord neuron.
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. Introduction
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Polyneuropathy is a disease involving multiple peripheral nerves injuries, which affects nerves
responsible for pain, movement, or both. It may also affect the autonomic nerves responsible for
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controlling functions such as digestion, bladder activity, blood pressure and heart rate. The National
Institute of Neurological Disorders and Stroke, NIH, USA estimates that approximately 20 million
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people in the United States have some form of peripheral neuropathy. Clinically, neuropathy can arise
from nerve partial or complete Wallerian axonal degeneration which is also a common feature of
traumatic, ischemic, inflammatory, toxic, metabolic, genetic, and neurodegenerative disorders
affecting the central and the peripheral nervous system, leading to failure of nerve conduction and
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neural functions. Nonetheless, the most commonly used agents for neuropathic pain treatment include
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tricyclic antidepressants, anticonvulsants, and serotonin-norepinephrine reuptake inhibitors. To date,
not a single drug that can stop or reverse the nerve injury or degeneration can be found in the clinic.
Moreover, to the best of our knowledge, there are no drugs, which promote nerve regeneration.5
A
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P
major weaknesses of an existing polyneuropathic drug development program is the inaccurate choice
of a neuronal cellular target. In many polyneuropathies, there is very little, if any, death of the
neuronal cell body. The primary acute pathology is distal axonal injury and degeneration. Yet, most
high-throughput cell free, target drug-based screens use neuronal molecular targets and signaling
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molecules
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and fail to take in consideration the phenotypic induction, or enhancement of new axonal
sprouts which can reestablish the nerve circuit.
Neuroregeneration is a concept, which encompasses neuronal growth-promoting and inhibitory cues,
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leading to neuroplasticity and axonal outgrowth. The principal morphological characteristics of
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neuritogenesis are branching of neurites followed by elongation of axons and dendritic arborization. It
has become apparent that damaged neurons do regenerate in an active process by different
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mechanisms under treatment with neurotrophins such as nerve growth factor (NGF) and brain derived
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neurotrophic factor (BDNF). Neuritogenic substances hold the promise of therapeutic efficacy in the
treatment of neuronal injuries by the virtue of their ability to stimulate outgrowth of neurites from
neuronal cells11 which, cause a readjustment in the normal neuronal functions and local circuits in the
damaged nervous system. Therefore, use of the neurotrophic factors seems to be an important step in
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the process of neuronal regeneration.
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Many enhancers of nerve regeneration, such as stem cells
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and neurotrophin growth factors
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were found to enhance axonal regeneration after nerve injury in
animal models and found safe and efficient in neuropathic therapy in placebo-controlled, randomized
clinical trials,1
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but none has entered the clinic.
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Most probably, the key reasons for this failure are
the lengthy and costly regulatory process needed to evaluate complex biologicals for human use, the
inability of neurotrophins to cross the blood-brain-barrier, the short-term survival of implanted stem
cells, etc. To overcome this hurdle, use of natural and synthetic small molecules eliciting neuritogenic
(neurotropic, neurite outgrowth) activity alone or in combination with neurotrophins (NGF, BDNF) is
currently been focused as an alternative approach. Numerous agents have demonstrated the potential
to enhance neuronal repair following spinal cord or peripheral nerve injury using neurite outgrowth as
a biomarker for axonal extension in primary cell cultures and neuronal cell lines.17
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Recent drug
discovery reports suggest that different synthetic compounds alone,1
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or in combination with
neurotrophins, cooperate to induce the outgrowth of neurites in neurotrophic assays that identify
molecules that stimulate differentiation-neurite outgrowth of neuronal cells1
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, suggesting that
synthetic molecules may harmonize very well for the treatment of neuronal polyneuropathies.
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Therefore, towards improving and accelerating drug development for nervous system disorders
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is a critical unmet clinical need for a safe drug that enhances peripheral nerve regeneration after injury
or the dysfunction that results from polyneuropathies.
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In a recent study, we reported the synthesis of an unique family of methylene-cycloalkylacetate
MCAs)-based small molecule, (often observed as scaffold of various compounds and drugs of natural
origin) neurotropic drug (inducing neurite/axonal outgrowth) and indicated that the alkene element,
integrated within the cycloalkylacetate core, was obligatory for the neurotropic activity.21,22 Additional
studies have led to a wide variety of potential pharmaceutical candidates that share the methylene-
(
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cycloalkylacetate (MCA) scaffolds. Present study further characterizes the neurotropic activities of
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these compounds, specifically a candidate labeled HU-MCA-13, and analyzes its safety properties
using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro
“
off-target” pharmacological evaluations. This MCA derivative deserves further preclinical
mechanistic pharmacological characterizations and therapeutic efficacy in in vivo animal models of
polyneuropathies towards development of a clinically relevant neurotropic drug.
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. Results and Discussion
Synthesis and chemical characterization of HU-MCA-13:
Based on recently reported protocol for general and collective syntheses of methylene-caycloalkyl
acetates, the scaffold of HU-MCA-13 (Scheme 1) was successfully constructed through the simple
and straightforward sequence of transformations: α,α′-double alkylation of 1-(cyclohex-1-en-1-
yl)pyrrolidine 1; olefination of ketone 2 (access to intermediate 3); and hydrolysis (see section
Materials and Methods). 21, 24
Neurotropic activities of MCA-13:
MCA-13 induced neurite outgrowth in PC12 dopaminergic cell cultures. In the first step, using an
established PC12 neurotropic in vitro assay, we confirmed the neurotropic effect of HU-MCA-13 (Fig.
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, first row). Thereafter, by immunostaining, we found that cell bodies and neurite outgrowths of PC12
cells treated with 5 µM of HU-MCA-13 for 7 days expressed β tubulin III (Fig 2A), a cytoskeletal
protein which is specifically localized to neuronal microtubule network and its expression correlates
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with the earliest phases of neuronal differentiation. Next, we investigated the neurite outgrowth
enhancing effect of HU-MCA-13 when applied concomitantly with NGF, a known neurotrophin (Fig.
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A). We found that HU-MCA-13 alone at 10 μM, enhanced neurite outgrowth to a D
days and 0.30 at 7 days of treatment. NGF alone, at a regular dose of 50 ng/ml enhanced neurite
value of 0.54 at 2 days and 0.4 at 7 days of treatment (Fig. 3A). To verify a possible
additive or synergistic effect, we treated the cultures with a combination of low dose of NGF (1 ng/ml,
generating 10% neurite outgrowth = D of 0.05) and HU-MCA-13 (1 μM, generating about 10-15%
neurite outgrowth = D of 0.04), and found after 2 and 7 days of treatment, D of 0.85 and 0.38
respectively, findings indicating a significant synergistic effect. Since NGF plays an important role
f
value of 0.36 at
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outgrowth to a D
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during polyneuropathy inflammatory process , this observation further strength the prediction of a
potential synergistic enhancement regenerative effect of HU-MCA-13 upon in vivo delivery to an
animal model of neuropathy. To verify whether NGF mediates HU-MCA-13 neurotropic effect by Trk
A receptor, we pretreated the cultures with both 10 μM HU-MCA-13 and 250 nM K252a, a well-
known Trk A inhibitor. Under these conditions, NGF neurotropic effect was abolished (data not
shown) but not the inhibitory effect of K252a on HU-MCA-13 induced neurotropic activity (Fig 3A).
These findings may suggest that the neurotropic effect of HU-MCA-13 is not involving NGF-Trk A
receptor signaling.
HU-MCA-13 induced neurite outgrowth in dorsal root ganglion (DRG) sensory neurons. In the
second step, we study the effect of HU-MCA-13 on ex-vivo sensory neuronal cultures obtained from
dissociated dorsal root ganglia (DRG), a model in which the neurons tend to extend neurites in culture
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at a high rate, and all neurite outgrowths are axons, as confirmed by immune staining for cytoskeletal-
specific proteins.27 The results indicated that in the control medium, there was very poor spontaneous
neurite outgrowth of a length of about 200 micrometer (Fig. 1 and Fig. 3B) and a low ratio of 2
between the area covered by neurites and that covered by DRGs (Fig. 3C). By contrast, NGF induced
a strong, 3.5-fold increase in neurite outgrowth to a value of 700 micrometer (Fig. 1 and Fig. 3B) and a
ratio of 8.5 between the area covered by neurites and that covered by the DRG ganglia (Fig. 3C).
Similarly, HU-MCA-13 induced a 2.5 fold increase in neurite outgrowth to a value of about 500
micrometer (Fig. 1 and Fig. 3B) and a ratio of 5.4 between the area covered by neurites and that
covered by the DRG ganglia (Fig. 3C). However, the neurite outgrowths induced by HU-MCA-13
were less mature than in NGF-treated ganglia as evident from their shorter length and the lower
expression of the cytoskeletal-tubulin protein staining by comparison to NGF-treated DRGs (Fig. 2 C
compared to D). These findings may suggest that the maturation of the axons induced by HU-MCA-13
requires a longer period than for NGF, an issue deserving further experimentation. These experiments
extend the neurotropic effect of MCA-13 observed with PC12 dopaminergic neuronal cultures to DRG
sensory neurons.
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HU-MCA-13 induced neurite outgrowth in adult rat spinal cord primary cultures. To study the
effect of HU-MCA-13 on adult rat spinal cord primary cultures in vitro, we developed a purification
procedure that yields enriched different types of spinal cord neuronal cultures. This neurotropic assay
was performed using vehicle 1% DMSO in DMEM as control and 10 μM HU-MCA-13. The results in
Fig. 1 bottom row and Fig. 3D clearly indicate that in control there was very poor spontaneous neurite
outgrowth of a total length of about 180 µm and NGF induced neurite outgrowth to a total length of
about 800 µm. Similarly, HU-MCA-13 induced a neurite outgrowth of 600 micrometer. Fig. 2B
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indicates cytoskeletal-tubulin protein expression in the cultured neurons. These findings further extend
the neurotropic effect of HU-MCA-13 to spinal cord neurons, in addition to DRGs and PC12 cells.
We also examined whether HU-MCA-13 would promote the survival of neurons from the spinal cord,
by counting the number of neurons remaining after 48 hours in culture. From our counts we estimated
that 58 ± 10 % of the neurons present at 3 hours after plating survived for 48 hours in the presence of
HU-MCA-13, compared with only 35 ± 5% for neurons cultured without HU-MCA-13. While this
result supports the conclusion that HU-MCA-13 increased in vitro the survival of a variety of different
neuronal types within the spinal cord, present results do not exclude the possibility that certain types of
spinal cord neurons might not respond to HU-MCA-13, or that some respond more than others do.
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Safety of HU-MCA-13:
Single-dose, acute tolerance of HU-MCA-13 in mice. This pilot study was conducted according to
guidance for industry
single dose acute toxicity
testing
for pharmaceuticals
(
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/single-dose-acute-
toxicity-testing-pharmaceuticals) to obtain acute information of the dose toxicity of HU-MCA-13 in
male mice. Male C57BL/6 mice were injected intravenously with 0.2 ml of HU-MCA-13 in a dose of
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50 mg/kg. Mice were monitored for the sensory-motor performance during the first week after
injection using several routine motor tests, the neurologic function severity score (NSS) was measured,
but no neurological losses were observed (Table 1, Supporting information). The mortality and
changes on body weight, clinical signs and gross observation, were monitored up to 30 days after
treatment. We could not find any mortality at this high dose of 250 mg/kg. No significant changes on
body weight were detected by comparing HU-MCA-13 250 mg/kg to 1% DMSO treated groups: they
started with 21 and ended with 27 gram body weight. The mice were examined for autonomic
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symptoms by measuring salivation, urinary delivery, pupillary constriction, hair contraction, etc,
however, no differences were observed between the HU-MCA-13 and DMSO treated mice.
The hematopoietic system is one of the most sensitive parameters to assess the toxicity of drugs in
humans and animals. Tail vein blood samples were taken from DMSO 1% treated control (n=3) and
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50 mg/kg HU-MCA-13-injected mice (n=4) after 24 hours from injection and submitted for
hematocrit cell counting and biochemical analysis. Blood analyses are depicted in Figure 4. No
difference in blood count (Fig. 4A), electrolytes (Fig. 4B) and coagulation parameters (Fig. 4C) was
noted between the DMSO and HU-MCA-13-injected mice. The coagulation biomarkers (international
normalized ratio, prothrombin time, activated partial thromboplastin time, fibrinogen) were not
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affected by the exposure to HU-MCA-13 (Fig.4C) and similar to control mice. Of special importance
are normal liver (ALP, alkaline phosphatase; ALT, alanine transaminase and AST, aspartate
transaminase) functions and normal kidney (creatinine and blood urea nitrogen) functions, indicating
sufficient organ integrity at the end of the experimental period. The data obtained was similar to
29
hematological parameters reported for this mice strain and clearly indicates lack of differences on
kidney and liver parameters (Fig.4D) between the two mice groups. There was a small significant
increase in HU-MCA-13-injected mice on bilirubin concentration, a finding deserving further
investigation. Cumulatively these blood analyses indicate acute tolerability of 250 mg/kg HU-MCA-13
upon IV injection in C57BL/6 male mice.
Prioritizing compounds with a lower chance of causing toxicity, early in the drug discovery
3
0
process, would help in the drug development process. With this consideration we conducted acute
31
(
single-dose) toxicity study to determine the short-term adverse pathological effects on major mice
organs of HU-MCA-13 when administered in mice in a single dose, for a period of 24 h. One group of
5 mice received HU-MCA-13 orally at a dose of 2000 mg/ kg, dissolved in a nano-emulsifying drug
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delivery system in a bolus of 0.4 ml/mouse. After 24 hours exposure, the animals were sacrificed,
and organs were harvest for pathology analyses. In general, in all organs no cytotoxic lesions were
found (Fig. 5). In three animals administered with HU-MCA-13, we found in the last part of the colon,
rectum and the adjacent anal unhaired skin a severe purulent, necrotizing inflammation. This finding
was not related to the tested compound and was considered as an accidental finding. This information
on the lack of acute toxicity of HU-MCA-13 may predict the safety of this compound in future
therapeutic evaluation.
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HU-MCA-13 safety by in vitro pharmacological profiling. “Off-target” activity refers to all other
targets for which a molecule has affinity with the outcome of activation, blockade, or modulation,
resulting in a functional effect. In many cases, the off-target activity of the molecule can be sub-
clinical and not pose a concern. On the other hand, the off-target activity may result in side effects of
the active agent that range from a minor nuisance to a severe adverse event, resulting with drug
33
withdrawal. Therefore, in vitro pharmacological profiling is increasingly being used earlier in the
drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the
3
4
development of a candidate drug. With this background, assessing the specificity of HU-MCA-13
early in its development using highly relevant and predictive functional assays, which allow more
informed decisions about its safety, ultimately lead to the development of an effective neurotropic
drug. Tacking in account these considerations, we performed different pharmacological assays using
DiscoverX’s SAFETYscan, accumulating data points regarding the potential functional interaction
(
activation, inhibition, lack of effect) of HU-MCA-13 with physiological targets that includes G
protein-coupled receptors (GPCRs), ion channels, transporters, nuclear hormone receptors, and some
enzymes (Supporting Information-S2- Figure S2-8), targets known to show a clear correlation with
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observed in vivo toxic effects.34 We choose to investigate a concentration of 10 µM HU-MCA-13
which was not toxic to neurons and found very active in stimulating neurite outgrowth. We set a
threshold of 25% for inhibition or potentiation of activity on all tested targets as summarized in Table
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. The maximal response upon treatment with 10 μM HU-MCA-13 indicated that this compound was
not interacting with the majority of the targets, with the exception of the following G-protein coupled
receptors (GPCRs) : agonistic activity (increased cAMP level) towards α2A-adrenergic receptor
ADRA2A (24.5%); Cannabinoid receptor CB
1
-CNR1 (50.1%) Cannabinoid receptor CB2-CNR2
(
49.6%) and antagonistic activity (inhibition of cAMP level by 26.9%) towards Histamine H2 receptor
HRH2. Moreover, HU-MCA-13 inhibited by 25% Cyclooxygenase-1 activity (Table 2). These
findings may predict that these GPCRs and COX-1 may represent potential molecular targets of MCA-
1
3, and that upon using toxic, overdoses of HU-MCA-13 in animal or human studies, physiological
effects will be observed on : blood pressure, adrenaline release, sedation, increase of GI motility and
insulin secretion (ADRA2 related), euphoria and dysphoria, anxiety, memory impairment and poor
concentration, analgesia, hypothermia, increase in weight loss, emesis, depression (CNR1 related),
increased inflammation and reduction of bone mass (CNR2 related) and effects on gastric acid
secretion, emesis, positive heart inotropic effects (HRH2 related) as well as gastric and pulmonary
bleeding, dyspepsia and renal dysfunction (COX-1 related).34
“
On-target” activity refers to the site of action of the test substance (e.g., target receptor or enzyme)
3
5
that results in the desired pharmacodynamic effect associated with the treatment of disease. Since the
mechanism of HU-MCA-13 induced neurotropic effect is unknown, present findings may also suggest
the involvement of ADRA2A, CB1-CNR1 and/or CB2-CNR2, HRH2 and COX-1 in HU-MCA-13-
induced neurite outgrowth. This hypothesis is based on the reports indicating that activation of
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ADRA2A36 and CB1-CNR137 in PC12 cell cultures and inhibition of COX-1 in cholinergic
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neuroblastoma NG108-15 cells triggered neurite outgrowth.
Prostaglandin E (PGE ) is one of the major lipid mediators produced by the arachidonic acid
cascade. Arachidonic acid is produced in neurons by phospholipases A family members and is
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converted to PGE
2
by cyclooxygenase (COX)-1 and -2. It was reported that NGF induced COX-1
expression and enzymatic activity in PC12 cells.39 PGE
can enhance neuronal differentiation
2
expressed by stimulation of neurite outgrowth in neuroblastoma cell lines such as mouse NG108-15
40
41
cells and mouse DRG neurons. Therefore, to evaluate the possibility that HU-MCA-13 affects the
PGE production in PC12 cells, we treated the cultures for 24 hr with either different concentrations of
HU-MCA-13 or 1 µg/ml LPS, the lipopolysaccharide bacterial product known to increase PGE
production, cultures’ media was collected and PGE was measured by ELISA (Fig. 6A). It is evident
that HU-MCA-13 increased PGE production in a dose-dependent fashion by 3-6-fold over that
produced by control, untreated cultures. In another experiment, we investigated the individual and
combined effect of 50 ng/ml NGF and 1 µM HU-MCA-13 on production of PGE after 24 h treatment
of the PC12 cell cultures (Fig. 6B). NGF significantly increased the production of PGE but the
combined effect with HU-MCA-13 was weekly additive. These findings further support the
involvement of PGE production in HU-MCA-13 induced neurotropic effect and are calling for
2
2
2
2
2
4
2
2
2
additional research to clarify how partial inhibition of COX-1 affects arachidonate metabolic pathway
in relation to MCA-13 induced neurotropic activity.
HU-MCA-13 safety by in vitro evaluation using PhosphoSens® CSox based kinase assays. The
initial focus was to measure the modulatory activity in vitro of MCA-13 on protein kinases which
were reported to be involved in induction of neurite outgrowth.18, 43 To do this, we took advantage
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of a powerful method to measure the activity of recombinant protein kinases using a homogeneous and
continuous (kinetic) format, where the level of chelation-enhanced fluorescence (CHEF) is directly
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proportional to the amount of phosphorylated, real-time sensors consisting of sulfonamido-oxine
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(
Sox) chromophore linked to a peptide or protein substrates of selective kinases. This assay is ideal
for elucidating drug mechanism of action and is increasingly being applied earlier in the drug
development workflow to address off target effects and/or the challenges and opportunities for next
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generation protein kinase inhibitors. Since neurotrophins induce neurite outgrowth by activating the
different tropomyosin kinase receptors (Trk), and small molecules agonists of Trk receptors can
selectively activate Trks,46 we first investigated the ability of HU-MCA-13 to modulate TRKA,
TRKB and TRKC phosphorylation activities (Supplement-Figure Figure S3-2). We found no effects of
HU-MCA-13 on TRKA and TRK B and a a slight inhibitory effect on TRKC (no effect on the initial
rate, so not inhibition, but rather a small delay in the later stage of the progress curve as it approaches
the maximum signal (RFU Max), which can reflect reduced stability of the kinase). The TRKs
constructs used in the assay contained only the intracellular kinase domain (amino acids 442-786) and
therefore presently we cannot exclude that HU-MCA-13 may interact with the TRKs extracellular
domain thereby inducing the conformational change required for the kinase domain
activation.However this possibility is less likely, considering that K252a Trk A inhibitor didn’t
abolished HU-MCA-13 neurotropic effect (Fig.3A). . Moreover, no effects of HU-MCA-13were found
on other kinases (ASK1, ERK2, JNK3, p38α, CDK5, KDR-VEGFR, AKT1, GSK3β, PDGFRβ,
PKACα, PKC, CK1, IKKβ) which stimulate neurite outgrowth. Also, no effects were observed of HU-
MCA-13 on kinases (EGFR, IGF1R, FGFR 1 and 2, CAMK2α,) which inhibit neurite outgrowth or
calcineurin phosphatase which dephosphorylates the neurite outgrowth stimulatory protein, cofilin.47
The inability of 5 µM HU-MCA-13 to inhibit/activate significantly any of the kinases or phosphatase
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tested further confirm the kinases data from DiscoverX’s SAFETYscan and indicate off target safety
of HU-MCA-13.
Although, the cellular signaling direct mechanisms responsible for the neurotropic action of HU-
MCA-13 compounds were not directly addressed in the present research, we would like to propose a
few hypotheses. In view of the strong neurotropic effect of HU-MCA-13 in the different neuronal
systems investigated (Figs 1-3), the first option to be considered is that this compound activates
directly the TrkA-NGF receptor, representing an agonist inducing receptor tyrosine kinase activation,
which in turn phosphorylates cytoskeletal and other neuronal substrates to induce the neurotropic
effect. This possibility is less likely, considering the inability of K252a-TrkA inhibitor to block HU-
MCA-13 neurotropic effect (Fig. 3) and the lack of HU-MCA-13 stimulatory effects on Trk receptors
as evaluated by in vitro phosphorylation using PhosphoSens® CSox based kinase assays (Figure S3-2
supporting information). Alternatively, present findings may also suggest the involvement of
ADRA2A, CB1-CNR1 and/or CB2-CNR2, HRH2 and COX-1 (Table 2) in HU-MCA-13-induced
neurite outgrowth. The third hypothesis considers that HU-MCA-13 compound affects the
cytoskeleton of the PC12 cells, either by interacting with the microtubules or tau proteins as evident
from β tubulin III immunostaining (Fig.2), and therefore, inducing the neurotropic activity. Based on
the finding that HU-MCA-13 increased PGE2 levels (Fig.6), the fourth most plausible hypothesis
suggests that prostaglandin PGE2 and/or other arachidonic acid metabolite contribute to HU-MCA-13
neurotropic effect. While other mechanisms can still be addressed for a comprehensive understanding
of HU-MCA-13 –induced neurotropic effect, current findings may provide starting clues to the
molecular mechanisms of HU-MCA-13-induced neurotropic effect. Future molecular,
pharmacokinetic and pharmacodynamic research is required to optimize HU-MCA-13 as a new drug
candidate for polyneuropathy therapy.
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In summary, present findings indicate that HU-MCA-13 analogue (3-(3-allyl-2
methylenecyclohexyl) propanoic acid is a promising neurotropic, safe, lead compound for further drug
development towards polyneuropathy therapy.
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. Materials and Methods
Neurotropic activity and safety. DMEM media, fetal calf (FCS) and horse (HS) serums, penicillin
and streptomycin were purchased from Biological Industries (Beit Haemek, Afula, Israel). Type IV
collagenase and DMSO was purchased from Sigma Aldrich Merck, St Louis, MO, USA. Tissue
culture grade mouse β-NGF, was purchased from Alomone Labs (Jerusalem, Israel). The mouse anti-
neuron-specific βIII-tubulin (clone TuJ-1) monoclonal antibody (catalog number: MAB1195) and the
PGE -specific, ELISA kit (Catalogue number KGE004B) were purchased from R&D Systems Inc.
2
Minneapolis, MN, USA. Cy3-conjugated goat anti-mouse antibody was purchased from Jackson
ImmunoResearch Labs, West Grove, PA, USA.
Synthesis. Unless otherwise stated, all reagents were purchased from commercial suppliers and used
without further purification. Reactions were monitored by thin-layer chromatography (TLC) on silica
gel 60-F254 aluminum plates (Merck) and/or gas chromatography-mass spectrometry (GCMS).
Visualization of compounds on TLC was accomplished by irradiation with UV light at 254 nm and/or
vanillin stain. GCMS Analysis was performed with ‘Agilent 7820A’ gas chromatograph equipped with
‘
Agilent 5975’ quadrupole mass selective detector, using Agilent HP-5MS capillary column (30 m,
.25 mm, 0.25 μm film). Column chromatography was performed using silica gel 60 (particle size
0
0.040-0.063 mm) purchased from Sigma-Aldrich. Proton and Carbon NMR spectra were recorded on
Varian Mercury 300 MHz spectrometer in deuterated solvent. Proton chemical shifts are reported in
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ppm (δ) relative to tetramethylsilane with the solvent resonance employed as the internal standard
1
3
(CDCl , δ 7.26 ppm). C Chemical shifts are reported in ppm from tetramethylsilane with the solvent
3
resonance as the internal standard (CDCl , δ 77.0 ppm). Data is reported as follows: chemical shift,
3
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multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration and coupling
constants (Hz). High resolution mass spectra were determined on a ThermoScientific LTQ Orbitrap
XL (FTMS). Infrared (IR) spectra were recorded on a ThermoFischer Scientific NICOLET iS10
spectrometer.
All compounds were prepared according to the general procedures reported in ref. 21 and 24.
3
-(3-allyl-2-oxocyclohexyl)propanenitrile (2): 1-(cyclohex-1-en-1-yl)pyrrolidine was prepared by
refluxing cyclohexanone (35.2 mmol, 3.45 g, 1.0 equiv.) and pyrrolidine (105.6 mmol, 8.63 mL, 3.0
equiv.) in dry toluene (35.0 mL,1 M), in the presence of catalytic amount of pTSA, till all water was
distilled by Dean-Stark apparatus. After removal of toluene and traces of pyrrolidine by vacuum
evaporation, to the crude compound was added dry CH
3
CN (35.0 mL) followed by dropwise addition
o
of Acrylonitrile (1.0 equiv., 35.2 mmol, 2.3 mL) and the mixture was stirred for 3 h at 40 C. DIEA
(
(
1.0 equiv, 35.2 mmol, 6.1 mL) was added as one portion, followed by slow addition of allyl bromide
1.0 equiv., 35.2 mmol, 3.0 mL). The mixture was stirred for 12 h at 80 ºC, then quenched with water
and refluxed for 1 h. Purification of the crude product by flash column chromatography (10% ethyl
acetate in hexane) yielded pure 3-(3-allyl-2-oxocyclohexyl)propanenitrile (2.24 g, 35% yield, light
1
orange oil). H NMR (300 MHz, CDCl
3
): δ 5.82-5.68 (m, 1H), 5.04-4.97 (m, 2H), 2.55-2.33 (m, 5H),
1
3
2.22-1.68 (m, 6H), 1.56-1.21 (m, 3H). C NMR (75 MHz, CDCl
3
): δ 212.0, 136.2, 136.2, 116.3, 50.5,
-1
4
9.1, 35.2, 34.6, 33.4, 25.4, 25.1, 15.2. IR (neat): 2933, 2245, 1706, 1640, 1447, 912 cm . HRMS
+
(
m/z) calcd for C12
H
17NONa ([M+Na] ): 214.1202; found: 214.1203.
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-(3-allyl-2-methylenecyclohexyl)propanenitrile (3): Methyltriphenylphosphonium bromide (24.3
mmol, 8.7 g) and potassium tert-butoxide (24.3 mmol, 2.7 g) were stirred at 40 ºC in dry THF (0.4 M)
for 2 h. A 3M solution of 3-(3-allyl-2-oxocyclohexyl)propanenitrile (12.2 mmol, 2.3 g) in dry THF
was added dropwise at 0 ºC and the mixture was stirred for 2 h at room temperature. Water was added,
and the aqueous layer was extracted twice with diethyl ether. The combined organic layers were dried
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(
Na
2
SO ), filtered, and the solvent was removed under reduced pressure. Purification of the residue by
4
flash column chromatography (10% ethyl acetate in hexane) yielded pure 3-(3-allyl-2-
1
methylenecyclohexyl)propanenitrile as a mixture of diastereoisomers (1.9 g, 86% yield, yellow oil). H
NMR (300 MHz, CDCl
3
): δ 5.87-5.73 (m, 1H), 5.06-4.99 (m, 2H), 4.74-4.58 (m, 2H), 2.44-2.32 (m,
1
3
3
H), 2.07-1.42 (m, 9H), 1.10-0.87 (m, 2H). C NMR (75 MHz, CDCl ): major diastereoisomer:
3
δ 154.3, 137.5, 137.4, 115.8, 107.6, 102.6, 43.8, 43.1, 37.5, 34.9, 28.2, 25.7, 15.2; minor
diastereoisomer, characteristic signals: δ 152.1, 120.0, 41.9, 39.7, 36.7, 33.4, 33.1, 27.5, 21.3, 15.3. IR
-1
(
(
neat): 2923, 2245, 1640, 1444, 1392, 1333, 994, 891 cm . HRMS (m/z) calcd. for C13
H
19NNa
+
[M+Na] ): 212.14097; found: 212.14099.
3-(3-allyl-2-methylenecyclohexyl)propanoic acid (MCA-13): A mixture of 3-(3-allyl-2-
methylenecyclohexyl)propanenitrile (1.0 equiv., 10.6 mmol, 2.0 g), ethanol (70 mL) and 65% aq.
NaOH (212.0 mmol, 8.5 g) was stirred at 80 ºC for 3 h. The mixture was concentrated under vacuum
and the residue was quenched with water. The aqueous solution was extracted with ether, acidified
with conc. HCl to pH=1 and then extracted again with ethyl acetate. The mixture was dried over
Na
R
2
R
SO , filtered and evaporated to yield 3-(3-allyl-2-methylenecyclohexyl)propanoic acid as mixture
R
4
R
of diastereoisomers (2.2 g, 98% yield, orange oil). 1
P
P
R
R
): δ 11.35 (bs, 1H),
3
5
0
1
3
.89 (m, 2H).
P
P
R
3
R
): major diastereoisomer: δ 180.8, 155.1, 137.7, 115.6, 102.3,
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3.9, 43.6, 37.2, 35.3, 35.0, 32.1, 27.3, 25.9; minor diastereoisomer, characteristic signals: δ 153.4,
37.5, 115.5, 106.6, 42.2, 39.8, 36.8, 33.5, 33.4, 32.3, 26.7, 21.3. IR (neat): 2924, 1705, 1640, 1414,
-1
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PC12 cells. PC12 cells were cultured in T-200 flasks in high glucose (4.5 gr/L) DMEM medium
supplemented with 7% FCS, 7% horse serum and 1% penicillin and streptomycin. Cells will be
0
maintained at 37 C in a humidified incubator containing 6 % CO
2
. All experiments were carried out in
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a GMP grade C clean room, according to ISO 7 requirements (10,000 particles/m ). For neurotropic
experiments, tissue culture Falcon plates were coated with 200 μg/ml collagen type 1 and placed under
UV light for 30 minutes for sterilization. Thereafter, one ml of PC12 cell suspension (5000 cells/well)
was applied in 12 or 24 well plate, respectively. The cultures were maintained in the incubator two
days before exposure to investigated compound. Each neurotropic experiment contained two controls.
The first consisted of untreated cells (negative control), representing the random effect: "noise signal"-
the ability of cells to spontaneously express neurite outgrowths which in seven days are of a length
less than two-fold cell diameter. Also, negative controls consisted of cultures treated with 1% DMSO,
solvent used to solubilize MCA-13. The positive controls represented neuronal cultures treated with 50
ng/ml NGF, indicating maximal neurite outgrowth that can be achieved in this model. The
experimental group consisted of PC12 cells treated with different concentrations of MCA-13 or NGF
and their combinations. In each experiment, after two and seven days, six cultures will be evaluated
for neurotropic effect. These consecutive observations allow measurement of the progress of neurite
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outgrowth elongation and the percentage of cells with neurites. In order to assess neurotropic effect,
the neurite outgrowth length in each culture was quantified. For this purpose, each culture was placed
under an inverted microscope and photographed by the attached camera. Each well was photographed
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at three to five representative areas. After acquiring the photos, they were analyzed by ImageJ, NIH
software. The neurite outgrowth was estimated by fractal dimension (D ), a statistical parameter that
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describes the fractional space (area and length) filled by neurite outgrowth. D ranged from 0 to 1.20.
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This method estimated the number of pixels covered by neurites/cells compared to empty space per
square field, and therefore plotting log (number of square boxes) vs. log (size in pixels) relationship
generated a linear curve with D representing the slope of the curve. Every photograph that was taken,
f
was opened in a Photoshop software and a new layer was generated on it. Using a 5-pixel wide digital
pencil tool, all the outgrowths were marked and the layer with the markers (outgrowth network) were
saved in a 0-255 gray scale. Thereafter, the saved layer was opened by ImageJ NIH software. The
software was "skeletonize" the layer, measuring the length and complexity of every outgrowth in a
).49
fractal box count and calculated the fractional dimension parameter (D
f
DRG neurons. To perform these experiments, 10 DRGs were excised under sterile conditions and
transferred to Dulbecco’s Modified Eagle Medium (DMEM) containing 0.25% Type IV collagenase in
at 37°C for 45 min, followed by a 20-min incubation with 0.025% trypsin in DMEM, to digest the
associated connective tissue surrounding the neurons. After plating the ganglia onto collagen (50
μg/ml)/laminin mixture (5 μg/ml)-coated tissue culture wells for 24 h, DRG neurons were cultured for
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additional 1-7 days in fresh Neurobasal A/B27 medium containing either 50 ng/ml NGF (positive
control), medium (DMSO 1%) negative control and 5 µM MCA-13. The living cultures were
photographed and thereafter, the ganglia were immunostained for β tubulin III and again photographed
using a fluorescent microscope. A researcher blinded to the experimental conditions performed image
analysis, and the neurite outgrowth length (in micrometers) was measured.50
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Spinal cord neurons. Spinal cords were isolated from adult rats (4–6 months old), and the meninges
were removed from the spinal cord. The spinal cord was then cut into small pieces and collected in
cold Hibernate A (Brainbits, Springfield, IL, USA), glutamine (0.5 mM), and B27 (Invitrogen,
Carlsbad, CA). Briefly, dissected pieces of adult rat spinal cord tissue were maintained in phosphate-
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2+
buffered saline (PBS) without Ca and Mg and pooled together and transferred to an enzymatic
dissociation media containing 20 IU/ml papain in Earle's balanced salt solution (Worthington
Biochemical, Freehold, NJ) and incubated for 30 min at 37 °C. After enzymatic dissociation, the
papain solution was aspirated and the tissue was mechanically triturated with a fire-polished Pasteur
pipette in 6 ml of complete media of fresh Hibernate A, glutamine (0.5 mM), and B27 containing 2000
IU/ml DNase and 10-mg/ml protease inhibitors. Thereafter, the cell suspension was layered over a 4
ml step gradient (Optipep diluted 0.505:0.495 [v/v] with Hibernate A–glutamine 0.5 mM–B27) and
then made to 15, 20, 25, and 35% (v/v) in Hibernate A–glutamine 0.5 mM–B27 followed by
centrifugation for 15 min, using 800 g, at 4° C. The top 7 ml of the supernatant was aspirated. The
next 2.75 ml from the major band was collected and diluted in 5 ml Hibernate A–B27 and centrifuged
at 600 × g for 2 min (Fig. 1). The pellet was suspended in Hibernate A–B27, and after a second
centrifugation, was suspended in the culture medium. The cell suspensions was plated on pre-coated
collagen IV/laminin mixture –coated 96-well plates (Becton-Dickinson, Bedford, MA) at a density of
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1
.0 × 10 cells/well. The spinal cord neurons were maintained for one day in culture and then treated
with 1% DMSO-DMEM or 10 μM MCA-13 for 7 days. The cultures were photographed, and Image
analysis was performed. Neurite outgrowth was measured and presented as mean length of a neurite
(
microns). To measure neuronal survival, 450 cells were counted in 10 randomly chosen microscope
fields of a known area in each of the three dishes per condition using a 20 x objective and phase-
contrast optics. Counts of adherent cells were carried out 3 hours after plating, and counts of neurite
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outgrowth-bearing cells were carried out on cells at 48 hours. Cell survival was estimated by
expressing the density of neurite outgrowth-bearing cells at 48 hours as a percentage of the density of
adherent cells at 3 hours. This method reduced the possibility that counts included non-neuronal cells,
which do not elaborate extensive neurite outgrowths but might result in a failure to count some
neurons at 48 hours if those cells failed to extend neurite outgrowths at this time point.
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Indirect immunofluorescence microscopy. All neuronal cells cultures were grown on glass
coverslips at 37°C and 5% CO
2
. Cells were then washed twice with PBS, fixed for 15 minutes with
3
.7% paraformaldehyde at room temperature, and permeabilized for 1 minute with 0.5% Triton X-100
in PBS on ice. Cells were then incubated for 3 hours at room temperature with 15 µg/ml anti-neuron-
specific βIII-tubulin mouse IgG2A (Clone # TuJ-1) monoclonal antibody diluted in PBS containing
1
0% normal goat serum. Cells were rinsed in PBS and labeled with Cy3-conjugated goat anti-mouse
antibody (1:100) for 2 hours at room temperature. Cells were then rinsed with PBS. Coverslips were
mounted on glass slides. All images were collected with an Olympus FV1000 confocal scanner
mounted on an IX-81 microscope using an UPlanApo 60×, NA 1.42 lens.
Cytotoxicity. Cell death was evaluated by morphological appearance of the cells and release of LDH
into the medium, in the absence and presence of different concentrations of synthetic compounds as
previously described.52
PGE
2
measurements. Aliquots of the culture media of PC12 cells treated for 48 h with either 1 μ
using an PGE -specific,
in pg/mL was
g/mL LPS , 50 ng/mL NGF or 5 µM MCA-13 were assayed for PGE
2
2
ELISA kit, according to the manufacturer’s instructions. The amount of PGE
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calculated from a PGE
2
standard curve, with a sensitivity of 41.4 pg/mL. Values are mean ± SD (n =
4
) Statistical differences between groups were determined by Student’ s t-test and ANOVA followed
by Bonferroni posttest and were considered significant when *p < 0.01 compared to control.
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Pathological Analysis of Major Organs. C57Bl/6 mice, 8 weeks old, obtained from Envigo animal
breeding center were used in the study. All experiments were approved by the Institutional Animal
Care and Use Committee and performed according to OECD guidelines for testing of chemicals.53
Organs were harvested from mice that were administered with MCA-13 for the detection of possible
toxicological lesions in the framework of a safety assessment. Organs (brain hyppocampus, heart,
lung, stomach, kidneys, small intestine, colon, liver, spleen), of five HU-MCA-13 treated and three
untreated mice, were harvested and fixed in 4% formaldehyde. Then, the organs were trimmed in a
standard position per organ and put in embedding cassettes. (6 cassettes per organ per mice). Paraffin
blocks were sectioned at approximately 3-5 microns thickness. The sections were applied on a glass
slide and stained with hematoxylin & eosin. Pictures were taken on microscope (Olympus BX60) at
magnification of X4 and X10 using microscope's Camera (Olympus DP73, serial NO. OH05504). The
slides were examined by an experienced pathologist (Dr. Dr. Loeb Emanuel). Microscopic findings
were classified with standard pathological nomenclature and severities of findings were graded on a
scale of minimal, mild, or severe. Grades of severity for microscopic findings were subjective;
minimal was the least extent discernible and severe was the greatest extent possible.
DiscoverX’s SAFETYscan methods. Described in the Supplement section.
PhosphoSens® CSox based kinase assays. Described in the Supplement section
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Statistics. Each experiment was performed in triplicates. Unless otherwise stated, by using SPSS
software, one-way ANOVA was performed for the fractional dimension of each compound, in order to
evaluate the neurotropic effect. In case of significance, Bonferroni post-hoc analysis was performed.
The results were considered significant when p<0.01.
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Associated Content
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
Sensory motor/neurological performance.
DiscoverX’s SAFETY scan methods and individual target results.
MCA-13 safety by in vitro evaluation using PhosphoSens® CSoxbased kinase and phosphatase
assays.
Author Information
Corresponding Authors
†
Dmitry Tsvelikhovsky * - The Institute for Drug Research, Division of Medicinal Chemistry, School
of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel.
E-mail: dmitryt@ekmd.huji.ac.il. Phone: (+972)2-675-7032. Fax: (+972)2-675-7076.
‡
Philip Lazarovici * - The Institute for Drug Research, Division of Pharmacology, School of
Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 9112102, Israel. E-
mail: philiplekmd.huji.ac.il. Phone: (+972)2-675-8729. Fax: (+972)2-675-7490.
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Author Contributions
Dmitry Tsvelikhovsky and Philip Lazarovici have directly participated in the conceptualization,
supervision, data curation, formal analysis of the study, writing, reviewing and editing of the
manuscript.
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Adi Lahiani, Dikla Hacham-Geula, David Lankri, Susan Cornell-Kennon, Erik M. Schaefer:
Methodology and Investigations.
All authors have read and approved the final version submitted.
Funding
DT, holds the Gerald Heller Chair in Pharmaceutical Chemistry, gratefully acknowledge the Israel
Science Foundation - ISF Research Grant (367/18) for the financial support. This work was also
supported in part by the Hebrew University of Jerusalem - Yissum Intramural Research Funds. PL
holds the Jacob Gitlin Chair in Physiology and is affiliated and supported by David R. Bloom Center
for Pharmacy, Dr. Adolf and Klara Brettler Center for Research in Molecular Pharmacology and
Therapeutics, and the Grass Center for Drug Design and Synthesis of Novel Therapeutics at the
Hebrew University of Jerusalem, Israel.
Notes
The authors declare no competing financial interest.
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References
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