Design, synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-migratory agents

Article abstract of DOI:10.1016/j.bmc.2018.01.003

Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary investigations of their anticancer activity demonstrated that several compounds have moderate antiproliferative effects on cancer cell lines with GI₅₀ values in the range of 13–50 μM. Furthermore, compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%, respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250 nM in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC₅₀ of Rac1 inhibition by lead compound EHop-016 of 1.1 μM, compound 11b demonstrates 4X improved in vitro efficacy.

Full text of DOI:10.1016/j.bmc.2018.01.003

Accepted Manuscript
Design, synthesis and biological evaluation of new carbazole derivatives as anti-
cancer and anti-migratory agents
Cornelis P. Vlaar, Linette Castillo-Pichardo, Julia I. Medina, Cathyria M.
Marrero-Serra, Ericka Vélez, Zulma Ramos, Eliud Hernández
PII:
S0968-0896(17)32272-1
https://doi.org/10.1016/j.bmc.2018.01.003
BMC 14157
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 November 2017
5 January 2018
8 January 2018
Please cite this article as: Vlaar, C.P., Castillo-Pichardo, L., Medina, J.I., Marrero-Serra, C.M., Vélez, E., Ramos,
Z., Hernández, E., Design, synthesis and biological evaluation of new carbazole derivatives as anti-cancer and anti-
migratory agents, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.003
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Design, synthesis and biological evaluation of new carbazole derivatives as anti-cancer and
anti-migratory agents
a
b
a
a
Cornelis P. Vlaar , Linette Castillo-Pichardo , Julia I. Medina , Cathyria M. Marrero-Serra , Ericka
a
a
a,
Vélez , Zulma Ramos and Eliud Hernández *
a
Department of Pharmaceutical Sciences, School of Pharmacy, University of Puerto Rico, San Juan, PR
00936
b
Department of Pathology and Laboratory Medicine, School of Medicine, Universidad Central del
Caribe, Bayamon, PR 00960
*
Corresponding author.
E-mail address: eliud.hernandez@upr.edu (E. Hernández); Tel: (787) 758-2525, ext. 5436
Abstract: Based on the efficacy of EHop-016 as an inhibitor of migration and Rac1 activation, a new
series of carbazole derivatives has been synthesized. Cytotoxic and anti-migratory effects of these
compounds were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. Preliminary
investigations of their anticancer activity demonstrated that several compounds have moderate
antiproliferative effects on cancer cell lines with GI50 values in the range of 13-50 µM. Furthermore,
compounds 3b and 11b inhibit migration activity of metastatic cell line MDA-MB-231 by 32% and 34%,
respectively. Compound 11b was shown to inhibit activation of the Rho GTPase Rac1 by 55% at 250 nM
in both MDA-MB-231 and MDA-MB-435 cell lines. Compared with the IC50 of Rac1 inhibition by lead
compound EHop-016 of 1.1 µM, compound 11b demonstrates 4X improved in vitro efficacy.
Keywords: Carbazole, EHop-016, Rac1, Breast Cancer, Migration, Metastasis, Rac1 inhibitor

1. Introduction
The major cause of death in breast cancer patients is the metastasis of primary tumor cells to
secondary tissues. Early detection of breast cancer, prior to metastasis, provides patients with a higher
probability of cure of their disease. To successfully invade a secondary site, a cancer cell completes a
series of steps including migration from the primary tumor, invasion of surrounding tissues and basement
1
membranes, entry (intravasation) and survival during circulation, and arrest at a distant target organ.
During cancer cell invasion, the migration of tumor cells through tissues frequently requires the
degradation of the extracellular matrix (ECM). In this process, known as invadopodia formation, an array
2
of several proteins play a key role. Invadopodia are actin-rich protrusive structures associated with
matrix degradation activity, and are believed to be important for tumor cells to be able to penetrate the
2
basement membrane of epithelia and blood vessels. The small GTPase Rac1, member of the Ras
superfamily of GTPases, has been implicated in the regulation of cellular migration and invasion and
3
-4
invadopodia formation in breast cancer cells. Rac1 is activated by GTP/GDP exchange factors (GEF)
5
that are regulated via a myriad of cell surface receptors. Therefore, therapeutic strategies that inhibit
binding of GEFs to Rac1 are a rational means to inhibit migration of cancer cells.
OCH3
O
O
S
N
N
OH
H
OCH3
N
N
H C
3
IG-105
HYL-6d
Cl
N
N
N
O
O
N
N
N
N
H
H
N
H C
O
3
EHop-016
Figure 1. Structure of representative anti-cancer carbazole derivatives.
MHY407
The carbazole skeleton is a key structural motif contained in a wide variety of synthetic and natural
6
,7
compounds with biological activities.
Carbazole derivatives have demonstrated diverse
7
8
9
10-11
pharmacological activities including antioxidant, anti-inflammatory, antibacterial, antitumor,
12
13
14
15
anticonvulsant, antipsychotic, antidiabetic, and larvicidal properties. The cytotoxic activity of
6,16
carbazole alkaloids has been correlated to their polycyclic, planar aromatic structure.

Selected examples of carbazole derivatives that have been evaluated for their anti-tumor potential
against several human tumor cell lines are represented in figure 1. The carbazole sulfonamide IG-105 is
an antimitotic agent that inhibits microtubule assembly through specific interactions within the tubulin
17
structure. Modelling studies suggested that the dimethoxypyridine and carbazole moieties bind to the
hydrophobic pocket of tubulin, while the sulfonamide group and the N atom of the carbazole group form
hydrogen bond interactions. Compound HYL-6d inhibits proliferation and migration in HUVEC cells
18
under pathological angiogenic conditions, critical factors in breast cancer progression and metastasis.
The epoxypropoxy carbazole derivative MHY407 effectively causes DNA damage by C-PARP
production, topoisomerase II inhibition and cell cycle arrest in the S phase by regulating cyclin D1, pRb,
1
9
and p21 levels. Our laboratory recently developed EHop-016, which was shown to reduce metastatic
20
cancer cell viability at a concentration of ≤5 µM.
Specifically, EHop-016 inhibits Rac1-Vav2
interaction with an IC50 = 1.1 μM. As a consequence, the Rac1-downstream effector PAK1 was inhibited
by ~60% at 2 μM, leading to reduction of in vitro lamellipodia formation and cell migration. Its activity
was as inhibitor of tumor growth and metastasis in was demonstrated in an in vivo mouse model of breast
21
cancer.
Molecular modeling suggested that EHop-016 binds to Rac1 via adoption of a “U-shaped”
2
0
conformation. We hypothesized that compounds with a more compact structural conformation that
closely adopt this “U-shaped” conformation, could improve inhibitory activity against Rac1. Previous
research has indicated that the carbazole group present in EHop-016 significantly contributes to Rac1
inhibitory activity. Therefore, we designed and synthesized several new series of EHop-016 derivatives
that maintain the carbazole group while mimicking this “U-shaped” conformation. Their cytotoxic and
anti-migratory activity against metastatic cancer cells was determined, and the most active migration
inhibitor was further evaluated for its Rac1 inhibitory activity.

2. Results and discussion
The main goal of this project is to discover novel anti-metastatic agents as identified by their potential
to inhibit cancer cell migration, while demonstrating limited off-target cytotoxicity. The carbazole and the
morpholinopropylamine substituents of EHop-016 are in a 1,3-relationship with respect to the pyrimidine
core (figure 1). It was reasoned, as also suggested via molecular dockings, that a 1,2-relationship would
be more likely to adapt a U-conformation, hypothesized to be favorable for Rac1 binding. Therefore,
several series of compounds were synthesized that replace the pyrimidine core with a pyridine, pyrazine,
benzene or cyclohexane group. This strategy enables positioning of the carbazole group in an ortho-
relationship with the second, modifying substituent. The synthetic procedures are described in schemes 1
to 3.
For all new compounds, the growth inhibitory activity against MCF-7 and MDA-MB-231 breast
22
cancer cells were tested using the Sulforhodamine B (SRB) assay . In addition, anti-migratory activity
2
3
was determined using the scratch-wound healing assay . In this assay, the relative migration of MDA-
MB-231 breast cancer cells in the presence of the novel compounds at a concentration of 10 µM was
compared to the migration in the presence of vehicle (0.02% DMSO). Representative photomicrographs
of the migration inhibition of compound 3b and 11b (see later) are represented in figure 2. It can be
observed that in the control, after 12 hours, wound healing is progressing considerably, and after 24
hours, the wound is basically healed. In contrast, in the presence of 3b or 11b, both after 12 and 24 hours,
the wound healing is significantly inhibited. Migration can only be observed in the metastatic cell line
MDA-MB-231, since the MCF-7 cells hardly migrate. The biological activities of the new compounds are
summarized in tables 1 to 3.

Figure 2. Inhibitory effect of 11b and 3b on MDA-MB-231 cells migration detected by wound-healing assay. MDA-MB-231
cells were treated with vehicle or with 11b and 3b at 10 µM and photomicrograph obtained at 0, 12, and 24 h.
2.1 Ortho amino-carboxamide-substituted EHop-016 derivatives
In the first two series of novel compounds (3a-d and 4a-f, scheme 1), we explored the introduction of
ortho amino-carboxamide substituents via replacement of the core pyrimidine group of EHop-016 with a
pyridine group. In these two series, the amino group is located at 2-position and the carboxamide group at
the 3-position of the pyridine ring. The carbazole group either forms an aromatic amine at the 2-position
or an amide at the 3-position. Both options place the key pharmacophores in a more compact ortho-
substituted relationship, hypothesized to provide compounds with increased activity. Compounds 3a-d
were synthesized via amide coupling of 2-chloronicotinic acid 1 with carbazole 2, followed by a CuI-
catalyzed coupling reaction with different amines to afford the corresponding 2-aminonicotinamide
derivatives 3a-d (scheme 1a). Compounds 4a-f were synthesized by nucleophilic aromatic substitution of

2-chloronicotinic acid 1 with carbazole 2 under microwave irradiation in water at 140 °C for 5 h in the
presence of 3 equiv. DIPEA (scheme 1b). The intermediate 2-aminocarbazole-nicotinic acid was reacted
via an amide coupling reaction with amines to obtain the corresponding 2-carbazolamine-nicotinamide
derivatives.
Scheme 1. General synthetic routes of 2-substituted-nicotinamide derivatives 3a-d and 4a-f. Reagents and conditions: (a) (i)
1
2
HOBt, EDAC, DMF, Et
water, 140 °C, 5h, (ii) HOBt, EDAC, DMF, Et
DMSO, 90 °C, 24 h, R-NH
3
N, rt; (ii) CuI, DIPEA, dioxane, 80 °C, 8-10 h, R = HNR R ; (b) Method A (i) DIPEA (3 equiv),
3
N, rt, R-NH . Method B (i) HOBt, EDAC, CH Cl , Et N, rt; (ii) CuI, Cs CO ,
2
2
2
3
2
3
2
.
From table 1 it can be observed that in the MCF-7 cancer cell line, compounds 4a and 4c-e showed
moderate antiproliferative activity with a GI50 in the range of 13.4-28.3 µM. In the MDA-MB-231 cell
line, compounds 3d and 4d-e inhibited cell proliferation with a GI50 in the range of 18-19.3 µM. The
remaining compounds in both series had a GI50 above 50 µM in both breast cancer cell lines tested. Thus,
in general, compounds of the second series with the aminocarbazole group at the 2-position appear to be
more cytotoxic than compounds in the first series. For comparison, the GI50 of EHop-016 in the MCF-7
and MDA-MB-231 cell lines was 14 and 15 µM respectively. The relative migration compared with
control of compounds 3a-d and 4a-f are summarized in table 1. Among the ten compounds available in
these two series, none of the compounds of the second series 4a-f inhibited migration significantly. While
compounds 4d and 4e were among the most cytotoxic compounds tested, they did not significantly inhibit
migration. In contrast, compound 3b inhibited migration with 32%, comparable with parent compound
EHop-016, which exhibited anti-migratory activity of 33% at 10 μM (and 17% at 5 µM). Compound 3d,

in which a propylene group is inserted between the morpholine and pyridine ring of 3b, shows a reduced
activity against cell migration as well as moderate growth inhibitory activity against the MDA-MB-231
cell line. When comparing EHop-016 and compound 3b, migration inhibition of MDA-MB-231 cells is
comparable, but the GI50 values are 15 µM and > 50 µM respectively. Therefore, compound 3b exhibits
reduced toxicity with similar anti-migratory potential compared to EHop-016.
Table 1. Growth inhibition and anti-migration activity for compounds 3a-d and 4a-f on MCF-7 and MDA-MB-231 cell lines.
a
GI50 (µM)
b,c
Cmpd
R =
Migration (%)
MCF-7
MDA-MB-231
O
3a
N
N
>50
>50
93 ±11.67
CH3
3
b
>50
>50
>50
68 ±10.54
N
O
3
c
>50
19
99 ±1.89
N
N
O
3
d
H
N
>50
28
76 ±7.87
N
O
4
a
H
N
>50
99 ±1.28
N
Boc
N
4
b
>50
27
>50
>50
18
95 ±9.80
93 ±2.01
91 ±14.10
99 ±4.12
N
N
H
4
c
N
N
H
N
4
d
13
N
N
H
4
e
14
18
N
N
H
OCH3
OCH3
4
f
>50
14
>50
15
92 ±8.55
N
OCH3
H
EHop-016
67 ±7.55
a
GI50 = compound concentration required to inhibit MDA-MB-231 proliferation by 50% after 24 h treatment. Values are expressed as the mean
of triplicate experiments, and standard deviation (SD) are <10%.

b
After 24 h, MDA-MB-231 cellular migration was determined by measuring the distance traveled from the edge of the scratch toward the center
of the scratch, relative to control.
c
Percent relative migration values at 10 µM are the average of three independent experiments.
2.2 Ortho diamino-substituted EHop-016 derivatives
The compounds described in the above paragraph have an amino group attached to the 2-position and
a carbonyl group attached to the 3-position of the pyridine ring. To further explore the ortho-substituted
pyridine series, we synthesized several derivatives in which both the 2- and the 3-position contain a
nitrogen atom, either as an amine or as a reverse amide group. The synthesis of these ortho-diamino-
substituted pyridine derivatives 7a-c and 11a-b is represented in scheme 2. Compounds 7a-c were
synthesized via reaction of 2-chloro-5-nitropyridine 5 with aminocarbazole 2 (scheme 2 a,b). Reduction
of the nitro group of 6, followed by acylation or alkylation provided compounds 7a-b and 7c respectively.
On the other hand, reaction of 5 with aliphatic amines and nitro group reduction provided intermediate 9.
Amide coupling with carbazole carboxylic acid 10 provided compounds 11a-b (scheme 2 c,d,e). Their
biological activities are summarized in table 2.
Scheme 2. General synthetic routes to 2,3-diamino-substituted pyridines 7a-c and 11a-b. Reagents and conditions: (a) THF,
Et
3
N, reflux, 2 h; (b) (i) SnCl
2
, HCl, EtOH, reflux, (ii) RCOCl, HOBt, EDAC, DMF, Et
3
N, rt (for 7a-b) or R-Cl, THF, Et
3
N,
1
2
reflux (for 7c); (c) R = HNR R , THF, Et
b).
3
N, reflux; (d) SnCl
2
, HCl, EtOH, reflux; (e) 10, HOBt, EDAC, DMF, Et N, rt (for 11a-
3
Among the five compounds synthesized, three compounds (7a-b and 11a) exhibited moderate anti-
proliferative activity (table 2). In MDA-MB-231 cancer cells, no growth inhibition was observed for
compounds 7c and 11b at concentrations ≤ 50 μM. Compound 11a contains the same structural elements
as compound 3d, but reverses the amide bond, which results in a slightly increased cytotoxicity towards

MCF-7, and reduced anti-migratory activity. Similarly, compound 11b reverses the amide bond of
compound 3b, and while both of these compounds do not inhibit growth of MCF-7 and MDA-MB-231
cell lines at a concentration ≤ 50 μM, 11b inhibits migration of MDA-MB-231 cells by 34% compared
with 32% for compound 3b. In compound 11a, a propylene group is inserted between the morpholine and
pyridine rings of 11b. Equivalent to the effects observed between compounds 3d and 3b, this
modification leads to increased growth inhibitory and reduced migration inhibitory activity. Compound
7b with an aromatic amide bond, and compound 7c with an aliphatic group at the 3-position have
moderate anti-migratory activity. From the compounds presented, 11b is the most active inhibitor of
migration so far, while it has no demonstrated growth inhibitory activity at concentrations ≤ 50 μM.
Table 2. Growth inhibition and anti-migration activity for compounds 7a-c and 11a-b on MCF-7 and MDA-MB-231 cell lines.
a
GI50 (µM)
1
2
b,c
Cmpd
R, R R =
Migration (%)
MCF-7
15
MDA-MB-231
23
7
7
7
a
b
c
99 ±4.29
N
O
O
41
23
31
81 ±11.12
81 ±8.27
N
O
>50
N
O
1
1
1a
1b
32
39
89 ±10.10
N
N
H
O
>50
>50
66 ±7.43
N
O
a
GI50 = compound concentration required to inhibit MDA-MB-231 proliferation by 50% after 24 h treatment. Values are expressed as the mean
of triplicate experiments, and standard deviation (SD) are <10%.
b
After 24 h, MDA-MB-231 cellular migration was determined by measuring the distance traveled from the edge of the scratch toward the center
of the scratch, relative to control.
c
Percent relative migration values at 10 µM are the average of three independent experiments.
2.3 Ortho dicarboxamide-substituted EHop-016 derivatives

In the final series investigated, compounds with ortho-dicarboxamide containing the carbazole group
that was strongly correlated with Rac1 inhibitory activity were synthesized. This was accomplished by
replacing the core pyrimidine group of EHop-016 with either a benzene, pyrazine or cyclohexane group.
Compounds 14a-h were synthesized via ring-opening reaction of the corresponding cyclic anhydride
building blocks with 3-amino-9-ethylcarbazole 2 in yields of 48 to 78% (scheme 3). The resulting ortho-
amidocarboxylic acids 13 were reacted in an amide coupling reaction with a variety of amines in order to
obtain the ortho-diamide products 14a-h in yields of 43 to 62%. The biological activities of the
compounds synthesized are summarized in table 3.
N
N
HN
O
HN
N
X
X
X
X
H N
2
X
X
+
O
O
O
a
R
b
O
O
H N
2
O
OH
HN
14a-h
R
12
2
13
Scheme 3. General synthetic route to ortho-diamide derivatives 14a-h. Reagents and conditions: (a) THF, rt 16 h; (b) HOBt,
EDC, THF, rt, 16 h.
In vitro analysis demonstrated that the ortho-dicarboxamide derivatives did not show growth inhibition
in MCF-7 and MDA-MB-231 cell lines (table 3). In addition, after 24 h of treatment at 10 μM using the
wound-healing assay in the MDA-MB-231 cell line, no migration inhibition could be observed. Hence,
the absence of any activity of these latter compounds, together with the fact that several compounds
described in the previous paragraphs show promising activity, demonstrates that there are clear Structure
Activity Relationships.

Table 3. Growth inhibition and anti-migration activity for compounds 14a-h on MCF-7 and MDA-MB-231 cell lines.
a
GI50 (µM)
b,c
Cmpd
R =
Migration (%)
MCF-7
MDA-MB-231
H
N
N
1
1
4a
4b
>50
>50
91 ±5.23
94 ±5.87
O
O
N
H
N
N
>50
>50
1
1
4c
H
N
>50
>50
>50
>50
99 ±4.50
N
H
N
4d
100 ±5.20
CH3
O
O
H
N
CH3
1
4e
>50
>50
99 ±2.57
H
N
1
1
4f
>50
>50
>50
>50
98 ±1.83
99 ±1.63
OH
H
N
N
4g
O
O
H
N
14h
N
>50
>50
100 ±0.56
Y = N-(9-ethyl-9H-carbazol-3-yl)
a
GI50 = compound concentration required to inhibit MDA-MB-231 proliferation by 50% after 24 h treatment. Values are expressed as the mean
of triplicate experiments, and standard deviation (SD) are <10%.
After 24 h, MDA-MB-231 cellular migration was determined by measuring the distance traveled from the edge of the scratch toward the center
b
of the scratch, relative to control.
c
Percent relative migration values at 10 µM are the average of three independent experiments.
2.4 Evaluation of compound 11b
From all synthesized compounds, 11b was the most potent inhibitor of migration, while at the same
time it did not show growth inhibitory effects. For this reason, this compound was selected to determine
Rac1 inhibitory activity via an ELISA-based Rac activity pulldown assay, and comparison of its

properties with EHop-016 are summarized in table 4. At 250 nM, 11b inhibits Rac activation by 55% in
both MDA-MB-231 and MDA-MB-435 cancer cells. Compared with EHop-016, with an IC50 = 1.1 µM,
11b is approximately 4X more potent as a Rac1 inhibitor. In addition, while EHop-016 significantly
inhibits growth of all three tested cell lines at ~15 µM, including the non-tumoral mammary epithelial
MCF-10A cell line, 11b does not affect the viability at concentrations of ~30-50 µM. Molecular docking
of 11b in Rac1, closely mimics the docking of EHop-016 in the expected U-bent conformation, with the
carbazole group occupying the hydrophobic pocket created by Val36 and Ala59. The central pyridine ring
of 11b is in a similar position of the pyrimidine core of EHop-016, and together with the morpholine
group is in close contact with the N-H group of the peptide bond between residues Val36 and Asp38. In
summary, compound 11b provides an improved Rac1 inhibitor with increased potency and reduced
toxicity, and promises to be a potentially useful molecular probe for the study of migration inhibition and
Rac activity.
Table 4. Comparison of the activities of compound 11b (A) with EHop-016 (B).
A
N
B
N
H
N
N
N
N
N
N
O
H
H
N
N
O
11b
EHop-016
O
MDA-MB-231: GI50 > 50 µM
MCF-7: GI50 > 50 µM
MCF-10A: No growth inhibition up to 36 µM
MDA-MB-231: GI50 = 15 µM
MCF-7: GI50 = 14 µM
MCF-10A: 50% growth inhibition at 10 µM
a
20
Rac1 inhibition
MDA-MB-435: 55% inhibition at 250 nM
MDA-MB-231: 55% inhibition at 250 nM
Rac1 inhibition
MDA-MB-435: IC50 = 1.1 µM
MDA-MB-231: IC50 = 3 µM
b
20
b
20
Molecular docking of 11b in Rac1
Molecular docking of EHop-016 in Rac1

a
Supplemental Figure S4
Supplemental Figure S3
b
3. Conclusions
In this research, we explored the Structure Activity Relationships of novel derivatives of the lead Rac1
inhibitor EHop-016. The new compounds maintain the carbazole group of EHop-016 that previously was
found to significantly contribute to Rac inhibition as a key structural feature. However, its central
pyrimidine core was replaced with isosteric groups that allowed the carbazole group to be positioned in an
ortho-relation with a wide diversity of substituents. Molecular dockings had suggested that ortho-
substituted compounds would more easily adapt the U-shaped conformation that was calculated for the
binding of EHop-016 to Rac1. The activity profile of the compounds was quite diverse. For example,
compounds 4d and 4e inhibited growth at ~15 µM, but did not significantly inhibit migration. On the
other hand, compounds 3b and 11b inhibited migration at 10 µM, comparable to EHop-016, but did not
inhibit growth at concentrations ≤ 50 μM in the cell lines tested. As the most potent inhibitor of
migration, activity of 11b was further investigated and found to inhibit Rac1 by 55% at a concentration of
250 nM. Thus, while 11b is a 4X more potent inhibitor of Rac1 than EHop-016, it has reduced cellular
toxicity. This will be beneficial for its use as a molecular probe for the study of Rac activity, or
potentially as an anti-metastatic cancer drug. Further studies of the mechanism of action are needed to
fully analyze the potential of this novel compound.
Acknowledgments
This project was supported by the American Association of Colleges of Pharmacy New Investigator
Award (to EH), PRINBRE (NIH-NIGMS 2P20GM103475-13) Pilot Project award (to LCP) and MBRS-
SCORE (NIH-NIGMS SC3GM1167313) grant (to CPV).
Supplementary data
Supplementary data associated with this article can be found in the online version.

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