Serendipitous Discovery of Leucine and Methionine Depletion Agents during the Search for Polyamine Transport Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b00568

Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies. In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Because S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an inability to biosynthesize spermidine, and spermine. Importantly, we found that this methionine-depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby obviating the need for a PTI. Using ³H-leucine (the gold standard for LAT-1 transport studies) and JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake of extracellular leucine but instead facilitated the efflux of intracellular leucine pools.

Full text of DOI:10.1021/acs.jmedchem.9b00568

Subscriber access provided by Murdoch University Library
Article
Serendipitous Discovery of Leucine and Methionine Depletion
Agents During the Search for Polyamine Transport Inhibitors
Chelsea Lynn Massaro, Jenna Thomas, Houssine Ikhlef,
Sharifa Dinara, Sara Cronk, Holly Moots, and Otto Phanstiel
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00568 • Publication Date (Web): 18 Feb 2020
Downloaded from pubs.acs.org on February 19, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Serendipitous Discovery of Leucine and Methionine Depletion Agents
During the Search for Polyamine Transport Inhibitors
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Chelsea Massaro, Jenna Thomas, Houssine Ikhlef, Sharifa Dinara, Sara Cronk, Holly Moots, and
Otto Phanstiel IV*
*Contacting author
Contact Information:
College of Medicine, University of Central Florida
1
2722 Research Parkway
Orlando, FL 32827
otto.phanstiel@ucf.edu
407-823-6545
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Targeting polyamine metabolism is a proven anticancer strategy. Cancers often escape the
polyamine biosynthesis inhibitors by increased polyamine import. Therefore, there is much
interest in identifying polyamine transport inhibitors (PTIs) to be used in combination therapies.
In a search for new PTIs, we serendipitously discovered a LAT-1 efflux agonist, which induces
intracellular depletion of methionine, leucine, spermidine, and spermine, but not putrescine. Since
S-adenosylmethioninamine is made from methionine, a loss of intracellular methionine leads to an
inability to biosynthesize spermidine and spermine. Importantly, we found that this methionine-
depletion approach to polyamine depletion could not be rescued by exogenous polyamines, thereby
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
obviating the need for a PTI. Using H-leucine (the gold standard for LAT-1 transport studies) and
JPH-203 (a specific LAT-1 inhibitor), we showed that the efflux agonist did not inhibit the uptake
of extracellular leucine, but instead facilitated the efflux of intracellular leucine pools.
2
ACS Paragon Plus Environment

Page 3 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
The native polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) are
1
essential growth factors in eukaryotic cells. At physiological pH, these polyamines are nearly
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fully protonated, allowing them to interact with biological polyanions in the cell including nucleic
acids, proteins, and phospholipids. Polyamines are involved in many biological processes
including cell replication, translation, transcription, and regulation of specific gene expression.1-2
In addition, they have roles in the regulation of cell proliferation, apoptosis, and tumorigenesis.
An association between high levels of polyamines and rapid proliferation of eukaryotic cells and
3
cancer was reported in 1968 by Russell and Snyder. Tumor cells in particular accumulate high
polyamine concentrations, particularly spermidine, and typically exhibit a high ratio of spermidine
to spermine.3-4 Depletion of intracellular spermidine and spermine has been shown to cause
5
growth arrest through the inhibition of translation. Moreover, depletion of these intracellular
polyamine pools affects a number of growth-regulating genes, which also contribute to the growth
arrest. Thus, maintenance of polyamine homeostasis is critical for cell viability and proliferation.6
Not surprisingly, many cancers are addicted to polyamines and targeting polyamine metabolism is
a validated anti-cancer strategy.1
Polyamine homeostasis is maintained through a balance of polyamine biosynthesis,
7
degradation, uptake and export from the cell (Figure 1). The first step in polyamine biosynthesis
is the formation of putrescine from ornithine by ornithine decarboxylase (ODC). The amino acid
L-ornithine itself can be generated from L-arginine (via arginase) or be imported from the plasma.
Due to its short half-life (10-30 minutes in mammalian systems), ODC is regulated at multiple
1
steps from transcription to post-translational modification. ODC activity is often upregulated in
8
human cancers relative to surrounding normal tissues in an effort to increase intratumoral
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
polyamine pools through the biosynthetic pathway. As such, ODC is a well-established cancer
target and α-difluoromethylornithine (DFMO) was developed as an irreversible inhibitor of ODC
7
that suppresses cancer development in animal models. Treatment with DFMO typically results
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
in rapid depletion of intracellular putrescine and spermidine, and growth arrest. Unfortunately,
cancers often circumvent DFMO by upregulating polyamine import to replenish their depleted
polyamine pools. Polyamine transport inhibitors (PTIs) have been developed to address this
7
DFMO escape pathway. For example, L3.6pl human pancreatic cancer cells treated with
DFMO+PTI (see example PTI structure in Figure 2) in the presence of exogenous spermidine
(
Spd, 1 µM) remained polyamine depleted and had decreased viability, whereas those treated with
DFMO+Spd were >90% viable and had increased intracellular polyamine pools.7
Spermidine and spermine biosynthesis requires the addition of an aminopropyl group onto
1
a putrescine or spermidine substrate, respectively. This aminopropyl group is derived from L-
methionine. Specifically, L-methionine is converted to S-adenosyl-L-methionine (SAM) via
methionine adenosyltransferase (MAT). SAM is then decarboxylated by S-adenosylmethionine
decarboxylase (AdoMetDC) to form S-adenosylmethioninamine, i.e., decarboxylated AdoMet.9
Two aminopropyltransferases, spermidine synthase (SRM) and spermine synthase (SMS), transfer
an aminopropyl moiety from S-adenosylmethioninamine to their respective substrates (putrescine
or spermidine) to form spermidine and spermine. Therefore, rapidly dividing cells must convert
some their intracellular methionine pools towards SAM and S-adenosylmethioninamine formation
to drive polyamine biosynthesis. In summary, polyamine biosynthesis is directly linked to L-
ornithine and L-methionine availability.
1
Polyamine catabolism involves spermine/spermidine N -acetyltransferase (SAT1), which
1
1
catalyzes the formation of N -acetylspermine and N -acetylspermidine by transferring the acetyl
4
ACS Paragon Plus Environment

Page 5 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
moiety from acetyl-coenzyme A (acetyl-CoA) to the N position of spermine or spermidine.
Acetylpolyamine oxidase (APAO) then catalyzes the conversion of these acetylated polyamines
2
to spermidine or putrescine, respectively, via oxidative cleavage. Alternatively, spermine oxidase
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SMOX) can directly convert spermine to spermidine.1 In addition to being converted to
(
spermidine or putrescine, the N-acetylated polyamine products of SAT1 reactions can also be
exported from the cells. In this regard, cells have the ability to maintain polyamine homeostasis
though modulation of polyamine biosynthesis, transport, and catabolism (Figure 1).8
Figure 1. Polyamine Metabolism and import of amino acids by LAT-1.
There has been strong interest in developing inhibitors of polyamine metabolism to control
cancer cell growth.10 In addition to DFMO (1), trans-4-methylcyclohexylamine (MCHA, 2) and
N-(3-aminopropyl)cyclohexylamine (CDAP, 3) have been developed as competitive inhibitors of
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the spermidine and spermine synthases, respectively11, 12 (see Figure 2). The transition state
analogues, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and adenosylspermidine, have
also shown promise as spermidine synthase inhibitors.13 However, cancer cells can
modulate/interconvert their polyamine pools or increase polyamine uptake to address these
interventions.12 PTIs like the one shown in Figure 2 (4) have been shown to be effective in
depleting cells of polyamines when used in combination with DFMO even in the presence of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
exogenous spermidine. Indeed, this combination of a polyamine biosynthesis inhibitor and a PTI
has been shown to significantly increase survival in an orthotopic mouse model of pancreatic
cancer using murine PanO2 cells.14
Beyond inhibiting the polyamine biosynthetic enzymes, another approach is to target the amino
acid pools from which the polyamines are derived. LAT-1/SLC3A2 (large neutral aminoacid
transporter 1) is a heterodimer comprised of a light subunit (SLC7A5, aka LAT-1) and a heavy
subunit (SLC3A2, Figure 1). This complex transports large neutral aminoacids such as leucine,
phenylalanine and methionine into mammalian cells. L-Leucine is used not only for protein
synthesis, but also serves as an intracellular signaling molecule, which can regulate cell growth
via stimulation of mTOR. Once activated, mTOR directly phosphorylates initiation factor 4E
binding protein (4E-BP1) and p70 ribosomal S6 kinase 1 (p70S6K) to facilitate growth.15
Activation of the mTOR pathway is found in many types of cancers and inhibitors of LAT-1
1
5
mediated transport (5-8) have been proposed as another anti-cancer strategy. Indeed, inhibition
of LAT-1 has been shown to suppress leucine uptake, mTOR signaling, and the growth of Panc-1
pancreatic cancer cells in vitro.15
6
ACS Paragon Plus Environment

Page 7 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Current inhibitors of polyamine metabolism (1-3), polyamine import (4) and LAT-1 (5-
8
).
In a search for compounds which decrease intracellular polyamine levels, we screened
several complex molecular libraries from the Torrey Pines Institute for Molecular Studies (TPIMS)
and identified a lead architecture A for further investigation (Figure 3). This screen involved
testing compounds generated at TPIMS for their ability to affect Chinese hamster ovary (CHO)
cell growth in the presence of compounds that target polyamine metabolism and import. Originally
designed to screen for PTIs, this assay serendipitously also identified compounds which affected
polyamine pools and growth by other mechanisms. In a subsequent study of 250 individual
compounds, we identified two promising hits (9 and 10 in Figure 3). This report describes the
synthesis and bioevaluation of these hit compounds, which appear to be efflux agonists of LAT-1
substrates (e.g., methionine and leucine).
L3.6pl pancreatic cancer cells treated in vitro with compound 10 (5 µM) were shown to
have significantly higher levels of glutamic acid, agmatine (a derivative of arginine), leucine,
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methionine, and phenylalanine, and ornithine in the supernatant and have significantly decreased
intracellular leucine, methionine, spermidine and spermine pools compared to an untreated control.
In summary, this work is significant as it provides a new way to reduce intracellular levels of
hydrophobic aminoacids. In one example, methionine restriction induced by 10 depletes
intracellular polyamine pools and reduces cell growth.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R1
R2
H
H
N
(S)
N
N
N
(S)
N
N
NH2
4
R3
4
N
(R)
N
(S)
H
H
H
N
R4
N
N
A
9
10
11: Ant-44 (3 HCl)
Figure 3. Lead architecture (A) identified from molecular library screening, top hits 9 and 10, and
1 (Ant-44, a fluorescent cytotoxic polyamine).
1
Results and Discussion
Synthesis. Since the original TPIMS molecular libraries were synthesized via solid-phase peptide
1
6
chemistry to provide small quantities for screening purposes, we developed a solution phase
synthetic approach to provide larger quantities of the top hits for further evaluation.
As a quick overview, our strategy (shown in Scheme 1 for compound 9) involved several peptide
coupling steps with HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium-3-oxide hexafluoro-phosphate) to create a linear triamide scaffold 17 with the
appropriate substituents. This triamide scaffold was then reduced with borane-THF to afford the
respective chiral triamine 18. Regioselective N-benzoylation of the terminal amine with N-
hydroxysuccinimide ester 19 provided a diamine scaffold 20, which could then be regiospecifically
8
ACS Paragon Plus Environment

Page 9 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
cyclized with oxalyl diimidazole to form diketopiperazine 21. Lastly, 21 could be reduced with
BH
3
/THF to form the desired target 9.
As an example, the synthesis of compound 9 is shown in Scheme 1. The starting materials
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3,3-dimethyl-butyric acid 12 and L-phenylalanine methyl ester hydrochloride 13 were used in the
initial coupling step to form amide 14 (95%), which was then hydrolyzed to form carboxylic acid
1
5 (99% yield). HATU-mediated coupling of acid 15 to L-phenylalanine amide 16 gave triamide
17 albeit in low yield (39%). Reduction of the triamide using BH /THF provided the triamine 18
52%). Subsequent N-benzoylation with N-hydroxysuccinimide ester of benzoic acid 19 gave the
desired benzamide 20 in 90% yield. Cyclization of 20 with oxalyl diimidazole provided the
diketopiperazine 21 (79% yield), which was reduced with BH /THF to give the desired compound
(65% yield). Overall, compound 9 was made in 9% yield over 7 steps.
3
(
3
9
Scheme 1a
aReagents: a) HATU, DIEA, DCM, rt, 95% yield; b) NaOH, MeOH, rt, 99% yield; c) HATU,
DIEA, DCM, rt, 39% yield; d) BH -THF, THF, reflux, 52% yield; e) DCM, rt, 90% yield; f) oxalyl
3
diimidazole (5 equiv), DCM, rt, 79% yield; g) BH -THF, THF, reflux, 65% yield
3
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 10 was synthesized using a modified approach to increase yield. As shown in Scheme
2
, isovaleric acid 22 and L-leucine ether ester hydrochloride 23 were coupled via HATU to form
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
amide 24 as the initial step in the synthesis of 10. Amidoester 24 was then converted to the
carboxylic acid 25 in 88% yield. To avoid the low yields encountered in the prior coupling of acid
1
5 with amide 16, we first coupled acid 25 to D-cyclohexylalanine methyl ester 26 to give
diamidoester 27 in 96% yield. Ammonia gas was used to convert the diamidoester 27 to triamide
8 in 82% yield. Note: this alternate approach to triamide formation (e.g., 28) was a significant
2
improvement over the related approach used to make triamide 17 in Scheme 1. The remainder of
the synthesis was carried out as described above to give the target compound 10 in 22% overall
yield after 8 steps. Note: the chiral integrity of the synthetic approach was maintained as evidenced
by comparison of the circular diichroism spectrum of 10 to its three diastereomers, which were
independently synthesized and will be reported in future work.
10
ACS Paragon Plus Environment

Page 11 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 2a
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
aReagents: a) HATU, DIEA, DCM, rt, 93% yield; b) NaOH, MeOH, rt, 88% yield; c) HATU,
DIEA, DCM, rt, 96% yield; d) NH (g), MeOH, rt, 82% yield; e) BH -THF, THF, reflux, 97%
3
3
yield; f) DCM, rt, 73% yield; g) oxalyl diimidazole (5 equiv), DCM, rt, 82% yield; h) BH
3
-THF,
THF, reflux, 60% yield
The bioevaluation of these compounds was then conducted using a series of assays
designed to compare the ability of each compound to target polyamine metabolism and influence
the transport of key amino acids and polyamines.
We first explored the ability of these polyamine-depleting compounds to affect polyamine
transport using established assays in our laboratory.
CHO K1 Studies. Wild type Chinese hamster ovary (CHO K1) cells were chosen to first study
the synthetic compounds’ impact on polyamine metabolism. The CHO K1 cell line has high
polyamine transport activity and was useful in screening the compounds for polyamine transport
inhibition. A dose-response curve was obtained for each compound to determine their toxicity in
CHO K1 cells after 48 h incubation. Compound 9 was non-toxic up to the highest dose tested (10
11
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
µM, see Figure S3). In contrast, compound 10 could be dosed for 48h up to 6 µM in CHO-K1 cells
without apparent impact on cell growth (Figure S3).
Inhibition of ODC by DFMO often leads to an increase in polyamine transport activity to
maintain intracellular polyamine homeostasis (Figure 1). The increased transport activity of
DFMO-treated cells was used to assess the PTI activity of these compounds by investigating the
ability of each compound to block the entry of a rescuing dose of spermidine (1 µM).17-18 Our
group has previously determined the 48 h IC50 value of DFMO in CHO K1 cells as 4.2 mM, as
well as the minimum amount of spermidine (Spd, 1µM) required to rescue the DFMO-treated CHO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
K1 cells back to >90% viability. These two parameters (4.2 mM DFMO and 1 µM Spd) remained
fixed throughout the assay. The third parameter was the candidate PTI compound which was used
in increasing doses up to its maximum tolerated concentration, MTC. The MTC is the maximum
dose the compound could be dosed individually and provide >90% cell growth relative to an
untreated control. Since non-toxic PTIs are expected to inhibit Spd entry, the cells treated with a
combination of DFMO, Spd, and PTI would be expected to resemble the DFMO-only treated
control. This assay allowed the potential PTIs to be tested, ranked, and compared.
In a 96-well plate format, CHO K1 cells were treated with the IC50 of DFMO (4.2 mM), a
fixed dose of Spd (1 µM), and increasing doses of the potential PTI compounds. The cells were
incubated for 48 h at 37°C. Results for compounds 9 and 10 are shown in Figure 4. The green
line in Figure 4 represents the % relative growth observed with the DFMO+Spd control, while the
red line represents the % relative growth for the DFMO-only control. The EC50 value is typically
defined here as the concentration of the compound needed to reduce the % relative growth to
halfway between the green and red lines, i.e. halfway between the DFMO+Spd and DFMO only
controls. Interestingly, neither of the compounds (9 or 10) successfully blocked the entry of the
12
ACS Paragon Plus Environment

Page 13 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
rescuing dose of Spd to the DFMO-treated CHO K1 cells and the EC50 could not be determined.
In summary, these CHO K1 experiments demonstrated that compounds 9 and 10 did not act as
effective PTIs in this cell line.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Inability of (a) Compound 9 and (b) Compound 10 to prevent Spd from rescuing DFMO-
14
ACS Paragon Plus Environment

Page 15 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
treated CHO K1 cells. CHO K1 cells (1000 cells/well) were incubated at 37°C for 48 h in the
presence of increasing doses of the respective compound in the presence of a fixed concentration
of DFMO (4.2 mM) and Spd (1 µM). The cells were pre-incubated with 1 mM aminoguanidine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
AG) for 24 h prior to compound addition. Column 1 shows the untreated CHO K1 control, while
columns 2-10 in Panel A and columns 2-7 in Panel B show the % relative cell growth when the
cells are dosed with either Spd (1 uM), DFMO (4.2 mM), or the compound alone at the different
concentrations. The remaining columns in each panel are % relative cell growth in the presence
of fixed concentrations of DFMO (4.2 mM) and Spd (1 µM) with increasing concentrations of the
compounds indicated in each panel. Performance differences by DFMO were due to the presence
of DMSO (0.15% by vol) used to dissolve compound 9 (Panel A), whereas compound 10 was
dissolved in PBS (Panel B). The data suggests that neither compound is toxic in this dose range
and neither performs as a PTI. Experiments were twice performed in triplicate and error bars show
the standard deviation from the mean for each experiment.
To further assess PTI activity, we also screened the candidate compounds (9 and 10) for
their ability to block Ant-44 entry (compound 11). Ant-44 is a cytotoxic homospermidine, a
1
9
anthracene-polyamine conjugate, previously synthesized in our lab. Ant-44 is taken up into CHO
K1 cells through the polyamine transport system (PTS). The selectivity for the PTS was
demonstrated through IC50 comparisons between the CHO cell line and a mutant CHO cell line
(
CHO-MG). The CHO-MG cell line is a polyamine-transport-deficient cell line and represented
cells with low PTS activity. Ant-44 displayed a nearly 150-fold preference for the CHO cell line
over the CHO-MG, suggesting that Ant-44 has high affinity for targeting cells with active
polyamine transport activity.19 Additionally, we have previously shown that the presence of
15
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
exogenous spermidine provides cell protection from the polyamine conjugate Ant-44 by
spermidine’s competitive access to cells via the PTS.20 Based on this result, we concluded that a
PTI, especially a polyamine-based PTI like compound 4 in Figure 2, would also block Ant-44
uptake.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We envisioned that a non-toxic PTI would inhibit the uptake of the cytotoxic polyamine
conjugate Ant-44 (11) and rescue cells from Ant-44 associated toxicity. For example, using the
IC50 dose of Ant-44, PTIs could be identified by measuring a compound’s ability to block Ant-44
entry and rescue cells back to >90% relative cell growth. Previous studies demonstrated that Ant-
4
4 (2.4 µM) significantly reduced cell viability in CHO K1 cells after 48 h incubation. This toxic
dose of Ant-44 was kept constant throughout the assay, while the candidate PTI was added in
increasing concentrations up to its MTC.
CHO K1 cells were treated in a 96-well plate with a toxic dose of Ant-44 (2.4 µM) alone
and dosed with the candidate compounds (9 and 10 at the non-toxic doses of 5 µM and 7 µM) and
incubated for 48 h at 37°C. Compounds 9 and 10 exhibited intriguing activity. Rather than rescue
the cells from Ant-44, the compounds seemed to significantly potentiate Ant-44’s toxicity. For
example, Ant-44 alone (2.4 µM) gave 23% relative cell growth, whereas Ant-44 in combination
with compounds 9 or 10 at 7 µM gave significantly reduced relative cell growth at 2.1% and 3%,
respectively, compared to the untreated control. Since neither 9 or 10 was toxic below 7 µM in
CHO K1 cells (Figure S3), this result implied potential synergism between these compounds and
Ant-44. At this point, our PTI discovery work flow was disrupted because we had identified new
structures, which gave the opposite result than expected.
16
ACS Paragon Plus Environment

Page 17 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
To further explore this novel effect, we modified our original screen to use a lower dose of
Ant-44 (0.5 µM) to improve the dose range of 9 and 10 that could be tested. In this regard, Ant-44
was dosed at 0.5 µM alone and in combination with increasing doses of compounds 9 and 10 up
to 5 µM. The CHO K1 cells were incubated for 48 h at 37°C, and the results are shown in Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
. The red line represents the % relative cell growth of the Ant-44 only control. The Ant44
potentiation assay EC50 value is defined as the concentration of the candidate compound required
to decrease the cell growth to half that of the Ant-44 only control. Both compounds 9 and 10 were
effective at decreasing cell growth, when used in combination with Ant-44 in a dose-dependent
fashion. Additionally, compounds 9 and 10 exhibited very low EC50 concentrations in CHO cells
in the presence of Ant-44 (0.5 µM), with EC50 values of 750 nM and 60 nM, respectively. We
noted that compound 10 was approximately 12.5 times more effective at potentiating Ant-44 than
compound 9 in CHO K1 cells.
17
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Potentiation of Ant‐44 by compounds 9 and 10 in CHO K1 cells, 48 h
1
20.00
100.00
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
80.00
60.00
40.00
20.00
0.00
Ant‐44 + [Compound 9]
Ant‐44 + [Compound 10]
Figure 5. Potentiation of Ant-44 toxicity by compounds 9 and 10 in CHO K1 cells. CHO K1 cells
were incubated for 48 h at 37°C with the respective compound and a fixed concentration of
cytotoxic Ant-44 (0.5 µM). Aminoguanidine (AG, 1 mM) solution was incubated with the CHO
K1 cells for 24 h prior to the addition of candidate compound. This was necessary to protect Ant-
44 from the amine oxidases present in the media containing fetal bovine serum. Column 1 is the
untreated CHO K1 control cells, column 2 shows the % relative cell growth (compared to the
untreated control) when dosed with Ant-44 alone at 0.5 µM, columns 3-16 have a fixed
concentration of Ant-44 (0.5 µM) with increasing concentrations of the candidate compounds as
indicated in each lane. Both compounds 9 and 10 are non-toxic to CHOK1 cells at the highest
concentration tested (5 µM). The Ant-44 potentiation EC50 values, defined as the concentration to
reduce the viability to half the Ant-44 only control, were 0.75 µM (9) and 0.06 µM (10),
18
ACS Paragon Plus Environment

Page 19 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
respectively. Experiments were twice performed in triplicate and error bars show the standard
deviation from the mean for each experiment.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To observe changes to the cell as a result of treatment with these compounds, the control
CHO K1 cells and the cells treated with Ant-44 (0.5 µM) and compound 10 at 5 µM in the
aforementioned 96-well plate experiment were observed under the microscope. As shown in
Supporting Information (Figure S1), the treated cells were not ruptured, and differed from the
control in terms of their number and rounded shape (in comparison to the more elongated control
cells). This data suggested that the treated cells were not growing. Intrigued by these observations,
we investigated the properties of these compounds in the L3.6pl human pancreatic cancer cell line.
L3.6pl Studies. Compounds 9 and 10 were evaluated in the metastatic human pancreatic cancer
7
cell line, L3.6pl. L3.6pl cells have a K-ras mutation, and high polyamine uptake activity. A
dose-response curve was obtained for each compound in L3.6pl cells to determine the 72h L3.6pl
IC50 value, defined as the dose at which L3.6pl cells gave 50% relative growth compared to the
untreated control. Compounds 9 and 10 had 72h IC50 values of 11.7 ± 0.9 µM and 5.9 ± 0.2 µM,
respectively. We noted that compounds 9 and 10 were more toxic to L3.6pl cells compared to
CHO K1 cells.
As performed previously with CHO K1 cells, L3.6pl cells were treated with compounds 9
and 10 and a fixed dose of Ant-44 to observe the potentiation effect. The 72 h IC50 dose of Ant-
4
4 in L3.6pl cells was previously determined by our group to be 4 µM. For this study, we used
half that dose to replicate the large ‘window’ of response used in the CHO experiments to look at
reduction in cell growth. In a 96-well plate, L3.6pl cells were dosed with a fixed concentration of
19
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Ant-44 (2 µM) and increasing doses of compounds 9 and 10. The cells were incubated for 72 h at
3
7°C, and the results are shown in Figure 6. Although both compounds were effective at reducing
cell growth, higher doses were required compared to CHO K1 cells. The EC50 for compound 9
was 3.27 ± 0.17 µM and compound 10 was 0.29 ± 0.1 µM. Both compounds were effective well
below their 72 h IC50 dose in L3.6pl cells. Similar to our observations in CHO K1 cells, compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0 was approximately eleven times more effective at increasing the potency of Ant-44 in L3.6pl
cells than compound 9.
Potentiation of Ant‐44 by Compounds 9 and 10 in L3.6pl cells, 72 h
1
20.00
100.00
80.00
60.00
40.00
20.00
0.00
Ant‐44 + [Compound 9]
Ant‐44 + [Compound 10]
Figure 6. Ability of compounds 9 and 10 to potentiate the effect of Ant-44 in L3.6pl cells. Cells
were incubated for 72 h at 37°C with the respective compound and Ant-44 (2 µM). A solution of
AG (250 µM) in phosphate buffered saline (PBS) was incubated with the cells for 24 h prior to
addition of compounds. Column 1 is the untreated L3.6pl control cells, column 2 shows the %
cell viability when dosed with Ant-44 alone at 2 µM, columns 3-16 have a fixed concentration of
20
ACS Paragon Plus Environment

Page 21 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Ant-44 (2 µM) with increasing concentrations of the candidate compounds as indicated in each
lane. Note: compounds 9 and 10 were nontoxic when tested alone at 5 µM and 1 µM, respectively.
To understand why Ant-44 becomes more potent in the presence of these compounds,
especially in the presence of compound 10, we designed an experiment to relate inhibition of cell
growth to intracellular polyamine and Ant-44 levels. One explanation for the enhanced potency
was that compound 10 increased polyamine import and, as a result, may have increased
intracellular Ant-44 levels. To test this hypothesis, we dosed L3.6pl cells with a fixed
concentration of Ant-44 (2 µM) alone and in combination with increasing concentrations of
compound 10 to explore how this combination therapy affected intracellular polyamine pools and
Ant-44 import. These results are displayed in Figure 7.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Single and combination therapies in L3.6pl cells with Ant-44 and compound 10 after
7
2h incubation. Polyamine and Ant-44 levels (expressed as nmoles/mg protein) and relative %
cell growth versus an untreated control were observed after 72 h incubation at 37°C. The untreated
control was run in parallel and polyamine levels determined in duplicate and % cell growth in
triplicate. Ant-44 was dosed at a fixed concentration of 2 µM and compound 10 at increasing
concentrations. Cell viability tracked well with total intracellular polyamine levels (sum of
putrescine, spermidine and spermine).
Neither Ant-44 (2 µM) or compound 10 at 1 µM alone significantly reduced cell growth as
measured by the MTS assay. The intracellular level of Ant-44 was essentially unchanged, where
22
ACS Paragon Plus Environment

Page 23 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the Ant-44 only control gave 2.15 ± 0.10 nmol Ant44/mg protein and when compound 10 at 1 µM
was present at 2.08 ± 0.05 nmol Ant-44/mg protein. If compound 10 was acting as a polyamine
import agonist, then we would expect the level of Ant-44 in the cells to increase with increasing
doses of compound 10. However, this was not observed.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Since compound 10 was neither acting as a PTI nor as a polyamine import agonist, how
could it lead to intracellular polyamine depletion? The decreased levels of intracellular polyamines
(putrescine, spermidine, and spermine) when L3.6pl cells are dosed with Ant-44 in combination
with compound 10 at 1 µM suggested that compound 10 may act via a different mechanism. To
test this hypothesis, we dosed L3.6pl cells with increasing concentrations of compound 10 without
Ant44. If compound 10 was acting on polyamine pools, one should see a reduction in total
polyamine levels as well as decreased cell viability in a dose dependent manner upon increasing
levels of 10. Therefore, we determined the toxicity of compound 10 by itself over a time range
(24, 48, and 72h exposure, Figure S2 in Supporting Information) and measured intracellular
polyamine levels after 72h exposure to 10 (Figure 8). The results of these latter experiments are
shown in Figure 8.
Compound 10 demonstrated increasing toxicity to L3.6pl cells over extended periods of incubation
(
24, 48, and 72h, Figure S2). As shown in Figure 8, after 72h of incubation of L3.6pl cells with 10
o
at 37 C, the intracellular polyamine levels of spermidine and spermine were significantly reduced,
whereas the putrescine content was relatively unaffected.
This was interesting because typically another polyamine depletion approach (DFMO+PTI
treatment) led to an absence of putrescine and a significant reduction in spermidine pools, while
the spermine pools were maintained (or even increased). We recently performed a systematic
23
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
study of polyamine biosynthesis inhibitors (using DFMO, MCHA and CDAP) in L3.6pl cells and
revealed the plasticity of polyamine homeostasis in these pancreatic cancer cells. The cells
maintained viability as long as either the spermine or spermidine pools were maintained near basal
levels and as long as the total polyamine pools were ≥ 40% of the untreated control. This suggested
that these cells maintain an excess pool of polyamines to help offset changes in intracellular
polyamine levels. For example, L3.6pl cells displayed 100% relative growth compared to an
untreated control in the presence of the SMS inhibitor (CDAP, 100 µM) and had no detectable
spermine.10 Since compound 10 gave specific depletion of both spermidine and spermine pools
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Figure 8), we speculated that it works through a different mechanism than DFMO+PTI.
Figure 8. Intracellular polyamine levels (expressed as nmoles polyamine/mg protein) in L3.6pl
cells dosed with increasing concentrations of compound 10 after cells were incubated for 72 h at
24
ACS Paragon Plus Environment

Page 25 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
7°C. The untreated control was run in parallel and polyamine levels determined in duplicate via
N-dansylation and HPLC. The data was averaged and reported as nmol polyamine (PA)/mg
protein.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
For example, DFMO+PTI treated L3.6pl cells were shown to have no detectable putrescine. As
shown in Figure 8, however, putrescine levels were relatively untouched (83% remaining) by
compound 10 (7 µM) compared to the untreated control. Why then do these cells lose 50% of their
spermidine pools and 30% of their spermine pools in the presence of compound 10?
One way to affect both spermidine and spermine pools is to inhibit methionine import, which in
turn would inhibit the formation of the decarboxylated S-adenosylmethionine needed to provide
the aminopropyl fragment required for spermidine and spermine synthesis. Since methionine,
leucine and phenylalanine all use the LAT1/SLC3A2 transporter to enter cells, we hypothesized
that 10 was inhibiting LAT mediated amino acid import.
To test this hypothesis, we measured the amino acid concentrations in the supernatants of L3.6pl
cells incubated with compound 10 at 2 µM and 5 µM for 72h compared to an untreated control (0
µM). This was straightforward to perform as these cells are adherent and one can directly sample
the supernatant. Even though the cells were all plated initially at the same cell density, some
conditions were toxic to the cells and reduced the number of cells available to metabolize amino
acids in the media. We controlled for this feature by first sampling the supernatant and then
harvesting the attached cell population. The harvested cells were washed, counted, and then lysed
open to obtain samples of their intracellular contents. The remaining cell pellet after lysis was
quantified for protein content (see Experimental section) to provide the mg of protein associated
25
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with each sample. The results are shown in Table 1 and are expressed as pmol of analyte/mg
protein in an effort to account for changes in cell population over the course of the experiment.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
ACS Paragon Plus Environment

Page 27 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Concentration (pmoles/mg protein) of specific metabolites collected in the supernatant
of L3.6pl cells grown for 72h at 37 C in the presence of increasing doses of compound 10a
o
Compd Putrescine Spermidine
Acetyl
spermidine
1936 ± 1365 6152 ± 5596
Spermine
Acetyl
spermine
77 ± 43
Glutamic acid
23412 ± 3983
20231 ± 649
34033 ± 540
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
0
2
5
µM 977 ± 522
µM 938 ± 70
µM 2224 ± 98
339 ± 340
131 ± 26
167 ± 32
1649 ± 18
1706 ± 29
2532 ± 406
2690 ± 889
49 ± 10
36 ± 4
Compd Agmatine
Arginine
Leucine
Methionine Ornithine Phenylalanine
1
0
0
µM
2 µM
µM
101
± 142
297
22121
± 10585
18396
± 70
43243
± 2047
18009
± 9061
15055
± 1043
202092
± 23817
22834
± 1984
17686
± 2432
64036
± 5454
10588
± 3212
9249
± 306
15951
± 118
14299
± 7194
11954
± 828
160462
± 18910
±
163
5
485
±
176
a
samples were run in duplicate.
As shown in Table 1, high levels of specific amino acids were observed in the presence of
compound 10 (5 µM). The highest levels were observed with leucine (Leu), phenylalanine (Phe)
and methionine (Met), i.e., the known LAT-1 substrates. Having gathered initial support for LAT
as the potential target of 10, we then determined the amount of Leu and Met remaining in cells
after 72h incubation with compound 10. These results are shown in Table 2.
27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 71
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Intracellular concentrations after L3.6pl cell lysis (pmol/mg protein) after 72h incubation
in the absence and presence of compound 10a
Compound 10
Leucine
(pmol/mg protein)
232 ± 10
Methionine
(pmol/mg protein)
167 ± 67
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(µM)
0
2
5
7
152 ± 8
94 ± 3
77 ± 1
226 ± 21
41 ± 23
Not detected
As shown in Table 2, a dose-dependent decrease in both leucine and methionine was observed.
Taken together, Tables 1 and 2 suggested that compound 10 influences the transport of large
neutral aminoacids and resulted in significant depletion of methionine and leucine pools in these
cells.
LAT-1 is a known antiporter, wherein intracellular glutamine is exchanged with extracellular
LAT-1 substrates like leucine and methionine. As a result of these data, we hypothesized that
compound 10 may act as a LAT import inhibitor or as a LAT efflux agonist. We note that leucine
and β-cyclohexylalanine (a reduced form of phenylalanine) are used in the synthesis of 10. As a
result, the molecular design of 10 (Figure 3) contains isobutyl and cyclohexylmethyl substituents
which resemble the side chains of the natural substrates of LAT-1 (leucine and phenylalanine).
We speculate that these features may provide 10 with special affinity for the hydrophobic
recognition sites on LAT-1; an idea that will be pursued in future work. 21
We note that virtually all known LAT-1 import inhibitors have amino acid motifs within their
2
2
structure and have large hydrophobic side chains (see Figure 2). In this regard, compound 10
provides a new scaffold for targeting LAT via a decorated piperazine motif. Instead of presenting
a hydrophobic amino acid to LAT (see LAT1 inhibitors in Figure 2), compound 10 presents the
28
ACS Paragon Plus Environment

Page 29 of 71
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
hydrophobic side-chains of the natural substrates (or mimics thereof) on a piperazine scaffold. We
speculated that compound 10 may outcompete the native amino acids by presenting four of these
recognized side chain ‘arms’ from a single platform. We note that this multi-prong substrate
approach has worked in other import systems, where for example, a tri-substituted system
outperformed a one- or two-armed system to more efficiently inhibit polyamine import.7, 23
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Intrigued by these possibilities, we investigated potential mechanisms of action for how compound
10 depletes intracellular methionine and leucine stores. One possibility is that 10 inhibited the
LAT-mediated uptake of these amino acids, so we investigated whether compound 10 inhibited
3
the entry of exogenous H-leucine into L3.6pl human pancreatic cancer cells. We elected to use
3H-leucine for these uptake studies to conform to the published methods in this area. As shown in
Figures 9-12, these results were compared to the known LAT-1 inhibitor, JPH-203.24
o
In control experiments run at two key temperatures, 4 C (a condition where active transport is
o
inhibited) vs. 37 C (a temperature at which active transport occurs) in L3.6pl cells, we
3
demonstrated that 60% of the bound H leucine pool was actively transported into cells and 40%
was tightly bound presumably to the outside of cells (and could not be washed off upon repeated
o
washing steps with 4 C buffer). This 40/60 ratio was determined by comparing the total
o
o
radioactivity counts associated with the washed cell pellets of the 4 C experiment and the 37 C
3
experiment. Note: In both experiments, the unbound H-leucine was removed via three separate
o
3
washing steps of the cells with 4 C buffer. Having a good understanding of where the H-labeled
leucine partitioned during the assay and the source of background signals, we then verified the
3
dose dependent decrease in the uptake of H Leucine using the known LAT 1 inhibitor JPH-203
Figure S3, Supporting Information). 24
(
29
ACS Paragon Plus Environment