Puromycin based inhibitors of aminopeptidases for the potential treatment of hematologic malignancies

Article abstract of DOI:10.1016/j.ejmech.2017.07.048

Substantial progress has been described in the study of puromycin and its analogs for antibiotic properties. However, the peptidase inhibitory activity of related analogs has not been explored as extensively. Specifically, inhibiting aminopeptidases for a

Full text of DOI:10.1016/j.ejmech.2017.07.048

Accepted Manuscript
Puromycin based inhibitors of Aminopeptidases for the potential treatment of
hematologic malignancies
Rohit Singh, Jessica Williams, Robert Vince
PII:
S0223-5234(17)30570-6
10.1016/j.ejmech.2017.07.048
EJMECH 9611
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 February 2017
Revised Date: 14 July 2017
Accepted Date: 22 July 2017
Please cite this article as: R. Singh, J. Williams, R. Vince, Puromycin based inhibitors of
Aminopeptidases for the potential treatment of hematologic malignancies, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.048.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Puromycin Based Inhibitors of Aminopeptidases for the
Potential Treatment of Hematologic Malignancies
Rohit Singh*, Jessica Williams, Robert Vince*
Center for Drug Design, Academic Health Center, University of Minnesota, Minneapolis, MN 55455,
USA.
Email addresses: singh109@umn.edu, vince001@umn.edu
KEYWORDS Aminopeptidases; Puromycin–Sensitive Aminopeptidase (PSA); Aminopeptidase N (APN);
Amino Acid Deprivation Response (AADR); Acute Myeloid Leukemia (AML); Acute Lymphoblastic
Leukemia (ALL).
ABSTRACT Substantial progress has been described in the study of puromycin and its analogs for
antibiotic properties. However, the peptidase inhibitory activity of related analogs has not been explored
as extensively. Specifically, inhibiting aminopeptidases for achieving antitumor effect has been sparsely
investigated. Herein, we address this challenge by reporting the synthesis of a series of analogs based on
the structural template of puromycin. We also present exhaustive biochemical and in vitro analyses in
support of our thesis. Analyzing the structure–activity relationship revealed a steric requirement for
maximum potency. Effective inhibitors of Puromycin–Sensitive Aminopeptidase (PSA) are disclosed
here. These potential therapeutic agents display superior in vitro antitumor potency against two leukemic
cell lines, as compared to known inhibitors of aminopeptidases.
INTRODUCTION
The structural similarities between puromycin (1, Figure 1) and the aminoacyl adenyl terminal of
aminoacyl–tRNA allow it to terminate the protein synthesis process by causing premature release of a
polypeptide from the ribosome [1, 2]. Tremendous progress in the study of puromycin and related
structures for the optimization of their antibiotic effects has been described over past several decades [3,
1

ACCEPTED MANUSCRIPT
4
]. However, the antibiotic efficacy of puromycin and related derivatives suffered due to their inability to
distinguish between protein synthesis in normal healthy cells and compromised, diseased cells [5–7]. In
prior studies we have considerably reported upon the structure–activity relationship for puromycin related
compounds as antibiotic agents [8–11]. Herein, we present our studies on the lesser–explored property of
puromycin–based compounds as inhibitors of peptidases [12]. We sought to restrict inhibition of protein
synthesis in the design of the desirable compounds to obtain better toxicity–profiles for the studied
analogs. Given that Puromycin–Sensitive Aminopeptidase (PSA) is among the most important
aminopeptidases being studied for implications on leukemia [13, 14], we have explored the efficacy
against PSA enzyme as a measure of the potency of synthesized compounds. Various analogs with
structures based on the puromycin template were synthesized and studied for the inhibition of
aminopeptidases.
Figure 1. Puromycin (1), Bestatin (2), and Tosedostat (3)
Aminopeptidases represent a class of metalloenzymes (usually zinc) that mediate the cleavage of amino
acids near the N–terminal of peptides [15]. The pleiotropic nature of aminopeptidase enzymes manifests
with broad implications in various medical conditions such as Alzheimer’s disease [16], Parkinson’s
disease [17], angiogenesis [18], diabetes [19], and Huntington’s disease [20]. These metalloenzymes also
play a crucial role in antigen presentation on the major histocompatibility complex (MHC) class I
molecules [21]. However, the most direct implication of aminopeptidases has been noted in the
modulation of cancerous malignancies [22–24]. The upregulation of aminopeptidases and amino acid
2

ACCEPTED MANUSCRIPT
transporters [25] in cancer cells provides support to this thesis [26]. The inhibition of aminopeptidase
mediated intracellular peptide degradation has been utilized to block the supply chain of recycled free
amino acids [27]. Depletion of amino acids in cells stimulates amino acid deprivation response (AADR)
[28]. For example, the induction of nutrient stress by administering asparagine synthetase inhibitors has
been described as a viable option for combating acute lymphoblastic leukemia (ALL) [29]. Interestingly,
compared to normal cells, malignant cells have higher sensitivity to the absence of specific amino acids,
leaving them vulnerable to apoptosis and a diminished rate of proliferation [30]. Seminal work by
Wheatly and coworkers highlighted this difference along with the mechanism for this selectivity by
analyzing the effects of nutrient deprivation on various malignant cell lines. While human fibroblasts
showed ability to reach quiescence (G ) under nutritionally stressful conditions, cell lines with malignant
0
phenotypes tried to continue the cell cycle leading to mitotic catastrophe and cell death [31]. This absence
of stringent control at the G1 checkpoint of the cell cycle in malignant cell lines affords a potential to be
exploited in selectively targeting them via the modulation of their nutrition supply. This should allow
healthy cells to continue to proliferate unaffectedly. Krige and coworkers later reported utilizing this
principle in the development of a new inhibitor of aminopeptidases for the treatment of acute myeloid
leukemia (AML) [32]. Krige et al included the well–known aminopeptidase inhibitor, bestatin (2, Figure
1
) for comparison while presenting the discovery of the prodrug, tosedostat (3, Figure 1) [33]. Tosedostat
is currently in phase II of clinical evaluation for its efficacy in the treatment of refractory and relapsed
AML [34].
This development notwithstanding, the chemotherapeutic treatment for leukemia has stagnated over the
last 40 years [35]. Main treatment options necessitate aggressive chemotherapy to target leukemic cells
[36–39]. Several limitations of current treatment options include the use of anthracycline drugs [38], and
severe adverse effects [39] including tumor lysis syndrome [40]. Interpatient genomic heterogeneity adds
further constraints to the targeted therapy approach [41]. In theory, such undesirable outcomes in the
treatment of leukemia could be avoided by adopting approaches targeting leukemogenesis, rather than
3

ACCEPTED MANUSCRIPT
later stages of tumor cell proliferation [42]. Towards that aim, we have focused our attention on the role
that various aminopeptidases play in the hydrolysis of several intra– and extra–cellular proteins for
providing recycled amino acids for the synthesis of new proteins, or for metabolism to provide
energetically favorable outcomes [43].
RESULTS AND DISCUSSION
Chemistry. Based on the previous studies focused on puromycin and related structures in our laboratories
[8, 44, 45], we envisioned the utility of puromycin aminonucleoside (PAN) as a versatile template for
obtaining structural analogs targeting the activity of aminopeptidases. As outlined in Scheme 1, all
inhibitor scaffolds presented herein were synthetically accessed with PAN as the starting precursor. With
the aim of being able to provide aminopeptidase inhibitors via a synthetic route that would be amenable to
furnishing the desirable compounds in a user–friendly, and commercially viable manner, the first inhibitor
4
(Figure 2) was generated via condensation of PAN with N–Boc–protected L–phenylalanine in a classic
dicyclohexyl carbodiimide (DCC) mediated process with N–hydroxy succinimide (NHS) additive [46].
Figure 2. Puromycin based inhibitors of aminopeptidases
Removal of the protective group was achieved within minutes by anhydrous trifluoroacetic acid (TFA).
Possible hydrolysis of the glycosidic bond necessitates the transformation to be performed under strict
anhydrous conditions. The reaction proceeds at room temperature to efficiently deprotect the amine
functionality within 10 minutes. Purification of the desired final compounds was accomplished by in
vacuo removal of excess TFA, along with azeotropic assistance from dry acetonitrile. The resultant
–
–
residue was solubilized in methanol and passed through a column of freshly prepared (Cl to OH )
4

ACCEPTED MANUSCRIPT
Amberlite resin. Finally, purification by flash chromatography as detailed in supporting information,
furnished the desirable compounds. Additional set of aminopeptidase inhibitors was prepared with the
introduction of sulfur moiety in the molecule (Scheme 1). 6–Dimethylamino–9–[3’–(S–benzyl–L–
cysteinylamino)–3’–deoxy–β–D–ribofuranosyl purine compound 8, and the S–diphenylmethyl congener 9
were prepared according to the reported procedures from our laboratories [47]. To continue exploration of
the effect of additional bulk on sulfur moiety, S–triphenylmethyl analog 10 was prepared.
Furthermore, it is known that D–isomers of puromycin derivatives usually do not contribute to the
inhibition of protein synthesis phenomenon [48]. We sought to exploit this property to limit any possible
protein synthesis inhibition while studying aminopeptidase inhibition properties of the synthesized
compounds. To that end, a complimentary set of D–isomer congeners with a variable number of phenyl
rings on sulfur was prepared (Scheme 1, compounds 11–13).
a
Scheme 1. Synthesis of cysteine derivatives for the inhibition of aminopeptidases
a
–
Reagents and conditions: (a) DCC, NHS, DMF, rt, 12 h; (b) TFA, 10 min, rt, Amberlite IRA 410 (OH) .
To gain further insights into the importance of the nucleosidic fragment in the structure of the
inhibitors of aminopeptidases, we synthesized the non–nucleoside compound 17 (Scheme 2). The amine–
propanol fragment in 17 was devised to represent the 3’–, 4’–, 5’– carbons and 5’–hydroxy fragment of
the parent compound as shown in blue color in Scheme 2. N–Boc–protected S–benzhydryl–L–cysteine
was coupled with 3–amino–1–propanol in an EDC/HOBt mediated reaction. The coupling product 16 was
deprotected via the use of 4N HCl in dioxane solution to obtain the desirable final product 17.
5

ACCEPTED MANUSCRIPT
a
Scheme 2. Synthesis of non–nucleoside analog for the inhibition of aminopeptidases
a
Reagents and conditions: (a) EDC, HOBt, DMF, rt, 18 h; (b) 4 N HCl, dioxane, rt, 1 h.
Dose–Response Studies Against Puromycin–Sensitive Aminopeptidase (PSA). PSA is a ubiquitous
metallopeptidase encoded by the NPEPPS gene with subcellular distribution in the cytosol and nucleus
[49]. Comprising of a 919 amino acid sequence, it has a broad substrate specificity, and is responsible for
the release of N–terminal amino acids from a wide array of peptides, amides, and arylamides. The
inhibition of PSA in a biochemical assay was studied and dose response curves obtained for all the
analogs (Table 1, and Figure 3). First, PSA was cloned from cDNA, and purified in the lab (see
Experimental Section). Assays were performed at room temperature in a buffer of 25 mM Tris, pH 7.5.
The reaction was started with the addition of alanine–4–methoxy–2–naphthylamide (Ala–4–MNA)
substrate. The accumulation of 4–MNA was measured by exciting at 340 nm and reading the fluorescence
at 425 nm at room temperature for 30 minutes.
a
Table 1. Bioactivity studies of the inhibitors of aminopeptidases
Protein
Synth.
Inhib. (%)
Cpd
ID
Vero Cell
CC50
HL60
EC50
MOLT4
EC50
b
Structure
PSA IC50 APN (%)
APN IC50
c
6

ACCEPTED MANUSCRIPT
Protein
Synth.
Inhib. (%)
Cpd
ID
Vero Cell
CC50
HL60
EC50
MOLT4
EC50
b
Structure
PSA IC50
APN (%)
APN IC50
c
2
0
.8 ±
.16
0.055 ±
0.007
0.17 ±
0.0078
1
9.7 ± 2.6
15.7%
41 ± 6.6
99%
3
0
.5 ±
.88
0.80 ±
0.077
2
3
77.6%
82.9%
28%
35%
25 ± 4.1
>100
>100
0
0
.26 ±
.036
0.68 ±
0.21
13 ± 3.2 4.7 ± 1.6 2.1 ± 0.3
3
0
.2 ±
.24
0.41 ±
0.02
0.39 ±
0.029
4
5.0 ± 1.3
0.0%
ND
59%
0
0
.94 ±
.18
1.1 ±
0.088
0.88 ±
0.067
0.19 ±
0.008
0.18 ±
0.017
8
83.4%
30%
7

ACCEPTED MANUSCRIPT
Protein
Synth.
Inhib. (%)
Cpd
ID
Vero Cell
CC50
HL60
EC50
MOLT4
EC50
b
Structure
PSA IC50
APN (%)
APN IC50
c
0
0
.0074 ±
.0009
0.42 ±
0.032
1.3 ±
0.063
9
92.5%
0%
> 100
32 ± 3.7
1
0
.4 ±
.33
0.28 ±
0.0098
1
0
59.0%
0%
> 100
>100
31 ± 2.3
1
1
53 ± 4.0
73.8%
3.2 ± 2.9
0%
17 ± 3.8 46 ± 4.6 14 ± 2.7
D–Isomer
4
0
.8 ±
.72
1
2
58.7%
5.6 ± 4.9
0%
46 ± 14 34 ± 3.1 11 ± 1.8
D–Isomer
8

ACCEPTED MANUSCRIPT
Protein
Synth.
Inhib. (%)
Cpd
ID
Vero Cell
CC50
HL60
EC50
MOLT4
EC50
b
Structure
PSA IC50
APN (%)
APN IC50
c
0
0
.27 ±
.03
1
3
>100
89.1%
0%
15 ± 2.0 10 ± 6.3 14 ± 2.8
D–Isomer
1
7
53 ± 13
0.0
ND
0%
>100
>100
>100
a
b
c
All IC , CC , and EC values are in µM. Percent Inhibition @10 µM. Protein Synthesis Inhibition Percent
25 µM. Not Determined.
5
0
50
50
d
@
The effects of steric constraints in the structural manifold were explored. Compared to compound
8
, the presence of extra aromatic rings in compounds 9 and 10 led to an enhancement of their potency in
impeding PSA activity. Notably, the diphenylmethyl substituent (9) enhanced the anti–PSA activity to
nanomolar levels. Further introduction of additional steric bulk to the molecular structure led to a decrease
in potency. The trityl compound 10 exhibited a 204–fold loss in potency as compared to 9. These results
suggest the requirement of an optimum size of steric bulk in this class of inhibitors of aminopeptidases. As
compared to the L–isomeric analogs, the corresponding D–isomers of mono–, di–, and tri– phenyl ring
containing derivatives (11, 12, and 13, respectively) did not display desirable anti–PSA potency.
9

ACCEPTED MANUSCRIPT
Figure 3. Dose–response curves for the inhibition of PSA
Among the non–nucleoside compounds, while bestatin (2) displayed IC50 value of 3.5 µM,
tosedostat (3) inhibited PSA with submicromolar concentration. The synthesized non–nucleoside
derivative 17 did not exhibit effective inhibition of PSA. Even though the comparison of the
aminopropanol fragment in 17, with the corresponding 3’– to 5’–hydroxy fragment of the parent
compound, is rendered tenuous due to the lack of structural constraints in 17, the diminished potency for
this compound (53 µM) as compared to the corresponding nucleoside compound 9 (0.007 µM) strongly
indicates that advantageous anti–aminopeptidase properties are induced by the presence of nucleosidic
motif in these inhibitors of aminopeptidases.
Anti–aminopeptidase Enzyme Studies Against Aminopeptidase N (APN). Apart from PSA,
Aminopeptidase N (APN) is another enzyme relevant to this study. In reference to its subcellular
distribution, APN is also known as membrane aminopeptidase. Encoded by the ANPEP gene, it exists as a
2
+
homodimer that requires one Zn cofactor per subunit. This broad–specificity aminopeptidase displays
preference for the cleavage of neutral and basic amino acids from the N–terminal of the peptides.
Initially, all compounds were tested for their anti–APN activity at 10 µM in a single–concentration
assay in Recombinant Human Aminopeptidase N protein purchased from R&D Systems. Incubation of the
enzyme and the inhibitors in Tris buffer, pH 7.5, was followed by initiation of the reaction by the addition
of alanine–7–amino–4–methylcoumarin (Ala–AMC) substrate. The accumulation of AMC was measured
by exciting at 380 nm and reading the fluorescence at 460 nm at room temperature for 20 minutes. Percent
inhibition was calculated for each inhibitor at 10 µM.
10

ACCEPTED MANUSCRIPT
Figure 4. Anti–APN dose–response curves for selected compounds
Dose–response curves were obtained for selected APN–active compounds (Figure 4). Compound 9
displayed the best anti–APN effect at 10 µM (92.5%). Submicromolar activity was recorded for several
compounds including 9 and 10. The lack of APN inhibition by non–nucleoside compound 17 underscored
the relevance of the presence of the nucleoside motif for effective inhibition of aminopeptidases. In
comparison with the inhibition of PSA, the synthesized derivatives were generally found to be less potent
inhibitors of APN.
Toxicity–profile Studies. Since the design rationale of synthesized molecules was based on impeding the
action of aminopeptidases, without inducing toxicity towards normal cells, we sought to study the ability
of synthesized derivatives in inhibiting protein synthesis. These analogs were tested for the inhibition of
translation and post–transcriptional modification of GFP derived from the control plasmid included in the
1
–Step Human In Vitro Protein Expression Kit from Thermo Scientific. The prepared analogs were tested
at 25 µM with puromycin included as a negative control. Preincubation of the compounds at 30 °C for 30
minutes was followed by measuring the GFP production at the same temperature for 2 hours by using a
Molecular Devices SpectraMax i3 spectrophotometer with excitation at 482 nm, and recording emission at
5
18 nm. Notably, some of the most active compounds against PSA and/or APN (compounds 8 through 13)
did not affect protein synthesis, affording better toxicity–profiles for these derivatives. Tosedostat (3)
displayed 35% inhibition of protein synthesis at 25 µM concentration.
Furthermore, to address the concern that the observed positive in vitro activity results might be
stymied by the propensity of such assays to display false positives due to the inherent noxiousness of the
tested compounds, all analogs were evaluated against Vero cells in an MTT assay. Some of the most
11

ACCEPTED MANUSCRIPT
PSA–active compounds in this study (9 and 10, Table 1) did not inhibit the growth of Vero cells and
furnished CC50 values greater than 100 µM. This effect seems to emanate from the higher steric
constraints afforded by the bulky cysteinyl substituents for these compounds [50]. Amongst the known
inhibitors of aminopeptidases, while bestatin did not display harmful effects against Vero cells (CC >100
50
µM), the toxicity–profile of tosedostat (CC = 13 µM) raises concerns about the selectivity it furnishes in
5
0
achieving its therapeutic objectives.
While several factors, such as changes in pH, apoptosis, undesirable inhibition of other enzymes, etc.,
could be responsible for toxicity against Vero cells, our results also eliminate the possibility of protein
synthesis inhibition being the cause for this effect for compounds 11, 12, and 13.
Antitumor Cell Proliferation Studies. Encouraged by the reactivity and toxicity profiles of the tested
analogs, we proceeded to study the effects of these compounds in tumor cell lines. As mentioned earlier,
studies by Wheatly and coworkers demonstrated that inducing nutrient stress via depriving cells of
essential amino acids could be utilized to target various malignant phenotypes [30]. Human promyelocytic
leukemia (HL60), and human acute lymphoblastic leukemia (MOLT4) cells were identified as most
susceptible to this effect. The cells were maintained in RPMI 1640 growth media supplemented with 10%
FBS, 1% Penicillin/Streptomycin and 1% Glutamax–1. For both cell lines, the measurement of cell
viability was carried out using a modified method of Mosmann based on 3–(4,5–dimethylthiazol–2–yl)–
2
,5–diphenyltetrazolium bromide (MTT) [51]. Compounds were diluted in culture medium, added to the
wells in triplicate, and incubated for a further 72 hours at 37 °C in a 5% CO /95% air humidified
2
atmosphere, followed by removal of media and addition of freshly prepared MTT (at 1 mg/mL in serum–
free, phenol red–free RPMI 1640 media). The MTT was removed after 3 hours and formazan crystals
were solubilized with isopropanol. Plates were read on a Molecular Devices SpectraMax i3
spectrophotometer at 570 nm for formazan and 690 nm for background subtraction.
The observed EC50 values revealed significant antitumor activity against these cell lines for several
analogs (Table 1). The parent puromycin molecule (1) seemed to inhibit the proliferation of both HL60
and MOLT4 cells. This result is attributable to the protein synthesis inhibitory action of puromycin.
12

ACCEPTED MANUSCRIPT
Excellent activity of compound 9 against PSA translated into single–digit micromolar efficacy against the
ALL cell line (MOLT4). Another addition of an aromatic ring in the structural motif led to further
decrease in potency with S–triphenylmethyl analog (10) found to be inactive in both the cell lines. Among
other structural congeners, the D–isomer analogs were found to be inactive. Conceivably, the lack of
potency for non–nucleoside compound 17 against PSA and APN, translated into attenuated activity
against these malignant cell lines. These SAR trends will be indispensable in refining the structural
features required for better potency in assays targeting AML (HL60) and ALL (MOLT4) and other
hematologically compromised cell lines.
Molecular Modeling and Docking Studies
To gain a better understanding of the observed bio–activity results, molecular modeling analysis was
performed for the representative compounds. Compounds 8, 9, and 10 were docked into the active site of
APN. To assess the importance of the purine ring in these inhibitors of aminopeptidases, the non–
nucleoside analogue 17 was also included in docking studies. To that end, the X–ray crystallographic
structure of human APN in complex with the known aminopeptidase inhibitor bestatin (PDB code: 4FYR)
[54] was imported into the workspace and refined with Protein Preparation Wizard [55, 56] implemented
in Maestro 11.0.015.
Initially at the preprocessing stage, missing hydrogen atoms in the structure were added, and disulfide
bonds were created. Water molecules beyond 5 Å from hetero groups were removed. Next, the creation of
zero–order bonds to metals was followed by the generation of metal binding states. Furthermore, missing
side–chains and loops were added using Prime to obtain a comprehensive protein structure. Additional
refinement performed on chain A entailed the optimization of hydrogen bonding network by using
PROPKA [56]. Next, the structure of the protein was minimized using the Optimized Potentials for Liquid
Simulations–3 (OPLS3) force field [57] by converging heavy atoms to RMSD of 0.3 Å.
The receptor grid generation tool in Maestro (Schrodinger Inc.) was used to define an active site in the
3
refined protein structure. The grid was set to cover all the residues within 20 Å box centered on the
2
+
bestatin ligand (2) with the Zn metal cofactor as a constraint during docking.
13

ACCEPTED MANUSCRIPT
Compounds 8, 9, 10, and 17 were sketched using Maestro and subjected to LigPrep [58] to generate
conformers incorporating metal states in the pH range of of 7 ± 3 to be utilized for the docking process.
2
+
All the dockings were performed using Glide [59] in Extra Precision (XP) mode [60] with Zn metal
cofactor as a constraint. To soften the potential of non–polar parts of the ligands, the van der Waals radii
of ligand atoms were scaled by a factor of 0.80 with a partial charge cut–off of 0.15. All the ligands were
sampled as flexible entities for docking. Strain correction terms were applied and finally, post–docking
minimization was performed on the generated poses. The model was validated by docking bestatin (2) in
the receptor grid and comparing it to the co–crystal structure as reported in PDB 4FYR. All docking
experiments were performed with Glide Docking of the Schrodinger Suite software [61]. Highest ranked
poses for compounds 8, 9, 10, and 17 docked in the binding pocket of APN are presented here (Figure 5).
(a)
(b)
14

ACCEPTED MANUSCRIPT
(c)
(d)
Figure 5. Top scoring poses of representative compounds in the active site of APN (PDB code: 4FYR) as
2
+
receptor. Zn cation is depicted as teal sphere. Amino acid residues in black are labeled in accordance to
the APN crystal structure (4FYR). Docked compounds are depicted in violet: Predicted binding modes of
(a) compound 8 (b) compound 9 (c) compound 10 (d) compound 17.
Structural analysis of the best poses from the docking studies shows that while compounds 9 and 10
align themselves to the active site of the receptor in a congruent manner, compound 8 is positioned in a
significantly different fashion. The positional congruity of purine and furanosyl nucleosidic fragments of 9
and 10 is evident from their overlay (Figure 6, a). Conceivably, both compounds create similar contacts
with surrounding amino acids chains of APN. Inclusion of 8 in this overlay reveals divergent interactions
of 8 with the amino acid chains of the protein (Figure 6, b).
(a) ¶
(b)
15

ACCEPTED MANUSCRIPT
Figure 6. An overlay of representative nucleoside inhibitors of aminopeptidases (8, 9, and 10) in the
active site of aminopeptidase N: (a) Overlay of 9 (orange) and 10 (azure) (b) Inclusion of compound 8
(
magenta) in the overlay reveals different binding than 9 (orange) and 10 (azure)
Examination of ligand interaction diagrams for these docking analyses (See Figure S1 in the Supporting
Information) lends support to this hypothesis. Despite several favorable H–bond and –cation interactions
π
of Arg381, the ligand–receptor complex of 8 is unable to overcome its destabilization by His388 and
Tyr477. While Tyr477 presents steric challenges for the protonated free amine, His388 clashes with the
hydrogens on –5’ and –4’ positions of the sugar. The distance of zinc cation from the nearest atom of the
ligand (3.17 Å) is too high for it to allow metal ligation. Interestingly, for both compounds 9 and 10,
His388 provides favorable π–π stacking interaction without presenting any steric clashes with both these
ligands. Phe472 also contributes via Aromatic–H bonds with both 9 and 10. Both the compounds form
multiple hydrogen bonds with solvent molecules (H O1118, and H O1315). Additionally, compound 9
2
2
benefits from making H–bonds with H O1176. Furthermore, the zinc cation sits at a distance of 2.36 Å
2
and 2.24 Å respectively, from the 5’–hydroxyl group of 9 and 10. This favorable proximity allows both
the ligands to coordinate with the metal center. This provides an additional mode for the activation of the
hydrolytic water molecule for these ligands.
Compound 17 was included in this study to gauge the effect of exclusion of nucleosidic scaffold in these
inhibitors of aminopeptidases. Docking analysis of compound 17 revealed poor binding by the ligand.
Absence of purine base in the molecular scaffold keeps the molecule devoid of favorable
–cation stacking opportunities.
π–π, as well as
π
Docking scores for these modeling experiments represent calculated estimation of the binding energy for
the ligand–receptor complex. The calculated docking scores for these studies corroborate the structural
features noted above and provide support for the observed activity of these compounds in our bioassays.
With the best docking score (–8.509 kcal/mol) among the studied analogues, compound 9 justifies the
observed inhibitory activity against APN. Docking score for compound 10 (–7.953 kcal/mol) once again
verifies its close proximity with 9, in both the binding pocket, and furnishing proportionate bioactivity.
16

ACCEPTED MANUSCRIPT
With docking score of 0.689 kcal/mol, compound 8 justifies its inferior inhibitory activity against APN.
Efforts to obtain crystallographic structural information on PSA are currently ongoing in our laboratories.
CONCLUSIONS
Inhibitors of aminopeptidases based on the structural template of puromycin were prepared and studied
for their potency against two broad–specificity aminopeptidases – PSA and APN. Clear trends from the
SAR data confirmed a noteworthy correlation of the steric bulk with the corresponding anti–
aminopeptidase potency. The steric requirement allows the inhibitory efficacy to reach maxima but
continuing the increase in steric bulk after crossing that threshold, leads to a comparative attenuation of
potency. Compounds 8 and 9 display exceptional anti–aminopeptidase potency against the tested
aminopeptidases. Compounds 9 and 10 did not inhibit protein synthesis and also did not show toxicity
against Vero cells. The apparent better potency of compound 8 against the cancer cell lines is attributable
to its inherent toxicity (30% protein synthesis inhibition @ 10
µM; Vero cell CC = 0.88 ± 0.067 µM).
50
These data clearly support compound 9 as the compound of interest for further studies.
By providing a new avenue for medicinal agents based on the structural motif of puromycin, this work
should augment the research efforts towards a more streamlined approach to inhibiting aminopeptidases.
Further studies with more structural variations, correlation of activity in biochemical assays with cell–
based assays, and analysis of more aminopeptidases are currently being carried out in our laboratories.
EXPERIMENTAL SECTION
Chemistry. General Procedures. All commercial chemicals were used as supplied unless otherwise
indicated. Dry solvents were either purchased or dispensed under argon from an anhydrous solvent system
(J. C. Meyer) using `two packed columns of neutral alumina or molecular sieves. Flash chromatography
®
was performed on Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica)
columns and the indicated mobile phase. All moisture sensitive reactions were performed under an inert
1
13
atmosphere of dry argon with oven–dried glassware (150 °C). H–NMR and C–NMR spectra were
recorded on a Varian 600 MHz or a Varian 400 MHz spectrometer. Proton chemical shifts are reported in
17

ACCEPTED MANUSCRIPT
ppm from an internal standard of residual chloroform (7.26 ppm) or methanol (3.31 ppm), and carbon
chemical shifts are reported using an internal standard of residual chloroform (77.0 ppm) or methanol
(49.1 ppm). Proton chemical data are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad, dd = double doublet, ddd = doublet of doublet of
doublets, td = triplet of doublets, qd = quartet of doublets), coupling constant, integration. High–resolution
mass spectra were obtained on Agilent TOF II TOF/MS instrument equipped with either an ESI or APCI
interface. Melting points were measured on an electrothermal Meltemp–II manual melting point apparatus
and are uncorrected. Analysis of sample purity was performed on an Agilent Technologies 1200 Series
HPLC system equipped with an Agilent 1260 Infinity Evaporative Light Scattering Detector, using an
Agilent Microsorb MV 100–5 C18 column (4.6 mm × 250 mm, 5 µM particle size). HPLC conditions:
solvent A = H O, solvent B = acetonitrile; flow rate = 1.0 mL/min; compounds were eluted with the
2
following mixtures: 0–2 minutes (gradient, 5% acetonitrile in H O to 10% acetonitrile in H O); 2 to 25
2
2
minutes (gradient, 10% acetonitrile in H O to 100% acetonitrile); 25 to 30 minutes (isocratic, 100%
2
acetonitrile); 30–32 minutes (gradient, 100% acetonitrile to 5% acetonitrile in H O). Purity was
2
determined by total absorbance at 254 nm. All tested compounds have a purity ≥95%. TLC analysis was
done on Uniplate silica gel plates (scored 10 x 20 cm, 250 microns) from Analtech Inc.
General procedure (A) for the coupling of amino acids with puromycin aminonucleoside: To a dry
Schlenk tube purged with argon and equipped with a magnetic stirbar were loaded puromycin
aminonucleoside (PAN, 100.0 mg, 0.34 mmol, 1 equiv.) and N–hydroxy succinimide (115.1 mg, 0.37
mmol, 1.1 equiv.). Appropriate N–protected amino acid substrate (0.37 mmol, 1.1 equiv.) was added to the
flask under a cone of argon and the flask sealed with a rubber septum. Anhydrous dimethylformamide
(DMF, 3.4 mL, 10 mL per mmol) was added to the reaction vessel via a syringe and the mixture stirred
while being cooled to 0° C in an ice–bath. Dicyclohexyl carbodiimide (DCC, 76.3 mg, 0.37 mmol, 1.1
equiv.) was then added to the reaction mixture and contents stirred at 0 °C for 15 minutes. The ice bath
was then removed and reaction contents allowed to stir at room temperature for 18 hours. The reaction
progress was monitored by TLC analysis. After the completion of reaction, the precipitated side–product
18

ACCEPTED MANUSCRIPT
dicyclohexyl urea was filtered out using a Büchner funnel. The filter paper and the insolubles were
washed with copious amounts of ethyl acetate. Filtrate was collected and removed in vacuo to yield the
desirable product in crude form. Purification of the crude product was performed by flash chromatography
®
utilizing Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns,
and mobile phase eluents as described below.
General procedure (B) for the deprotection of amine: To a dry Schlenk tube purged with argon and
equipped with a magnetic stirbar was loaded the N–protected starting material (1 equiv.) and the tube
sealed with a rubber septum. An excess of anhydrous trifluoroacetic acid (TFA, 12 mL/mmol) was added
to the reaction vessel via a syringe and the contents stirred at room temperature for 10 minutes under
argon. After 10 minutes, the volatiles were removed in vacuo. To ensure a complete removal of TFA, the
contents in reaction tube were azeotroped thrice with dry acetonitrile (1 x volume of TFA used) to obtain
–
the product in crude form. A resin column of Amberlite IRA–410 OH resin was freshly prepared as
–
described: Amberlite IRA–410 (20–25 mesh) Cl form was weighed (5 gm per 100 mL of sample solution)
and loaded on a column. 1 N solution of NaOH was prepared fresh by dissolving appropriate amount of
low chloride NaOH in deionized water and passed through the column (20 x volume of resin). The
–
–
–
3 3
exchange of Cl with OH ions was monitored by HNO /AgNO test for the presence of Cl ions in the
–
eluent. Once the Cl ions were not detectable in the eluent, deionized water was passed through the
column and the pH of the eluent monitored by pH–paper until it came down to 9. Next, the column was
equilibrated by passing methanol through it (3 x volume of resin). The residue from the reaction vessel
–
was redissolved in methanol and passed through this freshly prepared Amberlite IRA–410 OH resin
column. The column was washed with copious amount of methanol (10–15 x volume of resin). Removal
of methanol in vacuo furnished the product in crude form. Purification of the crude product was performed
®
by flash chromatography utilizing Teledyne ISCO’s Combiflash Rf system equipped with normal phase
RediSep (silica) columns, and mobile phase eluents as described below.
19

ACCEPTED MANUSCRIPT
(S)–2–Amino–
N–((2S,3S,4R,5R)–5–(6–(dimethylamino)–9H–purin–9–yl)–4–hydroxy–2–
(hydroxymethyl)tetrahydrofuran–3–yl)–3–phenylpropanamide (4). The crude product obtained by
following general procedure B as described above, was purified by flash chromatography utilizing
®
Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns with
methanol/chloroform eluent mixture (neat CHCl for 8 minutes, to 20% MeOH/CHCl gradual gradient
3
3
over the next 40 minutes) furnished compound 4 as white solid in 13.8 mg (68%) yield. Melting point:
1
1
7
3
2
1
84–186 °C;
R
f
= 0.31 (MeOH:CHCl
3
/ 1:9); H–NMR (600 MHz, CD
3
OD):
= 5.2 Hz, 2H), 3.84 (t,
= 7.2 Hz, 1H), 3.48–3.33 (m, 6H), 2.89 (dd, = 13.2, 7.4 Hz, 1H),
171.6, 169.0, 164.8, 161.8, 137.7,
29.7, 129.0, 128.2, 126.4, 117.0, 90.5, 86.2, 83.4, 76.5, 73.7, 62.7, 60.8, 56.3, 50.4, 41.0; HRMS (ESI+):
δ
8.24 (s, 1H), 8.10 (s, 1H),
.20 (q,
.68 (d,
J
= 7.4 Hz, 2H), 7.17–7.11 (m, 3H), 5.85 (s, 1H), 4.46 (t,
= 12.4 Hz, 1H), 3.55 (t,
J
J
= 1.5 Hz, 1H),
J
J
J
1
3
.76 (dd,
J = 13.3, 7.1 Hz, 1H); C–NMR (151 MHz, CD OD): δ
3
m/z calculated for C H N O [M + H]+ 442.2197, found 442.2215 (error –4.01 ppm).
2
1
28
7
4
(R
)–2–Amino–
N
–((2
S,3
S,4
R
,5R
)–5–(6–(dimethylamino)–9
H–purin–9–yl)–4–hydroxy–2–
(hydroxymethyl)tetra hydrofuran–3–yl)–3–(tritylthio)propanamide (10). The crude product obtained
by following general procedure B as described above, was purified by flash chromatography utilizing
®
Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns with
methanol/chloroform eluent mixture (neat CHCl for 10 minutes, to 5% MeOH/CHCl gradual gradient
3
3
over the next 35 minutes, to 10% MeOH/CHCl by step gradient for 10 minutes) furnished compound 10
3
as light yellow powder in 98.2 mg (65%) yield. Melting point: 188–192 °C; Rf = 0.27 (MeOH:CHCl3 /
1
1
6
4
2
1
:19); H–NMR (400 MHz, CD OD):
δ
8.31 (d,
= 7.6, 5.7 Hz, 4H), 5.99 (d,
.08 (m, 1H), 3.96–3.91 (m, 1H), 3.89 (ddd, = 9.1, 4.8, 1.7 Hz, 1H), 3.86–3.82 (m, 1H), 3.48 (s, 6H),
.61 (dd, = 12.9, 8.1 Hz, 1H), 2.51 (dd, = 11.7, 3.8 Hz, 1H); C–NMR (101 MHz; CDCl
54.8, 151.39, 148.4, 144.33, 144.22, 141.2, 137.3, 129.5, 129.0, 128.05, 127.89, 127.2, 126.9, 91.2, 84.8,
J
= 5.1 Hz, 1H), 8.19 (d,
J = 4.2 Hz, 1H), 7.41–7.36 (m,
3
H), 7.30–7.26 (m, 6H), 7.21 (dd,
J
J
= 2.4 Hz, 1H), 4.59–4.50 (m, 2H), 4.12–
J
1
3
J
J
3
): δ 179.8,
20

ACCEPTED MANUSCRIPT
6
7.2, 62.1, 57.1, 53.5, 51.16, 51.13, 38.6, 34.6, 31.6, 29.67, 29.63, 25.2, 22.6, 14.1; HRMS (ESI+): m/z
+
calculated for C H N O S [M + H] 640.2700, found 640.2681 (error –0.12 ppm).
3
4
37
7
4
(S)–2–Amino–3–(benzylthio)–N–((2S,3S,4R,5R)–5–(6–(dimethylamino)–9H–purin–9–yl)–4–
hydroxy–2–(hydroxy methyl) tetrahydrofuran–3–yl)propanamide (11). The crude product obtained by
following general procedure B as described above, was purified by flash chromatography utilizing
®
Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns with
methanol/chloroform eluent mixture (neat CHCl for 10 minutes, to 20% MeOH in CHCl gradual
3
3
gradient over the next 25 minutes) furnished compound 11 as white powder in 31.7 mg (37%) yield.
1
Melting point: 158–162 °C; Rf = 0.32 (MeOH:CHCl / 1:19); H–NMR (400 MHz, CD OD):
δ
8.33 (s,
= 2.9
= 12.6, 1.7 Hz, 1H), 3.77 (s, 2H), 3.76–
3
3
1
H), 8.18 (s, 1H), 7.35–7.32 (m, 2H), 7.26 (dd,
Hz, 1H), 4.65–4.58 (m, 2H), 4.18 (t, = 3.3 Hz, 1H), 3.91 (dd,
.67 (m, 2H), 3.48 (s, 6H), 2.87 (dd, = 13.7, 5.4 Hz, 1H), 2.71 (dd,
101 MHz, CD OD): 154.7, 151.5, 149.2, 139.1, 138.1, 137.8, 128.71, 128.70, 128.64, 128.42, 128.25,
J = 13.7, 6.2 Hz, 2H), 7.21–7.18 (m, 1H), 6.02 (d, J
J
J
1
3
3
J
J = 13.8, 7.5 Hz, 1H). C–NMR
(
3
δ
1
28.11, 128.06, 128.02, 126.70, 126.60, 120.2, 104.3, 90.6, 83.9, 73.6, 61.0, 53.4, 50.6, 37.6, 35.8, 35.1;
+
HRMS (ESI+): m/z calculated for C H N O S [M + H] 488.2074, found 488.2043 (error 6.46 ppm).
2
2
29
7
4
(S)–2–Amino–3–(benzhydrylthio)–N–((2S,3S,4R,5R)–5–(6–(dimethylamino)–9H–purin–9–yl)–4–
hydroxy–2–(hydroxy methyl)tetrahydrofuran–3–yl)propanamide (12). The crude product obtained by
following general procedure B as described above, was purified by flash chromatography utilizing
®
Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns. The use
of methanol/dichloromethane eluent mixture (neat DCM for 15 minutes, to 20% MeOH in DCM gradual
gradient over the next 20 minutes) furnished compound 12 as white powder in 28.2 mg (51%) yield.
1
Melting point: 192–194 °C; Rf = 0.23 (MeOH:DCM / 1:19); H–NMR (400 MHz, CD OD):
δ
8.26 (s,
= 2.3
= 6.8 Hz, 1H),
= 13.3, 5.5 Hz, 1H), 2.55 (dd,
3
1
H), 8.11 (s, 1H), 7.34 (d,
Hz, 1H), 5.20 (s, 1H), 4.52 (t,
.83–3.75 (m, 2H), 3.66 (dd,
J
= 7.7 Hz, 4H), 7.19 (q,
= 6.5 Hz, 1H), 4.48 (dd,
= 12.7, 2.5 Hz, 1H), 3.40 (s, 6H), 2.67 (dd,
J
= 7.0 Hz, 4H), 7.12–7.08 (m, 2H), 5.93 (d,
J
J
J
= 5.6, 2.6 Hz, 1H), 4.08 (d,
J
3
J
J
21

ACCEPTED MANUSCRIPT
1
3
J
3
= 13.4, 7.0 Hz, 1H); C–NMR (101 MHz, CD OD): δ 175.0, 154.7, 151.5, 149.1, 141.5, 128.13, 128.03,
1
27.99, 126.8, 120.2, 90.6, 84.0, 73.7, 61.0, 54.0, 53.4, 50.4, 37.0, 31.3, 22.3, 13.0; HRMS (ESI+): m/z
+
calculated for C H N O S [M + H] 564.2387, found 564.2391 (error –0.62 ppm).
2
8
33
7
4
(S)–2–Amino–
N–((2
S,3
S,4R
,5
R
)–5–(6–(dimethylamino)–9
H–purin–9–yl)–4–hydroxy–2–
(hydroxymethyl)tetrahydrofuran–3–yl)–3–(tritylthio)propanamide (13). The crude product obtained
by following general procedure B as described above, was purified by flash chromatography utilizing
®
Teledyne ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns with
3 3
methanol/chloroform eluent mixture (neat CHCl for 10 minutes, to 20% MeOH in CHCl gradual
gradient over the next 40 minutes) furnished compound 13 as white powder in 42.2 mg (39%) yield.
1
Melting point: 180–182 °C; Rf = 0.42 (MeOH:CHCl / 1:19); H–NMR (400 MHz, CD OD):
δ 8.24 (s,
3
3
1
1
H), 8.09 (s, 1H), 7.31 (d,
.5 Hz, 1H), 4.47 (q, = 5.3 Hz, 2H), 4.05 (t,
12.6, 3.0 Hz, 1H), 3.39 (s, 6H), 3.00 (t, = 6.2 Hz, 1H), 2.44 (qd,
174.60, 154.70, 151.53, 149.10, 144.59, 137.80, 129.43, 129.19, 127.70, 127.45,
J
= 7.6 Hz, 5H), 7.18 (t,
J
= 7.6 Hz, 4H), 7.10 (t,
J
= 7.1 Hz, 2H), 5.91 (d,
J
=
J
J
= 2.5 Hz, 1H), 3.80 (dd,
J
= 12.5, 1.6 Hz, 1H), 3.64 (dd,
J
1
3
=
J
J = 14.3, 6.3 Hz, 2H); C–NMR (101
MHz, CD OD):
δ
3
1
26.58, 126.33, 120.15, 90.73, 90.52, 83.94, 73.62, 73.57, 66.44, 61.01, 53.76, 53.67, 50.37, 36.72;
+
HRMS (ESI+): m/z calculated for C34
H
37
N
7
O
4
S [M + H] 640.2700, found 640.2714 (error –2.11 ppm).
(R)–2–amino–3–(benzhydrylthio)–N–(3–hydroxypropyl) propanamide (17)
To a 100 mL round bottom flask equipped with a magnetic stirbar was loaded tert–butyl (R)–(3–
benzhydrylthio)–1–((3–hydroxypropyl) amino)–1–oxopropan–2–yl)carbamate (50.2 mg, 0.11 mmol, 1
(
equiv.). An excess of 4 N HCl in dioxane solution (5 mL) was added to the reaction vessel and the
contents stirred at room temperature for 1 hour. The reaction progress was monitored by TLC analysis.
After 1 hour, the volatiles were removed in vacuo. The residue thus obtained was dissolved in chloroform
3
(8 mL), then washed with deionized water (3 x 10 mL) and NaHCO (2 x 10 mL), and brine (10 mL). The
organic layer was dried over anhydrous MgSO and filtered. The removal of solvent in vacuo furnished
4
the product in crude form. The crude product was purified by flash chromatography utilizing Teledyne
®
ISCO’s Combiflash Rf system equipped with normal phase RediSep (silica) columns. The use of
22

ACCEPTED MANUSCRIPT
3 3
methanol/chloroform eluent mixture (neat CHCl for 5 minutes, to 20% MeOH in CHCl gradual gradient
over the next 22 minutes) furnished compound 17 as light yellow oil in 18.2 mg (48%) yield. Rf = 0.25
1
(
MeOH:CHCl / 1:19); H–NMR (400 MHz; CD OD):
δ
7.32 (t,
= 6.2 Hz, 2H), 3.20 (dd,
= 13.3, 6.6 Hz, 1H), 1.61 (dt,
174.4, 141.44, 141.41, 130.2, 129.3, 128.5, 128.17, 128.14, 128.09, 128.02,
26.88, 126.86, 125.7, 59.0, 54.1, 53.9, 36.9, 36.1, 31.7; HRMS (ESI+): m/z calculated for C19
J
= 5.5 Hz, 4H), 7.19 (t,
J
= 6.7 Hz, 4H),
3
3
7
2
.12–7.10 (m, 2H), 7.07–7.02 (m, 1H), 5.15 (s, 1H), 3.48 (t,
H), 2.60 (dd, = 13.3, 6.1 Hz, 1H), 2.48 (dd,
J
J
= 3.4, 1.7 Hz,
1
3
J
J
J = 13.0, 6.5 Hz, 2H). C–
NMR (101 MHz; CD OD):
δ
3
1
H
25
N
2
O
2
S
+
[
M+H] 345.1637, found 345.1718 (error 40.8 ppm).
Biology. Cloning of Puromycin−Sensitive Aminopeptidase (PSA). PSA (gene name NPEPPS) was
amplified from human cDNA with primers designed to clone into pEXP−His MBP using the InFusion kit
by Clontech Laboratories Inc. The forward primer contained XhoI and TEV cleavage sites as well as a 15
base−pair
extension
required
for
the
InFusion
enzyme
(GGCCGCCGTACTCGAGGAAAACCTGTATTTTCAGGGCTCCTTCATGCCGGAGAAGAGGCCC).
The reverse primer contained a HindIII site in addition to a 15 base−pair extension required for the
InFusion enzyme (CCACCCTTCCAAGCTTTCACACTGTGGG TGGTGAGGC). The resulting PCR
product was cloned into linearized pEXP−HisMBP cut with XhoI and HindIII with the addition of the
InFusion enzyme. A plasmid with His−6−MBP was chosen for affinity purification on a nickel column as
well as the assistance of MBP for enhanced solubility. The resulting plasmid, pEXP−HisMBP−NPEPPS,
expresses a fusion protein of TEV cleavable NPEPPS with an
enhancer.
N−terminal His−tagged MBP solubility
Expression and purification of PSA. pEXP−HisMBP−NPEPPS was transformed into BL21 STAR (DE3)
E. coli cells for protein expression. 2 x 5 ml overnight seed cultures grown in LB broth were used to
inoculate 1 liter LB containing ampicillin. Cultures were grown to an OD600 of 0.6 with shaking at 37 °C
(~3 hours) followed by induction with 0.5 mM IPTG and shaking overnight at 16 °C. The cells were
centrifuged at 5,000 x g for 10 minutes, the media was discarded and the cell pellets were allowed to stay
at −20 °C overnight. Cell pellets were resuspended in 45 mL lysis buffer (50 mM HEPES, 300 mM NaCl,
23

ACCEPTED MANUSCRIPT
1
0 mM imidazole, pH 8.0) and sonicated on ice with four bursts (2 min, 20% duty cycle, power level 8)
with a 1 min break between each burst. The lysate was centrifuged at 50,000 x g for 10 min then 1 mL of
0% Ni−NTA (Qiagen) was added to the cleared supernatant. After incubating at 4 C for 1 hour rotating
5
°
end over end, the lysate was loaded onto a column and the flow through was collected. The column was
washed with 16 mL of wash buffer (50 mM HEPES, 300 mM NaCl, 20 mM imidazole, pH 8.0) and eluted
with 2.5 mL of elution buffer (50 mM HEPES, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted
protein solution was desalted on a PD−10 column into TEV cleavage buffer (50 mM Tris·HCl, pH 8.0, 0.5
mM EDTA, 300 mM NaCl, 1 mM DTT). TEV protease was added to a final concentration of 5 mg/ml
and the proteins were allowed to cleave overnight at 4 C. The cleaved proteins were purified by adding
°
5
00 L of 50% Ni−NTA resin and incubating at 4° C for 1 hour to bind the His−tagged MBP and
µ
His−tagged TEV protease. The lysate was again loaded onto a column and the flow through was collected
which now contained cleaved PSA protein. The column was washed with 4 x 1 mL of TEV cleavage
buffer to wash off any weakly bound cleaved protein keeping the washes separate. The column was eluted
with 2.5 mL of elution buffer and the protein concentration of all fractions was determined using Bradford
reagent. The flow through and the first wash were combined and concentrated with a 3,000 molecular
weight cutoff spinfilter to 0.424 mg/mL giving a final concentration of 4.3
Dose–Response Studies of PSA. Puromycin–sensitive aminopeptidase (PSA) inhibition assays were
performed as described previously [52]. Briefly, 40 nM PSA was incubated with 0.8 L of inhibitor and
5 mM Tris, pH 7.5, in 80 L total volume in a black 384–well plate in duplicate. The reaction was started
with the addition of alanine–4–methoxy–2–naphthylamide (Ala–4–MNA) substrate (Sigma) at a final
concentration of 190 M, which was the lab calculated K (results not shown). The accumulation of 4–
µM.
µ
2
µ
µ
m
MNA was measured by exciting at 340 nm and reading the fluorescence at 425 nm at room temperature
for 30 minutes on a Molecular Devices SpectraMax i3 plate reader. Steady state velocities were used to
determine IC values by fitting the velocities vs. inhibitor concentration to the sigmoidal concentration–
5
0
response curve (variable slope) using GraphPad Prism.
24

ACCEPTED MANUSCRIPT
Anti–aminopeptidase Enzyme Studies for APN (Single Concentration studies at 10 µM). Aminopeptidase
5
3
N assays were performed as described previously. Aminopeptidase N was purchased from R&D
Systems. Amino–4–methylcoumarin (Ala–AMC) was purchased from Bachem. Briefly, 0.1 g/ L APN
was incubated with 1 L of 1 mM inhibitor in 50 mM Tris buffer, pH 7.5, in 100 L volume in a black
84–well plate in duplicate. The reaction was started with the addition of Ala–AMC substrate at a final
µ
µ
µ
µ
3
concentration of 100 µM. The cleavage accumulation AMC was measured by exciting at 380 nm and
reading the fluorescence at 460 nm on a Molecular Devices SpectraMax i3 plate reader for 20 minutes at
room temperature. Percent inhibition is calculated in GraphPad Prism software using slopes in the linear
range for each inhibitor at 10
µM.
Anti–aminopeptidase Enzyme Studies for APN (Dose–Response Activity). Aminopeptidase N enzyme
5
3
assays were performed as described previously. Briefly, 0.1
inhibitor in 50 mM Tris buffer, pH 7.5, in 50 L volume in a black 384–well plate in duplicate.
Compounds were tested at 3x dilutions from 100 M final concentration. The reaction was started with the
addition of Ala–AMC substrate at a final concentration of 100 M. The accumulation of AMC was
µg/ µL APN was incubated with 1 µL
µ
µ
µ
measured by exciting at 380 nm and reading the fluorescence at 460 nm on a Molecular Devices
SpectraMax i3 plate reader for 30 minutes at room temperature. Steady state velocities were used to
determine IC values by fitting the velocities vs. inhibitor concentration to the sigmoidal concentration–
5
0
response curve (variable slope) using GraphPad Prism.
Cell Proliferation Studies HL60, and MOLT4. Both HL60 and MOLT4 cells were obtained as part of the
NCI–60 Human Tumor Cell Lines Screen and were maintained in growth media: RPMI 1640
supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1% Glutamax–1. Cells were seeded in 96–
4
well Costar plates at 25 x 10 cells/ml and cultured at 37 °C in humidified 5% (v/v) CO
2
incubator for 24
hours. Compounds were diluted in culture medium, added to the wells in triplicate, and incubated for a
further 72 hours after which time the media was removed and MTT (Sigma) was added in RPMI phenol
red free media. MTT was removed after 3 hours and formazan crystals were solubilized with 200
µL of
isopropanol. Plates were read on a Molecular Devices SpectraMax i3 spectrophotometer at 570 nm for
25

ACCEPTED MANUSCRIPT
formazan and 690 nm for background subtraction. EC50 values were calculated by fitting the data in
GraphPad Prism software.
Protein Expression Inhibition Assay. Protein expression inhibition was tested using the Thermo Scientific
1
–Step Human In Vitro Protein Expression Kit (#88882) that enables the translation and post–
transcriptional modification of full–length proteins from mRNA or plasmid templates. The experiment
was set up according to the manufacturer’s directions in a 384 well black plate and the control plasmid
expressing GFP was added at a 3x dilution in 5% DMSO. Compounds were tested in duplicate with
puromycin added to the plate as a negative control. The plate was pre–incubated at 30 °C for 30 minutes
then GFP production was measured by exciting at 482 nm and reading the fluorescence at 518 nm at 30
°
C for 2 hours on a Molecular Devices SpectraMax i3 spectrophotometer. Percent inhibition is calculated
in GraphPad Prism software using slopes in the linear range for each inhibitor at 10 M.
µ
Cell Cytotoxicity (Vero cells). Vero cells were obtained from the American Type Culture Collection and
maintained in growth media: MEM supplemented with 10% FBS, 1% Penicillin/Streptomycin and 1%
Glutamax–1. Cytotoxicity assays and data analyses were performed as detailed above for cell proliferation
studies.
ASSOCIATED CONTENT
Supporting
Information.
The
following
files
are
available
free
of
charge.
The Supporting Information is available free of charge on the
1
13
Spectral data, including H–NMRs and C–NMRs of the synthesized compounds (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*
E–mail addresses: singh109@umn.edu (RS); vince001@umn.edu (RV)
Telephone numbers: +1 (612) 624–2146 (RS); +1 (612) 624–9911 (RV)
26

ACCEPTED MANUSCRIPT
Author Contributions
All authors have given approval to the final version of the manuscript.
Funding Sources
This research was supported by the funds from the Center for Drug Design at the University of Minnesota.
ACKNOWLEDGMENT
This research was supported by the funds from the Center for Drug Design at the University of Minnesota.
ABBREVIATIONS
PSA, puromycin–sensitive aminopeptidase; APN, aminopeptidase N; AML, acute myeloid Leukemia;
ALL, acute lymphoblastic leukemia; AADR, amino acid deprivation response; PAN, puromycin
aminonucleoside; DCC, dicyclohexyl carbodiimide; NHS,
N
–hydroxy succinimide; TFA, trifluoroacetic
Ala–AMC, alanine–7–amino–4–
acid; Ala–4–MNA, alanine–4–methoxy–2–naphthylamide;
methylcoumarin; MTT, 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyl tetrazolium bromide; TLC, thin layer
chromatography.
REFERENCES
1
.
Daluge, S.; Vince, R. Synthesis and antimicrobial activity of a carbocyclic puromycin analog. 6–
Dimethylamino–9–[R–[2R–hydroxy–3R–(p–methoxyphenyl–l–alanylamino)]cyclopentyl] purine. J. Med.
Chem. 1972, 15, 171–177.
2
.
Nathans, D. Puromycin inhibition of protein synthesis: Incorporation of puromycin into peptide
chains. Proc. Natl. Acad. Sci. U. S. A. 1964, 51, 585–592.
Cuyas, E.; Martin–Castillo, B.; Corominas–Faja, B.; Massaguer, A.; Bosch–Barrera, J.; Menendez,
3
.
J. A. Anti–protozoal and anti–bacterial antibiotics that inhibit protein synthesis kill cancer subtypes
enriched for stem cell–like properties. Cell Cycle 2015, 14, 3527–3532.
27

ACCEPTED MANUSCRIPT
4
.
Tworowski, D.; Klipcan, L.; Peretz, M.; Moor, N.; Safro, M. G. Universal pathway for post
transfer editing reactions: Insights from the crystal structure of TtPheRS with puromycin. Proc. Natl.
Acad. Sci. U. S. A. 2015, 112, 3967–3972.
5
.
Maquigussa, E.; Arnoni, C. P.; Pereira, L. G.; Boim, M. A. Calcitriol ameliorates renal damage in
a pre–established proteinuria model. Mol. Med. Rep. 2015, 12, 1009–1015.
Davidoff, A. N.; Mendelow, B. V. Unexpected cytokinetic effects induced by puromycin include a
G2–arrest, a metaphase–mitotic arrest, and apoptosis. Leuk. Res. 1992, 16, 1077–1085.
6
.
7
8
.
.
Vazquez, D. Inhibitors of protein synthesis. FEBS Lett. 1974, 40, S63–S84.
Vince, R.; Daluge, S.; Brownell, J. Carbocyclic purmocyin: Synthesis and inhibition of protein
biosynthesis. J. Med. Chem. 1986, 29, 2400–2403.
Vince, R.; Almquist, R. G.; Ritter, C. L.; Daluge, S. Chloramphenicol binding site with analogues
of chloramphenicol and puromycin. Antimicrob. Agents Chemother. 1975, , 439–443.
0. Vince, R.; Isakson, R. G. An acyclic puromycin analog. 6–Dimethylamino–9–[2–hydroxy–3–(
methoxyphenyl–L–alanylamino)propyl]purine. J. Med. Chem. 1973, 16, 37–40.
1. Peterson, M. L.; Vince, R. Synthesis and biological evaluation of 4–purinylpyrrolidine
nucleosides. J. Med. Chem. 1991, 34, 2787–2797.
2. Schnebli, H. P.; Phillipps, M. A.; Barclay, R. K. Isolation and characterization of an enkephalin–
degrading aminopeptidase from rat brain. Biochim. Biophys. Acta 1979, 569, 89–98.
3. Löwenberg, B.; Morgan, G.; Ossenkoppele, G. J.; Burnett, A. K.; Zachée, P.; Dührsen, U.;
9
.
8
1
p–
1
1
1
Dierickx, D.; Müller–Tidow, C.; Sonneveld, P.; Krug, U.; Bone, E.; Flores, N.; Richardson, A. F.;
Hooftman, L.; Jenkins, C.; Zweegman, S.; Davies, F. Phase I/II clinical study of tosedostat, an inhibitor of
aminopeptidases, in patients with acute myeloid leukemia and myelodysplasia. J. Clin. Oncol. 2010, 28
,
4
1
333–4338.
4. Tehler, U.; Nelson, C. H.; Peterson, L. W.; Provada, C. J.; Hilfinger, J. M.; Lee, K.–D.; McKenna,
C. E.; Amidon, G. L. Puromycin–sensitive aminopeptidase: An antiviral prodrug activating enzyme.
Antivir. Res. 2010, 85, 482–489.
28