7-Substituted pterins provide a new direction for ricin A chain inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.05.025

Ricin is a potent toxin found in castor seeds. The A chain, RTA, enzymaticlly depurinates a specific adenosine in ribosomal RNA, inhibiting protein synthesis. Ricin is a known chemical weapons threat having no effective antidote. This makes the discovery of new inhibitors of great importance. We have previously used 6-substituted pterins, such as pteroic acid, as an inhibitor platform with moderate success. We now report the success of 7-carboxy pterin (7CP) as an RTA inhibitor; its binding has been monitored using both kinetic and temperature shift assays and by X-ray crystallography. We also discuss the synthesis of various derivatives of 7CP, and their binding affinity and inhibitory effects, as part of a program to make effective RTA inhibitors.

Full text of DOI:10.1016/j.ejmech.2011.05.025

European Journal of Medicinal Chemistry 46 (2011) 3608e3615
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
7-Substituted pterins provide a new direction for ricin A chain inhibitors
*
**
Jeff M. Pruet, Karl R. Jasheway, Lawrence A. Manzano, Yan Bai, Eric V. Anslyn , Jon D. Robertus
Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A1590, Austin, TX 78712, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Ricin is a potent toxin found in castor seeds. The A chain, RTA, enzymaticlly depurinates a specific
adenosine in ribosomal RNA, inhibiting protein synthesis. Ricin is a known chemical weapons threat
having no effective antidote. This makes the discovery of new inhibitors of great importance. We have
previously used 6-substituted pterins, such as pteroic acid, as an inhibitor platform with moderate
success. We now report the success of 7-carboxy pterin (7CP) as an RTA inhibitor; its binding has been
monitored using both kinetic and temperature shift assays and by X-ray crystallography. We also discuss
the synthesis of various derivatives of 7CP, and their binding affinity and inhibitory effects, as part of
a program to make effective RTA inhibitors.
Received 3 February 2011
Received in revised form
10 May 2011
Accepted 11 May 2011
Available online 20 May 2011
Keywords:
Ricin inhibitors
Pterin
Published by Elsevier Masson SAS.
7-Substituted derivatives
Luciferase assay
1. Introduction
The X-ray structure of RTA has been solved and refined [6]. Site-
directed mutagenesis and substrate binding in crystalline RTA
Ricin is an example of a type 2 ribosome inactivating protein
(RIP). Such proteins are composed of two subunits, the A chain and
B chain. The A chain (RTA) is involved in the inactivation of the
ribosome itself while the B chain assists in the uptake of the protein
into cells [1]. It is the B chain that results in the increased cyto-
toxicity of the type 2 ribosome inactivating protein, relative to type
1 RIPs. Once in the cytoplasm, RTA depurinates a specific adenosine
on ribosomal RNA [2]. This inhibits protein synthesis and results in
the death of the target cell.
provided key information leading to the accepted mechanism of
depurination [7]. This information has in-turn assisted the design
and development of small molecules that could potentially func-
tion as an antidote by binding to the RTA active site and inhibiting
activity [3,8].
The Schramm group has taken a different approach to inhibitor
design. They constructed stem-loop oligonucleotides which were
effective in the nM range, although this inhibition requires a low pH
and appears nonfunctional at physiologic pH [9]. They have
recently used locked, cyclic polynucleotides to inhibit RTA and the
related RIP saporin, and have obtained a crystal structure of the
complex [10]. In general, nucleic acid based compounds have
limited cell uptake, are often subject to degradation, and therefore
have limited “drugability”.
Computer based virtual screening of commercially available
small molecule libraries has been used to expand the list of
potential RTA inhibitors that can function at physiological pH. A
number of chemical platforms have been identified for inhibitor
design, including pterin, purine, and pyrimidine based inhibitors
[3,8b,11]. Pteroic acid (PTA) was one such compound, showing
Ricin can be toxic at doses as low as 0.1e1 mg/kg, depending on
the mode of administration [3]. Ricin is extracted from castor seeds,
and can be easily purified from the by-products of castor oil
manufacturing [4]. Due to its ease of manufacture, as well as the
very high toxicity, ricin represents a real threat as a chemical
warfare agent. For example, one of the first well-known uses of
ricin as a weapon was in the “umbrella tip” assassination of Georgi
Markov by the KGB in 1978 [5]. This threat is compounded further
due to the absence of a known antidote.
modest inhibition in an in vitro translation assay IC50 of 600
mM.
This assay involves translation of the protein luciferase, which gives
a luminescent response with luciferin. Active RTA halts the protein
synthesis, and the activity of potential inhibitors is determined by
measuring the recovery of luciferase counts at different concen-
trations of the inhibitor. The crystal structure of the complex
defined the active site of RTA. It showed that the protein had a large
Abbreviations: RTA, ricin toxin A; PTA, pteroic acid; 7CP, 7-carboxy pterin; 7AP,
7-carbamoyl pterin; 7HP, 7-hydrazide pterin; DSF, differential scanning fluorom-
etry; BSA, bovine serum albumin.
* Corresponding author. Tel.: þ1 512 471 0068; fax: þ512 471 7791.
** Corresponding author. Tel.: þ1 512 471 3175.
E-mail addresses: anslyn@austin.utexas.edu (E.V. Anslyn), jrobertus@mail.
utexas.edu (J.D. Robertus).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.05.025

J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
3609
binding site for its ribosome target, but this cleft could be sub-
divided. Adenine binds in a well defined pocket called the speci-
ficity site. This pocket initially adopts a closed form and thus
binding requires the Tyr80 residue of RTA to rotate 45ꢀ to the open
form [7]. This site has evolved to bind aromatic rings with an
exocyclic amine and a particular pattern of hydrogen bond donors
and acceptors [8b]. The observed binding showed the feasibility of
designing compounds that could bind in this pocket, but which if
properly diversified, could make additional contacts that would
provide drug-like specificity and affinity. Although the pterins have
limited solubility, their ability to interact with a number of specific
groups in the RTA active site makes them an attractive drug design
platform. We therefore began an exploration of derivatized pterins
with improved inhibitor characteristics. The X-ray structure
Scheme 2. Synthesis of amide derivatives.
suggests that pterin positions
pendants that might reach the RTA surface and make favorable
interactions.
6 and 7 could accommodate
3. Results and discussion
The active site of RTA contains various hydrogen bond donors
and acceptors, aiding in the binding of the natural substrate
adenine. It was previously seen that the pterin ring of pteroic acid
and neopterin participate in a larger number and stronger inter-
actions within the active site, compared to adenine [8b,14]. As all
current pterin-based inhibitors found from commercial libraries
had relied on the 6-position for functionalization, we diversified
our library by testing the regioisomeric 7-substituted derivatives,
a task greatly assisted by our new synthetic means [13].
An early effort centered on attaching a soluble carboxylate to
the pterin platform. It was previously observed that 6-carboxy
pterin had little or no detectable inhibition and showed no
electron density when soaked into RTA crystals [8b]. However,
the newly synthesized, regioisomeric 7-carboxy pterin, 7CP, is
one of the more potent small molecule inhibitors of RTA we have
observed. This led to a paradigm shift in the direction of our
pterin-based synthesis, as most pterin chemistry focuses on the
6-substituent due to similarities to naturally occurring folic acid
[12]. We discuss herein the results of 7CP in an in vitro trans-
lation assay, a differential scanning fluorimetry (DSF) assay, as
well as the X-ray structure of this new inhibitor bound into the
active site of RTA. We also discuss the construction of new
7-substituted pterins and their subsequent results in these
Beginning our study with 7-carboxy pterin (8), we immediately
saw success with the in vitro luciferase-based assay, shown in Fig. 1.
As shown, the IC50 for 7CP is about 230 mM, superior to that of
pteroic acid and neopterin, and vastly superior to 6-carboxy pterin
[8b]. In general, proteineligand complexes are more thermally
stable, and will usually have a higher melting temperature, Tm.
Therefore binding of 7CP to RTA can be observed using differential
scanning fluorimetry, DSF [15]. As shown in Fig. 2, the RTAꢁ7CP
complex has a Tm about 2 ꢀC higher than RTA alone; the standard
deviation of Tm measures is 0.1e0.3 ꢀC. Experimental results for the
inhibitor binding experiments are accumulated in Table 1.
various assays, with many new compounds resulting in
mM
inhibition. We report two key X-ray structures in this paper; the
full crystallographic description of all these structures will soon
be released independently.
Compound 8 (7CP) was soaked into RTA crystals and X-ray data
collected to 1.29 Å resolution. Fig. 3 shows the electron density for
the bound 7CP and Fig. 4 shows details of the interaction with the
protein. As seen in previous structures of RTA in complex with
pterin derivatives, the 2-amino group of the pterin ring donates
one hydrogen bond each to the carbonyl oxygen atoms of Val81
and Gly121, and the 4-oxo and N5 atoms accept hydrogen bonds
from Arg180 [14]. The tautomeric form of the pterin with a proton
on N1 is favored so that N1 can donate a hydrogen bond to
a carboxyl oxygen of Gly121 and N3 can accept a hydrogen bond
from the amido-N of Val81. An additional hydrogen bond, which
was not seen in the previous 6-substituted pterins, is made
between the amido-N of Tyr123 and the carbonyl oxygen of the 7-
carboxy group. This extra hydrogen bond is retained in all
subsequent derivatives based on this platform, likely contributing
2. Chemistry
Many of the pterin-based compounds evaluated were synthe-
sized directly via a previously reported acyl radical insertion reac-
tion (3-7, Scheme 1) [13]. The simple pterin core (2), which was
prepared via condensation of glyoxal with 1, was taken up in
aqueous sulfuric acid and treated with iron sulfate, tert-butyl
hydroperoxide, and an acyl source to provide compounds 3-7 in
yields ranging from 22 to 48%. Quantitative hydrolysis of 3 with
NaOH at 80 ꢀC provided 7CP (eg, 8, Scheme 1).
N-Substituted carbamoyl structures (9-15) were synthesized via
treatment of the methyl ester of 7CP (eg. 3) with the corresponding
amine nucleophile in refluxing methanol in
(Scheme 2). Compound 4 can also be constructed in this manner, as
an alternative to the acyl radical reaction.
a sealed tube
Scheme 1. Synthesis of 7CP (8) and derivatives.

3610
J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
Compounds 11 and 13 were synthesized to investigate the effect
of aromatic rings with electron rich and poor substituents, using
3,4,5-trimethoxybenzyl amine and 4-fluorobenzyl amine respec-
tively. Compound 13 showed a marked improvement over the alkyl
amide, giving an IC50 of 570 mM in the translation assay, and a Tm
shift of 2 ꢀC in the DSF experiment. Efforts to obtain a crystal
structure for this compound have been unsuccessful to date, arising
from an inability to find an appropriate cryoprotectant for this
particular ligand. Compound 11 did not show any appreciable
inhibition of RTA in the translation assay, and only shifted the Tm
by 1 ꢀC. This finding may suggest a binding preference towards
electron deficient substituents, or that the size of the trimethox-
yphenyl group may preclude favorable interaction with the protein.
Compounds 10 and 12 explore heterocycle pendants, using
aminomethyl pyridine and furfuryl amine as the respective
N-substituents. Compound 10 has an IC50 of 500 mM, and shows
a Tm shift of 1 ꢀC. Its binding was also observed in a 1.26 Å X-ray
structure. The compound had diminished electron density around
the pyridine ring, indicating the ring is not in a fixed position and
appears to rotate about its long axis. Efforts are underway to
diversify this pendent to provide more specific interactions within
the active site, and thereby improve binding. Compound 12 showed
Fig. 1. RTA Inhibition by 7CP. RTA activity is plotted as a function of 7CP concentration.
The data were fitted to a hyperbolic decay function and the IC50 was determined to be
230 mM.
an IC50 of 380 m
M, and a Tm shift of 2 ꢀC was observed in the DSF
to the general increase in potency of this series of pterin-based
inhibitors over previous ones.
assay, indicating similar binding affinity to that of 7CP. This
compound has been successfully seen in complex with RTA through
X-ray data at 1.89 Å. In the crystal structure, the furan group is
oriented such that its oxygen is facing the hydroxyl group on Tyr80,
however the distance between the pair is too long, at approxi-
mately 4 Å, for a hydrogen bonding interaction. As seen in Fig. 5, the
furan is close to the so-called “second pocket” of RTA. This pocket is
on the opposite side of the Tyr80 ring from the specificity pocket.
The second pocket has been confirmed to be the binding site for
guanine base in the GAGA tetraloop substrate adjacent to the
adenine targeted for depurination [10]. Efforts are underway to
create pendants extending from the 5 position on the furan ring
that could potentially occupy this second pocket. An electrostatic
surface map of the specificity pocket is shown in Fig. 6 illustrating
possible requirements for these modifications.
Having seen success with the carboxylic acid at position 7, we
turned our attention to amide derivatives, starting initially with the
unfunctionalized 7-carbamoyl pterin (7AP, eg 4). Transitioning
from the carboxylic acid, which is anionic at physiological pH, to the
neutral amide reduced the solubility of the pterin. Pterins are
notoriously insoluble in most any solvent, often requiring protec-
tion of the exocyclic amine, which would undoubtedly block
binding to RTA due to the very specific interactions needed with
this amine [7d,8]. However, compound 4 exhibited a 1 ꢀC Tm shift
by DSF, and X-ray data of the RTA/7AP complex at 1.75 Å confirmed
binding to the active site, as shown in Table 1. Unfortunately this
amide did not show a strong response in the in vitro luciferase-
based assay, which is attributed to its poor solubility in the
HEPES buffer solution necessary to run the assay.
Despite the apparent decrease in activity of the simplest amide
derivative of 7CP, we continued to explore larger amides that might
make favorable contacts with RTA. Compound 9 was chosen to
explore simple alkyl amides; this compound showed an IC50 of
1.6 mM in the in vitro assay. Although it is a poorer inhibitor than
7CP, it is still an improvement over the 6-substituted neopterin and
previously reported guanine-based inhibitors [3,8b]. Compound 9
was also crystallized with RTA, and a 1.26 Å X-ray structure
confirmed it bound into the active site, with the pterin group
making the expected interactions.
These results, along with those seen with 11 and 13, can again be
taken to indicate a trend favoring the binding of electron deficient
pendants to the 7CP platform. This conclusion is supported by the
binding strength of the electron deficient benzoic acid moiety seen
previously for pteroic acid [8b]. There may be important steric
restraints on the inhibitors synthesized to this point, in that smaller
amide derivatives gave better results than larger ones. Compound
14 was synthesized to determine if steric bulk could be better
accommodated if located further from the relatively small speci-
ficity pocket. Indeed, this compound had an IC50 of 210
mM, and
Fig. 2. DSF profile for RTA and a 7CP-RTA complex. Panel A shows the fluorescence as a function of temperature for RTA (solid line) and with 7CP (dashed line). Panel B shows the
negative differential of the data from panel A with RTA as black circles and the complex with open circles; the minimum of each curve marks the Tm for the unfolding.

J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
3611
Table 1
Summary of results.
a
Entry
Structure
Name
IC50
ΔTm
Resolution
8
7-carboxy pterin (7CP)
200
m
M
2
1
ꢀC
1.29 Å
1.75 Å
1.26 Å
1.26 Å
NA
4
7-carbamoyl pterin (7AP)
No Inhibition
1.6 mM
ꢀC
9
N-methyl-7-carbamoyl pterin
0.4 ꢀC
10
11
12
13
14
15
N-(methylamino pyridinyl)-7-carbamoyl pterin
N-(3,4,5-trimethoxybenzyl)-7-carbamoyl pterin
N-(furanylmethyl)-7-carbamoyl pterin
N-(4-fluorobenzyl)-7-carbamoyl pterin
N-(2-(phenylamino) ethyl)-7-carbamoyl pterin
7-hydrazide pterin (7HP)
500
mM
1
1
2
2
3
ꢀC
ꢀC
ꢀC
ꢀC
ꢀC
No Inhibition
400
600
200
500
m
m
m
m
M
1.89 Å
NA
M
M
1.75 Å
1.97 Å
M (35%)
<1s
5
6
7-propionyl pterin
700
500
m
M
2
ꢀC
1.35 Å
7-acetyl pterin
mM (20%)
1.3 ꢀC
1.26 Å
NA
7
7-(p-methoxybenzoyl) pterin
No Inhibition
<1s
a
When 50% inhibition was not reached, % inhibition given in parenthesis at maximum concentration.
shifted the Tm of RTA by 3 ꢀC, even stronger than that observed for
7CP. The crystal structure of this complex shows that the aniline NH
donates a hydrogen bond to the side-chain of Asn 209. This is one of
the few polar interactions seen for the pendent groups of this
inhibitor series. However, as observed for compound 10, only weak
electron density was observed for the aromatic ring, suggesting it is
mobile. We conclude from the results of the amide derivatives that
smaller, electron deficient substituents are preferred when in close
proximity to the pterin head-group, though larger groups can be
successfully tolerated with proper spacer length.
Examination of the crystal structure of 7AP (eg. 4) indicated
a water molecule forms a hydrogen bonded bridge from the amide
NH2 to the carboxylate of Glu177 of RTA. With this in mind, we
synthesized a hydrazide derivative (7HP, 15), postulating that the
additional NH2 would replace this water molecule and enhance the
binding compared with 7AP. 7HP was soaked into RTA crystals, and

3612
J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
Fig. 5. Surface representation of RTA in complex with furanomethyl carbamoyl pterin
(Compound 12). While the pterin occupies the specificity pocket (A), the furan is in
position to be derivatized at the 5 position to extend pendants around Tyrosine 80 and
into the second pocket (B). In the surface representation of RTA, white represents
hydrogen, green represents carbon, blue represents nitrogen, and red represents
oxygen (PDB ID 3PX9). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 3. 7-carboxy-pterin in complex with RTA. Density from the FOeFC map, shown as
a blue mesh, is contoured at 3
s around the ligand. Active site residues are labeled for
orientation (PDB ID 3PX8). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
ray analysis at 1.35 Å resolution. Compound 7 had a much bulkier
pendent; it failed to give any detectable inhibition of RTA and could
not be soaked into RTA crystals. Its insolubility and presumed poor
binding by the restrained methoxybenzoyl made this a poor candi-
date for further development. While compounds such as 10, 13, and
14 all have similar sized pendants, they contain at least one sp3
carbon linking the amide to otherchemical moieties. This methylene
appears to allow for a more flexible binding geometry to the RTA
active site. Compound 7 has a planar linking group, but lacks the
flexible methylene in the linker. This restriction appears great
enough to outweigh any favorable binding interactions by the
pendent. The results from the ketone series are in agreement with
the trend seen with the amides above; compounds with electron
donating groups and bulky substituents in close proximity to the
pterin core have diminished binding.
X-ray data collected to 1.97 Å, confirmed that this compound was
bound into the RTA active site. However, it did not displace the
water bound to Glu177. That water repositioned by about 0.4 Å and
the water bridge to the hydrazide nitrogen remained intact. The
7HP compound has a maximum solubility of 500 mM in the HEPES
buffer used for the in vitro translation assay. At this concentration, it
inhibited RTA by 35%, indicating an IC50 below 1 mM range.
Although not a strong inhibitor, this modification was a clear
improvement over compound 4.
Finally, as part of our study we sought to examine how replace-
mentof carboxylic acid derivatives with simple ketones would affect
the binding of the pterin platform. Compounds 5-7 had been con-
structed previously, and allow for the investigation of van der Waals
interactions as well as the effect of steric bulk. However, ketone
derivatives suffer the same solubility problems seen when tran-
sitioning from carboxylic acids to amides. Compound 6, baring
4. Summary
a simple methyl ketone, was soluble to 500 mM for the in vitro assay,
Pterins are able to bind in the RTA specificity pocket and make
more, and stronger, hydrogen bonds to the protein than does the
however only 20% inhibition was observed in the translation assay.
This compound was also seen in an X-ray crystal bound in the RTA
active site in the same basic orientation as 7CP. Compound 5 was
a better inhibitor, having an IC50 of 700 mM, similar to pteroic acid,
and a Tm shift of 2 ꢀC. Once again this pterin was observed in the X-
Fig. 6. Electrostatic surface map of the RTA active site. Red represents electron rich
regions, and blue indicates electron deficient or positive charge. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Fig. 4. Stereo view of 7CP bound to the active site of RTA. Hydrogen bonds are shown
as black dashed lines.

J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
3613
substrate adenine [8b]. This chemical specificity makes it an
attractive inhibitor design platform despite its poor solubility. The
platform can, in principle, be diversified to provide inhibitors with
better solubility and specificity. In this paper we examined the
utility of adding substituents at position 7 of the pterin. A carbox-
ylate at position 6 had previously abolished binding and this site
was not explored further [8b]. A carboxylate at position 7, in
contrast, was a good inhibitor of RTA. A simple amide at position 7
renders the compound much less soluble and a poorer inhibitor. It
is possible to make larger derivatives which are not necessarily
more soluble, but where additional contacts with RTA provide
comparable binding affinity. Amide derivatives with a methylene
linker appear to be able to adjust to the RTA active site. For example,
N-(furanylmethyl)-7-carbamoyl pterin has activity close to that of
7CP and places the furan ring in a position where further deriva-
tization should be able to reach a second binding pocket on RTA. In
principle, future compounds that use this potential may have both
improved binding specificity and solubility. Nearly all of the new
compounds showed a marked improvement in activity over neo-
pterin, and a number of these are also superior to pteroic acid [8b].
This work highlights the importance of 7-substitued pterin analogs
in the design of ricin inhibitors.
Addition of 1 mL THF resulted in product precipitation. The yellow
solid was filtered off and washed with cold methanol followed by
THF, then dried under vacuum to give 42.3 mg (85% yield) 9.
mp > 350 ꢀC(dec) 1H NMR (DMSO-d6, 400 MHz)
d
8.82 (s, 1H), 2.81
(d, J ¼ 4.8, 3H); 13C NMR (DMSO-d6, 125 MHz)
d 162.1, 160.4, 155.3,
154.7, 145.2, 136.9, 131.9, 26.3; HRMS-ESI (m/z): [M þ Na]þ calcd for
(C8H8N6O2Na)þ 243.06009; found 243.05983.
5.1.2. N-(methyl pyridinyl)-7-carbamoyl-pterin (10)
This compound was synthesized, using aminomethyl pyridine,
through an analogous method as that used for 9, using 50 mg of 3.
After the reaction was complete by LCMS analysis, it was cooled and
added to 50 mL cold water and refrigerated, whereby a yellow solid
formed. After filtration and drying, 40 mg (59% yield) of 10 was
recovered. mp ¼ 300 ꢀC(dec) 1H NMR (DMSO-d6, 400 MHz)
d 9.52
(t, J ¼ 5.9, NH), 8.9 (s, 1H), 8.63 (d, J ¼ 5.2, 1H), 8.0 (t, J ¼ 7.7, 1H), 7.55
(d, J ¼ 7.9, 1H), 7.49 (t, J ¼ 7.3, 1H), 4.71 (d, J ¼ 5.5, 2H); 13C NMR
(DMSO-d6, 125 MHz) d 163.3, 160.3, 155.9, 155.5, 154.3, 147.2, 145.8,
140.8, 136.8, 131.9, 123.6, 123.0, 42.7; HRMS-ESI (m/z): [Mþ1]þ
calcd for (C13H12N7O2)þ 298.10470; found 298.10461.
5.1.3. N-(3,4,5-trimethoxybenzyl)-7-carbamoyl-pterin (11)
This compound was synthesized with 3,4,5-trimethoxybenzyl
amine through an analogous procedure as used for 9, using
30 mg of 3. After precipitation with THF, filtration, and drying,
38 mg (76% yield) of 11 was recovered. mp ¼ 335 ꢀC(dec) 1H NMR
5. Experimental section
5.1. Synthesis
(DMSO-d6, 400 MHz)
d
9.33 (t, J ¼ 6.2, NH) 8.84 (s, 1H), 6.66 (s, 2H),
All reagents used were of commercial quality and were obtained
from Aldrich Chemical Co. and Fisher Scientific and were used as
received. 1H (400 MHz) and 13C (125 MHz) NMR spectra were
recorded in DMSO-d6 on a Varian spectrometer using the solvent as
reference. Chemical shifts are given in parts per million (ppm).
LCeMS data was recorded on an Agilent 6130 Quadrupole instru-
ment. High resolution mass spectrometry was performed with
a Varian 9.4T QFT-ESI ICR system. All solvents were removed by
4.4 (d, J ¼ 6.3, 2H), 3.72 (s, 6H), 3.6 (s, 3H); 13C NMR (DMSO-d6,
125 MHz)
d 164.1, 160.5, 156.0, 154.5, 152.7 (2C), 147.8, 136.7, 136.4,
134.7, 131.6, 104.9 (2C), 59.9, 55.8 (2C), 42.7; HRMS-ESI (m/z):
[M þ H]þ calcd for (C17H19N6O5)þ 387.14114; found 387.14119.
5.1.4. N-(furanylmethyl)-7-carbamoyl-pterin (12)
This compound was synthesized with furfuryl amine through an
analogous procedure as used for 9, using 30 mg of 3. After precipi-
tation with THF, filtration, and drying, 34.5 mg (92% yield) of 12 was
rotary evaporation under vacuum using
a standard rotovap
equipped with a dry ice condenser. All filtrations were performed
with a vacuum. Purity of all final compounds was determined to be
>95% on a Shimadzu HPLC with a 4.6 ꢂ 150 mm Phenomenex
recovered. mp > 350 ꢀC(dec) 1H NMR (DMSO-d6, 400 MHz)
d 9.26 (t,
J ¼ 6.2, NH), 8.86 (s,1H), 7.57 (d, J ¼ 2.8,1H), 6.42e6.35 (m,1H), 6.28
(d, J ¼ 2.8, 1H), 4.50 (d, J ¼ 6.4, 2H); 13C NMR (DMSO-d6, 125 MHz)
gemini 5
mm C18 column. The eluents were A, water, and B,
d 162.4, 159.5, 153.5, 152.0, 151.7, 147.4, 142.2, 138.2, 131.6, 110.5,
acetonitrile. Gradient elution from 0% B to 30% B over 20 min with
a final hold at 90% B for 5 min. Total run time was 25 min.
2-Aminopteridin-4-one (2), 7-methoxycarbonyl-pterin (3),
7-carbamoyl-pterin (4), 7-propionyl-pterin (5), 7-acetyl-pterin (6)
and 7-(p-methoxybenzyl)-pterin (7) were all prepared through
their literature procedures [12a,13].
7-carbamoyl-pterin (7AP eg. 4) can also be prepared by sus-
pending 3 in ammonia saturated methanol, in a sealed conical vial,
and heating to 80 ꢀC overnight with stirring. Addition of a small
amount of 0.5 M HCl, to neutralize, followed by filtration gave
a yellow solid which, after washing with methanol and drying, was
identical to the sample prepared through the previous method [13].
7-carboxy-pterin (7CP eg. 8) was prepared by hydrolysis of 3 in
0.5 M NaOH at 80 ꢀC overnight. The pH of the homogeneous
solution was then adjusted to 5 with 2 M HCl, and the yellow
precipitate filtered off and washed several times with water. This
was dried under vacuum to provide 8 in quantitative yield. This
compound had identical 1H and 13C NMR to those reported in the
literature [16].
107.2, 35.9; HRMS-ESI (m/z): [M þ H]þ calcd for (C12H11N6O3)þ
287.08871; found 287.08873.
5.1.5. N-(4-fluorobenzyl)-7-carbamoyl-pterin (13)
This compound was synthesized with 4-fluorobenzyl amine
through an analogous procedure as used for 9, using 20 mg of 3.
After precipitation with THF, filtration, and drying, 21 mg (74%
yield) of 13 was recovered. mp > 350 ꢀC(dec) 1H NMR (DMSO-d6,
400 MHz)
d
9.48 (t, J ¼ 6.6, NH), 8.86 (s, 1H), 7.38 (dd, J ¼ 5.3, 9.0,
2H), 7.14 (t, J ¼ 8.9, 2H), 4.47 (d, J ¼ 6.8, 2H); 13C NMR (DMSO-d6,
125 MHz) d 162.9, 161.9, 160.3, 154.4, 147.7, 136.6, 135.4, 135.3, 131.6,
129.4, 129.3, 115.0, 114.8, 41.7; HRMS-ESI (m/z): [M þ H]þ calcd for
(C14H12N6O2F)þ 315.10003; found 315.10003.
5.1.6. N-(2-(phenylamino) ethyl)-7-carbamoyl pterin (14)
This compound was synthesized with N-phenyl-ethylenedi-
amine through an analogous procedure as used for 9, using 20 mg
of 3. After precipitation with THF, filtration, and drying, 17.5 mg
(60% yield) of 14 was recovered. mp ¼ 330 ꢀC(dec) 1H NMR (DMSO-
d6, 400 MHz)
d
8.95 (t, J ¼ 6.1, NH), 8.81 (s, 1H), 7.00 (dd, J ¼ 8.5, 7.3,
5.1.1. N-methyl-7-carbamoyl-pterin (9)
2H), 6.55 (d, J ¼ 7.7, 2H), 6.45 (t, J ¼ 7.2, 1H), 5.71 (t, J ¼ 5.6, 1H) 3.43
To a suspension of 50 mg (0.23 mmol) 3 in 2 mL MeOH in
a sealed conical vial was added 0.3 mL methylamine. This was
stirred overnight at 60 ꢀC resulting in a homogeneous yellow
solution, at which point the reaction was complete by LCMS.
(dd, J ¼ 13.0, 6.5, 2H), 3.13 (dd, J ¼ 12.6, 6.1, 2H); 13C NMR (DMSO-d6,
125 MHz) d 162.9, 160.4, 156.0, 154.4, 148.6, 147.6, 136.6, 131.6, 128.9
(2C), 115.6, 111.9 (2C), 42.2, 38.3; HRMS-ESI (m/z): [M þ Na]þ calcd
for (C15H15N7O2Na)þ 348.11794; found 348.11795.

3614
J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
5.1.7. 7-hydrazide-pterin (7HP eg. 15)
1 mM EDTA, 4.1% PEG MW 8000) containing a dilution of micro-
seeds, and allowed to equilibrate in hanging drops of 10 l over
500
l reservoirs of mother liquor at 4 ꢀC. Microseed stocks were
prepared by adding an intact monoclinic RTA crystal to 500 l of
This compound was synthesized with 98% hydrazine through an
analogous procedure as used for 9, using 30 mg of 3. After
precipitation with THF, filtration, and drying, 18 mg (60% yield) of
14 was recovered. mp ¼ 290 ꢀC(dec) 1H NMR (DMSO-d6, 400 MHz)
m
m
m
mother liquor and vortexing the solution with a Seed Bead
(Hampton Research). Dilutions of the seed stock yielding optimum
quality crystals were determined for each new seed stock.
The inhibitors were dissolved in modified mother liquor (75 mM
BES pH 7.5, 4.1% PEG MW 8000) at an estimated final concentration
of 1.5 mM. RTA crystals were transferred to hanging drops of
modified mother liquor with inhibitor, and were allowed to soak for
at least 48 h prior to data collection. The crystals were transferred
briefly to drops identical to the soaking condition with added
cryoprotectant, mounted in a cryoloop (Hampton Research), and
flash frozen in liquid nitrogen. In some cases, the presence of
1.5 mM concentrations of different inhibitors changes the interac-
tion between the cryoprotectant and the crystal such that the
crystal is destroyed or the suppression of ice crystal formation is no
longer sufficient, therefore a cryoprotectant other than what was
used for native RTA crystals must be used. For 7CP, 15% glycerol was
used, and for compound 12, 10% PEG MW 400, 10% PEG MW 3350
was used. After flash freezing, crystals were either mounted
immediately in a nitrogen cold stream for data collection on the
home source, or they were stored in a dewar for shipping to the
Advanced Light Source.
d
8.9 (s, 1H), 7.34 (s, NH2); 13C NMR (DMSO-d6, 125 MHz)
d 161.2,
160.1, 155.6, 154.5, 145.7, 136.6, 132.6; HRMS-ESI (m/z): [M þ Na]þ
calcd for (C7H7N7O2Na)þ 244.05534; found 244.05536.
5.2. Luciferase translation assay
An in vitro translation assay was adapted from the Promega
original protocol for their Rabbit Reticulocyte Lysate System as
previously described [8a]. The compounds to be tested were solu-
bilized in 0.05 N KOH or 100% DMSO prior to their inclusion in the
assay. After mixing RTA and various concentrations of compounds
in the presence of BSA, translation reactions were initiated by
mixing a portion of the RTA/compound mixture with reticulocyte
lysate, and translation mix (amino acids, RNasin ribonuclease
inhibitor, luciferase control mRNA). For reactions in which
compounds were solubilized in DMSO, two distinct 30 min room
temperature incubations were performed. The first was done upon
mixing the various concentrations of compounds with BSA, and the
second when RTA was added. The reactions were incubated for
90 min at 30 ꢀC, after which they were stopped by freezing them
at ꢃ20 ꢀC. After thawing at room temperature, 2.5
ml reaction
mixture was mixed with 40
ml Luciferase Substrate Reagent
5.5. X-ray data collection and structure determination
(Promega), and their luminescence was measured on a Perkin
Elmer Envision luminometer (Waltham MA).
Diffraction data for the RTA-Compound 12 complex were
collected at 100 K on an R-AXIS IVþþ image plate detector (Rigaku)
with X-rays generated from a Rigaku MicroMax007 rotating anode
generator operated at 40 kV, 30 mA. Data for the RTA-7CP complex
were collected at 100 K on beamline 5.0.3 at the Advanced Light
Source (Lawrence Berkeley National Laboratory). Diffraction images
were processed and data reduced using HKL2000 [18]. For both
structures, the unit cell parameters were isomorphous with the
native monoclinic RTA crystals [17]. Data for the RTA-7CP complex
were collected to 1.29 Å resolution, including 227,142 observations
of 61,933 reflections, and the RTA-Compound 12 data were
collected to 1.89 Å with 73,906 observations of 18,685 reflections.
The data sets were scaled with Rmerge ¼ 4.2% and Rmerge ¼ 5.0% for
RTA-7CP and RTA-Compound 12 respectively.
For each concentration of inhibitor to be tested, reactions were
run both in the presence and absence of RTA. To calculate the RTA
activity, corrected for interference with the assay by the tested
compounds, the percent differences in luminescence for the cor-
responding reaction pairs were divided by the percent difference
for the controls with no inhibitor present. Values for IC50 were
calculated by fitting the plot of RTA activity vs. inhibitor concen-
tration to a hyperbolic decay function. An example plot is shown in
Fig. 1.
5.3. Differential scanning fluorimetry
Experiments were performed using a LightCycler 480 Real Time
PCR instrument (Roche) in a 96 well plate format. The samples were
prepared as 100
aliquoted into a LightCycler 480 multi-well plate. The samples
contained RTA at a final concentration of 2 M for both experi-
mental and control wells, the inhibitor was at a nominal final
concentration of 500 M for the experimental wells, and both sets
ml master mixes from which 30 ml triplicates were
m
Table 2
X-ray data data collection and refinement statistics for RTA/7CP and RTA/(12)
complexes.
m
were buffered in 100 mM HEPES pH 7.5, 125 mM NaCl. After mixing
the inhibitor with the RTA, a 30 min room temperature incubation
was done prior to the addition of fluorescent dye. SYPRO Orange
(Invitrogen) was added to each sample well to a final concentration
of 5X from a freshly prepared working stock of 100X. Samples were
heated from 25 ꢀC to 80 ꢀC in a LightCycler with a ramp rate of
Statistics
RTA$7CP
RTA$(12)
a (Å)
b (Å)
c (Å)
42.7
67.6
49.4
112.8
42.4
67.3
49.5
112.5
b
(deg)
Space group
Resolution (Å)
No. observed
Rmerge (last shell) (%)
I/dI (last shell) (%)
Completeness (last shell) (%)
Redundancy
No. reflections
P21
P21
45.69e1.29
227142
4.2 (27.6)
36.8 (3.2)
95.3 (91.3)
3.7
45.56e1.89
73906
5.0 (43.2)
44.9 (3.5)
95.6 (89.5)
3.7
0.04 ꢀC/s. Fluorescence of SYPRO Orange
(lex
¼
498 nm,
lem ¼ 610 nm) was measured at a frequency of 15 acquisitions per
ꢀC. The Tm for each sample was calculated from the first derivative
plot of fluorescence versus temperature. An example plot is shown
in Fig. 2.
61933
20.6
18685
19.5
Rwork (%)
5.4. Crystallization of RTA-inhibitor complexes
Rfree (%)
23.7
0.028/2.24
0.060
0.953
0.940
23.9
0.022/1.96
0.157
0.961
0.949
Rms deviation from ideality (Å,deg)
Estimated overall coordinate error(Å)
Correlation coefficient FOeFC
Correlation coefficient FOeFC FREE
Monoclinic RTA crystals were grown as described previously
[17]. Briefly, RTA at a concentration of 3 mg/ml was mixed at a 1:1
ratio with mother liquor (75 mM TriseHCl pH 8.9, 10 mM BME,

J.M. Pruet et al. / European Journal of Medicinal Chemistry 46 (2011) 3608e3615
3615
[3] D.J. Miller, K. Ravikumar, H. Shen, J.K. Suh, S.M. Kerwin, J.D. Robertus, J. Med.
Chem. 45 (2002) 90.
[4] D.R. Franz, N.K. Jaax, in: Textbook Of Military Medicine: Medical Aspects Of
Chemical And Biological Warfare, Office of the Surgeon General, Department
of the Army, United States of America, 1997.
[5] A. a. F. Loyd, M., in London Times, November 16 ed., London, England, 2001.
[6] (a) D. Mlsna, A.F. Monzingo, B.J. Katzin, S. Ernst, J.D. Robertus, Protein Sci. 2
(1993) 429;
Models were generated by molecular replacement as described
earlier [8a]. Briefly, the native RTA structure (PDB code 1RTC) was
modified such that Tyrosine 80 was replaced by Alanine, and was
used as the starting model for molecular replacement using MOL-
REP [19] from the CCP4 suite [20]. Refinement and electron density
map generation were carried out by REFMAC [21] and manual
model building and refinement, including the building of Tyrosine
80 and the ligand into the electron density, were done using COOT
[22]. Omit maps with amplitudes FoeFc were generated by REFMAC
using the refined complex structures, from which the ligand had
been removed, for phasing. A summary of the processing and
refinement statistics is shown in Table 2.
(b) W. Montfort, J.E. Villafranca, A.F. Monzingo, S.R. Ernst, B. Katzin,
E. Rutenber, N.H. Xuong, R. Hamlin, J.D. Robertus, J. Biol. Chem. 262 (1987)
5398.
[7] (a) A. Frankel, P. Welsh, J. Richardson, J.D. Robertus, Mol. Cell Biol. 10 (1990)
6257;
(b) Y. Kim, D. Mlsna, A.F. Monzingo, M.P. Ready, A. Frankel, J.D. Robertus,
Biochemistry 31 (1992) 3294;
(c) Y. Kim, J.D. Robertus, Protein Eng. 5 (1992) 775;
(d) A.F. Monzingo, J.D. Robertus, J. Mol. Biol. 227 (1992) 1136.
[8] (a) Y. Bai, A.F. Monzingo, J.D. Robertus, Arch. Biochem. Biophys. 483 (2009) 23;
(b) X. Yan, T. Hollis, M. Svinth, P. Day, A.F. Monzingo, G.W. Milne, J.D. Robertus,
J. Mol. Biol. 266 (1997) 1043.
Acknowledgments
[9] X.-Y. Chen, P.J. Berti, V.L. Schramm, J. Am. Chem. Soc. 122 (2000) 1609.
[10] M.C. Ho, M.B. Sturm, S.C. Almo, V.L. Schramm, Proc. Natl. Acad. Sci. U S A 106
(2009) 20276.
[11] (a) Y. Bai, B. Watt, P.G. Wahome, N.J. Mantis, J.D. Robertus, Toxicon (2010);
(b) P.G. Wahome, Y. Bai, L.M. Neal, J.D. Robertus, N.J. Mantis, Toxicon 56
(2010) 313.
This work was supported by National Institute of Health (NIH)
grant AI 075509, by Robert A. Welch Foundation grant F1225, and
by the College of Natural Sciences support to the Center for Struc-
tural Biology.
[12] (a) P. Waring, W.L.F. Armarego, Aust. J. Chem. 38 (1985) 629;
(b) E.C. Taylor, R.N. Henrie, R.C. Portnoy, J. Org. Chem. 43 (1978) 736.
[13] J.M. Pruet, J.D. Robertus, E.V. Anslyn, Tetrahedron Lett. 51 (2010) 2539.
[14] X. Yan, P. Day, T. Hollis, A.F. Monzingo, E. Schelp, J.D. Robertus, G.W. Milne,
S. Wang, Proteins 31 (1998) 33.
[15] F.H. Niesen, H. Berglund, M. Vedadi, Nat. Protoc. 2 (2007) 2212.
[16] U. Ewers, H. Gunther, L. Jaenicke, Chemische Berichte-Recueil 106 (1973)
3951.
Appendix. Supplementary data
The supplementary data associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2011.05.025.
References
[17] J.D. Robertus, M. Piatak, R. Ferris, L.L. Houston, J. Biol. Chem. 262 (1987) 19.
[18] Z. Otwinowski, W. Minor, Methods Enzymol. 27 (1997) 307.
[19] A. Vagin, A. Teplyakov, J. Appl. Cryst. 30 (1997) 1022.
[20] CCP4, Acta Cryst D5 (1994) 760.
[21] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Acta Crystallogr. D Biol. Crystallogr.
53 (1997) 240.
[1] (a) J.M. Lord, L.M. Roberts, J.D. Robertus, Faseb J. 8 (1994) 201;
(b) J.D. Robertus, A.F. Monzingo, in: M.W. Parker (Ed.), Protein Toxin Structure,
R.G. Landes Company, Austin, 1996, p. 253.
[2] (a) Y. Endo, K. Tsurugi, J. Biol. Chem. 262 (1987) 8128;
(b) M.P. Ready, Y. Kim, J.D. Robertus, Proteins 10 (1991) 270.
[22] P. Emsley, K. Cowtan, Acta Crystallogr. D Biol. Crystallogr. 60 (2004) 2126.