Peptide-conjugated pterins as inhibitors of ricin toxin A

Article abstract of DOI:10.1021/jm3016393

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of ricin toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC₅₀ values in the low micromolar range.

Full text of DOI:10.1021/jm3016393

Article
pubs.acs.org/jmc
Peptide-Conjugated Pterins as Inhibitors of Ricin Toxin A
Ryota Saito,*,† Jeff M. Pruet,‡ Lawrence A. Manzano,‡ Karl Jasheway,‡ Arthur F. Monzingo,§
Paul A. Wiget,‡ Ishan Kamat,‡ Eric V. Anslyn,*,‡ and Jon D. Robertus*,‡
†Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi 274-8510, Japan
‡Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A5300, Austin, Texas 78712, United
States
§Institute for Cellular and Molecular Biology, University of Texas at Austin, 2500 Speedway A4800, Austin, Texas 78712, United
States
ABSTRACT: Several 7-peptide-substituted pterins were
synthesized and tested as competitive active-site inhibitors of
ricin toxin A (RTA). Focus began on dipeptide conjugates, and
these results further guided the construction of several
tripeptide conjugates. The binding of these compounds to
RTA was studied via a luminescence-based kinetic assay, as
well as through X-ray crystallography. Despite the relatively
polar, solvent exposed active site, several hydrophobic
interactions, most commonly π-interactions not predicted by
modeling programs, were identified in all of the best-
performing inhibitors. Nearly all of these compounds provide
IC50 values in the low micromolar range.
particular the adenine binding “specificity” pocket, but in
general specific interactions will require suitably polar
inhibitors, inevitably leading to a penalty in desolvation upon
binding. To date, successful small molecule RTA inhibitors are
most commonly based upon pterins, guanines, pyrimidines, and
stem-loop oligonucleotides.3,8 Schramm and his colleagues have
developed transition state analogues in an RNA framework that
have low Kd values at acidic pH around 4.9
The use of pterins as RTA inhibitors began with pteroic acid
(PTA), giving an IC50 of 600 μM and a binding mode nearly
identical to that predicted by virtual screening programs.8b
Despite the modest potency, PTA remained the strongest RTA
inhibitor in the pterin series for over 10 years. This changed
when a preference for 7-substituted pterins was discovered,
with 7-carboxy-pterin (7CP) showing an IC50 of 230 μM.8a
Several amide derivatives of 7CP were explored in an attempt
to build off the initial lead structure. Optimization was slow
because of the notorious insolubility associated with the pterin
heterocycle, as traditional coupling techniques proved fruitless
because of solvent restrictions.8a,10 Structures of a previous key
pterin-based RTA inhibitors are shown in Figure 1 below.
A great improvement in the complexity and rate of
production of new pterin amides came through the use of
1,8-diazabicyclounde-7-cene (DBU), which we discovered
converts 7-methoxycarbonyl-pterin (1) to the methanol-soluble
DBU salt and further activates the carbonyl in situ to catalyze
amidation.11 No longer limited in the complexity of accessible
1. INTRODUCTION
The cytotoxin ricin, found in castor beans, is a prototypical type
2 ribosome inactivating protein.1 As such, it contains a catalytic
A chain, ricin toxin A (RTA), which depurinates a specific
adenosine in rRNA, and a lectin B chain that assists in cellular
uptake.2 Ricin poisoning can occur through a variety of
methods. As an example, when dispersed in aerosol form, it is
toxic at doses below 1 μg/kg body weight.3 Further adding to
its threat, ricin can be easily extracted from castor beans and the
byproducts of castor oil extraction. The low toxic dosage and
ease of acquisition make ricin a potential weapons threat, and
indeed ricin has been used as a weapon, most notably in the
assassination of Georgi Markov.4 Furthermore, there were
numerous cases of attempted use of ricin in the first decade of
the 21st century, including contaminated letters sent to
Washington.5
Countermeasures for ricin have included vaccines, inhibitors
of cellular trafficking, and competitive active-site inhibitors. The
last of these has been the method of choice in our work, as
widespread vaccination carries many practical challenges, and
the target for the cellular trafficking inhibitors is often not
understood, complicating rational drug design.6 The structure
of ricin, and RTA specifically, is known and has been refined to
high resolution. The mechanism of action is well understood
from structural and mutagenesis work.7 With this sound
foundation, RTA is a reasonable candidate for rationally
designed active-site inhibitors. There is one major concern
with such a design project, however. The native substrate is the
ribosome, and the RTA binding site is, not surprisingly, large
and mostly polar. There are some hydrophobic areas, in
Received: November 6, 2012
Published: December 7, 2012
© 2012 American Chemical Society
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Figure 1. Key structures of previous RTA inhibitors.
a
Scheme 1. Synthesis of Dipeptide Conjugates 2−8
a
See Table 1 for full structures.
a
Scheme 2. Synthesis of Tripeptide Conjugates 9−16
a
See Table 2 for full structures.
pterin amides, we synthesized a variety of expanded furanyl and
triazole linked pterins.11a While most derivatives exhibited IC50
values in the range 150−400 μM, several derivatives showed
IC50 in the 15−70 μM range and the improvements attributed
to the triazole ring forming a stronger polar contact with the
Tyr-80 residue. Many of the optimization strategies were aimed
at accessing the so-called “secondary binding pocket” of
RTA.6a,9 In the ligand-bound form of ricin, the side chain of
Tyr-80 rotates from a “closed” conformation, blocking access to
the specificity pocket, to an open form. This allows access to
the specificity pocket and divides the active site into two
pockets, with the secondary binding pocket accommodating an
adjacent guanosine on RNA.9
construction of 7-methoxycarbonyl-pterin (1), which was
prepared via acyl radical insertion.10 Compound 1 was
suspended in a minimal amount of dry methanol, and
solubilized through the addition of DBU, which also catalyzes
the amidation reaction.11 Commercially available dipeptides
(Scheme 1) or tripeptides (Scheme 2) were then added in a
slight excess (2 equiv). Through use of at least 4 equiv of DBU
the carboxylic acid of the peptides is deprotonated, while
sufficient DBU remains for the dual role of solubility and
nucleophilic catalysis. Yields for the reactions ranged from 29%
to 98%.
3. RESULTS AND DISCUSSION
In the optimization of drug candidates, peptides are of
particular interest because of their commercial availability and
the diverse structural characteristics provided by individual
amino acids.12 We postulated that the use of peptides would be
a convenient method of mapping the active site of RTA for
potential favorable interactions. We report herein the use of di-
and tripeptide conjugated pterins with the general design
depicted in Schemes 1 and 2, with an emphasis on aromatic
side chains, as competitive binding inhibitors of RTA. Within
the series, the best structure showed an IC50 of 6 μM.
3.1. Dipeptide Conjugated Pterins. Previous results from
our laboratory showed that bulky aromatic groups in the
immediate proximity of the pterin headgroup were not well
accommodated in the active site, while groups with extended
linker length could better access favorable binding interaction-
s.8a With those results in mind, we opted to begin exploring
dipeptides rather than single amino acids attached to the pterin
core. Strengthening this design plan, modeling by the virtual
docking programs GOLD and ICM showed dipeptides were an
appropriate length to move toward the secondary binding
pocket. The first amino acid, which we shall refer to as P1,
appeared by modeling to solely serve the role of a simple
spacer. Three representative P1 amino acids were chosen:
glycine, alanine, and serine. Glycine represents a simple linker.
2. CHEMISTRY
The synthesis of all compounds used in this study was carried
out as shown in Schemes 1 and 2. Synthesis began with the
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Alanine provides a more hydrophobic linker and introduces a
source of conformational restrictions due to α-substitution.
Serine serves as a polar linker capable of hydrogen bonding. For
the second amino acid, P2, emphasis was placed on aromatic
side chains, which could in principle stack with the Tyr-80
residue that separates the two pockets.
Containing a glycine−phenylalanine dipeptide, compound 2
represents the simplest of structures that meet the above
criteria for our inhibitor design. Screening compound 2 against
RTA in a luciferase-translational assay, we observed an IC50 of
20 μM (Figure 2) (see Experimental Details). This represents a
Figure 2. Dose−response curve for compound 2 with RTA. RTA
activity was determined through normalization of luciferase counts in
the presence and absence of 2. Percent activity is plotted as a function
of inhibitor concentration.
Figure 3. (a) Binding of 2 to RTA showing unexpected edge-to-face
interaction. (b) Comparison of inhibitors with Gly-Gly and Gly-Phe
pendants. The RTA active site is shown as the thinner bonds and the
ligands with thicker bonds. Carbons of 2 are light, while those of the
Gly-Gly ligand are dark. Waters are shown as spheres.
10-fold improvement in potency over the lead structure 7-
carboxy-pterin (7CP), which had an IC50 of 200 μM.8a Because
we were intrigued by the nature of the improved potency, a
solution of 2 was soaked into preformed RTA crystals and X-
ray data were collected. The binding mode of 2 is shown in
Figure 3. As seen previously with pterin/RTA complexes, the
pterin makes all the expected interactions with RTA. These
include a tautomerization of the lactam NH, allowing for a
hydrogen bond from the N1 position of the pterin to the
carbonyl of Gly-121, and the N3 position accepting a hydrogen
bond from Val-81. Furthermore, an amide NH from the
peptide forms a hydrogen bond bridge with a water molecule to
the side chain of Glu-177. In addition to these favorable polar
interactions, an unexpected edge-to-face interaction between
the aromatic side chain of the inhibitor’s P2 group with the
invariant Trp-211 residue of RTA was observed. To
accommodate the space filled by the bulky aromatic ring, the
Tyr-80 residue has rotated by roughly 45° compared to that
seen for previous 7-substituted pterins. This π-interaction was
not predicted by the docking programs GOLD and ICM,
highlighting the utility of empirically mapping the active site for
favorable interactions.
well as changes in solvation of both the ligand and the protein.
In short, caution is required in interpreting how changes in this
series of ligands affect the observed IC50 values. One valid
assumption is that when all the atoms of a bound ligand can be
seen in the X-ray structure, it suggests that specific interactions
are made that can compensate for the entropic cost of freezing
out bond rotations within that ligand.
The IC50 for 7CP is about 200 μM, that for a pterin
derivative bearing solely a Gly pendant is about 600 μM (data
not shown), and that for the Gly-Phe derivative 2 is 20 μM. All
three make the same strong interactions between the pterin and
the RTA specificity pocket; however, it appears that attaching
only the P1 Gly linker is energetically unfavorable, likely for
entropic reasons. However, adding the P2 Phe residue
substantially improves binding. Presumably the favorable
edge-to-face interaction can subsidize the cost of freezing the
inhibitor and also contributes enough favorable interaction to
drive the binding to a 30-fold increase over the monopeptide
derivative. We can also compare compound 2, with its Gly-Phe
pendant, to one with Gly-Gly. Such a compound has an IC50 of
300 μM; its binding is superimposed on 2 in Figure 3b. The
pterin binding is identical and the pendent chains are similar.
The C-terminal carboxylates occupy the same volume of space.
The added phenyl side chain of 2 makes the edge-to-face
interaction with the side chain of Trp211 as described above.
Note that the side chain of Tyr 80 is displaced upward in 2 to
In an analysis of the effects of chemical modification on
ligand binding, it is important to recall that a change in the free
energy of binding of just 1.4 kcal/mol changes binding affinity
by a factor of 10. A 3-fold change in affinity is the result of a
difference of just 0.6 kcal/mol. The ΔG of binding reflects
differences in enthalpy, typified by changes in hydrogen bond
strength, and also reflects entropy. The latter is difficult to
assess, as it involves configurational entropy of the ligand as
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make space for the ligand phenyl group. The phenyl group at
P2 of compound 2 also displaces two waters, labeled W1 and
W2, present in the Gly-Gly complex. It is plausible that the
enthalpically favorable edge-to-face interaction combined with
the entropically favorable release of water drives the binding of
2, which is about 2 kcal/mol more favorable than that of the
Gly-Gly ligand.
Table 1. Summary of Dipeptide Conjugates
Encouraged by the above result, we synthesized compounds
3 and 4, which vary in the π-donor ability of the aromatic
ring.13 The tyrosine-terminated compound 3 performed quite
well in the in vitro assay, showing an IC50 of 6 μM. X-ray
analysis revealed that compound 3 shared an identical binding
mode as 2, displaying the same edge-to-face interaction. The
indole ring of tryptophan has been identified as one of the best
aromatic π-donors and thus would be expected to further
strengthen the edge-to-face interaction, thereby strengthening
inhibitor binding.13 However, the P2 tryptophan compound 4
showed slightly diminished potency compared to the previous
two structures, with an IC50 of around 40 μM. Examination of
the crystal structure of the RTA/4 complex revealed a
somewhat disordered binding, as the pterin ring was firmly
locked in place and clearly visible, but the peptide showed very
limited density, indicating nonspecific interactions. Some
evidence of the placement of the tryptophan side chain was
observed through displaced water molecules previously seen in
the active site. While 4 was an unexpectedly weaker inhibitor
than 2 or 3, it is still superior to the vast majority of RTA
inhibitors that function at physiological pH.3,8a,b,d
Our next goal was to change the linkers for the phenylalanine
and tryptophan based compounds as a means to explore side
chain steric and hydrogen bonding effects. Compounds 5−8
were synthesized and tested for their activity against RTA. The
use of an alanine as P1 produced stronger inhibitors than
serine, as compounds 5 and 6 had IC50 values of around 20 μM
while 7 and 8 had IC50 values closer to 100 μM (see Table 1).
In this way, the slightly less potent P2 tryptophan compound 4
was improved through the use of alanine (e.g., 6). Several
attempts were made to synthesize an Ala-Tyr linked pterin, as it
was an attractive target given the above results for alanine and
the potency of 3. However, the alanine series proved more
challenging synthetically. It appears the branched α-carbon
severely diminishes reactivity, and compounds 5 and 6 could
only be synthesized through use of DMSO as a cosolvent at
elevated temperatures. The serine series does not suffer such
steric limitations, presumably because of the side chain
hydroxyl further activating the pterin carbonyl via hydrogen
bonding. The use of numerous forcing conditions never proved
to be successful in the synthesis of an Ala-Tyr derivative.14
While inhibition results from the serine-linked compounds (7,
8) were less than satisfactory, some interesting structural
features were observed when these compounds were soaked
into RTA crystals and X-ray data collected. The hydroxyl group
of P1 serine forms a hydrogen bond with the side chain of Asn-
209, which appears to pull the dipeptide “out of place” from
making the edge-to-face interaction just described for 2 and 3.
This likely explains the decreased potency and suggests that the
gain of a P1 hydrogen bond contributes less to the binding
affinity than the hydrophobic π-interaction. Interestingly, in the
case of the serine−tryptophan conjugated pterin (8), the “out
of place” positioning led to the ability to make a cation−π
interaction with Arg-258 (Figure 4). However, this alternative
π-interaction cannot compensate for the loss of the edge-to-face
interaction. It is possible the glycine−tryptophan conjugated
Figure 4. X-ray structure of the RTA/8 complex showing the
cation−π interaction.
pterin (4) showed disordered binding due to the indole ring
being partially involved in a cation−π interaction and partially
in the edge-to-face interaction.
To accommodate the anionic phosphate backbone in the
natural RNA substrate, the linker space between the two
binding pockets of RTA is lined with positive charge with three
exposed arginine residues; in addition to the catalytic R180,
these include arginines 134, 215, and 258.6a It is of note that
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compound 8 represents the first clear interaction with an
arginine residue in the space between the two binding pockets
and it is a cation−π interaction. To date, none of the terminal
carboxylates of the dipeptides formed a salt bridge with a
guanidinium of arginine. While the π-interactions observed in
this study are rooted in electrostatics, one might not expect
such a polar active site to show greater affinity for ligands
seeking interactions more commonly associated with hydro-
phobic environments. When comparing the cation−π inter-
action of compound 8 to the edge-to-face interactions of 2 or 3,
the diminished activity of 8 likely stems from the enthalpic
penalty of desolvating a charged arginine, a penalty that was not
compensated by the π-interaction. Desolvation of an aromatic
side chain, however, occurring for the edge-to-face interaction,
should not carry a significant enthalpic penalty, leading to a net
gain in binding strength for 2 and 3.
Table 2. Summary of Tripeptide Conjugates
3.2. Tripeptide Conjugated Pterins. The results from the
dipeptide series indicated that access to the second pocket
would require a longer peptide pendant. With the terminal
carboxylate of the dipeptides not involved in any strongly
contributing interaction, further diversification to tripeptides
could potentially give rise to increased binding affinity. Our
efforts with the tripeptide derivatives can be divided into two
main categories: those that build directly off results seen in the
dipeptide series and those suggested by virtual modeling of
potential inhibitors. Compounds 9−13 were synthesized
toward the first goal, while compounds 14−16 fit the latter
direction (Scheme 2). Alanine was not explored as a linker in
this series because of the synthetic challenges encountered in
the dipeptide series. The results are summarized in Table 2.
The Gly-Phe-Phe conjugated pterin 9 exhibited an IC50 of 15
μM, the same level of potency as the parent dipeptide 2. X-ray
analysis for the RTA/9 complex revealed the P2 aromatic side
chain was again making the energetically favorable edge-to-face
interaction, while the terminal P3 aromatic side chain formed a
slipped-stacked π-interaction with the P2 benzene ring (Figure
5). As the terminal end of the ligand is merely interacting with
the ligand itself and not the protein, it is not surprising that 9
should have similar potency as 2. In a relatively solvent-exposed
area, the P3 ring is best able to interact with the inhibitor and
not the protein target.
Compounds 10 and 11 are structurally similar to 9 but vary
in the π-density at P3 and also have a potential for P3 to make a
favorable polar interaction within the active site. The P3
tyrosine-terminated compound 10 showed activity on par with
that of 2 and is only slightly less potent than 9. The tryptophan-
terminated pterin (11) had an IC50 of 35 μM, weaker still than
the parent tripeptide conjugate 9. Efforts to obtain suitable
crystal structures for both 10 and 11 have been unsuccessful to
date.
In an effort to bypass the intramolecular slipped stack seen
for 9, the tripeptide conjugate 12 was synthesized containing a
Gly-Leu-Phe pendant (Scheme 2). By replacing the P2 amino
acid with leucine, we should replace the edge-to-face interaction
with a generic hydrophobic interaction, reduce the P2−P3
stacking, and perhaps allow P3 to bind deeper in the RTA
second pocket. Compound 12 displayed an IC50 of 35 μM.
Although slightly weaker than 9, 12 still represents a relatively
strong interaction. The Gly-Leu-Tyr conjugate 13 was also
synthesized. It had an IC50 of around 20 μM. Crystallographic
data were collected for the complexes of RTA with both of
these modified tripeptide conjugates. In both cases the leucine
side chain was clearly observed filling the space once occupied
Figure 5. Surface representation of the RTA/9 complex, showing
edge-to-face and slipped-stacked π-interactions. The RTA surface is
shown as an electrostatic map.
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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 mm × 150
mm Phenomenex Gemini 5 μm C18 column. The eluents were A,
consisting of water + 0.1% TFA, and B, consisting of acetonitrile +
0.1% TFA. Gradient elution was from 5% B to 50% B over 25 min
with a final hold at 90% B for 5 min. Total run time was 30 min.
5.1. Synthesis. 7-Methoxycarbonyl-pterin (1). 1 was synthe-
sized by our previously reported acyl-radical insertion.10
General Procedure for Synthesis of Peptide Conjugated
Pterins. To a slurry of 7-methoxycarbonyl-pterin (1) in MeOH (0.5−
1.0 mM) was added 4 equiv of DBU to give a clear solution. An
amount of 2 equiv of the corresponding peptide was then added to the
mixture, which was stirred at room temperature for 4−24 h. Reaction
completion was judged by LC−MS analysis. After the reaction was
complete, dilute aqueous HCl was added dropwise to afford a yellow
precipitate. The solid was collected by filtration and washed several
times with water and methanol to provide the product. In instances of
remaining starting material or the hydrolyzed byproduct 7-carboxy-
pterin (7CP), the crude solid was redissolved in 0.5 mM aqueous
NaOH and loaded onto an LH-20 gel filtration column and eluted
with 0.1 M NaCl. The pure fractions were pooled and precipitated
with HCl, followed by filtration to provide the pure final product.
N-{N-(Pterin-7-yl)carbonylglycyl}-L-phenylalanine (2). This
compound was synthesized in 59% yield using the above method.
1H NMR (400 MHz, DMSO-d6) δ/ppm 12.80 (s, 1H), 11.59 (s, 1H),
by the aromatic side chains of 2, 3, and 9; however, neither
structure displayed strong density for the terminal P3 amino
acid. Without the driving force to slip-stack, the aromatic ring
of P3 must rotate freely and may occupy numerous partially
stabilizing geometries.
Modeling was undertaken to identify a tripeptide conjugate
with the best potential of accessing the secondary binding
pocket. Despite tryptophan being the least promising aromatic
amino acid explored with the dipeptides, it was predicted to be
the most promising as a third amino acid in accessing the
second pocket by π-stacking with Tyr-80. In the choice of
dipeptide linkers to a tryptophan, serine appeared best in the
virtual docking studies. While serine-based compounds gave the
poorest performance in the dipeptides, modeling suggested
promise when serine was used for a tripeptide conjugate. Thus,
we chose to examine variations on glycine and serine as the first
two amino acids of a family of tripeptides (e.g., Gly-Ser for 14,
Ser-Gly for 15, and Ser-Ser for 16). As summarized in Table 2,
these structures showed inhibition in the range of 55−100 μM,
worse than that seen for the dipeptide conjugates 2−6 and all
previous tripeptides conjugates. Indeed these results were partly
anticipated by earlier results with serine.
While it is easy to simply view the results from compounds
14−16 as an example of the inaccuracies of virtual drug
modeling, it is more interesting when viewed in the context of
the apparent preference toward hydrophobicity over hydrogen
bonding seen previously. While there is some difference in the
terminal aromatic group, the primary difference between
compound 13 and the poorer performing compound 14 is
replacement of the hydrophobic isopropyl group with a
hydroxyl moiety, and yet the more hydrophobic structure is 3
times more potent. This further highlights a stark difference in
the design of pterin inhibitors over previous reports that
attributed improved potency to increased hydrogen
bonding.8a,11a
8.86 (t, J = 5.9 Hz, 2H), 8.84 (s, 1H), 8.32 (d, J = 7.9 Hz, 1H), 7.28−
7.12 (m, 5H), 7.01 (br s, 2H), 4.44 (ddd, J = 8.5, 7.9, 5.1 Hz, 1H),
3.96 (dd, J = 16.7, 5.9 Hz, 1H), 3.88 (dd, J = 16.7, 5.9 Hz, 1H), 3.03
(dd, J = 13.8, 5.1 Hz, 1H), 2.87 (dd, J = 13.8, 8.5 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ/ppm 172.7, 168.2, 162.8, 160.4, 156.1, 154.5,
147.2, 137.4, 136.5, 131.9, 129.2 (2C), 128.2 (2C), 126.5, 53.6, 42.1,
36.8. HR-ESIMS m/z [M − H]−: calcd for (C18H16N7O5)− 410.121
84; found 410.121 52.
N-{N-(Pterin-7-yl)carbonylglycyl}-L-tyrosine (3). This com-
1
pound was synthesized in 56% yield using the general method. H
NMR (400 MHz, DMSO-d6) δ/ppm 12.76 (br s, 1H), 11.77 (s, 1H),
9.20 (s, 1H), 8.90 (t, J = 5.9 Hz, 1H), 8.87 (s, 1H), 8.23 (d, J = 8.0
Hz), 7.12 (br s, 2H), 6.99 (dt, J = 8.5, 2.0 Hz), 6.62 (dt, J = 8.5, 2.0
Hz), 4.37 (ddd, J = 8.4, 8.2, 5.1 Hz, 1H), 3.98 (dd, J = 16.8, 5.9 Hz,
1H), 3.90 (dd, J = 16.8, 5.9 Hz, 1H), 2.93 (dd, J = 13.8, 5.1 Hz, 1H),
2.78 (dd, J = 13.8, 8.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ/
ppm 172.9, 168.1, 162.8, 160.5, 155.9, 154.6, 147.2, 136.5, 131.9, 130.1
(2C), 127.4, 115.0 (2C), 109.6, 53.9, 42.1, 36.1. HR-ESIMS m/z [M −
H]−: calcd for (C18H16N7O6)− 426.116 75; found 426.116 25.
4. CONCLUSION
In recent years we have seen a 2 order of magnitude
improvement in the potency of pterin-based RTA inhibitors
(600 μM for pteroic acid, 6 μM for 3). This optimization has
been facilitated by the rapid generation of diverse structures
through the use of di- and tripeptide conjugated pterins. The
majority of compounds reported here had IC50 values at or
below 50 μM. As such, these are some of the best competitive
RTA inhibitors capable of functioning at physiological pH.
Much of the success of these compounds over previous pterin
inhibitors appears to stem from an unexpected preference
toward hydrophobic and π-interactions, despite the primarily
polar surface of the active site. These structures should guide
future development of pterin-based RTA inhibitors and can be
transitioned to various forms of peptide isosteres for further
optimization.
N-{N-(Pterin-7-yl)carbonylglycyl}-L-tryptophan (4). This com-
1
pound was synthesized in 99% yield using the general method. H
NMR (400 MHz, DMSO-d6) δ/ppm 12.72 (br s, 1H), 11.67 (s, 1H),
10.86 (d, J = 2.0 Hz, 1H), 8.89 (t, J = 5.8 Hz, 1H), 8.87 (s, 1H), 8.30
(d, J = 7.8 Hz, 1H), 7.53 (dt, J = 7.9, 1.0 Hz, 1H), 7.32 (dt, J = 8.0, 1.0
Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 7.05 (br s, 2H), 7.04 (ddd, J = 8.0,
7.0, 1.0 Hz, 1H), 6.96 (ddd, J = 7.9, 7.0, 1.0 Hz, 1H), 4.51 (td, J = 7.8,
5.2 Hz, 1H), 4.00 (dd, J = 16.7, 5.8 Hz, 1H), 3.93 (dd, J = 16.7, 5.8 Hz,
1H), 3.19 (dd, J = 14.6, 5.2 Hz, 1H), 3.05 (dd, J = 14.6, 7.8 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ/ppm 173.1, 168.0, 162.6, 160.3,
155.9, 154.4, 147.1, 136.4, 135.9, 131.8, 127.1, 123.6, 120.7, 118.2,
118.0, 111.2, 109.4, 52.9, 42.0, 27.0. HR-ESIMS m/z [M − H]−: calcd
for (C20H17N8O5)− 449.132 74; found 449.132 04.
N-{N-(Pterin-7-yl)carbonyl-L-alanyl}-L-phenylalanine (5). This
compound was synthesized in 55% yield using a modified version of
the above method, whereby the reaction temperature was raised to 80
5. EXPERIMENTAL DETAILS
1
°C in a sealed vessel DMSO as a cosolvent. H NMR (400 MHz,
All reagents used were of commercial quality and were obtained from
Aldrich Chemical Co. and Fisher Scientific and were used as received.
Peptides were purchased through GenScript and BAChem and used as
DMSO-d6) δ/ppm 12.80 (br s, 1H), 11.61 (s, 1H), 8.85 (s, 1H), 8.61
(d, J = 7.9 Hz, 1H), 8.47 (d, J = 8.0 Hz), 7.27−7.14 (m, 5H), 7.08 (br
s, 1H), 4.53 (dq, J = 7.9, 7.0 Hz, 1H), 4.46 (ddd, J = 9.2, 8.0, 5.0 Hz,
1H), 3.09 (dd, J = 13.8, 5.0 Hz, 1H), 2.91 (dd, J = 13.8, 9.2 Hz, 1H),
1.32 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ/ppm
172.6, 171.4, 161.7, 160.3, 155.9, 154.5, 146.9, 137.4, 136.3, 132.0,
129.1 (2C), 128.1 (2C), 126.4, 53.5, 48.1, 36.6, 18.8. HR-ESIMS m/z
[M − H]−: calcd for (C19H18N7O5)− 424.137 49; found 424.136 92.
1
received. H and 13C 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). LC−MS data were recorded on an
Agilent 6130 quadrupole instrument. High resolution mass spectrom-
etry was performed with a Varian 9.4 T QFT-ESI ICR system. All
solvents were removed by rotary evaporation under vacuum using a
325
dx.doi.org/10.1021/jm3016393 | J. Med. Chem. 2013, 56, 320−329

Journal of Medicinal Chemistry
Article
N-{N-(Pterin-7-yl)carbonyl-L-alanyl}-L-tryptophan (6). This
compound was synthesized in 30% yield using the same modified
procedure reported for 5. H NMR (400 MHz, DMSO-d6) δ/ppm
1H), 11.59 (s, 1H), 10.87 (s, 1H), 8.86 (s, 1H), 8.83 (t, J = 5.9 Hz,
1H), 8.36 (d, J = 7.8 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 7.8
Hz, 1H), 7.33 (1H, d, J = 7.9 Hz, 1H), 7.24−7.13 (m, 8H), 7.06 (dd, J
= 7.1, 7.9 Hz, 1H), 6.98 (dd, J = 7.1, 7.8 Hz, 1H), 4.61 (ddd, J = 4.1,
8.5, 9.2 Hz, 1H), 4.50 (ddd, J = 5.4, 7.8, 8.1 Hz, 1H), 3.93 (dd, J = 5.7,
16.8 Hz, 1H), 3.84 (dd, J = 5.7, 16.8 Hz, 1H), 3.20 (dd, J = 5.4, 14.7
Hz, 1H), 3.07 (dd, J = 8.1, 14.7 Hz, 1H), 3.02 (dd, J = 4.1, 13.8 Hz,
1H), 2.74 (dd, J = 9.2, 13.8 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) δ/ppm 173.1, 170.9, 167.9, 162.7, 160.3, 155.7, 154.4, 147.1, 137.6,
136.6, 136.0, 131.8, 129.3 (2C), 127.9 (2C), 127.2, 126.2, 123.7, 120.9,
118.4, 118.1, 111.3, 109.6, 53.6, 52.9, 42.2, 37.6, 27.0. HR-ESIMS m/z
[M − H]−: calcd for (C29H26N9O6)− 596.201 15; found 596.201 19.
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-leucinyl]-L-phenylala-
nine (12). This compound was synthesized in 97% yield using the
1
12.73 (br s, 1H), 11.63 (s, 1H), 10.83 (d, J = 2.4 Hz, 1H), 8.85 (s,
1H), 8.64 (d, J = 7.8 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 7.53 (dt, J =
7.9, 1.0 Hz, 1H), 7.31 (dt, J = 8.0, 1.0 Hz, 1H), 7.16 (d, J = 2.4 Hz,
1H), 7.05 (br s, 2H), 7.03 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.94 (ddd, J
= 7.9, 7.0, 1.0 Hz, 1H), 4.61−4.53 (m, 1H), 4.52 (ddd, J = 8.2, 8.0, 5.0,
1H), 3.20 (dd, J = 14.8, 5.0 Hz, 1H), 3.06 (dd, J = 14.8, 8.2 Hz, 1H),
1.34 (d, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ/ppm
172.9, 171.4, 161.6, 160.2, 155.9, 154.4, 146.9, 136.6, 135.9, 131.9,
127.1, 123.6, 120.8, 118.2, 118.0, 111.3, 109.5, 52.9, 40.0, 26.9, 18.7.
HR-ESIMS m/z [M − H]−: calcd for (C21H19N8O5)− 463.148 39;
found 463.148 04.
1
general procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 8.80 (s,
N-{N-(Pterin-7-yl)carbonyl-L-serinyl}-L-phenylalanine (7).
1H), 7.20−7.09 (m, 5H), 4.35−4.27 (m, 2H), 3.87 (d, J = 5.7 Hz,
2H), 2.87 (m, 2H), 1.50 (m, 1H), 1.33 (m, 2H), 0.79 (dd, J = 12.6, 6.5
Hz, 6H); 13C NMR (125 MHz, DMSO-d6) δ/ppm 173.3, 172.2, 168.2,
163.2, 158.5, 155.1, 138.0, 137.7, 136.8, 132.3, 129.5 (2C), 128.5 (2C),
126.8, 109.9, 53.9, 51.2, 42.6, 41.4, 36.9, 24.5, 23.5, 22.1. HR-ESIMS
m/z [M + H]+: calcd for (C24H29N8O6)+ 525.220 46; found 525.220
28.
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-leucinyl]-L-tyrosine
(13). This compound was synthesized in 84% yield using the general
method. 1H NMR (400 MHz, DMSO-d6) δ/ppm 9.18 (s, 1H), 8.88 (t,
J = 5.5 Hz, 1H), 8.12 (dd, J = 13.6, 8.0 Hz, 2H), 6.99 (d, J = 8.5 Hz,
2H), 6.61 (d, J = 8.5 Hz, 2H), 4.41−4.24 (m, 2H), 3.95 (d, J = 7.6 Hz,
2H), 2.94−2.74 (m, 2H), 1.56 (m, 1H), 1.39 (m, 2H), 0.84 (dd, J =
12.1, 6.6 Hz, 6H); 13C NMR (150 MHz, DMSO-d6) δ/ppm 172.7,
171.6, 167.7, 162.7, 160.3, 155.8, 154.4, 147.1, 138.4, 131.5, 129.9
(2C), 127.4, 114.9 (2C), 53.7, 52.8, 50.7, 42.1, 35.8, 23.9, 23.0, 21.6.
HR-ESIMS m/z [M + H]+: calcd for (C24H29N8O7)+ 541.215 37;
found 541.215 31.
This compound was synthesized in 68% yield using the general
procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.81 (br s, 1H),
1
11.83 (s, 1H), 8.86 (s, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.37 (d, J = 8.3
Hz, 1H), 7.70−6.60 (m, 7H), 5.07 (br s, 1H), 4.53 (dt, J = 8.2, 5.1 Hz,
1H), 4.46 (td, J = 8.3, 5.2 Hz, 1H), 3.71 (dd, J = 11.1, 5.1 Hz, 1H),
3.66 (dd, J = 11.1, 5.1 Hz, 1H), 3.07 (dd, J = 13.8, 5.2 Hz, 1H), 2.91
(dd, J = 13.8, 8.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ/ppm
172.6, 169.2, 162.2, 160.2, 156.0, 154.8, 146.9, 137.4, 136.3, 132.0,
129.2 (2C), 128.2 (2C), 126.4, 61.7, 55.0, 53.6, 36.8. HR-ESIMS m/z
[M − H]−: calcd for (C19H18N7O6)− 440.132 40; found 440.131 91.
N-{N-(Pterin-7-yl)carbonyl-L-serinyl}-L-tryptophan (8). This
compound was synthesized in 61% yield using the general procedure.
1H NMR (400 MHz, DMSO-d6) δ/ppm 12.74 (br s, 1H), 11.78 (br s,
1H), 10.86 (d, J = 2.3 Hz, 1H), 8.88 (s, 1H), 8.61 (d, J = 8.1 Hz, 1H),
8.38 (d, J = 7.9 Hz, 1H), 7.52 (dd, J = 7.8, 1.0 Hz, 1H), 7.30 (dt, J =
7.8, 1.1 Hz, 1H), 7.21 (br s, 2H), 7.16 (d, J = 2.3 Hz, 1H), 7.03 (ddd, J
= 8.2, 7.8, 1.0 Hz, 1H), 6.94 (td, J = 8.2, 7.8, 1.1 Hz, 1H), 4.62−4.46
(m, 2H), 3.78−3.65 (m, 2H), 3.19 (dd, J = 14.7, 5.4 Hz, 1H), 3.07
(dd, J = 14.7, 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ/ppm
173.0, 169.2, 162.1, 160.2, 155.1, 154.5, 146.9, 136.6, 136.0, 132.0,
127.3, 123.8, 120.9, 118.4, 118.2, 111.4, 109.5, 61.8, 54.9, 53.1, 27.1.
HR-ESIMS m/z [M − H]−: calcd for (C21H19N8O6)− 479.143 30;
found 479.143 15.
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-phenylalanyl]-L-phe-
nylalanine (9). This compound was synthesized in 70% yield using
the general procedure. 1H NMR (400 MHz, DMSO-d6) δ/ppm 12.77
(br s, 1H), 11.62 (s, 1H), 8.86 (s, 1H), 8.83 (t, J = 5.7 Hz, 1H), 8.40
(d, J = 7.8 Hz, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.30−7.13 (m, 10H),
7.09 (br s, 2H), 4.58 (ddd, J = 9.4, 8.5, 4.2 Hz, 1H), 4.44 (ddd, J = 8.8,
7.8, 5.3 Hz, 1H), 3.94 (dd, J = 16.7, 5.7 Hz, 1H), 3.82 (dd, J = 16.7, 5.7
Hz, 1H), 3.08 (dd, J = 13.9, 5.3 Hz, 1H), 3.00 (dd, J = 13.9, 4.2 Hz,
1H), 2.93 (dd, J = 13.9, 8.8 Hz, 1H), 2.73 (dd, J = 13.9, 9.4 Hz, 1H);
13C NMR (100 MHz, DMSO-d6) δ/ppm 172.7, 170.9, 167.8, 162.7,
160.4, 156.1, 154.5, 147.2, 137.6, 137.4, 136.5, 131.9, 129.3 (2C),
129.1 (2C), 128.2 (2C), 128.0 (2C), 126.5, 126.3, 53.6 (2 × CH2Ph),
42.2, 37.6, 36.7. HR-ESIMS m/z [M − H]−: calcd for (C27H25N8O6)−
557.190 25; found 557.190 16.
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-serinyl]-L-tryptophan
(14). This compound was synthesized in 29% yield using the general
1
procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.68 (s, 1H),
11.61 (s, 1H), 10.86 (d, J = 2.4 Hz, 1H), 8.93 (t, J = 5.8 Hz, 1H), 8.88
(s, 1H), 8.13 (t, J = 7.6 Hz, 2H), 7.52 (dd, J = 7.8, 1.1 Hz, 1H), 7.32
(dt, J = 7.6, 1.1 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 7.09−7.00 (m, 3H),
6.97 (ddd, J = 8.0, 7.6, 1.1 Hz, 1H), 4.87 (br s, 1H), 4.48 (td, J = 7.6,
5.5 Hz, 1H), 4.41 (dt, J = 7.6, 5.8 Hz, 1H), 4.01 (d, J = 5.8 Hz, 2H),
3.59 (dd, J = 10.9, 5.8 Hz, 1H), 3.54 (dd, J = 10.9, 7.6 Hz, 1H), 3.18
(dd, J = 14.7, 5.5 Hz, 1H), 3.07 (dd, J = 14.7, 7.6 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ/ppm 172.9, 169.6, 168.1, 162.7, 160.3, 155.9,
154.3, 147.1, 136.4, 135.9, 131.8, 127.1, 123.7, 120.7, 118.2, 118.0,
111.2, 109.4, 61.6, 54.8, 52.8, 42.2, 26.9. HR-ESIMS m/z [M − H]−:
calcd for (C23H22N9O7)− 536.164 77; found 536.164 59.
N-[N-{N-(Pterin-7-yl)carbonyl-L-serinyl}glycyl]-L-tryptophan
(15). This compound was synthesized in 34% yield using the general
1
procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.69 (br s, 1H),
11.66 (s, 1H), 10.85 (d, J = 2.4 Hz, 1H), 8.88 (s, 1H), 8.69 (d, J = 7.8
Hz, 1H), 8.42 (t, J = 5.9 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.51 (dd, J
= 8.0, 1.1 Hz), 7.35−7.28 (m, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.05
(ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.97 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H),
5.24 (s, 1H), 4.52 (ddd, J = 7.8, 5.9, 5.7 Hz, 2H), 4.45 (ddd, J = 8.3,
8.0, 5.3 Hz, 1H), 3.81 (dd, J = 16.8, 5.9 Hz, 1H), 3.67 (dd, J = 16.8, 5.7
Hz, 1H), 3.17 (dd, J = 14.7, 5.3 Hz, 1H), 3.17 (d, J = 5.9 Hz, 2H), 3.01
(dd, J = 14.7, 8.3 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ/ppm
173.2, 169.6, 168.6, 162.4, 160.4, 156.0, 154.6, 147.0, 136.4, 136.1,
132.0, 127.2, 123.8, 120.9, 118.4, 118.1, 111.4, 109.6, 61.7, 55.2, 53.1,
42.0, 27.1. HR-ESIMS m/z [M − H]−: calcd for (C23H22N9O7)−
536.164 77; found 536.164 71.
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-phenylalanyl]-L-tyro-
sine (10). This compound was synthesized in 78% yield using the
1
general procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.67 (s,
1H), 11.59 (s, 1H), 9.20 (s, 1H), 8.85 (s, 1H), 8.83 (t, J = 5.8 Hz, 1H),
8.31 (d, J = 7.8 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.22−7.12 (m, 5H),
7.10 (br s, 2H), 7.02 (d, J = 8.5 Hz, 2H), 6.65 (d, J = 8.5 Hz, H), 4.58
(ddd, J = 4.2, 8.4, 9.4 Hz, 1H), 4.35 (ddd, J = 5.4, 7.8, 8.6 Hz, 1H),
3.93 (dd, J = 5.8, 16.6 Hz, 1H), 3.83 (dd, J = 5.8, 16.6 Hz, 1H), 3.00
(dd, J = 4.2, 13.9 Hz, 1H), 2.94 (dd, J = 5.4, 14.1 Hz, 1H), 2.81 (dd, J
= 8.6, 14.1 Hz, 1H), 2.73 (dd, J = 9.4, 13.9 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ/ppm 173.1, 171.2, 168.3, 163.1, 160.8, 156.1,
154.7, 147.4, 137.7, 136.9, 132.0, 130.3 (2C), 129.5 (2C), 128.3 (2C),
127.7 (2C), 126.6 (2C), 115.3, 54.2, 53.9, 42.5, 37.7, 36.2. HR-ESIMS
m/z [M − H]−: calcd for (C27H25N8O7)− 573.185 17; found 573.185
21.
N-[N-{N-(Pterin-7-yl)carbonyl-L-serinyl}-L-serinyl]-L-trypto-
phan (16). This compound was synthesized in 36% yield using the
1
general procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.66 (s,
1H), 11.66 (s, 1H), 10.85 (d, J = 2.4 Hz, 1H), 8.88 (s, 1H), 8.67 (d, J
= 7.9 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.51
(dd, J = 7.8 Hz, 1H), 7.32 (dd, J = 7.8, 1.0 Hz, 1H), 7.16 (d, J = 2.4
Hz, 1H), 7.07 (br s, 2H), 7.05 (ddd, J = 8.2, 7.8, 1.0 Hz, 1H), 6.97
(ddd, J = 8.2, 7.8, 1.0 Hz, 1H), 5.30 (br s, 1H), 4.89 (br s, 1H), 4.64
N-[N-{N-(Pterin-7-yl)carbonylglycyl}-L-phenylalanyl]-L-tryp-
tophan (11). This compound was synthesized in 78% yield using the
1
general procedure. H NMR (400 MHz, DMSO-d6) δ/ppm 12.67 (s,
326
dx.doi.org/10.1021/jm3016393 | J. Med. Chem. 2013, 56, 320−329

Journal of Medicinal Chemistry
Article
a
Table 3. Crystallographic Data
2
pterin-Gly-Gly
P41212
8
9
space group
P41212
P41212
P41212
cell constants (Å)
a = b = 68.0, c = 140.7
a = b = 68.2, c = 140.8
a = b = 67.8, c = 140.7
a = b = 67.7, c = 140.6
resolution (Å)
50.−1.52 (1.55−1.52)
50.−1.89 (1.92−1.89)
20.−1.54 (1.57−1.54)
50.−1.70 (1.73−1.70)
Rmerge (%) (outer shell)
⟨I/σI⟩ (outer shell)
0.098 (0.957)
7.3 (2.1)
99.8 (100.0)
51 425
13.8
0.131 (0.630)
6.3 (2.2)
100.0 (100.0)
27 776
13.8
0.056 (0.509)
13.3 (4.4)
99.7 (98.6)
49 375
13.1
0.091 (0.863)
10.4 (2.3)
91.5 (100.0)
33 847
12.3
completeness (%) (outer shell)
unique reflections
redundancy
no. of residues
263
263
268
283
no. of protein atoms
no. of ligand atoms
2083
2097
2122
2090
30
23
35
41
no. of solvent atoms
Rworking
397
283
221
337
0.201
0.230
21.1
0.202
0.236
29.6
0.201
0.239
20.4
0.209
0.236
24.7
Rfree
average B factor for protein atoms (Å2)
average B factor for ligand atoms (Å2)
average B factor for solvent atoms (Å2)
rmsd from ideality, bond (Å)
rmsd from ideality, angle (deg)
% residues in most favored region
20.5
34.6
17.0
34.0
34.5
38.4
27.8
34.6
0.027
2.212
98.1
0.007
0.951
98.9
0.031
2.625
97.7
0.006
1.009
98.5
% residues in additional allowed region
1.5
1.1
1.2
1.5
a
Values in parentheses correspond to values of highest resolution shell.
(dt, J = 7.8, 5.2 Hz, 1H), 4.51−4.33 (m, 2H), 3.82 (dd, J = 11.0, 5.2
Hz, 1H), 3.70 (dd, J = 11.0, 5.2 Hz, 1H), 3.66−3.53 (m, 2H), 3.17
(dd, J = 14.6, 5.2 Hz, 1H), 3.04 (dd, J = 14.6, 7.8 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ/ppm 173.1, 169.7, 169.4, 162.2, 160.3, 156.0,
154.6, 146.9, 136.3, 136.0, 132.1, 127.2, 123.8, 120.9, 118.4, 118.1,
111.4, 109.5, 62.0, 61.5, 55.1, 54.8, 53.2, 27.0. HR-ESIMS m/z [M −
H]−: calcd for (C24H24N9O8)− 566.175 33; found 566.175 16.
5.2. In Vitro Luciferase Assay. The compounds to be tested were
solubilized in 0.05 N KOH prior to their inclusion in the assay. After
RTA and various concentrations of compounds were mixed 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). The mixtures were incubated for 90 min at 30 °C,
after which the reactions were stopped by freezing the mixtures at −20
°C. After a thaw at room temperature, 2.5 μL of reaction mixture was
mixed with 40 μL of luciferase substrate reagent (Promega), and their
luminescence was measured on a Perkin-Elmer Envision luminometer
(Waltham MA).
For each concentration of inhibitor to be tested, reactions were run
in both 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
corresponding 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 concentration
to a hyperbolic decay function.
HF rotating anode generator operated at 40 kV, 30 mA. Diffraction
data from crystals soaked with 2, pterin-Gly-Gly, and 8 were collected
at 100 K at the Advanced Light Source beamline 5.0.2 at the Lawrence
Berkeley National Laboratory. Diffraction images were processed and
data reduced using HKL2000.15 The structures of the RTA-compound
complexes were solved by molecular replacement with MOLREP16
using the structure of RTA (PDB accession code 1RTC)7c as the
search model. Model building was carried out using Coot (Emsley et
al., 2010).17 Refinement of models was done using Refmac 5.5010918
and PHENIX.19 There were several rounds of refinement followed by
manual rebuilding of the model. To facilitate manual rebuilding, a
difference map and a 2Fo − Fc map, σA-weighted to eliminate bias
from the model,20 were prepared. 5% of the diffraction data were set
aside throughout refinement for cross-validation.21 MolProbity was
used to determine areas of poor geometry and to make Ramachandran
plots.22
Crystallographic data for the complexes are presented in Table 3.
Coordinates of the refined model of RTA complexed with 2, pterin-
Gly-Gly, 8, and 9 have been deposited in the Protein Data Bank with
accession codes 4HUO, 4HV7, 4HV3, and 4HUP, respectively.
ASSOCIATED CONTENT
Accession Codes
PDB codes for the refined model of RTA complexed with 2,
pterin-Gly-Gly, 8, and 9 are 4HUO, 4HV7, 4HV3, and 4HUP,
respectively.
■
5.3. X-ray Crystallography. Tetragonal RTA crystals were grown
at 4 °C using the sitting drop method from 7 mg/mL RTA and 1.3 M
ammonium sulfate, 0.1 M sodium malonate, pH 5.0. For the purpose
of determining the structure of RTA in complex with a compound, a
crystal was transferred to a reservoir containing 1.5 mM compound in
artificial mother liquor (1.3 M ammonium sulfate, 0.1 M sodium
malonate, pH 5.0). Prior to data collection, a crystal was transferred
briefly to a drop of 1.3 M ammonium sulfate, 1.3 M malonate, pH 6,
for cryoprotection. The crystal mounted in a cryoloop (Hampton
Research, Laguna Niguel, CA) was flash frozen in liquid nitrogen and
mounted in the cold stream on the goniostat.
AUTHOR INFORMATION
Corresponding Author
*For R.S.: phone, +81-47-472-1926; e-mail, saito@chem.sci.
toho-u.ac.jp. For E.V.A.: phone, +1-512-471-0068; fax, +512-
471-7791; e- mail, anslyn@austin.utexas.edu. For J.D.R.: phone,
+1-512-471-3175; e- mail, jrobertus@mail.utexas.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health
(NIH) Grant AI 075509, by Robert A. Welch Foundation
■
X-ray diffraction data were collected from a crystal soaked with 9 at
100 K on an R-AXIS IV++ image plate detector (Rigaku, The
Woodlands, TX) with X-rays generated by a Rigaku MicroMax-007
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(10) Pruet, J. M.; Robertus, J. D.; Anslyn, E. V. Acyl radical insertion
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Tetrahedron Lett. 2010, 51 (18), 2539−2540.
Grant F1225, and by the College of Natural Sciences support to
the Center for Structural Biology. Assistance for this work was
provided by the Macromolecular Crystallography Facility, with
financial support from the College of Natural Sciences, the
Office of the Executive Vice President and Provost, the Institute
for Cellular and Molecular Biology, the University of Texas at
Austin. The Berkeley Center for Structural Biology is supported
in part by the National Institutes of Health, National Institute
of General Medical Sciences, and the Howard Hughes Medical
Institute. The Advanced Light Source is supported by the
Director, Office of Science, Office of Basic Energy Sciences, of
the U.S. Department of Energy under Contract DE-AC02-
05CH11231.
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ABBREVIATIONS USED
■
RTA, ricin toxin A; PTA, pteroic acid; 7CP, 7-carboxy-pterin;
DBU, 1,8- diazabicyclounde-7-cene; P1, first amino acid; P2,
second amino acid; P3, third amino acid
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