Crystallographic evidence that TAN-1057 and the blasticidin S family of antibiotics
inhibit translation by stabilizing a distorted binding mode of P-site tRNA
Poster F2-1871
51st ICAAC
Chicago, IL, USA
September 17-20, 2011
New Haven, CT 06511 Phone: 203-624-5606
www.Rib-X.com
WIMBERLY, B.T., IPPOLITO, J.A., KANYO, Z.F., DE VIVO, M., O’DOWD, H., DUFFY, E.M.
Rib-X Pharmaceuticals, Inc., New Haven, CT USA
Abstract
Results
Results
Background
The natural products TAN-1057 and blasticidin S inhibit catalysis of peptide bond formation on the 50S ribosomal subunit. A crystal structure of
blasticidin S bound to the Haloarcula marismortui 50S subunit (H50S) revealed two blasticidin S ligands bound to the 50S P-loop, the binding site for the
C74C75A76 end of peptidyl-tRNA (P-tRNA). These results suggested that the mechanism of action is competition between blasticidin S and the CCA
from P-tRNA. However, blasticidin S actually stimulates binding of oligonucleotide analogs of P-tRNA. To clarify the role of tRNA in the mechanism of
action, we have determined ribosome-bound crystal structures of blasticidin S and TAN-1057 in the presence of oligonucleotide analogs of tRNA.
Methods
Antibiotics and oligonucleotides were soaked into pre-formed 50S crystals. X-ray data collection, processing, and refinement followed standard methods.
If oligonucleotide models of tRNA are omitted during a soak of TAN, difference electron density for two TAN ligands is visible at the same sites seen
for BLS (Figure 3). As with BLS, both TAN ligands base-pair with the conserved P-loop residues G2284 and G2285. The density is better defined for
the inhibitor base-paired to G2284, consistent with the initial BLS structure [3].
Figure 3 - X-ray crystal structure of TAN bound to the Haloarcula marismortui 50S ribosomal subunit. As
with BLS, two TAN ligands bind the P-loop in the absence of oligonucleotide substrate analogs.
Difference electron density is drawn in black. Note the relatively poorer density for the lower ligand
base-paired to G2285.
Results
When oligonucleotide analogs of tRNA are co-soaked with TAN-1057 or blasticidin S, density is seen for only a single antibiotic base-paired to G2284
(H50S numbering), and strong density is also seen for the C74C75A76 tRNA analog. The antibiotic intercalates between the bases of C75 and A76, and C75
base-pairs with G2285. C75 occupies the site populated by the second, less strongly bound antibiotic ligand seen in oligonucleotide-free soaks.
Significantly, the TAN or BLS ligand binds the P-loop intimately associated with the CCA oligonucleotide, stacking between the bases of C75 and
A76. This “gapped” CCA binding mode is the same for both TAN and BLS (Figure 4A,B). The gapped binding mode seen here in H50S is also seen
in crystal structures of P-loop inhibitors bound to bacterial 50S or 70S, and it is independent of the order of addition of the CCA and inhibitor
ligands (data not shown).
The gapped binding mode is significantly distorted relative to the CCA binding mode seen in normal translation. C75 is displaced by the inhibitor
base-paired to C75's normal partner G2284; C75 forms a base-pair instead with G2285. A76 binds a site that largely overlaps its normal binding site,
but the nucleotide is rotated away from its normal orientation to the extent that the covalently attached peptide would be displaced over 5
angstroms away from its usual position. It is possible that because of this displacement from the active site, no electron density is visible for the
amino acid portion of the peptidyl tRNA substrate analog 5’ CCA-N-acetyl-Phe used in the BLS experiment.
These structural results are in excellent agreement with the biochemical data available for TAN and BLS. The extensive contact between the
P-loop inhibitors and the CCA oligonucleotide would seem to be consistent with the observed stimulation of binding of peptidyl tRNA or
oligonucleotide analogs thereof [1]. This extensive CCA/inhibitor interface also provides a structural basis for the relatively high binding affinity of
BLS. The inhibitor-induced stabilization of the binding of tRNA to the P site is also consistent with the stabilization of polysomes by BLS and other
P-loop inhibitors (Rib-X unpublished data). In addition, the intimate association of these P-loop inhibitors with a significantly distorted tRNA may
also provide a better framework for understanding the slow two-stage kinetics observed for inhibition of translation by BLS [9]. The displacement
of A76 from the active site by several angstroms also provides a structural basis for the greatly slowed (but not completely abolished) rate of
peptide bond formation [9].
Conclusions
TAN-1057 and the blasticidin S family are proposed to inhibit translation by stabilizing a distorted mode of P-tRNA binding with just one bound
antibiotic ligand intercalated between C75 and A76 of P-tRNA. This binding mode provides a more complete and consistent explanation for the
biochemical data and SAR of the TAN-1057 and blasticidin S families. These crystal structures have inspired a Rib-X program (RX-04) to target this
clinically unexploited P-loop site with the goal of discovering a novel, potent Gram-negative antibacterial.
Introduction
Conclusions
The rise of drug resistance among established pathogens has created an urgent need for improved antibacterials. In an effort to broaden spectrum
and to avoid existing resistance mechanisms, novel structural scaffolds that either inhibit the function of clinically unexploited targets or show a novel
mechanism of inhibition of established targets are sought. This work aims to qualify the mechanism of action of compounds suggested to bind to the
P-site of the large ribosomal subunit and to assess the suitability of that site for the design of novel antibiotics.
• X-ray crystallography has provided new insights into the structural basis for the mechanism of action of blasticidin S and TAN-1057, two natural
products that bind the P-loop, a clinically unexploited target site in the ribosome.
• Both inhibitors are proposed to stop translation in the same manner, by stabilizing a distorted mode of P-tRNA binding with one antibiotic
ligand sandwiched between C75 and A76 of P-tRNA.
Figure 1 - Chemical structures of the natural products (A) Blasticidin S (BLS) and
(B) TAN-1057 (TAN).
O
H2N
H
N
N
NH
OH
NH
O
NH2 O
N
O
A
Blasticidin S
H2N
N
NH2
N
H
N
NH2 O
O
NH
N
N
H
O
NH2
B
TAN-1057
Figure 2 - The crystal structure of BLS bound to
the P-loop of the Haloarcula marismortui 50S
ribosomal subunit [3]. BLS has been proposed to
compete with the CCA end of P-site tRNA. In the
absence of tRNA substrate, two BLS ligands bind
to the adjacent P-loop residues G2284 and G2285
(H.marismortui numbering). The upper ligand
paired to G2284 is better ordered.
Blasticidin S (BLS, Figure 1) is a natural product in the cytosamine family known to strongly inhibit
peptide-bond formation [1,2]. A crystal structure has been reported of BLS bound to the
Haloarcula marismortui 50S (H50S) ribosomal subunit, localizing it to the region where the peptidyl
tRNA binds [3]. In the crystal structure, difference electron density is apparent for two molecules
of BLS, with one ligand exhibiting stronger density than the other. The better-ordered BLS ligand
base-pairs to G2284, while the other base-pairs to an adjacent residue, G2285 (Figure 2). These
RNA residues are the most highly conserved part of the “P loop”, the binding site for the 3’
C74C75A76-peptide portion of peptidyl tRNA. Thus BLS was proposed [3] to mimic C74 and C75 of
the P-site tRNA and to compete with the binding of the tRNA CCA end to the P site, thereby
preventing the formation of a new peptide bond. However, it has been shown [1] that BLS
stimulates rather than inhibits ribosomal binding of oligonucleotide models of peptidyl-tRNA, the
normal substrate in the P-site. In order to elucidate the role of the tRNA 3’ CCA end during
inhibition by these natural products, we have determined the crystal structures of BLS in the
presence of CCA-containing oligonucleotides. Furthermore, we have determined the crystal
structures of TAN-1057 (TAN, Figure 1), another natural product with similar core features [4], in
both the presence and the absence of CCA-containing oligonucleotides. This was pursued to add
confidence to the emerging picture of ligand binding in the P-site.
Methods
BLS was purchased from Sigma and used without further purification. TAN was synthesized in-house. Oligonucleotides were purchased from
Dharmacon. Inhibitors and oligonucleotides were soaked simultaneously into pre-formed H50S crystals grown and cryocooled as described
previously [5]. X-ray diffraction data were collected at synchrotron sources. Images were integrated and scaled using the HKL2000 suite [6]. CNX
[7] was used for molecular replacement and refinement, and O [8] was used for visualization.
In contrast, crystal structures of either TAN or BLS determined in the presence of oligonucleotide models of the tRNA 3' CCA end contain only
one bound inhibitor molecule (Figure 4). The binding site corresponds to the better-ordered site seen in the absence of oligonucleotides, the site
that allows base-pairing with G2284. The binding pose of the BLS and TAN ligands in this site is essentially independent of the presence or absence
of added CCA-containing oligonucleotides.
Figure 4 - BLS and TAN stabilize a distorted mode of CCA binding to the P-loop.
(A) X-ray crystal structure of BLS bound to the Haloarcula marismortui 50S ribosomal subunit in the presence of the peptidyl tRNA substrate analog 5’ CCA-N-acetyl-Phe. Only one
BLS ligand is seen, which is base-paired to G2284, just as the better ordered ligand does in oligonucleotide-free soaks.The N-acetylated amino acid is not shown as it is disordered.
(B) X-ray crystal structure of TAN bound to the Haloarcula marismortui 50S ribosomal subunit in the presence of the tRNA substrate analog ACCA.
• The crystal structures of blasticidin S and TAN-1057 have inspired a Rib-X program (RX-04) to target this unexploited P-loop site with the goal
of discovering a novel antibacterial scaffold that can be tuned for potency against multidrug-resistant Gram-positive and Gram-negative pathogens.
References
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