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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5319_Библиотеки_им_академика_М_И_Перельмана.pdf
A Case Study: Structure-Based
Inhibitor Design for tRNAGuanine Transglycosylase
Contents
21.1 Shigellosis: Disease and Therapeutic Options – 325
21.2 Blocking Pathogenesis on the Molecular Level – 325
21.3 The Crystal Structure of tRNA-Guanine
Transglycosylase as aStarting Point – 326
21.4 A Functional Assay to Determine Binding Constants – 326
21.5 LUDI Discovers the First Leads – 329
21.6 Surprise: AFlipped Amide Bond and aWater Molecule – 330
21.7 Hot Spot Analysis and Virtual Screening Open the
Floodgate to New Ideas for Synthesis – 331
21.8 The Filling of Hydrophobic Pockets and
Interference with aWater Network – 332
21.9 With aSalt Bridge: Finally Nanomolar! – 334
21.10 Surprise: The Enzyme is Only Functional as aDimer – 338
21.11 Site-directed Mutagenesis:
What Binds the Dimer Together – 340
21.12 When Nothing Else Works: Chemical Poking
at the Contact Interface – 342
21.13 Only Serendipity Can Help: Dierent
Crystal Form—New Dimer – 343
21.14 Tracking the Dynamic Transformation
with the Appropriate Spins – 343
© The Author(s), under exclusive license to Springer-Verlag GmbH, DE, part of Springer Nature 2024
G. Klebe, Drug Design, https://doi.org/10.1007/978-3-662-68998-1_21
21.15 When Sulfur Accidentally Oxidizes and Starts aFragment
Design Project in aNew Arrangement – 346
21.16 A Fragment Opens aTransient Pocket and Suggests
the Design of Bacteria-specic Inhibitors – 349
21.17 Many Ways to aSmart Antibiotic Against Shigellosis – 350
21.18 Synopsis – 352
Bibliography and Original Papers – 353
. • Blocking Pathogenesis on the Molecular Level
The numerous examples in the last part of this book
(Chaps.22–32), many of which have successfully led
to marketed products, are preceded by an exemplary
case study. The main purpose of this chapter is to show
how the inhibition of a target protein can be achieved
by different approaches and how initial ligands can be
candidates for further medicinal chemistry optimization. Using the tRNA-modifying enzyme tRNA-guanine transglycosylase (TGT) as an example, we present
the options for inhibitor development that result from
the iterative application of several design cycles and the
biophysical and crystallographic characterization of the
structure-based design methods described in the previous chapters (Chaps.7, 8, 13, 14, and20). The example
refers to work done by the research group of François
Diederich at the ETH in Zurich, Switzerland, as well as
to work performed by the author’s research group at the
University of Marburg, Germany. Because the work was
accomplished in an academic environment, it was possible to use different tools of structure-based design and
to pursue some of the more fundamental problems in the
context of the project.
21.1 Shigellosis: Disease and Therapeutic
Options
Shigella dysentery is asevere diarrheal illness that is
caused by Shigella bacteria. These bacteria are ingested
with contaminated water or food and adhere to epithelial
cells in the intestinal mucosa. They are extremely contagious: 10–100 bacteria are enough to cause an infection.
Worldwide, shigellosis represents aserious problem. Almost 270 million cases are reported annually, of which
over amillion are fatal. The disease is widespread in developing countries, but over half amillion cases are also
annually reported in industrialized countries. Above all,
the disease ourishes under conditions of inadequate hygiene and poor water quality as is found in war, natural
catastrophes, famine, and in refugee camps. Dysentery
is aparticular problem in Africa where it can occur concomitantly with AIDS.
As with any bacterial infectious disease, shigellosis
can be treated with antibiotics. The infections that occur
in industrialized countries are cured in this way. Unfortunately, Shigella, which is very similar to the Esche-
richia coli that naturally occurs in the intestinal ora, has
atendency to become resistant to antibiotics very quickly.
Moreover, antibiotic therapy also kills the naturally occurring bacteria of the intestinal ora, and this also produces diarrheal symptoms and severe dehydration in the
patients. This can lead to alife-threatening disruption of
electrolyte homeostasis, particularly in small children.
Therefore, specic therapeutic approaches that suppress
the pathogenicity of Shigella are sought.
21.2 Blocking Pathogenesis
on the Molecular Level
Shigella infect the epithelial cells of the colon. Once in
contact with these cells, they secrete invasins through
acomplex secretion apparatus. The invasins create apore
in the host cell membrane. As this invasin pore remains
connected to the secretion apparatus, acontinuous channel is formed. Through this channel, further virulence
factors are transported directly from the bacterial cytoplasm into the host cell cytoplasm. There, the virulence
factors cause the bacteria to be engulfed by aspecic type
of endocytosis, that is through an internalization of the
cell membrane, which completes the uptake of uid and
particles into the cell. Initially, the ingested bacterium is
still enclosed in an envelope (atype of endosome), but
this dissolves within afew minutes. The bacteria are then
free to move around in the cytoplasm of the host cell and
use the infected cell for further replication.
The genes for the components of the secretion apparatus as well as the genes for the invasins and other
virulence factors are located on avery large plasmid. The
expression of all these genes requires the transcription
factor VirF, whose gene is also located on this plasmid.
In order for the protein encoded by the virF gene to be effectively synthesized on the ribosome, aspecically modied tRNA base is required. The tRNA is aribonucleic
acid of about 80nucleotides (. Fig.32.18, Sect.32.7).
It is terminally loaded with an amino acid that is dened
by its specic base triplet in the central loop, called the
anticodon loop. When the gene information is translated
from mRNA, acorresponding tRNA is bound to the
ribosome for each amino acid encoded there, which is deposited as abase triplet. This tRNA carries the required
amino acid so that the correct residue is incorporated
into the nascent peptide chain of the resulting protein.
The modied base required for the efcient biosynthesis
of the VirF transcription factor is located at position34
(the so-called wobble position) of certain tRNAs. They
are loaded with the amino acid aspartate, asparagine,
histidine, or tyrosine. If this modication is missing,
only asmall amount of VirF will be produced. Shigella
bacteria then produce very little of the invasins needed to
infect colon epithelial cells. Their pathogenicity is, therefore, greatly reduced.
Bacteria have enzymes that can make these changes in
tRNA. In the rst step, aguanine 21.1 is cut out of the
tRNA molecule at position34 and replaced with an altered base, preQ1 21.2 (. Fig.21.1). This step is catalyzed
by the enzyme tRNA-guanine transglycosylase (TGT).
The exchanged base in the tRNA is further modied in
the next step of an enzymatic cascade to yield the base
queuine as the nal product. Inhibitors of the bacterial
TGT, therefore, represent aspecic therapeutic principle
for selectively targeting the pathogenicity of Shigella. In
contrast to therapy with broad-spectrum antibiotics, the
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
bacteria are not killed, but the disease-causing infection
of the epithelial cells is prevented. Higher developed eukaryotic organisms such as humans also possess such
an enzyme. Unlike bacteria, which use ahomodimeric
enzyme, the eukaryotic enzyme is aheterodimer. In addition, higher organisms do not convert preQ1 to the end
product queuine, but incorporate queuine directly into
tRNA.
21.3 The Crystal Structure
of tRNA-Guanine Transglycosylase
as aStarting Point
First, the crystal structure determination of TGT in complex with preQ1 was determined from arelated species.
This species shows an exchange of aPhe for aTyr in the
active site, which is immaterial for substrate or ligand
binding. Later, the structure complexed with apart of
the tRNA was elucidated (. Fig.21.2). According to
these structures, base exchange occurs via the following
reaction pathway (. Fig.21.3). Initially, the tRNA with
the covalently attached guanine binds to the enzyme. The
base with its ribose moiety is pulled out of the tRNA
molecule and is specically recognized by Asp 102, Asp
156, Gln 203, Gly 230, and Leu 231. The reaction starts
with anucleophilic attack at carbon C1 of the ribose
ring. The C1–N bond is cleaved, and aproton is transferred from the contact-mediating water to guanine. The
released base, together with the formed hydroxide ion,
leaves the binding pocket. Subsequently preQ1 is taken
up by the same binding site. For this, the peptide bond
between Leu 231 and Ala 232 must ip over. The nitro-
gen atom at position9 of preQ1 then relays aproton to
the nitrogen atom at position3 and carries out anucleophilic attack on the ribose, which is covalently attached
to Asp 280. Once the new bond to the tRNA is formed,
the chemically altered tRNA leaves the enzyme. Asp 102
is critically involved in the recognition process of the
bound base.
21.4 A Functional Assay to Determine
Binding Constants
The base-exchange reaction is accomplished in two steps.
In principle, both steps can be blocked by inhibitors. This
must be considered in afunctional assay. In the rst step,
the unmodied tRNA is bound (. Fig.21.4). Sufciently
large inhibitors could competitively prevent this step. After the tRNA is covalently attached to the enzyme, the
guanine base is released and leaves the protein. Next,
preQ1 binds. Apotential inhibitor can also compete with
this uptake into the binding site, but must not be much
larger than guanine or preQ1. In this way small inhibitors display adifferent inhibition prole than structurally
larger inhibitors.
Radioactively labeled guanine is used to measure inhibition. If this guanine is added to the tRNA, the TGT
will catalyze its incorporation, and the tRNA molecule
will become radioactively labeled. If the tRNA is separated at xed intervals, and the incorporated radioactivity is measured, the reaction kinetics of the incorporation
process and, therefore, the catalytic rate of the enzyme
can be followed. If potential inhibitors are added, fewer
TGT molecules will be available for the transformation,
21
. Fig. 21.1 The enzyme tRNA-guanine transglycosylase (TGT) cat-
alyzes the exchange of guanine 21.1 for preQ1 21.2 in tRNA (left).
Next, the further modication of this base to queuine, which is incor-
porated in the tRNA is achieved by other enzymes. The exchange of
the base takes place in the wobble position of the anticodon loop of
the tRNA (right)
ab
cd
. • The Crystal Structure of tRNA-Guanine Transglycosylase as aStarting Point
. Fig. 21.2 The crystal structure of TGT with a portion of the
tRNA. The protein adopts aTIM-barrel fold. The tRNA binds to the
protein near the catalytic center with the bases U33, G34, and U35,
and the base (gray) to be exchanged at position34 is completely rotated out from the tRNA molecule (upper part). Aview into the binding
site is shown (below). The already-incorporated, modied base preQ1
is held in place in the guanine-recognition pocket (orange) by Asp
102, Asp 156, Gly 230, and Leu 231. The ribose moiety is arranged in
asmall hydrophobic pocket (blue). Uracil33, preceding guanine34,
lies in the green-colored part of the binding pocket, and the uracil35
residue, following guanine34, lies in the red-colored binding areas.
(7 https://sn.pub/TTqICn)
ab
cd
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
. Fig. 21.3 Mechanism of the base exchange reaction in the trans-
glycosylase. The tRNA with guanine34 is bound and awater molecule mediates the contact with the nitrogen atom at the 7-position.
Asp 280 attacks the C1 carbon of the ribose ring as anucleophile(a).
The C1–N bond is cleaved and guanine is released by accepting aproton from the adjacent water molecule(b). Together with the resulting
hydroxide ion, it leaves the binding pocket. In the same binding site,
preQ1 is now incorporated by ipping the peptide bond between Leu
231 and Ala 232 over(c). The proton at position9 of preQ1 is transferred to the nitrogen atom at position3. This creates aguanidium-like
moiety that is stabilized by the carboxylate groups of Asp 102 and
Asp 156. The deprotonated nitrogen atom at position9 then nucleophilically attacks the ribose moiety which is covalently attached to Asp
280. Anew bond to the tRNA is formed(d). This releases the modied
tRNA, which leaves the enzyme. The incorporated preQ1 base releases
its proton into the surrounding solvent
21
and incorporation rate is reduced. This can be seen in
the observed enzyme kinetics. Inhibition constants can
be determined by detailed evaluation of the kinetics. It
can also be determined separately whether inhibitors interact competitively with the binding tRNA or whether
they also compete with the exchange of the small base.
If asmall inhibitor occupies the preQ1 binding pocket
after the tRNA is already bound and the guanine base
has been removed, it will prevent the tRNA from further
reaction. This is called noncompetitive inhibition. If the
binding of such asmall inhibitor occurs before the tRNA
binds to the enzyme, it will act competitively with respect
to the entire tRNA, just like alarger inhibitor. Overall,
small inhibitors that ll only the guanine/preQ1 binding
. • LUDI Discovers the First Leads
. Fig. 21.4 The base-exchange reaction takes place in two steps. Inhibitors can compete with the binding of the complete tRNA (left, dark
gray) as well as the exchange of the small nucleobase (middle, light gray)
pocket are “mixed” inhibitors. They inhibit the enzyme in
two different ways and are, therefore, more efcient than
larger inhibitors at the same binding strength.
21.5 LUDI Discovers the First Leads
In the beginning of the project, only the structure of
the binary complex of TGT with preQ1 was known.
The two-step inhibition mechanism explained in the
last section was also unknown at the time. During the
course of the project Bernhard Stengl managed to clarify the details of this process. Ulrich Grädler used the
binary TGT•preQ1 structure as areference and initiated
asearch for potential inhibitors with LUDI (ade novo
design program; Sect.20.10). He was able to nd hits in
achemical catalog. The compounds listed in . Fig.21.5
were proposed. Among them, 21.3 proved to be amicromolar inhibitor. Acrystal structure could be determined
with this hit (. Fig.21.6). There was great delight when
4-aminophthalic acid hydrazide 21.3 was shown to bind
to the enzyme exactly as LUDI had predicted.
Next, LUDI was consulted to predict further groups
for the inhibitor that would ll in the as-yet unoccupied areas in the binding pocket. On the one hand, an
expansion of the ring system by an additional aromatic
ring was proposed. On the other hand, the placement
of anitrogen-containing heterocycle at the unoccupied
interaction site near Asp 102 and Asp 280 was considered. Hans-Dieter Gerber synthesized derivatives 21.4–
21.6 (. Fig.21.6). Compounds 21.4 and 21.5 achieved
10-times better inhibition of the enzyme in the assay than
21.3. The results were quite different with the heterocyclic
derivative 21.6. It was signicantly worse than the initial
lead structure. Ulrich Grädler was able to solve the crystal
structures with these inhibitors, which exhibited the expected binding mode. It was shown in the structure with
21.6 that the heterocycle falls very near the terminal amide
. Fig. 21.5 Proposals for the rst lead structures by LUDI. Among them, 21.3 proved to be atwo-digit micromolar inhibitor
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
21
. Fig. 21.6 Crystal structure of TGT with 21.3, the rst hit from
LUDI. The agreement between the predicted (left) and the nal experiment is almost perfect. LUDI indicated additional interaction centers
in the lower part of the binding pocket that had not yet been used
(right). Therefore, starting from 21.3, the inhibitors 21.4 and 21.5,
which bind better by afactor of10, were synthesized. The two derivatives 21.6 and 21.7, which were extended with aheterocycle, occupy
the still unused additional interaction sites even better. However, the
two derivatives showed decreased binding afnity, presumably due to
group of Asn70. It was then obvious that 21.6 needed
an additional amino group to build an additional contact
with the protein. This synthesis was accomplished, and the
crystal structure with 21.7 in fact did show the expected
binding mode with the additional H-bond. However, even
this derivative was less potent than the original lead structure 21.3. Amore detailed analysis of the structural data
showed that the appended heterocycle in 21.6 and 21.7
is disordered, and ahydrogen bond between the exocyclic amino group and the carbonyl group of Leu 231 is
very long. The heterocycle was incorporated based on the
idea that it would be benecial to have acharged group
that can also form hydrogen bonds to the two neighboring aspartate groups. These two groups were assumed to
adopt adeprotonated state. Then apositive charge on
the triazole group would be ideal for an interaction. But,
which protonation state do these groups adopt? ApKa
measurement was carried out on arelated model compound. Asmall-molecule crystal structure determination
was undertaken on acrystal grown under the same buffer
conditions as the protein complex was crystallized. Both
experiments showed that the heterocycle exists without
acharge, that is, both of the neighboring nitrogen atoms
are deprotonated. Although it is not obligatory that the
repulsive interactions with the two neighboring aspartate residues 102
and 280, since the heterocycles do not have the desired positive partial
charge at the site of action. (7 https://sn.pub/R6M5Uw)
same protonation state is found in the protein’s binding
pocket, this model appears to be plausible to explain the
decreasing binding afnity of 21.6 and 21.7: An uncharged
triazole ring between the two negatively charged aspartate groups must experience arepulsive interaction with
at least one of the two acidic groups. This could reconcile
the decreasing binding afnity, the observed disorder, and
the elongated H-bond to the carbonyl group of Leu 231.
21.6 Surprise: AFlipped Amide Bond
and aWater Molecule
Novo Nordisk kindly provided an additional compound,
21.8 that emulates the original interaction pattern of the
initial lead structure (. Fig.21.7). Upon docking this
derivative, however, it was shown that the distance between the polar nitrogen atom in the central pyridazinone ring and the carbonyl group in Leu 231 was too
large. Nevertheless, the compound was a micromolar
hit. The crystal structure that was determined with the
related derivative 21.9 delivered an explanation. The pep-
tide bond, which, for mechanistic reasons, acts as aswitch
between two conformations, takes on adifferent orienta-
. • Hot Spot Analysis and Virtual Screening Open the Floodgate to New Ideas for Synthesis
. Fig. 21.7 Left Analogue 21.8 should also emulate the interaction
pattern of the original lead structure. If this derivative is placed in
the binding pocket (purple), the distance between the polar nitrogen
atoms in the central pyridazinone ring and the carbonyl group on Leu
231 seems to be too large for an H-bond. Nonetheless, 21.8 binds to
tion! When ipped, the NH functional group is found in
the binding pocket. The contact between this NH group
and the polar nitrogen atom in the ligand is mediated by
an interstitial water molecule. Because the details of the
above-described enzymatic mechanism were not known
at that time, the ipping of the peptide bond switch could
not have been predicted. Furthermore, the incorporation
of awater molecule was abig surprise. It underscores the
importance of repeatedly determining crystal structures
with newly found lead structures.
21.7 Hot Spot Analysis and Virtual
Screening Open the Floodgate
to New Ideas for Synthesis
How can multiple binding modes be made avirtue out of
necessity? Ruth Brenk used the protein conformers in the
structure with 21.3 as well as the geometry in the complex
with 21.9 to carry out ahot spot analysis (Sect.17.10).
The result of this analysis is shown in . Fig.21.8. Avir-
tual screening (Sect.7.6) was performed with the generated pharmacophore and this produced aplethora of
alternative molecular scaffolds (. Fig.21.9) to occupy
the protein with micromolar afnity. Right The crystal structure that
was determined with the very similar inhibitor 21.9 (orange) shows two
surprises: The peptide bond rotates its orientation and now directs its
NH group towards the binding pocket, and a water molecule (red
sphere) mediates the interaction with the ligand!
the guanine-binding site (. Fig.21.2). Many of the hits
that were discovered in this way proved to be micromolar
inhibitors. They afforded many new ideas for synthetic
entry points to develop new inhibitors. Of these the
pyridazinone (trione, 21.10), pteridine (21.11), 6-aminoquinazolinone (21.12), and particularly the lin-benzoguanine scaffold (21.13), which was studied with the group
at ETH Zurich, were investigated in detail.
Let us turn to the distribution of hot spots in the
binding pocket. The new lead structures all interact at
sites in the “upper part” of the binding pocket. However, an additional favorable binding area, capable of
interacting with donor properties as well as hydrophobic moieties, is indicated in the “lower left part” of the
binding site next to the two aspartic acid residues Asp
102 and Asp 280. These binding sites were not used in
previous design. Considering the binding mode of the
bound tRNA (. Fig.21.2), the ribose sugar moiety at
position34 is accommodated in this region. The hot spot
analysis suggests a hydrophobic molecular fragment.
Afavorable site for an H-bond donor is located slightly
above. This region corresponds to the binding site between the two aspartic acids, where the two heterocyclic
derivatives 21.6 and 21.7 have already been placed. An-
ab
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
. Fig. 21.8 Hot spot analysis shows preferred binding areas for
ahydrogen-bond donor (left), acceptor (center), and ahydrophobic
group (right). In addition, it was shown that the polar groups of 21.9
(cf. . Fig.21.7) fall into the preferred binding area. In the bottom left
corner of the binding pocket (near the binding site of the ribose moiety . Fig.21.2, blue), other binding areas are indicated that were addressed in subsequent design steps
. Fig. 21.9 A variety of suggestions from virtual screening, some
examples of which were experimentally tested and found to be micromolar inhibitors. In particular, the pyridazinone (triones, 21.10), the
pteridine (21.11), the 6-aminoquinazolinone (21.12), and the lin-ben-
other favorable area for an acceptor group is indicated at
the rim of this pocket. The 2′ and 3′ hydroxyl groups of
the tRNA ribose moiety are placed in this area.
zoguanine scaffolds (21.13) served as the rst possible lead structures
for further synthesis and optimization. By addition of suitable substituentsR, numerous derivatives could be synthesized
itors with such side chains were designed and led to the
derivatives displayed in . Fig.21.10. Disappointingly,
these showed only amodest improvement. In addition
to the synthesis of the pteridines and aminoquinolinones
developed in Marburg, the lin-benzoguanines 21.13 were
21
21.8 The Filling of Hydrophobic Pockets
and Interference with aWater
Network
advanced by Emanuel Meyer and Simone Hörner at the
ETH in Zurich. But unfortunately, no really striking
improvement in afnity could be found for any of the
derivatives listed in . Fig.21.10. They occupy the small
Agolden rule in drug design is that the occupancy of an
empty hydrophobic pocket with alipophilic group leads
to an increase in afnity (Sect.4.9). Accordingly, inhib-
hydrophobic pocket between Val45, Leu 68, and Asn70,
as planned and as shown in the crystal structure with
21.14. Bernhard Stengl and Tina Ritschel took another