A rapid synthesis of low-nanomolar divalent LecA inhibitors in four linear steps from d-galactose pentaacetate

Article abstract of DOI:10.1039/d0cc03490h

Chronic infections with Pseudomonas aeruginosa are associated with the formation of bacterial biofilms. The tetrameric P. aeruginosa lectin LecA is a virulence factor and an anti-biofilm drug target. Increasing the overall binding affinity by multivalent

Full text of DOI:10.1039/d0cc03490h

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A rapid synthesis of low-nanomolar divalent LecA
inhibitors in four linear steps from D-galactose
pentaacetate†
Cite this:DOI: 10.1039/d0cc03490h
Received 15th May 2020,
Accepted 17th June 2020
abc
d
a
Eva Zahorska,
Sakonwan Kuhaudomlarp,
Saverio Minervini,
a
d
a
bce
Sultaan Yousaf, Martin Lepsik,
Thorsten Kinsinger, Anna K. H. Hirsch,
DOI: 10.1039/d0cc03490h
d
abc
Anne Imberty
and Alexander Titz
*
rsc.li/chemcomm
10–15
Chronic infections with Pseudomonas aeruginosa are associated with reach binding affinities in the mid to low micromolar range.
the formation of bacterial biofilms. The tetrameric P. aeruginosa lectin In Nature, the rather weak lectin–carbohydrate binding is often
LecA is a virulence factor and an anti-biofilm drug target. Increasing the overcome by increasing valency, and thus enhancing apparent
1
6,17
overall binding affinity by multivalent presentation of binding epitopes affinity.
Likewise, a boost in target-binding affinity was
6,18
can enhance the weak carbohydrate–ligand interactions. Low- achieved with multivalent inhibitors of LecA and LecB.
Since
nanomolar divalent LecA ligands/inhibitors with up to 260-fold LecA is a tetramer and pairs of binding sites are geometrically
valency-normalized potency boost and excellent selectivity over favorably oriented, simultaneously binding divalent inhibitors can
19,20
human galectin-1 were synthesized from D-galactose pentaacetate boost binding affinity through favorable binding entropy.
and benzaldehyde-based linkers in four linear steps.
Notably, Pieters and co-workers have developed divalent LecA inhi-
bitors based on complex and rigid repeating units of carbohydrate-
Pseudomonas aeruginosa has been classified as a priority-1 pathogen triazole spacers with potent binding affinities ranging from 12 to
2
1–23
by the World Health Organization due to its high antimicrobial 220 nM,
while a divalent inhibitor with a more flexible linker
24
resistance and the lack of new drugs to treat multidrug-resistant reaches an affinity of 80 nM. In another report, an oligoproline-
strains. New strategies against these bacterial infections are being spaced digalactoside bound to LecA with K
1
25
d
of 71 nM.
2
explored to overcome the current antimicrobial-resistance crisis.
In this work, we aimed to develop divalent LecA ligands with a
The so-called anti-virulence therapy aims to neutralize bacterial focus on drug-like properties, synthetic accessibility and linker
virulence factors instead of increasing the selection pressure simplicity enabling future lead optimization. Spacer length and
imposed by targeting essential cellular functions with antibiotics flexibility are important factors contributing to the overall potency
6
and thereby circumvents the advent of new resistances whilst of multivalent inhibitors. An optimized linker connecting two
3,4
preserving commensal bacteria. This strategy is investigated for neighboring binding sites within one LecA tetramer and avoiding
P. aeruginosa infections by targeting its tetravalent lectins LecA and unwanted cross-linking between different LecA tetramers is desired
2,5,6
LecB.
Both proteins are virulence factors regulated by quorum (Fig. 1a). b-Linked aryl aglycons increase the binding strength
sensing, mediate bacterial host-cell adhesion and are essential of galactosides to LecA by establishing CH–p interactions with
7–9
15
structural components of P. aeruginosa biofilms. Whereas the His50. The co-crystal structure of LecA with phenyl b-D-galactoside
best L-fucose/D-mannose-based LecB antagonists bind in the nano- (PDB code: 5d21) showed possible growth vectors in meta- and
26
molar range, monovalent D-galactose-based LecA inhibitors only para-position at the phenyl aglycon (Fig. 1b).
Protein-templated dynamic combinatorial chemistry (DCC)
is an elegant method for the identification of potent ligands
from a combinatorial library of building blocks with suitable
a
b
Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical
Research Saarland, Helmholtz Centre for Infection Research, 66123 Saarbr u¨ cken,
Germany. E-mail: alexander.titz@helmholtz-hzi.de
2
7,28
linking chemistry in presence of a given protein.
To apply
this method to LecA, we introduced hydrazides at the para- or
meta-position of phenyl b-D-galactoside in order to allow for
acylhydrazone formation in DCC. For this purpose, we chose
two galactoside building blocks with meta- or para-attached
hydrazides, 1m and 1p, and varied linker length, rigidity and
number of rotatable bonds by systematically increasing the
number of methylene units in the corresponding benzaldehyde
Deutsches Zentrum f u¨ r Infektionsforschung (DZIF), Standort Hannover-
Braunschweig, 38124 Braunschweig, Germany
c
Department of Pharmacy, Saarland University, 66123 Saarbr u¨ cken, Germany
Universit ´e Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France
Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research
Saarland, Helmholtz Centre for Infection Research, 66123 Saarbr u¨ cken, Germany
Electronic supplementary information (ESI) available. See DOI: 10.1039/
d
e
†
d0cc03490h
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of LecA: since the addition of LecA to the library caused precipita-
tion, all divalent molecules were individually synthesized in absence
of protein. Acylhydrazone formation of aldehydes A–F with excess
hydrazide 1m or 1p under acidic conditions yielded the mono- and
divalent LecA ligands A5m–F5m and A5p–F5p. The reduced solubility
of the meta-series compared to the para-series and more difficult
purifications could explain the lower yields despite nearly quantita-
tive turnover during the individual reactions.
All synthesized galactosides, A5m–F5m and A5p–F5p, were
then analyzed in the previously established competitive LecA
1
1
binding assay based on fluorescence polarization (Fig. 2).
Monovalent meta-ligand A5m (IC50 = 21.6 Æ 4.5 mM) was twice
as potent as its para-isomer A5p (IC50 = 55.5 Æ 4.4 mM). The
divalent ligands B5m–F5m and B5p–F5p showed a very similar
profile in the competitive binding assay with very similar IC50
values in the single-digit micromolar range and a very steep Hill
Fig. 1 Design of divalent LecA inhibitors accessible through a short synthetic
route with acylhydrazone coupling chemistry. (a) Possible binding modes of
divalent LecA inhibitors with desired linkage for two adjacent binding sites. (b)
The crystal structure of phenyl b-D-galactoside in complex with LecA (pdb code:
5
(
d21) and distances between two ligands within one pair of binding sites in LecA slope of the fit. These observations are likely a result of reach-
from meta to meta: 23 Å, from para to para: 25 Å). (c) Building blocks of LecA
ing the lower assay limit since the low affinity of the fluorescent
primary ligand (Kd = 7.4 mM) required a relatively high LecA
concentration of 20 mM. Therefore, ligand affinities with
orders-of-magnitude higher potencies than the primary compe-
titively displaced ligand cannot be reliably determined.
inhibitors: m/p-hydrazinecarbonylphenyl b-D-galactopyranoside (1m, 1p) and
benzaldehydes A–F. Bis-benzaldehyde linkers B–F with systematic variation of
length and number of rotatable bonds to optimize distance and flexibility.
spacers B–F. The corresponding monovalent control A was
To overcome the competitive binding assay’s limitation, we
included (Fig. 1c).
analyzed all inhibitors in a direct LecA binding experiment using
Benzaldehydes A and B were commercially available and surface plasmon resonance (SPR). In case of the monovalent
bis-benzaldehydes C–F were obtained in one step using 4- inhibitors A5m and A5p, rapid changes in the binding response
hydroxybenzaldehyde in a double nucleophilic substitution during the association and dissociation phases were observed,
reaction on aliphatic a,o-di-halogenated C1–C4 hydrocarbons under indicating fast association/dissociation kinetics of the monovalent
microwave irradiation (Scheme 1). Lewis acid-promoted glycosyla- inhibitors to immobilized LecA (Fig. 3a and b). Due to this fast
tion of methyl meta- or para-hydroxybenzoate with b-D-galactose association/dissociation behavior, kon and koff for their interaction
pentaacetate (2) yielded glycosides 3m and 3p in 69% and 47% with LecA could not be accurately determined and affinity analysis
yield, respectively. Removal of the acetates under Zempl ´e n condi- was performed instead of determining K at steady-state binding. In
d
tions gave galactosides 4m and 4p quantitatively. Subsequent ester SPR, the monovalent ligands A5m and A5p have K values of 4.9 Æ
d
hydrazinolysis resulted in hydrazides 1m and 1p in very good yields. 0.1 and 5.6 Æ 0.3 mM, respectively (Table 1). The binding affinities
These building blocks were then used in DCC reactions in presence for those monovalent compounds were validated using isothermal
titration microcalorimetry (ITC) as an orthogonal method and
similar values to SPR data were obtained (ITC: A5m K = 2.7 Æ
d
1
.3 mM, A5p K
d
= 6.1 Æ 0.5 mM, Table 1 and Fig. S1, ESI†). In both
analyses, a meta-substitution of the phenyl aglycon in A5m resulted
in higher affinities to LecA compared to the para-isomer A5p.
Scheme 1 Synthesis of divalent LecA ligands and their monovalent ana-
logs. Reagents and conditions: (i) 4-hydroxy benzaldehyde, K CO , DMF,
0 1C, microwave, 3–10 h; (ii) methyl m/p-hydroxybenzoate, BF
CH Cl , 0 1C – r.t., o.n.; (iii) NaOMe, MeOH, r.t., o.n.; (iv) NH NH
2
3
7
3
ÁEt
ÁH
2
2
O,
O,
Fig. 2 Evaluation in a competitive binding assay. One representative titration
is shown for each series (right) – steep titration slopes for divalent inhibitors
indicate the lower assay limit was reached. Aver. and std. dev. from at least 3
independent titrations of triplicates each. n.d. = not determined.
2
2
2
2
MeOH, 70 1C, o.n. (v) formic acid, DMSO, r.t., 4 h, for D5m: DMSO/MeCN,
for F5m: H O/MeCN.
2
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from the para- and meta-series, B5p and C5m, together with
their respective monovalent counterparts (A5p and A5m), were
analyzed by SPR for their interaction with human galectin-1.
Neither the monovalent (at 250 mM) nor the divalent com-
pounds (at 25 mM) had a detectable interaction with the
immobilized galectin-1 (Fig. S3, ESI†).
Since we did not succeed in obtaining crystal structures of LecA
complexed with the divalent inhibitors, we carried out modeling on
pairs of para- and meta-compounds, selecting the high-affinity
binder C5m and its para-counterpart C5p; and the longer, less
active E5m and E5p. The dynamics of these four compounds were
simulated in the free state and in the modeled complex with LecA.
In the free state, the ligands adopted a broad range of semi-
extended to fully extended conformations characterized by
Gal:C1ÁÁÁGal:C1 distances ranging from 20 to 31 Å at a cutoff of
Fig. 3 SPR analyses of the interaction between the monovalent inhibitors
(a) A5m and (b) A5p. The sensorgrams are shown on the left panel and the
affinity analyses on the right. (c) Sensorgrams of the most potent divalent
inhibitors from meta-series (C5m) and (d) from para-series (B5p) at five
different concentrations (0, 10, 50, 100, 200 nM).
5% frequency (Fig. 4a, solid curves). A distance close to the
In contrast to the monovalent hydrazides, the SPR sensor- crystallographic value of 29 Å (PDB: 5d21) is desired for optimal
grams of the divalent inhibitors clearly indicated much slower divalent binding of LecA. In the longer compounds, E5m and E5p,
association/dissociation of the compounds from LecA, demon- there was a small population (around 5%) of folded conformations
strating the benefit of divalent binding and enabling determi- (see the peaks at 5 Å in Fig. 4a). The unfolding of these conforma-
d
nation of kinetic parameters (kon and koff) as well as K (calculated tions prior to LecA binding may be in part responsible for their
from koff/kon, Fig. 3c, d and Fig. S2, ESI†). The divalent inhibitors slower on-rate (kon).
B5m–F5m and B5p–F5p showed a strong increase in potency into
The four ligands modeled in complex with LecA showed a
the low nanomolar range (Table 1). In the para-series, compound narrow distribution of the Gal:C1ÁÁÁGal:C1 distances (Fig. 4a,
B5p was the most potent ligand (K
d
= 10.8 Æ 1.0 nM), with a 520- dashed lines). C5m, C5p and E5p sampled distances of 28.9 Æ
fold increase (260-fold valency-corrected) compared to its mono- 0.4 Å, 28.1 Æ 0.9 Å, and 28.2 Æ 0.7 Å, respectively, close to the 29 Å
valent congener A5p (Kd = 5600 Æ 300 nM). The divalent para- observed in crystal structures, thus indicating that the linker
ligands showed a slight decrease in potency with increasing spacer lengths are well-suited to bridge two LecA monomers. The narrow
length, resulting from gradually decreasing kon and increasing koff range for C5m (28–31 Å) may explain the lower affinity of shorter
(
Table 1). In the meta-series, similar trends were absent. C5m with B5m. In contrast, C5p and E5p have larger range (26–31 Å) and
one central methylene unit had the optimal length and showed a shortening is beneficial such as in B5p. E5m with LecA displayed a
of 18.9 Æ 1.6 nM. Shortening or increasing spacer length higher mean distance of 30.2 Æ 0.8 Å (range of 28 to 33 Å). Such
resulted in reduced affinities and surprisingly the second longest longer meta linker pushes the two LecA monomers slightly apart,
compound E5m was the least efficient inhibitor (K
of 80.7 Æ which is not favourable. The T-shaped CH–p interaction between
1.4 nM). Comparing across the meta-series, the kon values were His50 and the phenyl aglycon was observed with higher frequency
surprisingly 2–4 times smaller for compounds C5m and E5m. The for the meta compounds C5m and E5m compared to their para
off values were gradually increasing going from C5m–F5m. The koff analogues C5p and E5p (Fig. 4b). On the opposite, the para ligands,
for B5m was 5-times higher than that of C5m. C5p and E5p, preferentially adopted an inverted V-shape (Fig. 4c
K
d
d
1
k
We then studied compound selectivity in binding experi- and d) in which their phenyl aglycons sampled a variety of
ments towards human galectin-1, a homodimeric lectin that arrangements with respect to His50 (parallel, diagonal, T-shape).
specifically recognizes b-galactoside containing glycans such as
To conclude, we designed and synthesized highly potent
2
9
Me-b-lactoside (K
d
= 187 mM). The most potent inhibitors divalent LecA inhibitors in four linear chemical steps from
a
Table 1 Affinity and kinetic analyses of the LecA-inhibitor interactions determined by SPR and ITC
meta series
para series
3
À1 À1
off (Â10À3
À1
3
À1 À1
off (Â10À3
À1
k
on (Â10 M
s
)
k
s
)
K
d
(nM)
r.p.
k
on (Â10 M
s
)
k
s
)
K
d
(nM)
r.p.
A5m
—
—
—
—
4900 Æ 100
2700 Æ 1300
27.3 Æ 1.6
18.9 Æ 1.6
23.8 Æ 7.1
80.7 Æ 11.4
33.4 Æ 2.3
1
—
90
130
103
30
73
A5p
—
—
—
—
5600 Æ 300
6100 Æ 500
10.8 Æ 1.0
20.5 Æ 0.2
21.4 Æ 1.0
22.5 Æ 9.0
50.1 Æ 1.2
1
—
259
137
131
124
56
b
b
B5m
C5m
D5m
E5m
F5m
196 Æ 6
59 Æ 3
120 Æ 41
45 Æ 4
104 Æ 53
5.34 Æ 0.28
1.11 Æ 0.08
2.67 Æ 0.38
3.64 Æ 0.35
3.39 Æ 1.5
B5p
C5p
D5p
E5p
F5p
152 Æ 3
114 Æ 2
121 Æ 9
103 Æ 2
79 Æ 5
1.64 Æ 0.13
2.33 Æ 0.02
2.57 Æ 0.20
2.19 Æ 0.45
3.98 Æ 0.16
a
Averages and std. dev. from three independent experiments. Relative potencies (r.p.) were calculated compared to monovalent compound in each
b
series (A5m and A5p) and valency-normalized. ITC determination.
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Conflicts of interest
There are no conflicts to declare.
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