An i-biofilm Agen s agains Pseudomonas ae uginosa: A S uc u e−
Ac i i y Rela ionship S udy o C‑Glycosidic LecB Inhibi o s
Roman Somme ,
†,‡
Ka ha ina Rox,
‡,§
S e anie Wagne ,
†,‡
Di k Hauck,
†,‡
Sa ah S. Hen ikus,
†,‡,#
Shelby Newsad,
†,‡
Ta jana A nold,
‡,§
Thomas Ryckmans,
∥
Ma k B ons up,
‡,§
Anne Imbe y,
⊥
Annabelle Va o ,
⊥
Rol W. Ha mann,
‡,#,¶
and Alexande Ti z*
,†,‡,#
†
Chemical Biology o Ca bohyd a es, Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land (HIPS), Helmhol z Cen e o
In ec ion Resea ch, D-66123 Saa b ucken, Ge many
‡
Deu sches Zen um u In ek ions o schung (DZIF), S ando Hanno e , D-38124 B aunschweig, Ge many
§
Chemical Biology, Helmhol z Cen e o In ec ion Resea ch, D-38124 B aunschweig, Ge many
∥
Roche Pha maceu ical Resea ch and Ea ly De elopmen , Roche Inno a ion Cen e Basel, CH-4070 Basel, Swi ze land
⊥
Uni . G enoble Alpes, CNRS, CERMAV, F-38000 G enoble, F ance
#
Depa men o Pha macy, Saa land Uni e si y, D-66123 Saa b ucken, Ge many
¶
D ug Design and De elopmen , Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land (HIPS), Helmhol z Cen e o In ec ion
Resea ch, D-66123 Saa b ucken, Ge many
*
SSuppo ing In o ma ion
ABSTRACT: Biofilm o ma ion is a key mechanism o an imic obial esis ance. We ha e ecen ly epo ed wo classes o o ally
bioa ailable C-glycosidic inhibi o s o he Pseudomonas ae uginosa lec in LecB wi h an ibiofilm ac i i y. They p o ed efficien in
a ge binding, we e me abolically s able, non oxic, selec i e, and po en in inhibi ing o ma ion o bac e ial biofilm. He e, we
designed and syn hesized six new ca boxamides and 24 new sul onamides o a de ailed s uc u e−ac i i y ela ionship o wo
clinically ep esen a i e LecB a ian s. Sul onamides gene ally showed highe inhibi ion compa ed o ca boxamides, which was
a ionalized based on c ys al s uc u e analyses. Subs i u ions a he hiophenesul onamide inc eased binding h ough ex ensi e
con ac s wi h a lipophilic p o ein pa ch. These me abolically s able compounds showed a u he inc ease in po ency owa d he
a ge and in biofilm inhibi ion assays. In gene al, we es ablished he s uc u e−ac i i y ela ionship o hese p omising
an ibiofilm agen s and showed ha modifica ion o he sul onamide esidue bea s u u e op imiza ion po en ial.
■INTRODUCTION
Pseudomonas ae uginosa is an oppo unis ic G am-nega i e
bac e ium wi h high clinical impo ance and classified as a
c i ical p io i y 1 pa hogen by he WHO in 2017.
1−4
Especially
o cys ic fib osis (CF) pa ien s, ch onic in ec ions esul in
ecu en pneumonia, sepsis, and lung damage.
5
Challenges in
ea ing P. ae uginosa in ec ions esul om i s in insic
an imic obial esis ance and acqui ed esis ances ha o en
lead o mul id ug- esis an MDR o XDR s ains.
6
In addi ion,
he bac e ium’s an imic obial ole ance is u he enhanced by
he sel - o ma ion o biofilms, a p o ec i e enclosu e agains
hos immune de ense and an ibio ic ea men .
7,8
Because
bac e ia esiding in a biofilm a e up o 1000- old mo e esis an
owa d an ibio ics,
7
a ge ing biofilm o ma ion has been an
eme ging he apeu ic app oach in ecen yea s o o e come he
esis ance p oblem ( e iewed in e s 9−11).
The wo i ulence ac o s LecA
12
and LecB
13
(ini ially called
PA-IL and PA-IIL
14
) a e egula ed by quo um sensing
15
and
ha e decisi e oles in biofilm o ma ion. I is cu en ly
an icipa ed ha bo h e a alen ca bohyd a e-binding p o eins
c oss-link glycoconjuga es on hos cells o issue wi h bac e ial
lipopolysaccha ide and exopolysaccha ides o s abilize he
ma ix and in eg i y o he biofilm.
9,16
Thus, blocking his
p ocess wi h exogenous compounds could p e en he
o ma ion o e en des oy es ablished biofilms.
Recei ed: July 11, 2019
Published: Sep embe 25, 2019
A icle
pubs.acs.o g/jmc
Ci e This: J. Med. Chem. 2019, 62, 9201−9216
© 2019 Ame ican Chemical Socie y 9201 DOI: 10.1021/acs.jmedchem.9b01120
J. Med. Chem. 2019, 62, 9201−9216
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Bo h ca bohyd a e-binding p o eins, so-called lec ins, we e
fi s isola ed om he clinical isola e P. ae uginosa PAO1 by
Gilboa-Ga be e al.
14,17,18
Onehu dle o he apeu ic
in e en ion is he ac ha P. ae uginosa has a high genomic
di e si y among diffe en isola es.
19−21
The p o ein sequence
o LecA is a he conse ed among P. ae uginosa s ains bu o
LecB isola es a e g ouped in o ei he PAO1- o PA14-like LecB
p o ein sequence amilies.
22,23
Despi e hese sequence
a ia ions in he wo LecB a ian s, hey su p isingly ha e a
conse ed binding specifici y o simila glycoconjuga es, which
pa es he way o he simul aneous a ge ing o a b oad ange
o clinical isola es wi h one single compound. In i s qua e na y
s uc u e, LecB o ms nonco alen homo e ame s whe e wo
Ca2+-ions a e p esen in each monome ,
23,24
media ing he
ecogni ion o i s ca bohyd a e ligands, L- ucose and D-
mannose (e.g., me hyl α-D-mannoside (1), Figu e 1). Because
LecB is localized ex acellula ly,
13
he G am-nega i e bac e ial
cell en elope which usually imposes a s ingen hu dle o
many an ibio ics wi h in acellula a ge s, is no p oblema ic
o a ge ing LecB.
Besides i s ole in biofilm o ma ion and bac e ial adhesion,
LecB was also shown o ca bohyd a e-dependen ly block
human cilia y bea ing,
25
in e e e wi h issue epai p ocesses
26
and, ecen ly, ac i a e B-cells.
27
Fu he mo e, a di ec
in ol emen o LecA and LecB in in ec ion and hos
coloniza ion by P. ae uginosa using a mu ine in ec ion model
e ealed he sui abili y o bo h lec ins as he apeu ic
a ge s.
28,29
Inhala ion o an ae osol o ucose and galac ose,
he ligands o LecB and LecA, esul ed in a educ ion o
bac e ial load in human in ec ed ai ways
30,31
and in mice.
32
Because ucosides display highe affini ies o LecB han
mannosides, inhibi o de elopmen gene ally cen e ed a ound
ucose-based inhibi o s p esen ed on a mul i alen scaffold o
u he inc ease affini y/a idi y.
33,34
Following his s a egy,
mul i alen glycopep ide dend ime s ha e been de eloped
which efficien ly inhibi he o ma ion and dispe se es ablished
biofilms o P. ae uginosa.
35
Ano he example o mul i alen
ucosides on a calixa ene scaffoldshowed e ypo en
nanomola affini ies o LecB bu su p isingly equi ed
millimola concen a ions (5 mM) o biofilm inhibi ion and,
in con as o he desi ed p ope ies, he compound induced
bac e ial agg ega ion.
28
The e o e, mul i alen p esen a ion o
ca bohyd a es could mimic bac e ial exopolysaccha ides and,
hus, s abilize he biofilm a he han inducing i s desi ed
disin eg a ion. In addi ion, hese mul i alen p esen a ions o
na i e ca bohyd a es may be immunogenic and in e e e wi h
he pa ien ’s immune sys em, e.g., by binding o he a ious
inna e immuni y pa e n ecogni ion ecep o s.
To o e come hese disad an ages in insic o mul i alen ly
displayed lec in ligands, we ha e emba ked on he de elop-
men o mono alen glycomime ic small molecules as
compe i i e inhibi o s o LecB.
36−41
Ou small molecules
possess d ug-like p ope ies ha esul ed in o al bioa ailabili y
o he wo es ed C-glycosides wi h sys emic dis ibu ion,
which is impossible o he high molecula weigh mul i alen
compounds.
We ha e s a ed wi h he weak LecB ligand me hyl α-D-
mannoside (1) and ans o med i in o C-6 modified amide
and sul onamide de i a i es ha led o an inc ease in po ency
up o a ac o o 20 (e.g., 2and 3,Figu e 1).
36,38
These
compounds showed good ecep o binding kine ics and p o ed
efficien in he p e en ion o bac e ial adhesion. Because ucose
and mannose a e ecognized by LecB, we me ged he necessa y
unc ional g oups ha we e shown o elici a ac i e
in e ac ions wi h he p o ein in o one molecule and ob ained
he fi s o ou C-glycosides lacking he O-glycosidic linkage.
37
A e he explo a ion o addi ional in e ac ions o hep ose-
39
o - fluo oglycomime ics
40
wi h LecB, we hen combined he
ini ial C-glycosides wi h he amido- and sul onamido
subs i uen s o ob ain he glycomime ics 4−7.
41
Especially
he sul onamides 6and 7displayed a o able p ofiles in a ge
binding po ency and selec i i y, ADME/Tox pa ame e s and
Figu e 1. Design app oach o C-glycosidic LecB inhibi o s and ex ension o he s uc u al space o ex ended SAR s udies o compounds 4−37.
De i a i es o me hyl α-D-mannoside 1−3and hei inhibi o y po ency o he binding wi h LecBPAO1.
36
C-Glycosides simul aneously de i ed o D-
mannosides and L- ucosides a e hyb id- ype LecB ligands 4and 5and 6and 7.
41
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o al bioa ailabili y in a mu ine pha macokine ics model.
Impo an ly, 6and 7possessed excellen an ibiofilm ac i i y
in a P. ae uginosa biofilm o ma ion assay as moni o ed by
con ocal fluo escence ligh mic oscopy.
He e,weha esignifican ly inc eased he numbe o
de i a i es o he C-glycoside ligands o LecB o 30 new
de i a i es yielding a de ailed s uc u e−ac i i y ela ionship
o his class o d ug-like an ibiofilm compounds. We ha e
de e mined key in e ac ions o he new mo e po en
de i a i es om c ys allog aphic analyses, deduced he
s uc u al basis o he inc eased binding po ency o he
PA14 a ian o LecB, and add essed a new binding pocke on
he p o ein su ace. The C-glycosidic dime hyl hiophene
sul onamide 22 was iden ified as on unne wi h good
po ency in a biofilm assay and excellen ADME/Tox
p ope ies.
■RESULTS AND DISCUSSION
Design. C-Glycosides o he amide se ies, e.g., 4and 5, and
he sul onamide se ies, e.g., 6and 7, ha e p e iously been
epo ed by us as po en glycomime ic inhibi o s o LecB
(Figu e 1).
41
To expand he s uc u e−ac i i y ela ionship o
hese po en compound classes, we aimed o u he di e si y
he amide and sul onamide subs i uen s and explo e hei
in e ac ion wi h LecB.
In he amide se ies, he cinnamide and dime hoxycinnamide
de i a i e o a mannoside and i s C-glycoside analogue a e
be e binde s han benzamides o alipha ic amides.
36,38,41
We
he e o e aimed a analyzing a po en ial igidifica ion o he
cinnamide by ing closu e be ween he o ho-posi ion o he
phenyl g oup and he α-ca bon by in oducing he e oa om
linke s in benzo hiophene-, benzo u an-, and indole-2-ca box-
amides (9−11). These subs i u ions affec igidi y, hyd ogen-
bond dona ing o accep ing p ope ies, and o al pola su ace
a ea o he esul ing molecules while main aining he o iginal
cinnamide pha macopho e. Fu he mo e, we subs i u ed he
double bond in he cinnamoyl g oup wi h 5-membe ed
he e ocycles, i.e., a hiazole and a hiophene (12,13).
To add ess he e y po en sul onamide se ies o C-
glycosides and expand he highly o ally bioa ailable hiophenyl
de i a i e 7, we included a numbe o diffe en 5-membe ed
he e ocycles (14−28), e.g., u an, oxazole, py azoles, and he
egioisome o he o iginal hiophene. In addi ion, a ious
subs i uen s we e a ached o hose he e ocycles, and he ocus
was se o me hyl g oups ha showed good po ency inc ease in
he p e ious mannose-se ies (IC50s o LecBPA14: phenyl-
sul onamide 16 μM→ ime hylphenylsul onamide 1 μM, see
Figu e 2, compounds 47 and 3).
36
Inspec ion o he c ys al s uc u e
41
o he complex o 7wi h
LecB indica ed a possible second shallow cle be ween he wo
loops o med by esidues Val69−Asp75 and Glu95−Ala105
Figu e 2. Compe i i e binding assay o inhibi o s wi h LecBPAO1 and LecBPA14 based on fluo escence pola iza ion. Means and s anda d de ia ions
we e de e mined om a minimum o h ee independen expe imen s. n.s.: no soluble a 1 mM in TBS/Ca con aining 1% DMSO. IC50 alues o
1−7,38, and 41−46 wi h LecBPAO1 and LecBPA14 we e p e iously published.
23,36−38,41
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wi hin each o he hiophene (Suppo ing In o ma ion, Figu e
S1). The e o e, we designed sho space s o di ec ly a ached
o he hiophene moie y in posi ion 5 wi h he aim o e he
addi ional subs i uen s a ge ing his cle (29−37).
Syn hesis o C-Glycoside LecB Inhibi o s. Fo he
syn hesis o he β-C-glycosides 8−28,weob ained he
p ecu so 39 om L- ucose (38) by Hen y addi ion o
ni ome hane wi h in si u ing closu e o he β-anome
ollowed by a educ ion o yield amine 40 (Scheme 1).
37,41,42
Then, di e sifica ion o gi e amide o sul onamide subs i u ed
LecB an agonis s was gene a ed by coupling wi h diffe en
elec ophiles (Scheme 1). The final coupling s ep yielded
amides 8−13 and sul onamides 14−28 in mode a e o good
yields (17−76%, o e 2 s eps).
To p obe he po en ial addi ional binding pocke on LecB
(see Suppo ing In o ma ion, Figu e S1), he b omo hiophene
28 was u he ans o med in palladium-ca alyzed Sonogashi a
c oss coupling eac ions o he subs i u ed alkyne de i a i es
29−36 in accep able yields o 24−74% o hese p o ec ing-
g oup ee syn heses. Z-S y yl 37 was ob ained om he
Sonogashi a p oduc 29 ollowing hyd ogena ion wi h a
Lindla ca alys in 59% yield.
Inhibi ion o LecB Ca bohyd a e Binding Func ion.
All syn hesized s uc u es we e hen analyzed o hei capaci y
o inhibi bo h ep esen a i e lec in a ian s o he wo
clinically ele an bac e ial s ain clades, LecBPAO1 and
LecBPA14, using es ablished compe i i e binding assays
23,36
(Figu e 2).
P e iously, he impac o modifica ions a he cinnamide
subs i uen in 2was ound negligible and a dime hoxy
subs i u ion (43,Figu e 2) only ma ginally inc eased
po ency.
38
Because igidifica ion and ex ension o he
cinnamide o a naph halene ca boxamide was also ole a ed
by he p o ein o he mannose-se ies,
38
we es ed new
de i a i es o C-glycosidic cinnamide 4: benzo hiophene (9),
benzo u an (10), and indole (11). Despi e he in oduc ion o
isos e ic changes in pola i y and hyd ogen bonding p ope ies,
9−11 showed simila o sligh ly dec eased ac i i ies in his
se ies wi h benzo hiophene 9as he bes inhibi o o bo h
LecB a ian s (IC50 4.28 and 2.34 μM, o LecBPAO1 and
LecBPA14, espec i ely). A educ ion in affini y which was
especially p onounced o he PAO1- ype lec in was also
obse ed o ca boxamide-linked hiazole 12. The co espond-
ing hiophene de i a i e 13 was insoluble unde he assay
condi ions.
In analogy o he mannose-se ies, C-glycosidic sul onamide
de i a i es 6and 7showed supe io affini ies o e he amide-
g oup (4,5). While a ela i ely ex ended SAR was desc ibed
o he cinnamides in he mannose se ies,
38
he p e iously
syn hesized mannose-de i ed sul onamides
36
occupy a na ow
Scheme 1. Syn hesis o he Amides 8−13 and Sul onamides 14−38
a
a
Reagen s and condi ions: (a) MeNO2, DBU, molecula sie es 3 Å, 1,4-dioxane, 50 °C, 3 d; (b)P /C, H2, HCl, MeOH, , 2 d; (c) acyl/sul onyl
chlo ide o ca boxylic acid/EDC·HCl, E 3N, DMF, 0 °C; (d) CuI, Pd(PPh3)2Cl2, RCCH, E 3N, DMF, 50 °C, 16−42 h; (e) 1 a m H2, Lindla ’s
ca alys , quinoline, , 46 h. Yields o 8−28 a e gi en o e wo s eps om he ni o de i a i e 39.
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s uc u al di e si y: subs i u ion o he phenyl moie y in 46
wi h me hyl g oups (→3) leads o inc eased affini ies and
isos e ic eplacemen o he phenyl subs i uen by hiophene
(46 →45) imp o ed po ency owa d LecB.
To ex end he SAR o his po en LecB inhibi o class, we
es ed a se o C-glycosidic 5-membe ed he e oa om-
subs i u ed sul onamides wi h addi ional subs i uen s a ying
in cons i u ion and he e oa om posi ion (14−28). Despi e he
a he high s uc u al di e si y o he sul onamide subs i uen s,
all es ed compounds po en ly inhibi ed bo h LecB a ian s in
he low mic o- o nanomola ange (IC50[LecBPAO1] 1.32−7.01
μM; IC50[LecBPA14] 0.20−1.04 μM). While no affec ed by
cons i u ional change o he he e oa om posi ion (7→21,o
19 →20), he affini y d opped sligh ly i he ing sul u was
subs i u ed by oxygen in u an de i a i es (7→14 o 22 →
15) o ni ogen in py azoles (18−20).
Enla gemen o he a oma ic co e by addi ion o alkyl g oups
o halogen subs i uen s in 7gene ally had a beneficial impac
on binding especially in he hiophene se ies wi h low mic o-
o nanomola ac i i ies o 22−25 o bo h LecB a ian s.
Dime hyl 22 and monome hyl 25 (IC50[LecBPAO1] 1.32−1.87
μM; IC50[LecBPA14] 0.20−0.33 μM) we e he bes ligands
among hese hiophenes, bo h p esen ing a me hyl g oup in
o ho-posi ion o he sul onamide linke . A simila phenomen-
on was obse ed o he py azoles, whe e he bes compound
18 (IC50[LecBPAO1] 2.42 μM; IC50[LecBPA14] 0.68 μM) also
had such an o ho-me hyl subs i uen compa ed o he less
ac i e de i a i es 19 and 20 (IC50[LecBPAO1] 5.88−7.01 μM;
IC50[LecBPA14] 0.73−0.96 μM), whose me hyl g oups we e in
diffe en posi ions.
Ex ension o he ini ial and unsubs i u ed hiophene 7 o
add ess he second cle on LecB yielded he se o 10 alkyne
and alkene de i a i es 29−37. Again, all compounds showed
po en inhibi ion o bo h lec in a ian s in he submic omola
ange o he PA14- ype and a 1−3μMaffini y ange o he
PAO1- ype. Phenylace ylene de i a i e 29 s ands ou as mos
po en inhibi o o bo h LecB a ian s (IC50[LecBPAO1] 1.52
μM; IC50[LecBPA14] 0.14 μM). Fu he modifica ions o 29
we e ole a ed by bo h p o eins al hough wi h a mode a e
educ ion in affini y o up o 2- old: eplacemen o he phenyl
wi h o he g oups (34,35) o elonga ed spacing (36) was
possible. The posi ion o a me hyl subs i uen a he phenyl
g oup in o ho-, me a-, o pa a-posi ion (30−32) did no ha e
an influence on ac i i y, and ans o ma ion o he ace ylene
in o Z-alkene 37 was also no al e ing po ency d ama ically.
The he modynamics o binding o he po en 2,5-dime hyl
hiophene de i a i e 22 o bo h LecB a ian s was hen u he
s udied by iso he mal i a ion calo ime y (Figu e 3,
Suppo ing In o ma ion, Table S1). The ligand showed a 1
ligand o 1 LecB monome binding s oichiome y and affini ies
o LecB in he low mic o- o nanomola ange, Kd= 1.2 μM
o PAO1 and Kd= 0.32 μM o PA14, and hus confi med he
ob ained IC50 da a. As obse ed o 7,
41
also his hiophene-
con aining ligand 22 showed an enhanced en halpy d i en
binding (ΔH−47.3 o −40.4 kJ/mol) compa ed o he
ca bocyclic de i a i es 4and 6, which was pa ially
compensa ed by dis a o ed en opic con ibu ions (−TΔS
13.5−3.3 kJ/mol).
S uc u e o LecB in Complex wi h Dime hyl hio-
phene 22. To analyze he impac o he me hyl subs i uen s in
his inhibi o class, we pe o med c ys alliza ion o dime hyl h-
iophene 22 in complex wi h LecBPA14 using hanging d op
coc ys alliza ion. The complex o he lec in c ys allized in he
P61 space g oup wi h ou p o ome s pe asymme ic uni . The
esul ing s uc u e was sol ed o 1.45 Å esolu ion, and all
ca bohyd a e-binding si es we e occupied wi h compound 22
(Figu e 4, Suppo ing In o ma ion, Table S2).
In p e ious s uc u es o LecB wi h sul onamide ligands, we
ha e obse ed wo diffe en o ame s o he sul onamides
esul ing in wo ypes o in e ac ion: a specific in e ac ion
41
wi h he p o ein as desc ibed o 6o 7wi h he cle enclosed
be ween he wo loops Val69−Asp75 and Asp96−Asp104 and
a likely unspecific in e ac ion
36
induced by c ys al con ac s o
he mannose-de i a i e 3. In e es ingly, in he s uc u e o
LecB wi h dime hyl hiophene de i a i e 22, bo h binding
poses can be obse ed (Figu e 4A,B,C). In his espec , one
binding mode o 22 esembles he s uc u e o LecB wi h
hiophene 7in a way ha he hiophene esidue is o ien ed
be ween he same loops o he p o ein wi hou dis u bing
c ys al con ac s (Figu e 4B, D). This suppo s he p e ious
a gumen a ion o he ele ance o his binding mode
compa ed o he second obse ed pose o 22, which is simila
o he epo ed s uc u e
36
o he mannose analogue 3whe e
c ys al packing in e ac ions likely a o ed he al e ed
o ien a ion o he sul onamide subs i uen (Figu e 4A).
Fu he mo e, he c ys al s uc u e o 7in complex wi h
LecBPA14 shows a igh coo dina ion o he hiophene moie y
o he CH2g oup o Se 97 (S-CH2dis ance 4.3 Å, o he
calcula ed S-CH2dis ance 3.3 Å, sum o H and S an de Waals
adii is 3.05 Å). This Se 97 is pa o he loop Asp96−Asp104
adjacen o he ca bohyd a e-binding si e. A highly simila
in e ac ion is seen in he complex o 22 and LecBPA14 (S-CH2
dis ance 3.9 Å, o he calcula ed S-CH2dis ance 3.2 Å, sum o
H and S an de Waals adii is 3.05 Å). The me hyl g oup o
22 in o ho-posi ion o he sul onamide linke en e s deeply
in o his s ill a he shallow pocke and es ablishes nume ous
lipophilic con ac s wi h Gly24, Val69, and he CH2o Asp96.
This ex ensi e in e ac ion pa e n se es as an explana ion o
he beneficial effec on he binding affini y o he o ho-me hyl
g oup.
Coo dina ion o he ca bohyd a e-de i ed ligands by
LecBPAO1 and LecBPA14 is la gely simila .
23
Sub le diffe ences
a e in oduced by he sequence a ia ions be ween hose wo
ype s ains and esul in he obse ed diffe ence in ac i i y o
Figu e 3. Iso he mal i a ion mic ocalo ime y o LecBPAO1 and
LecBPA14 wi h dime hyl hiophene 22. Means and s anda d de ia ions
we e de e mined om a minimum o h ee independen i a ions.
One ep esen a i e i a ion g aph is depic ed o LecBPAO1 only.
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he LecB a ian s. Sul onamides a e gene ally ecognized e y
well, and he PA14 a ian o LecB has a e y high affini y o
he sul onamides, 3−10- old highe han he PAO1 a ian .
In e es ingly, in he cinnamide se ies o he ca boxamides,
hese diffe ences a e anished and some imes selec i i y is e en
in e ed o he benefi o LecBPAO1 (e.g., 20 s 37 μM o 43
and 2.34 s 2.8 μM o 5, espec i ely). An explana ion o his
obse a ion esul s om he p esence o Se 97 in LecBPA14 ha
in oduces u he s e ic bulk compa ed o Gly97 in i s PAO1
ela i e.
The LecB s uc u es e eal his Gly o Se a ia ion o
LecBPA14 a posi ion 97 poin ing in o he ligand binding si e
Figu e 4. C ys al s uc u e o LecBPA14 wi h C-glycoside ligand 22 (1.45 Å esolu ion, PDB 5MAZ), (A) Obse ed binding pose o 22 wi h c ys al
con ac s. (B) Obse ed binding pose o 22 wi hou c ys al con ac s. (C) Supe posi ion o bo h obse ed binding poses. (D) Rele an binding pose
o 22 wi h 2Fobs −Fcalc elec on densi y displayed a 1σ. Ligands and amino acids o he ca bohyd a e ecogni ion domain (CRD) a e depic ed as
s icks colo ed by elemen s (C, g ay; N, blue; O, ed; S, yellow); p o ein su ace in anspa en blue and wo Ca2+-ions in he binding si es a e shown
as g een sphe es.
Figu e 5. (A) C ys al s uc u e o he complex o LecBPA14 wi h dime hyl hiophene 22 (PDB 5MAZ) e eals he hyd ophobic in e ac ion o he
o ho-me hyl g oup a ached o he hiophene esidue wi h a hyd ophobic pa ch on he p o ein su ace. Fu he mo e, he hiophene in e ac s wi h
he side chain CH2o Se 97. (B) C ys al s uc u e o he complex o LecBPAO1 wi h manno-cinnamide 2(PDB 5A3O) e eals he hyd ophobic
in e ac ion o he cinnamoyl g oup wi h a hyd ophobic pa ch o med by he loop ca ying Gly97 ha lacks he se ine side chain p esen in
LecBPA14. (C) supe posi ion o he wo s uc u es indica ing he s e ic clash be ween ca boxamide subs i uen s, con o ma ionally fixed h ough a
hyd ogen bond o hei NH g oup wi h he ca boxyla e o Asp96, and he bulk o he side chain o Se 97 in LecBPA14. This se es as an explana ion
o he inc eased selec i i y o he amides o LecBPAO1, which con as s he selec i i y o he sul onamides o LecBPA14. Elec on densi y 2Fobs −
Fcalc is displayed a 1σ. Ligands a e depic ed as s icks colo ed by elemen s (C, g ay; N, blue; O, ed; S, yellow); p o ein su ace in blue and wo
Ca2+-ions in he binding si es a e shown as g een sphe es.
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(Figu e 5). In case o LecBPAO1, his se ine is absen and a
glycine moie y occupies i s posi ion. The posi ion o he en i e
loop inco po a ing ei he se ine o glycine is no affec ed by
his subs i u ion. Now, he complex o LecBPAO1 wi h he
cinnamide o he mannose se ies (2,Figu e 5B) shows a igh
lipophilic in e ac ion o he cinnamide moie y wi h he CH2
g oup o glycine.
38
Fu he mo e, he cinnamide esidue is
addi ionally con o ma ionally fixed h ough a hyd ogen bond
o he cinnamide NH wi h he side chain o Asp96.
Because he ca boxamides a e con o ma ionally fixed in hei
o ien a ion h ough his hyd ogen bond o he amide-NH wi h
Asp96, hey would expe ience a s e ic epulsion om his
Se 97 in he PA14 a ian o LecB and he e o e possess he
obse ed less p onounced affini y inc ease compa ed o he
sul onamides wi h LecBPA14 (see o e lay in Figu e 5C). The
la e ones a e posi ioned diffe en ly due o he diffe en s e ic
o ien a ion o he linking sul onamide (plana and ans o he
ca boxamides, Figu e 5B, compa ed o h ee-dimensional and
cis o he sul onamides, Figu e 5A). The e o e, he selec i i y
diffe ences o LecBPA14 and LecBPAO1 o he cinnamide se ies
o he ca boxamides depend on he amino acid p esen a
posi ion 97.
Inhibi ion o Bac e ial Biofilm Fo ma ion. The wo
mos p omising compounds selec ed om he compe i i e
binding assay, dime hyl hiophene 22 and Sonogashi a p oduc
29, we e hen es ed in a biofilm assay. We used gene ically
modified P. ae uginosa PA14 cons i u i ely and in acellula y
exp essing he fluo escen p o ein mChe y om he pMP7605
plasmid,
43
ollowed by quan ifica ion o fluo escence using
con ocal ligh scanning mic oscopy (CLSM) as p e iously
desc ibed.
41
The p e equisi e o his in si u imaging assay ha
fluo escence in ensi y di ec ly co ela es wi h cell densi y was
es ablished ea lie .
41
The desi ed absence o bac e icidal o
bac e ios a ic effec s was also confi med o hese wo selec ed
compounds by measu ing o al fluo escence in ensi ies o he
bac e ial cul u es a e g ow h in he p esence o 100 μM
compounds o 23 h. Bac e ia eached compa able densi ies
wi h compounds as he DMSO con ol (Figu e 6).
Fo biofilm inhibi ion expe imen s, bac e ial cul u es we e
g own in he p esence o 100 μM compounds o 48 h when
biofilm mass was quan ified by CLSM (Figu e 7). The wo
sul onamide C-glycosides 22 and 29 showed e y po en
inhibi ion o P. ae uginosa biofilm o ma ion by >80% and
>90%, espec i ely. This s a is ically significan educ ion o
biofilm o ma ion compa ed o he DMSO con ol is
con as ing he nonsignifican effec o he na u al ca bohy-
d a e ligands me hyl α-D-mannoside (1) and me hyl α-L-
ucoside (42), p e iously epo ed (da a o he la e wo
compounds om Somme e al.
41
). This inac i i y o 1and 42
is in con as o hei good biophysical p o ein binding ac i i y
and may esul om con inuous deple ion in he complex
biofilm expe imen whe e bac e ial ac o s could deg ade hese
molecules and/o employ hese na u al glycosides as nu ien s,
whe eas he C-glycosides emain ac i e.
In Vi o Me abolic S abili y and Toxici y. In i o
me abolic s abili y o his compound class was assessed o a
numbe o sul onamide-based LecB inhibi o s in he p esence
o mouse o human li e mic osomes and mu ine plasma
(Table 1,Figu e 8A, Suppo ing In o ma ion, Table S3).
Compounds we e selec ed based on s uc u al di e si y o he
sul onamide subs i uen and included oxazole 17, dime hyl-
hiophene 22, me hyl hiophene 23, dichlo o hiophene 27, and
he Sonogashi a p oduc 29. A low in insic clea ance (CLin )
by mouse and human li e mic osomes o all es ed
compounds wi h alues a 10 μL/min/mg p o ein o below
e ealed a e y high s abili y (Table 1). In mu ine plasma,
hose fi e compounds we e also ully s able o e a pe iod o
120 min wi hou any de ec able deg ada ion (Figu e 8A).
These da a e eal a high deg ee o in i o me abolic s abili y
o his compound class and suppo ou ecen esul s on
sul onamide-linked C-glycosides bea ing ime hylphenyl 6and
Figu e 6. Bac e ial g ow h o mChe y-exp essing P. ae uginosa
quan ified by fluo escence in ensi y (FI) and no malized on he
DMSO con ol in he p esence o 100 μM lec in inhibi o s 22 o 29.
Figu e 7. Inhibi ion o biofilm o ma ion by P. ae uginosa a e 48 h
g ow h in he p esence o compounds 22 o 29. Depic ed da a o
me hyl α-L- ucoside (42), 1, and he DMSO con ol ha e been
published.
41
(A) Quan ifica ion o biofilm biomass. A e ages and
s anda d de ia ions o biofilm o ma ion om h ee independen
assays. S a is ical significance was calcula ed using he S uden ’s es .
(B) Raw da a o con ocal fluo escence mic oscopy 3D images show
one ep esen a i e z-s ack pe condi ion.
Table 1. Me abolic S abili y o Selec ed Sul onamides
agains Mouse and Human Li e Mic osomes
a
CLin
compd mouse human
17 <10 <10
22 <10 <10
23 10 <10
27 <10 <10
29 <10 <10
a
In insic clea ance (CLin )inμL/min/mg.
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hiophene 7 ha also showed a good me abolic s abili y agains
li e mic osomes and mu ine plasma.
41
The fi e hiophenes we e u he s udied, and oxici y o
compounds was assessed in i o using he immo alized
human hepa ocy e cell line Hep G2 (Figu e 8B). Only he
alkyne de i a i e 29 displayed a weak oxici y a he highes
concen a ion es ed esul ing in 64% hepa ocy e iabili y. No
oxici y was de ec ed o all o he compounds es ed up o a
concen a ion o 100 μM, which is consis en wi h p e ious in
i o da a on 6and 7and allows o gene ally classi y his
compound class as non oxic agains Hep G2 cells. Addi ionally,
he sul onamides 6and 7a e non oxic in a mu ine
pha macokine ics model as epo ed.
Binding o plasma p o eins educes he amoun o ee d ug,
which is he necessa y s a e o binding o he d ug’s a ge . On
he con a y, plasma p o ein binding can posi i ely esul in
p olonged plasma hal -li es (Figu e 9). The e o e, balancing
he plasma p o ein binding is necessa y, and we es ed his
p ope y o a se o selec ed LecB- a ge ing sul onamides, he
C-glycosides 6,7,17,22,23, and 29, and he O-glycosides 3
and 45 (which a e he O-linked analogues o he C-glycosides
6and 7). All es ed compounds showed app ox 70−80%
plasma p o ein binding wi h he excep ion o he alkyne 29,
which was highly bound by plasma p o eins. The wo
sul onamides ime hylphenyl 6and hiophenyl 7show a
plasma p o ein binding o 73% and 81%, espec i ely. Bo h
compounds had been analyzed o hei pha makokine ics in
mice, and he compound wi h highe plasma p o ein binding,
7, also showed an inc eased plasma hal -li e o 34 min s 17
min o 6.
41
■CONCLUSIONS
In summa y, wo clinically ele an a ian s o LecB we e
es ed wi h >30 newly syn hesized glycomime ic inhibi o s o
ex end he known s uc u e−ac i i y ela ionship o C-
glycosides. Po en inhibi ion o LecB o bo h s ain ypes
was obse ed, and sul onamides gene ally pe o med be e in
inhibi ion expe imen s han he ca boxamides. In e es ingly,
he affini y diffe ence o he ca boxamides o he wo lec ins
a ian s was a he small and usually only app oxima ely 2- old
be e o LecBPA14. In con as , he sul onamides displayed a
highe affini y inc ease o LecBPA14 o e LecBPAO1, and he
diffe ence was up o 10- old o he Sonogashi a p oduc s 29−
36.
The c ys al s uc u e o he dime hyl hiophene 22 wi h
LecBPA14 explains i s inc eased po ency due o a lipophilic
in e ac ion o he me hyl g oup in o ho-posi ion o he
Figu e 8. (A) S abili y o LecB ligands in mouse plasma. The e o ba s show he s anda d de ia ion o minimum h ee assays. (B) Toxici y o LecB
ligands o human li e Hep G2 cells. Measu ed OD560nm/670nm was no malized o he con ols. Un ea ed cells se ed as nega i e con ol
(no malized OD560nm/670nm = 1), and T i on X-100 ea ed cells se ed as posi i e con ol (no malized OD560nm/670nm = 0). The e o ba s show he
s anda d de ia ion o minimum h ee assays.
Figu e 9. Plasma p o ein binding o LecB inhibi o s C-glycosides 6−
29 and he O-glycosides 3and 45 and nap oxen as a con ol. Box
plo s show mean and confidence in e als.
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sul onamide wi h hyd ophobic p o ein esidues. Fu he mo e,
by compa ison o his LecBPA14/22 s uc u e wi h he
p e iously epo ed s uc u e o cinnamide 2wi h LecBPAO1,
he selec i i y diffe ences o LecBPA14 and LecBPAO1 o he
cinnamide se ies o he ca boxamides could be assigned o he
amino acid p esen a posi ion 97: se ine in LecBPA14 induces a
s e ic clash wi h he cinnamide esidue ha is absen in case o
Gly97 o LecBPAO1.
The wo p omising compounds, dime hyl hiophene 22 and
phenylace ylene bea ing hiophene 29, we e u he es ed in a
biofilm assay. Bo h compounds showed a s ong inhibi ion o
biofilm o ma ion by P. ae uginosa. Fu he compound p ofiling
o ADME and oxici y pa ame e s showed ha bo h
compounds and o he s o his class had highes me abolic
s abili y in mu ine plasma and wi h mouse and human li e
mic osomes. Howe e , he dime hyl hiophene 22 was finally
p io i ized as he on - unne due o a e y high plasma
p o ein binding and mainly he mode a e mammalian
cy o oxici y obse ed a 100 μM o he o he wise e y po en
ex ended s uc u e 29.
In gene al, C-glycosidic sul onamides showed highes
po ency owa d bo h LecB s ain- ypes and hei specific
s uc u e−ac i i y ela ionship was a ionalized by c ys al
s uc u e analyses. Ne e heless, he su ace exposed na u e
o he a he shallow binding si e whe e he sul onamide
subs i uen esides on LecB allows a ce ain deg ee o
modifica ion a he sul onamide subs i uen s. This will be a
beneficial ai o u u e op imiza ion o compounds o
a achmen o o he ca go molecules, e.g., imaging p obes
44
o
an ibio ics o a ge ed deli e y.
■EXPERIMENTAL SECTION
Chemical Syn hesis. Thin laye ch oma og aphy (TLC) was
pe o med on Silica Gel 60 coa ed aluminum shee s con aining
fluo escence indica o (Me ck KGaA, Da ms ad , Ge many) and
de eloped unde UV ligh (254 nm) and aqueous KMnO4solu ion o
a molybda e solu ion (a 0.02 M solu ion o ammonium ce ium sul a e
dihyd a e and ammonium molybda e e ahyd a e in aqueous 10%
H2SO4). P epacked Silica Gel 60 columns om In e chim and a
Teledyne Isco Combiflash R 200 sys em we e used o p epa a i e
medium p essu e liquid ch oma og aphy (MPLC). Nuclea magne ic
esonance (NMR) spec oscopy was pe o med on a B uke A ance
III 500 Ul aShield spec ome e o on a B uke A ance III 400
Ul aShield spec ome e a 500 MHz/400 MHz (1H), 126 MHz/101
MHz (13C), o 376 MHz (19F). Chemical shi s a e gi en in pa s pe
million (ppm) and we e calib a ed on esidual sol en peaks as
in e nal s anda d.
45
Mul iplici ies we e specified as s (single ), d
(double ), ( iple ), q (qua e ), o m (mul iple ). The signals we e
assigned wi h he help o 1H, 1H-COSY, and DEPT-135-edi ed 1H,
13C-HSQC expe imen s. Assignmen numbe ing o he C-glycoside
a oms and g oups co esponds o he numbe ing in ucose. High
esolu ion mass spec a (HRMS) we e ob ained on a B uke maxis 4G
h -QqToF spec ome e , and he da a we e analyzed using Da a-
Analysis (B uke Dal onics, B emen, Ge many). Comme cial
chemicals and sol en s we e used wi hou u he pu ifica ion.
Deu e a ed sol en s we e pu chased om Eu iso op (Saa b ucken,
Ge many). C-Glycoside 39 was syn hesized ollowing he p ocedu e
desc ibed by Phiasi ongsa e al.,
42
and educ ion owa d amine 40 was
desc ibed p e iously.
37
The pu i y o he final compounds was u he
analyzed by HPLC-UV, and all UV ac i e compounds had a pu i y o
a leas 95%. Ch oma og aphic sepa a ion was pe o med on a Dionex
Ul ima e 3000 HPLC (The mo Scien ific, Ge many) wi h UV
de ec ion a 254 nm using a RP-18 column (100/2 Nucleoshell
RP18plus, 2.7 μm, om Mache y Nagel, Ge many) as s a iona y
phase. LCMS g ade dis illed MeCN and double dis illed H2O we e
used as mobile phases. In a g adien un, an ini ial concen a ion o
5% MeCN in H2O was inc eased o 95% du ing 7 min a a flow a e
600 μL/min. The injec ion olume was 10 μL o 1 mM compound in
H2O/DMSO = 100:1.
Gene al P ocedu e o Amide and Sul onamide Couplings.
β-L- ucopy anosyl me hylamine (40) (1 equi ) and ie hylamine (1.5
equi ) we e dissol ed in d y DMF (30 mL pe g am subs a e) and
cooled o 0 °C. The co esponding chlo ide (1.2 equi ) dissol ed in
DMF (0.08 M) was added d opwise unde ni ogen. In he case o
ca boxylic acids, EDC·HCl (1.2 equi ) was added. The eac ion was
allowed o wa m o and was s i ed o u he 1−24 h. Sa u a ed
aqueous NH4Cl was added and ex ac ed wi h E OAc. The combined
o ganic laye s we e d ied o e Na2SO4,fil e ed, and concen a ed in
acuo. Unless o he wise indica ed, he esidue was pu ified by
ch oma og aphy on silica (CH2Cl2 o CH2Cl2/E OH = 10:1 o
CH2Cl2/MeOH = 10:1).
N-β-L-Fucopy anosylme hyl benzamide (8). Compound 8was
ob ained ollowing he gene al p ocedu e om 40 and benzoyl
chlo ide as a colo less solid (70.9 mg, 0.251 mmol, 55%). 1H NMR
(500 MHz, MMeOH-d4)δ7.82−7.81 (m, 2H, CHphenyl), 7.56−7.51
(m, 1H, A CH), 7.78−7.44 (m, 2H, A CH), 3.71 (m, 2H, CH2NH),
3.67−3.64 (m, 1H, H-4), 3.64−3.60 (m, 1H, H-5), 3.50−3.48 (m,
2H, H-1, H-3), 3.35−3.32 (m, 1H, H-2), 1.25 (d, J= 3.61 Hz, CH3).
13C NMR (126 MHz, MeOH-d4)δ170.9 (C = O), 135.9 (A C),
132.7 (A CH), 129.6 (A CH), 128.5 (A CH), 80.0 (C-2), 76.3 (C-
3), 75.8 (C-5), 73.7 (C-4), 69.9 (C-1), 42.6 (CH2), 17.2 (C-6) ppm.
HRMS calcd C14H20NO5+: 282.1336, ound 282.1345.
N-β-L-Fucopy anosylme hyl Benzo[b] hiophene-2- ca boxamide
(9). Compound 9was ob ained ollowing he gene al p ocedu e om
40 and benzo[b] hiophene-2-ca bonyl chlo ide as a yellowish solid
(12 mg, 0.036 mmol, 21%). 1H NMR (500 MHz, MeOH-d4)δ7.99
(s, 1H, A CH), 7.93−7.88 (m, 2H, A CH), 7.46−7.39 (m, 2H,
A CH), 3.77−3.72 (m, 1H, CH2NH), 3.70−3.61 (m, 3H, CH2NH,
H-4, H-5), 3.54−3.46 (m, 2H, H-1, H-3), 3.38−3.33 (m, 1H, H-2),
1.28 (d, J= 6.31 Hz, 3H, CH3). 13C NMR (126 MHz, MeOH-d4)δ
165.4 (CO), 142.6 (A C), 140.9 (A C), 140.0 (A C), 127.6
(A CH), 126.8 (A CH), 126.4 (A CH), 126.1 (A CH), 123.3
(A CH), 80.0 (C-2), 76.4 (C-3), 76.0 (C-5), 73.8 (C-4), 70.0 (C-
1), 42.8 (CH2), 17.3 (CH3). HRMS calcd C16H20NO5S+: 338.1057,
ound 338.1045.
N-β-L-Fucopy anosylme hyl Benzo[b] u an-2-ca boxamide (10).
Compound 10 was ob ained ollowing he gene al p ocedu e om 40
and benzo[b] u an-2-ca bonyl chlo ide as a colo less solid (32 mg,
0.10 mmol, 58%). 1H NMR (500 MHz, MeOH-d4)δ7.74−7.71 (m,
1H, A CH), 7.61−7.59 (m, 1H, A CH), 7.50 (d, J= 0.80 Hz, olefin-
H), 7.48−7.44 (m, 1H, A CH), 7.34−7.30 (m, 1H, A CH), 3.83−
3.79 (m, 1H, CH2NH), 3.67−3.61 (m, 3H, CH2NH, H-4, H-5),
3.50−3.48 (m, 2H, H-1, H-3), 3.37−3.33 (m, 1H, H-2), 1.28 (d, J=
6.31 Hz, 3H, CH3). 13C NMR (126 MHz, MeOH-d4)δ161.7 (C
O), 156.6 (A C), 150.0 (A C), 129.0 (A C), 128.4 (A CH), 125.0
(A CH), 123.9 (A CH), 113.0 (A CH), 111.6 (olefin-CH), 80.0 (C-
2), 76.4 (C-3), 76.0 (C-5), 73.8 (C-4), 70.3 (C-1), 42.2 (CH2) 17.3
(CH3). HRMS calcd C16H20NO6+: 322.1285, ound 322.1300.
N-β-L-Fucopy anosylme hyl 1H-Indole-2-ca boxamide (11).
Compound 11 was ob ained ollowing he gene al p ocedu e om
40,1H-indole-2-ca boxylic acid and EDC·HCl as a colo less solid (28
mg, 0.09 mmol, 31%). 1H NMR (500 MHz, DMSO-d6)δ11.57 (d, J
= 2.1 Hz, 1H, NHindole), 8.41 ( , J= 5.7 Hz, 1H, NHCO), 7.60 (d, J=
8.0 Hz, 1H, A CH), 7.43 (d, J= 8.3 Hz, 1H, A CH), 7.29−7.12 (m,
2H, 2 ×A CH), 7.03 (ddd, J= 8.0, 6.9, 1.0 Hz, 1H, A CH), 4.96 (dd,
J= 4.5, 1.5 Hz, 1H, OH), 4.68 (d, J= 5.2 Hz, 1H, OH), 4.39 (d, J=
4.9 Hz, 1H, OH-4), 3.70 (ddd, J= 13.9, 4.6, 2.4 Hz, 1H, CH2NH),
3.53−3.44 (m, 2H, H-5, H-4), 3.36−3.25 (m, 3H, CH2NH, H-2, H-
3), 3.24−3.18 (m, 1H, H-1), 1.12 (d, J= 6.3 Hz, 3H, H-6). 13C NMR
(126 MHz, DMSO-d6)δ161.5 (C = O), 136.4 (A C), 131.6 (A C),
127.1 (A C), 123.3 (A CH), 121.5 (A CH), 119.7 (A CH), 112.3
(A CH), 102.9 (A CH), 78.7 (C-1), 74.5 (C-2 o C-3), 73.8 (C-5),
71.6 (C-4), 68.7 (C-2 o C-3), 41.2 (CH2NH), 17.2 (C-6). HRMS
calcd C16H21N2O5+: 321.1445, ound 321.1429.
N-β-L-Fucopy anosylme hyl 2-Phenyl-1,3- hiazol-4-ca boxa-
mide (12). Compound 12 was ob ained ollowing he gene al
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