Anti-biofilm Agents against Pseudomonas aeruginosa: A Structure-Activity Relationship Study of C-Glycosidic LecB Inhibitors

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 Downloaded ia HELMHOLTZ CTR FOR INFECTION RSRCH on Oc obe 28, 2019 a 14:19:36 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. 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 Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9202 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 Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9203 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. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9204 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. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9205 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. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9206 (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. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9207 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. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9208 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 Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9209 (34) Be na di, A.; Jimenez-Ba be o, J.; Casna i, A.; De Cas o, C.; Da b e, T.; Fieschi, F.; Finne, J.; Funken, H.; Jaege , K.-E.; Lahmann, M.; Lindho s , T. K.; Ma adi, M.; Messne , P.; Molina o, A.; Mu phy, P. V.; Na i i, C.; Osca son, S.; Penades, S.; Pe i, F.; Pie e s, R. J.; Renaude , O.; Reymond, J.-L.; Richichi, B.; Rojo, J.; Sansone, F.; Scha e , C.; Tu nbull, W. B.; Velasco-To ijos, T.; Vidal, S.; Vincen , S.; Wennekes, T.; Zuilho , H.; Imbe y, A. Mul i alen glycoconju- ga es as an i-pa hogenic agen s. Chem. Soc. Re . 2013,42, 4709−4727. (35) Johansson, E. M. V.; C usz, S. A.; Kolomie s, E.; Bu s, L.; Kadam, R. U.; Caccia ini, M.; Ba els, K.-M.; Diggle, S. P.; Cama a, M.; Williams, P.; Lo is, R.; Na i i, C.; Rosenau, F.; Jaege , K.-E.; Da b e, T.; Reymond, J.-L. Inhibi ion and dispe sion o Pseudomonas ae uginosa bio ilms by glycopep ide dend ime s a ge ing he ucose- speci ic lec in LecB. Chem. Biol. 2008,15, 1249−1257. (36) Hauck, D.; Joachim, I.; F ommeye , B.; Va o , A.; Philipp, B.; Molle , H. M.; Imbe y, A.; Exne , T. E.; Ti z, A. Disco e y o wo classes o po en glycomime ic inhibi o s o Pseudomonas ae uginosa LecB wi h dis inc binding modes. ACS Chem. Biol. 2013,8, 1775− 1784. (37) Somme , R.; Exne , T. E.; Ti z, A. A biophysical s udy wi h ca bohyd a e de i a i es explains he molecula basis o mono- saccha ide selec i i y o he Pseudomonas ae uginosa lec in LecB. PLoS One 2014,9, No. e112822. (38) Somme , R.; Hauck, D.; Va o , A.; Wagne , S.; Aud ay, A.; P es el, A.; Molle , H. M.; Imbe y, A.; Ti z, A. Cinnamide de i a i es o D-mannose as inhibi o s o he bac e ial i ulence ac o LecB om Pseudomonas ae uginosa. Chemis yOpen 2015,4, 756−767. (39) Ho mann, A.; Somme , R.; Hauck, D.; S i el, J.; Go ke - Schne mann, I.; Ti z, A. Syn hesis o mannohep ose de i a i es and hei e alua ion as inhibi o s o he lec in LecB om he oppo unis ic pa hogen Pseudomonas ae uginosa. Ca bohyd . Res. 2015,412,34− 42. (40) Besh , G.; Somme , R.; Hauck, D.; Siebe , D. C. B.; Ho mann, A.; Imbe y, A.; Ti z, A. De elopmen o a compe i i e binding assay o he Bu kholde ia cenocepacia lec in BC2L-A and s uc u e ac i i y ela ionship o na u al and syn he ic inhibi o s. MedChemComm 2016, 7, 519−530. (41) Somme , R.; Wagne , S.; Rox, K.; Va o , A.; Hauck, D.; Wamho , E.-C.; Sch eibe , J.; Ryckmans, T.; B unne , T.; Rademache , C.; Ha mann, R. W.; B ons up, M.; Imbe y, A.; Ti z, A. Glycomime ic, o ally bioa ailable LecB inhibi o s block bio ilm o ma ion o Pseudomonas ae uginosa. J. Am. Chem. Soc. 2018,140, 2537−2545. (42) Phiasi ongsa, P.; Samoshin, V.; G oss, P. Hen y condensa ions wi h 4,6-O-benzylidenyla ed and non-p o ec ed D-glucose and L- ucose ia DBU-ca alysis. Te ahed on Le . 2003,44, 5495−5498. (43) Lagendijk, E. L.; Valido , S.; Lame s, G. E. M.; de Wee , S.; Bloembe g, G. V. Gene ic ools o agging G am-nega i e bac e ia wi h mChe y o isualiza ion in i o and in na u al habi a s, bio ilm and pa hogenici y s udies. FEMS Mic obiol. Le . 2010,305,81−90. (44) Wagne , S.; Hauck, D.; Ho mann, M.; Somme , R.; Joachim, I.; Mulle , R.; Imbe y, A.; Va o , A.; Ti z, A. Co alen lec in inhibi ion and applica ion in bac e ial bio ilm imaging. Angew. Chem., In . Ed. 2017,56, 16559−16564. (45) Go lieb, H. E.; Ko lya , V.; Nudelman, A. NMR Chemical shi s o common labo a o y sol en s as ace impu i ies. J. O g. Chem. 1997,62, 7512−7515. (46) Wilkins, M. R.; Gas eige , E.; Bai och, A.; Sanchez, J. C.; Williams, K. L.; Appel, R. D.; Hochs asse , D. F. P o ein iden i ica ion and analysis ools in he ExPASy se e . Me hods Mol. Biol. 1998,112, 531−552. (47) Kabsch, W. XDS. Ac a C ys allog ., Sec . D: Biol. C ys allog . 2010,66, 125−132. (48) Winn, M. D.; Balla d, C. C.; Cow an, K. D.; Dodson, E. J.; Emsley, P.; E ans, P. R.; Keegan, R. M.; K issinel, E. B.; Leslie, A. G. W.; McCoy, A.; McNicholas, S. J.; Mu shudo , G. N.; Pannu, N. S.; Po e on, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S. O e iew o he CCP4 sui e and cu en de elopmen s. Ac a C ys allog ., Sec . D: Biol. C ys allog . 2011,67, 235−242. (49) McCoy, A. J.; G osse-Kuns le e, R. W.; Adams, P. D.; Winn, M. D.; S o oni, L. C.; Read, R. J. Phase c ys allog aphic so wa e. J. Appl. C ys allog . 2007,40, 658−674. (50) Mu shudo , G. N.; Skubak, P.; Lebede , A. A.; Pannu, N. S.; S eine , R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 o he e inemen o mac omolecula c ys al s uc u es. Ac a C ys allog ., Sec . D: Biol. C ys allog . 2011,67, 355−367. (51) Emsley, P.; Lohkamp, B.; Sco , W. G.; Cow an, K. Fea u es and de elopmen o Coo . Ac a C ys allog ., Sec . D: Biol. C ys allog . 2010,66, 486−501. (52) Schneide , C. A.; Rasband, W. S.; Elicei i, K. W. NIH Image o ImageJ: 25 yea s o image analysis. Na . Me hods 2012,9, 671−675. (53) Heydo n, A.; Nielsen, A. T.; Hen ze , M.; S e nbe g, C.; Gi sko , M.; E sbøll, B. K.; Molin, S. Quan i ica ion o bio ilm s uc u es by he no el compu e p og am COMSTAT. Mic obiology 2000,146, 2395−2407. Jou nal o Medicinal Chemis y A icle DOI: 10.1021/acs.jmedchem.9b01120 J. Med. Chem. 2019, 62, 9201−9216 9216