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

8822 |Chem. Commun., 2020, 56, 8822--8825 This jou nal is ©The Royal Socie y o Chemis y 2020 Ci e his: Chem. Commun., 2020, 56, 8822 A apid syn hesis o low-nanomola di alen LecA inhibi o s in ou linea s eps om D-galac ose pen aace a e† E a Zaho ska, abc Sakonwan Kuhaudomla p, d Sa e io Mine ini, a Sul aan Yousa , a Ma in Lepsik, d Tho s en Kinsinge , a Anna K. H. Hi sch, bce Anne Imbe y d and Alexande Ti z * abc Ch onic in ec ions wi h Pseudomonas ae uginosa a e associa ed wi h he o ma ion o bac e ial bio ilms. The e ame ic P. ae uginosa lec in LecA is a i ulence ac o and an an i-bio ilm d ug a ge . Inc easing he o e all binding affini y by mul i alen p esen a ion o binding epi opes can enhance he weak ca bohyd a e–ligand in e ac ions. Low- nanomola di alen LecA ligands/inhibi o s wi h up o 260- old alency-no malized po ency boos and excellen selec i i y o e human galec in-1 we e syn hesized om D-galac ose pen aace a e and benzaldehyde-based linke s in ou linea s eps. Pseudomonas ae uginosa has been classi ied as a p io i y-1 pa hogen by he Wo ld Heal h O ganiza ion due o i s high an imic obial esis ance and he lack o new d ugs o ea mul id ug- esis an s ains. 1 New s a egies agains hese bac e ial in ec ions a e being explo ed o o e come he cu en an imic obial- esis ance c isis. 2 The so-called an i- i ulence he apy aims o neu alize bac e ial i ulence ac o s ins ead o inc easing he selec ion p essu e imposed by a ge ing essen ial cellula unc ions wi h an ibio ics and he eby ci cum en s he ad en o new esis ances whils p ese ing commensal bac e ia. 3,4 This s a egy is in es iga ed o P. ae uginosa in ec ions by a ge ing i s e a alen lec ins LecA and LecB. 2,5,6 Bo h p o eins a e i ulence ac o s egula ed by quo um sensing, media e bac e ial hos -cell adhesion and a e essen ial s uc u al componen s o P. ae uginosa bio ilms. 7–9 Whe eas he bes L- ucose/D-mannose-based LecB an agonis s bind in he nano- mola ange, mono alen D-galac ose-based LecA inhibi o s only each binding a ini ies in he mid o low mic omola ange. 10–15 In Na u e, he a he weak lec in–ca bohyd a e binding is o en o e come by inc easing alency, and hus enhancing appa en a ini y. 16,17 Likewise, a boos in a ge -binding a ini y was achie ed wi h mul i alen inhibi o s o LecA and LecB. 6,18 Since LecA is a e ame and pai s o binding si es a e geome ically a o ably o ien ed, simul aneously binding di alen inhibi o s can boos binding a ini y h ough a o able binding en opy. 19,20 No ably, Pie e s and co-wo ke s ha e de eloped di alen LecA inhi- bi o s based on complex and igid epea ing uni s o ca bohyd a e- iazole space s wi h po en binding a ini ies anging om 12 o 220 nM, 21–23 while a di alen inhibi o wi h a mo e lexible linke eaches an a ini y o 80 nM. 24 In ano he epo , an oligop oline- spaced digalac oside bound o LecA wi h K d o 71 nM. 25 In his wo k, we aimed o de elop di alen LecA ligands wi h a ocus on d ug-like p ope ies, syn he ic accessibili y and linke simplici y enabling u u e lead op imiza ion. Space leng h and lexibili y a e impo an ac o s con ibu ing o he o e all po ency o mul i alen inhibi o s. 6 An op imized linke connec ing wo neighbo ing binding si es wi hin one LecA e ame and a oiding unwan ed c oss-linking be ween diffe en LecA e ame s is desi ed (Fig. 1a). b-Linked a yl aglycons inc ease he binding s eng h o galac osides o LecA by es ablishing CH–pin e ac ions wi h His50. 15 The co-c ys al s uc u e o LecA wi h phenyl b-D-galac oside (PDB code: 5d21) showed possible g ow h ec o s in me a-and pa a-posi ion a he phenyl aglycon (Fig. 1b). 26 P o ein- empla ed dynamic combina o ial chemis y (DCC) is an elegan me hod o he iden i ica ion o po en ligands om a combina o ial lib a y o building blocks wi h sui able linking chemis y in p esence o a gi en p o ein. 27,28 To apply his me hod o LecA, we in oduced hyd azides a he pa a-o me a-posi ion o phenyl b-D-galac oside in o de o allow o acylhyd azone o ma ion in DCC. Fo his pu pose, we chose wo galac oside building blocks wi h me a-o pa a-a ached hyd azides, 1mand 1p, and a ied linke leng h, igidi y and numbe o o a able bonds by sys ema ically inc easing he numbe o me hylene uni s in he co esponding benzaldehyde a Chemical Biology o Ca bohyd a es, Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land, Helmhol z Cen e o In ec ion Resea ch, 66123 Saa b u ¨cken, Ge many. E-mail: alexande . i z@helmhol z-hzi.de b Deu sches Zen um u ¨ In ek ions o schung (DZIF), S ando Hanno e - B aunschweig, 38124 B aunschweig, Ge many c Depa men o Pha macy, Saa land Uni e si y, 66123 Saa b u ¨cken, Ge many d Uni e si e ´G enoble Alpes, CNRS, CERMAV, 38000 G enoble, F ance e D ug Design and Op imiza ion, Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land, Helmhol z Cen e o In ec ion Resea ch, 66123 Saa b u ¨cken, Ge many †Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: 10.1039/ d0cc03490h Recei ed 15 h May 2020, Accep ed 17 h June 2020 DOI: 10.1039/d0cc03490h sc.li/chemcomm ChemComm COMMUNICATION Open Access A icle. Published on 06 July 2020. Downloaded on 10/27/2020 8:31:51 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue This jou nal is ©The Royal Socie y o Chemis y 2020 Chem. Commun., 2020, 56, 8822--8825 | 8823 space s B–F. The co esponding mono alen con ol Awas included (Fig. 1c). Benzaldehydes Aand Bwe e comme cially a ailable and bis-benzaldehydes C–F we e ob ained in one s ep using 4- hyd oxybenzaldehyde in a double nucleophilic subs i u ion eac ion on alipha ic a,o-di-halogena ed C1–C4 hyd oca bons unde mic owa e i adia ion (Scheme 1). Lewis acid-p omo ed glycosyla- ion o me hyl me a-o pa a-hyd oxybenzoa e wi h b-D-galac ose pen aace a e (2) yielded glycosides 3mand 3pin 69% and 47% yield, espec i ely. Remo al o he ace a es unde Zemple ´ncondi- ions ga e galac osides 4mand 4pquan i a i ely. Subsequen es e hyd azinolysis esul ed in hyd azides 1mand 1pin e y good yields. These building blocks we e hen used in DCC eac ions in p esence o LecA: since he addi ion o LecA o he lib a y caused p ecipi a- ion, all di alen molecules we e indi idually syn hesized in absence o p o ein. Acylhyd azone o ma ion o aldehydes A–F wi h excess hyd azide 1mo 1punde acidic condi ions yielded he mono- and di alen LecA ligands A5m–F5mand A5p–F5p.The educedsolubili y o he me a-se ies compa ed o he pa a-se ies and mo e di icul pu i ica ions could explain he lowe yields despi e nea ly quan i a- i e u no e du ing he indi idual eac ions. All syn hesized galac osides, A5m–F5mand A5p–F5p, we e hen analyzed in he p e iously es ablished compe i i e LecA binding assay based on luo escence pola iza ion (Fig. 2). 11 Mono alen me a-ligand A5m(IC 50 = 21.6 4.5 mM) was wice as po en as i s pa a-isome A5p(IC 50 = 55.5 4.4 mM). The di alen ligands B5m–F5mand B5p–F5pshowed a e y simila p o ile in he compe i i e binding assay wi h e y simila IC 50 alues in he single-digi mic omola ange and a e y s eep Hill slope o he i . These obse a ions a e likely a esul o each- ing he lowe assay limi since he low a ini y o he luo escen p ima y ligand (K d = 7.4 mM) equi ed a ela i ely high LecA concen a ion o 20 mM. The e o e, ligand a ini ies wi h o de s-o -magni ude highe po encies han he p ima y compe- i i ely displaced ligand canno be eliably de e mined. To o e come he compe i i e binding assay’s limi a ion, we analyzed all inhibi o s in a di ec LecA binding expe imen using su ace plasmon esonance (SPR). In case o he mono alen inhibi o s A5mand A5p, apid changes in he binding esponse du ing he associa ion and dissocia ion phases we e obse ed, indica ing as associa ion/dissocia ion kine ics o he mono alen inhibi o s o immobilized LecA (Fig. 3a and b). Due o his as associa ion/dissocia ion beha io , k on and k off o hei in e ac ion wi h LecA could no be accu a ely de e mined and affini y analysis was pe o med ins ead o de e mining K d a s eady-s a e binding. In SPR, he mono alen ligands A5mand A5pha e K d alues o 4.9  0.1 and 5.6 0.3 mM, espec i ely (Table 1). The binding affini ies o hose mono alen compounds we e alida ed using iso he mal i a ion mic ocalo ime y (ITC) as an o hogonal me hod and simila alues o SPR da a we e ob ained (ITC: A5mK d =2.7 1.3 mM, A5pK d =6.10.5 mM, Table 1 and Fig. S1, ESI†). In bo h analyses, a me a-subs i u ion o he phenyl aglycon in A5m esul ed in highe affini ies o LecA compa ed o he pa a-isome A5p. Fig. 1 Design o di alen LecA inhibi o s accessible h ough a sho syn he ic ou e wi h acylhyd azone coupling chemis y. (a) Possible binding modes o di alen LecA inhibi o s wi h desi ed linkage o wo adjacen binding si es. (b) The c ys al s uc u e o phenyl b-D-galac oside in complex wi h LecA (pdb code: 5d21) and dis ances be ween wo ligands wi hin one pai o binding si es in LecA ( om me a o me a:23Å, ompa a o pa a: 25 Å). (c) Building blocks o LecA inhibi o s: m/p-hyd azineca bonylphenyl b-D-galac opy anoside (1m,1p)and benzaldehydes A–F. Bis-benzaldehyde linke s B–F wi h sys ema ic a ia ion o leng h and numbe o o a able bonds o op imize dis ance and lexibili y. Scheme 1 Syn hesis o di alen LecA ligands and hei mono alen ana- logs. Reagen s and condi ions: (i) 4-hyd oxy benzaldehyde, K 2 CO 3 , DMF, 70 1C, mic owa e, 3–10 h; (ii) me hyl m/p-hyd oxybenzoa e, BF 3 E 2 O, CH 2 Cl 2 ,01C – . ., o.n.; (iii) NaOMe, MeOH, . ., o.n.; (i ) NH 2 NH 2 H 2 O, MeOH, 70 1C, o.n. ( ) o mic acid, DMSO, . ., 4 h, o D5m: DMSO/MeCN, o F5m:H 2 O/MeCN. Fig. 2 E alua ion in a compe i i e binding assay. One ep esen a i e i a ion is shown o each se ies ( igh ) – s eep i a ion slopes o di alen inhibi o s indica e helowe assaylimi was eached.A e .ands d.de . oma leas 3 independen i a ions o iplica eseach.n.d.=no de e mined. Communica ion ChemComm Open Access A icle. Published on 06 July 2020. Downloaded on 10/27/2020 8:31:51 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online 8824 |Chem. Commun., 2020, 56, 8822--8825 This jou nal is ©The Royal Socie y o Chemis y 2020 In con as o he mono alen hyd azides, he SPR senso - g ams o he di alen inhibi o s clea ly indica ed much slowe associa ion/dissocia ion o he compounds om LecA, demon- s a ing he bene i o di alen binding and enabling de e mi- na ion o kine ic pa ame e s (k on and k off )aswellasK d (calcula ed om k off /k on ,Fig.3c,dandFig.S2,ESI†). The di alen inhibi o s B5m–F5mand B5p–F5pshowed a s ong inc ease in po ency in o he low nanomola ange (Table 1). In he pa a-se ies, compound B5pwas he mos po en ligand (K d =10.81.0 nM), wi h a 520- old inc ease (260- old alency-co ec ed) compa ed o i s mono- alen congene A5p(K d =5600300 nM). The di alen pa a- ligands showed a sligh dec ease in po ency wi h inc easing space leng h, esul ing om g adually dec easing k on and inc easing k o (Table 1). In he me a-se ies, simila ends we e absen . C5mwi h one cen al me hylene uni had he op imal leng h and showed a K d o 18.9 1.6 nM. Sho ening o inc easing space leng h esul ed in educed a ini ies and su p isingly he second longes compound E5mwas he leas e icien inhibi o (K d o 80.7  11.4 nM). Compa ing ac oss he me a-se ies, he k on alues we e su p isingly 2–4 imes smalle o compounds C5mand E5m.The k o alues we e g adually inc easing going om C5m–F5m.Thek o o B5mwas 5- imes highe han ha o C5m. We hen s udied compound selec i i y in binding expe i- men s owa ds human galec in-1, a homodime ic lec in ha speci ically ecognizes b-galac oside con aining glycans such as Me-b-lac oside (K d = 187 mM). 29 The mos po en inhibi o s om he pa a- and me a-se ies, B5pand C5m, oge he wi h hei espec i e mono alen coun e pa s (A5pand A5m), we e analyzed by SPR o hei in e ac ion wi h human galec in-1. Nei he he mono alen (a 250 mM) no he di alen com- pounds (a 25 mM) had a de ec able in e ac ion wi h he immobilized galec in-1 (Fig. S3, ESI†). Since we did no succeed in ob aining c ys al s uc u es o LecA complexed wi h he di alen inhibi o s, we ca ied ou modeling on pai s o pa a-andme a-compounds, selec ing he high-affini y binde C5mand i s pa a-coun e pa C5p;and helonge ,less ac i e E5mand E5p. The dynamics o hese ou compounds we e simula ed in he ee s a e and in he modeled complex wi h LecA. In he ee s a e, he ligands adop ed a b oad ange o semi- ex ended o ully ex ended con o ma ions cha ac e ized by Gal:C1Gal:C1 dis ances anging om 20 o 31 Å a a cu off o 5% equency (Fig. 4a, solid cu es). A dis ance close o he c ys allog aphic alue o 29 Å (PDB: 5d21) is desi ed o op imal di alen binding o LecA. In he longe compounds, E5mand E5p, he e was a small popula ion (a ound 5%) o olded con o ma ions (see he peaks a 5 Å in Fig. 4a). The un olding o hese con o ma- ions p io o LecA binding may be in pa esponsible o hei slowe on- a e (k on ). The ou ligands modeled in complex wi h LecA showed a na ow dis ibu ion o he Gal:C1Gal:C1 dis ances (Fig. 4a, dashed lines). C5m,C5pand E5psampled dis ances o 28.9  0.4 Å, 28.1 0.9 Å, and 28.2 0.7 Å, espec i ely, close o he 29 Å obse ed in c ys al s uc u es, hus indica ing ha he linke leng hs a e well-sui ed o b idge wo LecA monome s. The na ow ange o C5m(28–31 Å) may explain he lowe a ini y o sho e B5m. In con as , C5pand E5pha e la ge ange (26–31 Å) and sho ening is bene icial such as in B5p.E5mwi h LecA displayed a highe mean dis ance o 30.2 0.8 Å ( ange o 28 o 33 Å). Such longe me a linke pushes he wo LecA monome s sligh ly apa , which is no a ou able. The T-shaped CH–pin e ac ion be ween His50 and he phenyl aglycon was obse ed wi h highe equency o he me a compounds C5mand E5mcompa ed o hei pa a analogues C5pand E5p(Fig. 4b). On he opposi e, he pa a ligands, C5pand E5p, p e e en ially adop ed an in e ed V-shape (Fig. 4c and d) in which hei phenyl aglycons sampled a a ie y o a angemen s wi h espec o His50 (pa allel, diagonal, T-shape). To conclude, we designed and syn hesized highly po en di alen LecA inhibi o s in ou linea chemical s eps om Fig. 3 SPR analyses o he in e ac ion be ween he mono alen inhibi o s (a) A5mand (b) A5p. The senso g ams a e shown on he le panel and he affini y analyses on he igh . (c) Senso g ams o he mos po en di alen inhibi o s om me a-se ies (C5m) and (d) om pa a-se ies (B5p) a i e diffe en concen a ions (0, 10, 50, 100, 200 nM). Table 1 Affini y and kine ic analyses o he LecA-inhibi o in e ac ions de e mined by SPR and ITC a me a se ies pa a se ies k on (10 3 M 1 s 1 )k off (10 3 s 1 )K d (nM) .p. k on (10 3 M 1 s 1 )k off (10 3 s 1 )K d (nM) .p. A5m— — 4900 100 1 A5p— — 5600 300 1 — — 2700 1300 b — — — 6100 500 b — B5m196 6 5.34 0.28 27.3 1.6 90 B5p152 3 1.64 0.13 10.8 1.0 259 C5m59 3 1.11 0.08 18.9 1.6 130 C5p114 2 2.33 0.02 20.5 0.2 137 D5m120 41 2.67 0.38 23.8 7.1 103 D5p121 9 2.57 0.20 21.4 1.0 131 E5m45 4 3.64 0.35 80.7 11.4 30 E5p103 2 2.19 0.45 22.5 9.0 124 F5m104 53 3.39 1.5 33.4 2.3 73 F5p79 5 3.98 0.16 50.1 1.2 56 a A e ages and s d. de . om h ee independen expe imen s. Rela i e po encies ( .p.) we e calcula ed compa ed o mono alen compound in each se ies (A5mand A5p) and alency-no malized. b ITC de e mina ion. ChemComm Communica ion Open Access A icle. Published on 06 July 2020. Downloaded on 10/27/2020 8:31:51 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online This jou nal is ©The Royal Socie y o Chemis y 2020 Chem. Commun., 2020, 56, 8822--8825 | 8825 galac ose pen aace a e. These simple and apidly accessible di alen inhibi o s B5m–F5mand B5p–F5pha e compa able o supe io ac i i y o he p e iously epo ed and s uc u ally complex di- and mul i alen LecA ligands. Mono alen analogs A5mand A5pshowed binding o LecA in SPR and ITC expe i- men s in he low mic omola ange (K d = 2.7–6.1 mM). Di alen display o hese epi opes in B5m–F5mand B5p–F5pboos ed binding a ini y wi h LecA o low nanomola alues. Molecula dynamics simula ions ga e insigh s in o he in e play o linke geome y and leng h o an op imal di alen binding. To he bes o ou knowledge, compound B5pwi h a K d o 10.8 nM is he mos po en di alen LecA ligand epo ed o da e wi h con i med selec i i y o LecA o e galec in-1. Due o he simplici y o ou syn he ic design and eadily accessible build- ing blocks, u he ine uning and op imiza ion o d ug-like p ope ies can be eadily implemen ed. Fu u e op imiza ion o hese compounds a ge ing LecA may p o ide a ea men o bio ilm-associa ed P. ae uginosa in ec ions. The au ho s a e g a e ul o Di k Hauck (HIPS) o excellen echnical assis ance and Va sha R. Jumde (HIPS) o assis ing wi h DCC. M. L. acknowledges a EU H2020 Ma ie Sklodowska-Cu ie g an (795605). Compu a ions we e un on GRICAD in as uc u e, HPC-EUROPA3 p ojec (H2020-INFRAIA-2016-1-730897), and EPCC a he Uni e si y o Edinbu gh, Sco land, and HPC esou ces om GENCI-IDRIS (G an 2019-A0070711040). A. T. hanks he Eu opean Resea ch Council (ERC S a ing G an , Swee bulle s) and Deu sche Fo schungsgemeinscha (Ti756/5-1). S. Y. acknowl- edges a RISE ellowship, Ge man Academic Exchange Se ice DAAD. The wo k was suppo ed by he ANR/DFG F ench- Ge man GLYCOMIME p ojec (ANR-AAPG-2017, DFG Ti756/5-1). A. I. and S. K. acknowledge suppo om Glyco@Alps (ANR-15- IDEX02), Labex A cane/CBH-EUR-GS (ANR-17-EURE-0003). A. K. H. H. g a e ully acknowledges he ERC S a ing G an (757913). Con lic s o in e es The e a e no con lic s o decla e. No es and e e ences 1 WHO, h ps://www.who.in /news- oom/de ail/27-02-2017-who-publishes- lis -o -bac e ia- o -which-new-an ibio ics-a e-u gen ly-needed, accessed 9 Ap il 2020. 2 S. Wagne , R. Somme , S. Hinsbe ge , C. Lu, R. W. Ha mann, M. Emp ing and A. Ti z, J. Med. Chem., 2016, 59, 5929–5969. 3 A. E. Cla wo hy, E. Pie son and D. T. Hung, Na . Chem. Biol., 2007, 3, 541–548. 4 M. B. Cal e , V. R. Jumde and A. Ti z, Beils ein J. O g. Chem., 2018, 14, 2607–2617. 5 J. 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J. Nilsson, ChemBioChem, 2007, 8, 1389–1398. Fig. 4 Molecula dynamics simula ions o C5m,E5m;C5p;E5p: (a) dis ibu ion o Gal:C1Gal:C1 dis ances o compounds in he ee s a e (solid lines) and in complex wi h LecA (dashed lines). (b) Dis ibu ion o dis ances be ween His50 and he phenyl aglycon o ligands in complex wi h LecA (solid lines o LecA p o ein chain A, dashed lines o chain B). (c– ) Snapsho om ajec o ies o C5p(c), E5p(d), C5m(e), and wo snapsho s o E5m( ) indica ing he con o ma ional change o LecA. Communica ion ChemComm Open Access A icle. Published on 06 July 2020. Downloaded on 10/27/2020 8:31:51 AM. This a icle is licensed unde a C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence. View A icle Online