# The Parkinson's disease drug Tolcapone and analogues are potent glycomimetic lectin inhibitor of Pseudomonas aeruginosa LecA

Steffen Leusmann, Eike Siebs, Sakonwan Kuhaudomlarp, Annabelle Varrot, Anne Imberty, Bernd Kuhn, Christian Lerner, Uwe Grether, Alexander Titz

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Steffen Leusmann, Eike Siebs, Sakonwan Kuhaudomlarp, Annabelle Varrot, Anne Imberty, et al. The Parkinson's disease drug Tolcapone and analogues are potent glycomimetic lectin inhibitor of Pseudomonas aeruginosa LecA. Angewandte Chemie International Edition, 2025, pp.e202508864. 10.1002/anie.202508864. hal-05345236

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# The Parkinson's disease drug Tolcapone and analogues are potent glycomimetic lectin inhibitor of *Pseudomonas aeruginosa* LecA

Steffen Leusmann<sup>#1,2,3</sup>, Eike Siebs<sup>#1,2,3</sup>, Sakonwan Kuhaudomlarp<sup>4,5</sup>, Annabelle Varrot<sup>4</sup>, Anne Imberty<sup>4</sup>, Bernd Kuhn<sup>6</sup>, Christian Lerner<sup>6</sup>, Uwe Grether<sup>6</sup>, Alexander Titz<sup>1,2,3</sup>

keywords: drug repurposing, glycomimetics, catechols, lectins, inhibitors

## Abstract

The notorious pathogen *Pseudomonas aeruginosa* relies on the lectin LecA for host cell adhesion, invasion, and biofilm formation. Motivated by the pressing need for new antiinfective therapies caused by antimicrobial resistance, inhibitors of LecA are under investigation. Complementary to the use of carbohydrate-based inhibitors, we have previously identified catechols as weak but specific ligands of LecA, constituting a novel class of non-carbohydrate glycomimetics. Here, we present the catechol Tolcapone and derivatives as a new class of potent glycomimetics. By growing the initial millimolar fragment hits, we identified Tolcapone as a promising compound. To gain insight into the structure-activity relationship (SAR) of catechols as LecA binders more than 3,200 compounds of the Roche in-house library

<footnote n="1"><sup>1</sup> Chemical Biology of Carbohydrates (CBCH), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research, 66123 Saarbrücken, Germany</footnote>

<footnote n="2"><sup>2</sup> Department of Chemistry, PharmaScienceHub (PSH), Saarland University, 66123 Saarbrücken, Germany</footnote>

<footnote n="3"><sup>3</sup> Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig, 38124 Braunschweig, Germany</footnote>

<footnote n="4"><sup>4</sup> Univ. Grenoble Alpes, CNRS, CERMAV, 38000 Grenoble, France</footnote>

<footnote n="5"><sup>5</sup> Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand</footnote>

<footnote n="6"><sup>6</sup> Pharmaceutical Research and Early Development (pRED), Roche Innovation Center Basel, F. Hoffmann-La Roche AG, 4070 Basel, Switzerland</footnote>

<footnote><sup>#</sup> contributed equally</footnote>
were experimentally screened in a competitive binding assay at three concentrations. Of these, 48 compounds were chosen for further investigation, resulting in compounds equipotent to aryl galactosides, the current epithome of LecA inhibition. X-ray crystallography and saturation transfer difference NMR spectroscopy revealed conserved interactions of the catechol moiety in the glycan binding site of LecA and rationalized the observed SAR. Our findings demonstrate that it is possible to develop potent non-carbohydrate glycomimetic lectin inhibitors, paving the way for a new avenue of research that could yield innovative anti-infective drugs.

## Introduction

The antibiotic resistance crisis necessitates the development of new drugs and therapy regimes to fight the increasing threat of bacterial infections.[1] In 2019, antimicrobial resistance was associated with 5 million deaths globally.[2] This number is projected to increase to over 8 million deaths per year by 2050, coinciding with an annual loss of >45 million life years due to antimicrobial resistance.[3] To guide the development of novel antimicrobial drugs the World Health Organization classified carbapenem-resistant *Pseudomonas aeruginosa* as a pathogen of high concern.[4] The opportunistic pathogen *P. aeruginosa* especially threatens predisposed individuals, e.g., patients in intensive care units or those suffering from cystic fibrosis. Therapy of the resulting lung, wound, urinary tract or bloodstream infections is impeded by high rates of intrinsic and acquired antibiotic resistances.[5]

One innovative approach attracting growing attention is the development of antivirulence drugs, also called pathoblockers.[6] In contrast to antibiotics, these molecules do not affect bacterial survival fitness but merely aim to disarm the pathogens which may also reduce the risk for resistance development. The inhibition of bacterial adhesion, host cell invasion and biofilm formation, key mechanisms of infection and chronicity, is a prime target of the antivirulence approach.

During the infection cycle *P. aeruginosa* utilises one of its lectins, LecA, to bind to the mammalian glycolipid globotriaosylceramide (Gb3, CD77) to facilitate host cell adhesion and subsequent invasion.[7] In addition to host cell invasion, LecA is of crucial importance for biofilm formation as evidenced by deletion experiments.[8] Biofilms are a mechanism of adaptive resistance and act as diffusion barrier for antibacterial agents and the immune system.[5] At the same time, the biofilm also reduces access to nutrients resulting in metabolically less active bacteria, which are less prone to the activity of most antibiotics that
generally target active bacterial metabolism.[9] Biofilms consist of bacteria embedded in a
complex hydrogel of extracellular nucleic acids, proteins, lipids and exopolysaccharides.[10]
Soluble lectins such as LecA provide rigidity to the biofilm by crosslinking glycans of bacteria,
host cells and matrix components.[11]

The significant role of LecA for pathogenicity and the potential of LecA inhibition was
demonstrated in murine *in vivo* infection experiments.[12,13] The administration of LecA
inhibitors not only reduced lung damage and inflammation as well as bacterial dissemination,
but also showed synergistic effects with common antibiotics. Notably, these findings aligned
closely with clinical observations in patients suffering from severe *P. aeruginosa* lung
infections. Here, the inhalation of carbohydrate-containing aerosols decreased bacterial
colonization and infection severity.[14,15] Consequently, targeting LecA is considered a
promising strategy to break antimicrobial resistance in *Pseudomonas* infections.[11,16]

LecA is a homotetrameric lectin with affinity for D-galactosides.[17–19] Importantly,
binding to LecA is highly dependent on the complexation of a single calcium–ion present in
the carbohydrate binding site. The design of LecA inhibitors focussed on the derivatisation of
D-galactose present in its natural carbohydrate ligands such as Gb3. Addition of a β-linked
aromatic aglycon lowered the K_D value from 87 µM for D-galactose to 14 µM in para-
nitrophenyl β-D-galactoside (*pNPGal*).[20] Recently, Bruneau et al. reported on a benzophenone
modified galactoside reaching an affinity of 1 µM.[21] The improved potency of such aryl β-D-
galactosides has been attributed to CH–π interactions with His50 in close proximity to the
carbohydrate-binding site.[20] Furthermore, the multivalent presentation of binding epitopes is
a common strategy to improve the affinity of lectin ligands.[22] While nanomolar affinities can
be reached, the large size of multivalent glycosides prevents oral bioavailability and could lead
to immunogenicity. Furthermore, in the case of LecA inhibitors, multivalent binding could lead
to an undesired increase in biofilm cohesion and bacterial aggregation by undesired
crosslinking LecA tetramers with bacteria and host cells. Additionally, selectivity is a concern
due to the presence of numerous host lectins recognising the same carbohydrate epitope, e.g.,
the galectin family.[16] A notable exception to large multivalent ligands are divalent precision
ligands bridging the two neighbouring sites in one homotetramer of LecA,[23–26] which results
in nanomolar binders with promising antivirulence efficacy in diverse functional *in vitro*
assays.[27–29]

Following a different rationale to search for non-carbohydrate lectin inhibitors, we
recently reported on catechols as orthosteric, non-carbohydrate binders of calcium-dependent
lectins such as LecA.[30] Catechols are generally considered to be pan-assay interference
compounds (PAINS).[31] Nevertheless, a vigorous cascade of screening and hit validation confirmed electron-deficient catechols as LecA binders with a clear binding mode without unspecific reactivity. Due to their more druglike characteristics, molecules of this compound class likely possess favourable pharmacokinetic properties. Here, we report on our efforts to increase the affinity of the known weakly binding catechol-containing fragments towards LecA in order to establish a new class of potent glycomimetics.

# Results and Discussion

## Identification and Evaluation of Tolcapone as LecA inhibitor

In our previous work[30] on catechols as novel LecA binders two observations caught our attention: first, the affinity to LecA seemed to improve with increasing electron-deficiency of the catechol as evidenced by cyano and nitro derivatives; second, a benzophenone motif greatly benefitted the binding affinity. Therefore, we focussed our attention on commercially available nitro-catechols. Prominent derivatives are inhibitors of catechol-O-methyltransferase (COMT) and approved drugs for the treatment of Parkinson's disease. Four commercial COMT inhibitors were thus evaluated in a competitive binding assay based on fluorescence polarisation (FP) as previously reported (Table 1, Figure 1).[32]

While Nitecapone and Entacapone carry acyclic vinyl residues, Opicapone and Tolcapone are substituted with (hetero-)aromatic systems. Importantly, Tolcapone combines the strongly electron-withdrawing nitro group with a benzophenone motif. Interestingly, structurally similar Nitecapone and Entacapone were inactive at the highest tested concentration of 1.5 mM despite carrying the nitro-catechol pharmacophore. Opicapone showed an IC₅₀ value of 842 ± 134 µM, which is a significant improvement compared to the initial millimolar screening hits. Finally, Tolcapone showed the highest inhibition of LecA with an IC₅₀ value of 257 ± 40 µM.
<table><caption>**Table 1:** Approved COMT inhibitors were evaluated for their LecA inhibition in a competitive binding assay up to 1.5 mM. Averages and standard deviation from n ≥ 2 experiments are given.</caption><thead><tr><th>ID</th><th>Name</th><th>Structure</th><th>IC<sub>50</sub> [µM]</th></tr></thead><tbody><tr><td>1</td><td>Nitecapone</td><td><img src="" alt="Chemical structure of nitecapone"></td><td>no inhibition</td></tr><tr><td>2</td><td>Entacapone</td><td><img src="" alt="Chemical structure of entacapone"></td><td>no inhibition</td></tr><tr><td>3</td><td>Opicapone</td><td><img src="" alt="Chemical structure of opicapone"></td><td>842 ± 134</td></tr><tr><td>4</td><td>Tolcapone</td><td><img src="" alt="Chemical structure of tolcapone"></td><td>257 ± 40</td></tr></tbody></table>

To confirm the affinity of Tolcapone towards LecA in an orthogonal assay, the compound was then evaluated in a direct binding experiment by surface plasmon resonance (SPR). A K<sub>d</sub> of 181 ± 6 µM was determined which is in good agreement with the IC<sub>50</sub> of 257 ± 40 µM from the FP assay (Figure 1). Tolcapone significantly surpasses the affinity of our previous hits 4-nitrocatechol (K<sub>D</sub> = 560 ± 340 µM) and 3,4-dihydroxybenzophenone (K<sub>D</sub> = 3460 ± 410 µM),[30] which underlines the hypothesis of beneficial contributions of a nitro group and the benzophenone motif.
<figure><img src="image_2.png" /></figure>

**Fig. 1: (A)** The inhibition of LecA by COMT inhibitors was analysed in a competitive binding assay based on fluorescence polarisation (one representative dataset shown, error bars from technical triplicates). **(B)** SPR sensorgram and steady state analysis of binding of Tolcapone to LecA

*Binding mode of Tolcapone in complex with LecA by X-ray crystallography and STD NMR*

To rationalise the high affinity of Tolcapone to LecA we conducted X-ray crystallography studies. Crystals of LecA in complex with Tolcapone were obtained and solved at a resolution of 1.32 Å (pdb code 8GUV). In this structure, electron density could be unambiguously assigned to Tolcapone present in the carbohydrate binding sites of LecA. Interestingly, crystal contacts between protomers of neighbouring asymmetric units led to two different binding poses of Tolcapone (Figure 2). In addition, a third Tolcapone molecule is stacked between the two protein-bound ligands (Figure 2-A). A comparison with the co-crystal structure of LecA and 3-cyanocatechol (pdb code 6YO3) revealed that the catechol motif is always bound in a conserved manner with the phenyl ring and hydroxy groups occupying the same space independent of the binding pose of Tolcapone (Figure S2-A). Importantly, in both LecA-bound poses Tolcapone mimics the key calcium complexation and hydrogen bonding interactions to Asp100 and Asn107 established by OH3 and OH4 of the native ligand galactose (Figure S2-B).[19] Additionally, the backbone carbonyl and side chain of Thr104 as well as the backbone carbonyl of Tyr36 are located within hydrogen bonding distance (< 3.5 Å) of the catechol. With a pK<sub>a</sub> value of ~ 4.6 one of the hydroxy groups of Tolcapone is likely deprotonated in these interactions. While the pK<sub>a</sub> of one of the hydroxy groups in Tolcapone is ~ 4.6, a conclusion on the protonation state in the crystal cannot be drawn.

Notably, Tolcapone is horizontally flipped by 180° between the two observed poses. While the ketone is exposed to the solvent in one orientation (Figure 2-B), it points to the protein surface in the other (Figure 2-C).
<figure><img src="image_3.png" /><figcaption>**Fig. 2** The crystal structure of LecA complexed with Tolcapone (pdb code 8GUV). (A) Two different binding modes of Tolcapone were observed for carbohydrate binding sites of neighbouring asymmetric units (pink and blue). A third Tolcapone molecule is stacked between the two protein-bound ligands (green). (B) Binding pose I: the nitro group engages in water-mediated hydrogen bonding to the protein backbone and the ketone points away from the protein. (C) Binding pose II: the nitro group forms a hydrogen bond with Asn107 and the carbonyl group points towards the protein. Dashed lines indicated hydrogen bonding distance or metal complexation.</figcaption></figure>

In binding pose I, Tolcapone's nitro group extends into a pocket usually occupied by OH6 of galactose where the same water-mediated interaction to the protein backbone of Gln53 and Pro51 is formed (Figure 2-B). The same interaction was observed in the complex of LecA with 3-cyanocatechol (pdb code 6YO3).[30] While OH6 of galactose engages in two more hydrogen bonds with His50 and Gln53, these interactions were not observed for the nitro group of Tolcapone, in part because the side chain of Gln53 was not resolved. The carbonyl group of
Tolcapone points away from the protein towards the solvent with the *p*-tolyl ring likely engaging in weak hydrophobic interactions with the protein surface. The CH–π interaction with His50 which is the rationale for the high affinity of aryl galactosides is absent (distance ~7 Å).

In contrast, the carbonyl group of Tolcapone points towards the protein in binding pose II (Figure 2-C). Notably, the water molecule allowing the indirect hydrogen bonding to the protein backbone from pose I was not observed in this orientation. However, a superposition of both poses of Tolcapone suggests the ketone is in a suitable position to form this interaction in case the water molecule is present (Figure S2-A, cf. Fig. 6). Additionally, the ketone is located within 3.3 Å of the Nτ of His50. Due to an intramolecular hydrogen bond of His50 Nπ to the main chain carbonyl of Pro51, a protonated state of His50 is required for an additional interaction with Tolcapone. The flipped orientation furthermore allows hydrogen bond formation between the nitro group and Asn107. The pocket of LecA that is usually occupied by galactose-OH6 remains unoccupied. A rigid body movement of the loop around Gln53 is observed (up to 2.4 Å for Gln53-Cα), potentially caused by a steric effect of the tolyl residue or the crystal artifact of the third Tolcapone molecule. An overlay of the binding poses highlighting these changes is shown in Figure S3.

In the solid state, two different binding poses for Tolcapone were observed. To shed light on the situation in solution, we conducted saturation transfer difference (STD) NMR spectroscopy with LecA (Figure 3). Assignment of the resonances of Tolcapone was carried out with ¹H,¹³C-HMBC spectra and a titration with NaOD (Figure S4 – S5). In the STD-NMR spectrum, the strongest transfer of saturation was observed to the proton at position 4 of Tolcapone (assigned to 100% STD effect). Additionally, the difference spectrum shows the second strongest signal for the protons at position 3 (36%). Considering that this position contains two chemically equivalent protons, it is likely one proton receives higher saturation than the other one. These results are in agreement with binding pose II, where the proton at position 5 is further away from the protein and thus receives less saturation transfer. Taken together, pose II was identified as dominant orientation in solution. Binding of Tolcapone to LecA is therefore driven by the central interactions of the catechol moiety with the protein-bound calcium ion, additional hydrogen bonding established by the nitro group and a possible interaction of the carbonyl group with the protein. Furthermore, the tolyl residue presumably engages in hydrophobic interactions as seen by saturation transfer to its protons. Assuming a similar binding mode for the nitro-catechol, the vinyl-containing drugs Nitecapone and Entacapone lack a hydrogen bond acceptor in the same position as the ketone in Tolcapone.
Furthermore, possible clashes by their branched substituents with the protein surface could serve as an explanation for the lack of binding to LecA (Table 1).

<figure><img src="image_4.png" /><figcaption>**Fig. 3** Saturation transfer difference (STD) NMR spectroscopy of Tolcapone in presence of LecA. (A) Off resonance spectrum without protein saturation. (B) Resulting STD NMR spectrum and calculated STD effect for protons of the ligand. The peak at 2.5 ppm corresponds to residual DMSO.</figcaption></figure>
## Screening of tolcapone derivatives

Next, we screened a library of catechols from F. Hoffmann-La Roche, the developer of Tolcapone, in the competitive binding assay to establish a structure-activity relationship. To this end, two sets of compounds with a molecular cut-off of 500 Da were chosen: a first set of 342 compounds contained derivatives of tolcapone, while a second set of 3222 compounds comprised catechols covering a larger chemical space. Some compounds were part of both sets, which resulted in 3267 unique compounds tested for LecA inhibition. Tests were run in a 384-well-plate format at three concentrations (first set at 125 µM, 32 µM and 4 µM, second set at 100 µM, 25 µM and 3 µM) in presence of 10% DMSO and 250 µM 2-mercaptoethanol to prevent interference by a potential unspecific reactivity of electron-rich catechols as established earlier.[30] Inhibitory activity is reported as relative potency (% inhibition) compared to the known LecA binder *pNPGal* at 500 µM, which was defined as 100% inhibition. We investigated the structure-activity relationship (SAR) of this data using matched molecular pair analysis.[33] Key SAR trends are summarized in Figure 4.

The crucial role of calcium complexation by the free catechol was underlined by the complete lack of activity of (partially) protected compounds such as **5** with a methyl protected catechol. Furthermore, replacement of the nitro group by various other functionalities (**6 – 14**) resulted in a loss of activity with the notable exception of oxadiazole **13**. Especially the lower activity of nitrile **12** is noteworthy considering the cyano group is both strongly electron-withdrawing and a hydrogen bond acceptor. The lack of affinity by compounds containing other hydrogen bond acceptors shows that the contributions of the nitro group towards binding are likely twofold: it serves as hydrogen bond acceptor with Asn107 and reduces the electron density of the catechol which was previously shown to be crucial. In line with the lower affinity of Opicapone compared to Tolcapone, the replacement of the ketone with small heterocycles abolished the activity (**16 – 18**). To quantify the contributions of additional residues to the binding, acetophenone **19** can serve as a reference point. No gain of affinity is achieved by converting the ketone in **19** to esters or amides (**20 – 25**). Additionally, replacement of the phenyl ring with other (hetero-)cycles was generally disfavoured (**26 – 29**). Encouragingly, the most potent derivatives (e.g., **15**, Figure 4) showed almost identical activity as the carbohydrate-based reference *pNPGal* at 500 µM.
<figure><img src="image_5.png" /></figure>

<table><thead><tr><th>ID</th><th>Name</th><th>R<sub>1</sub></th><th>R<sub>2</sub></th><th>R<sub>3</sub></th><th>Activity [%]</th></tr></thead><tbody><tr><td>4</td><td>Tolcapone</td><td>H</td><td>H</td><td>NO<sub>2</sub></td><td>43.9 ± 2.1</td></tr><tr><td>5</td><td>RO0613272*</td><td>H</td><td>CH<sub>3</sub></td><td>NO<sub>2</sub></td><td>-5.3 ± 7.6</td></tr><tr><td>6</td><td>RO0441446</td><td>H</td><td>H</td><td>H</td><td>13.8 ± 3.9</td></tr><tr><td>7</td><td>RO0619831</td><td>H</td><td>H</td><td>OH</td><td>7.5 ± 4.9</td></tr><tr><td>8</td><td>RO0613662</td><td>H</td><td>H</td><td>NH<sub>2</sub></td><td>7.9 ± 3.4</td></tr><tr><td>9</td><td>RO0618738</td><td>H</td><td>H</td><td>COOH</td><td>2.1 ± 2.9</td></tr><tr><td>10</td><td>RO0482485</td><td>H</td><td>H</td><td>NHCOCH<sub>3</sub></td><td>20.1 ± 2.8</td></tr><tr><td>11</td><td>RO0614498</td><td>H</td><td>H</td><td>CONH<sub>2</sub></td><td>21.8 ± 3.4</td></tr><tr><td>12</td><td>RO0614283</td><td>H</td><td>H</td><td>CN</td><td>20.2 ± 4.8</td></tr><tr><td>13</td><td>RO0618764</td><td>H</td><td>H</td><td>Q<sub>2</sub>N<sub>2</sub>N=N</td><td>57.7 ± 3.8</td></tr><tr><td>14</td><td>RO0616633</td><td>H</td><td>H</td><td>N<sub>2</sub>N=N<sub>2</sub>N=N</td><td>0.8 ± 8.7</td></tr><tr><td>15</td><td>RO0622671</td><td>Cl</td><td>H</td><td>NO<sub>2</sub></td><td>96.7 ± 4.0</td></tr></tbody></table>

<figure><img src="image_6.png" /></figure>

<table><thead><tr><th>ID</th><th>Name</th><th>Residue</th><th>Activity [%]</th></tr></thead><tbody><tr><td>16</td><td>RO0404949</td><td>O-N<sub>2</sub>N<sub>2</sub></td><td>9.4 ± 7.5</td></tr><tr><td>17</td><td>RO0637684</td><td>S<sub>2</sub>N<sub>2</sub>N<sub>2</sub>N<sub>2</sub>ClH<sub>2</sub>N</td><td>15.2 ± 8.7</td></tr><tr><td>18</td><td>RO0400323</td><td>OH-N<sub>2</sub>N<sub>2</sub></td><td>1.9 ± 3.3</td></tr><tr><td>19</td><td>RO0190793</td><td>COCH<sub>3</sub></td><td>31.5 ± 3.8</td></tr><tr><td>20</td><td>RO0190794</td><td>COOC<sub>2</sub>H<sub>5</sub></td><td>14.0 ± 4.2</td></tr><tr><td>21</td><td>RO0182737</td><td>COOC<sub>2</sub>H<sub>5</sub></td><td>22.9 ± 1.8</td></tr><tr><td>22</td><td>RO5555827</td><td>CONH(4-F-Ph)</td><td>8.7 ± 3.3</td></tr><tr><td>23</td><td>RO555213</td><td>CONHBn</td><td>26.6 ± 4.2</td></tr><tr><td>24</td><td>RO0625962</td><td>O-N<sub>2</sub>N<sub>2</sub></td><td>10.0 ± 3.6</td></tr><tr><td>25</td><td>RO5555214</td><td>CO(1-piperidin)</td><td>5.0 ± 11.8</td></tr><tr><td>26</td><td>RO0404946</td><td>CO(2-imidazol)</td><td>16.8 ± 3.8</td></tr><tr><td>27</td><td>RO0415345</td><td>CO-c-hexane</td><td>23.4 ± 3.6</td></tr><tr><td>28</td><td>RO0406369</td><td>CO(2-benzimidazol)</td><td>26.7 ± 3.6</td></tr><tr><td>29</td><td>RO0403531</td><td>COC<sub>6</sub>H<sub>5</sub></td><td>41.8 ± 7.3</td></tr></tbody></table>

**Fig. 4** Selected examples highlight key lessons learned from the screening of 3267 catechols in the competitive binding assay. Activity is depicted as % inhibition at a compound concentration of 100 µM (125 µM when marked with *) relative to 500 µM pNPGal. Averages and standard deviations were calculated from 4 replicates.

Compounds displaying >50% inhibition were selected for a more detailed characterisation, while those with >105% inhibition were excluded as false positives due to precipitation. This resulted in 48 compounds selected for an extended dose-response analysis in the competitive binding assay based on fluorescence polarisation (Table S1). The purity of all compounds was quantified by LCMS and 43/48 test molecules showed a purity of >95%. One compound was excluded due to insufficient purity resulting in a final number of 47 compounds tested. The final DMSO concentration was increased to 20% because higher inhibitor concentrations were required to obtain full titration curves. 2-Mercaptoethanol was excluded from the assay since all compounds possess electron-deficient catechol moieties which previously did not lead to unspecific reactivity.

Trends observed in the initial screening were also reflected in the determined IC₅₀ values. In total, 26 of the 47 compounds showed a higher LecA inhibition than Tolcapone and
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seven compounds reached IC₅₀ values below 100 µM (Figure 5-A), which was preserved in the presence of detergent (0.01% Triton X-100) to account for false positives from possible compound aggregation (Table S2). The replacement of the nitro group in Tolcapone with an oxadiazole in **13** resulted in a higher IC₅₀ value compared to Tolcapone (346 ± 36 µM vs. 257 ± 40 µM), which was in contrast to the somewhat higher inhibition in the initial screening set (Fig. 4). The phenyl ring as a second aromatic system was generally preferred over other aromatic substituents. As one exception, brominated indole **30** (IC₅₀ = 43 µM) was among the best performing compounds. Here, the bromine substituent appeared important since the derivative lacking the halogen substituent showed a 10-fold reduced inhibition. Another interesting modification was the replacement of the ketone in Tolcapone with a diketone. Both compounds with this modification showed a higher affinity than Tolcapone (RO0619291, 127.7 µM and RO0621161, 152.8 µM; Table S1). Substitution at the second phenyl ring with small substituents was favoured in the ortho position over para and meta as highlighted by **33**. A possible explanation for this observation is a substituent-induced change in the angle of the two phenyl ring planes of benzophenone motif which may allow improved hydrophobic interactions with the protein surface. Compound **15** (IC₅₀ = 58 µM) bearing an additional chlorine substituent at the catechol ring may profit from the same effect. Furthermore, the electron-density of the catechol is further decreased by the electronegative chlorine and the small pocket in LecA usually occupied by galactose-OH6 could get partially occupied. Larger substituents at the distal phenyl ring were preferred in the para position over meta analogs, with an additional preference of esters over amides. This is evidenced by the two potent compounds **31** and **32**, which were among the most potent out of the tested compounds (IC₅₀ = 43 µM, IC₅₀ = 56 µM, respectively).

To confirm the high inhibition of LecA by these non-carbohydrate glycomimetics, **15** and **33** were then further tested in an orthogonal binding assay by isothermal titration calorimetry (Figure 5-B). As a control, the binding of *pNPGal* to LecA was determined and a *K*<sub>D</sub> = 10.1 µM was in good agreement with the value of 14.1 µM determined by Kadam et al.[20]. The thermodynamics of the *pNPGal* - LecA interaction are coined by a high binding enthalpy of -50.1 kJ/mol, which is offset by an entropy cost (-TΔS = +21.6 kJ/mol). Gratifyingly, the two analysed catechols showed *K*<sub>D</sub> values of 12 – 15 µM which is in the same range as the potent glycoside *pNPGal*. Interestingly, while their binding enthalpy is reduced (ΔH = 22.8 – 24.4 kJ/mol), the binding of both catechols is also driven by favourable entropic
contributions (-TΔS = -5.4 and -3.2 kJ/mol). This is a major difference compared to carbohydrate-based ligands and reflects the more hydrophobic character of the interaction.

The presence of two fluorine atoms in the high affinity ligand **33** enabled another orthogonal LecA binding experiment (Figure 5-C). In analogy to Denavit et al.,[34] ligand-observed ¹⁹F NMR spectra of 50 µM **33** were recorded in absence and in presence of LecA (100 µM). In the presence of LecA, a strongly reduced ¹⁹F-NMR peak intensity of **33** down to the noise level highlights potent binding to LecA. Importantly, the ligand's fluorine signal was restored when *pNPGal* was added as competitor molecule, indicating the specific and reversible catechol – LecA interaction.
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<table><thead><tr><th></th><th>pNPGal</th><th>15</th><th>33</th></tr></thead><tbody><tr><td>K<sub>D</sub> [µM]</td><td>10.1 ± 1.2</td><td>12.0 ± 2.9</td><td>15.0 ± 2.1</td></tr><tr><td>N</td><td>0.81 ± 0.09</td><td>0.92 ± 0.05</td><td>0.82 ± 0.03</td></tr><tr><td>ΔG [kJ/mol]</td><td>-28.6 ± 0.3</td><td>-28.2 ± 0.6</td><td>-27.6 ± 0.4</td></tr><tr><td>ΔH [kJ/mol]</td><td>-50.1 ± 2.3</td><td>-22.8 ± 1.5</td><td>-24.4 ± 2.2</td></tr><tr><td>-TΔS [kJ/mol]</td><td>+21.6 ± 2.6</td><td>-5.4 ± 2.1</td><td>-3.2 ± 2.5</td></tr></tbody></table>

<figure><img src="image_9.png" /></figure>

<table><thead><tr><th></th><th colspan="4">33</th></tr><tr><th></th><th>-</th><th>+ pNPGal</th><th>+ LecA</th><th>+ LecA + pNPGal</th></tr></thead><tbody><tr><th>Peak intensity [%]</th><td>106</td><td>100</td><td>1</td><td>79</td></tr></tbody></table>

**Fig. 5:** (A) Catechols with an IC₅₀ below 100 µM determined in the competitive binding assay, (B) ITC titration data and thermodynamic parameters of catechols **15** and **33** in comparison to the modified carbohydrate *pNPGal*, (C) Binding of **33** (50 µM) to LecA (100 µM) by ligand-observed ¹⁹F NMR spectroscopy and its competitive displacement with addition of *pNPGal* (1 mM).
X-ray crystallography of LecA in complex with Tolcapone derivatives

Additional X-ray crystallography studies were conducted with selected derivatives of Tolcapone. Co-crystal structures of **33** (pdb code 9I7Z) and **15** (pdb code 9I80) were solved at a resolution of 1.85 Å and 1.95 Å, respectively (Figure 6). In contrast to Tolcapone, here only single binding poses were observed for both compounds and no crystal contacts were found between neighboring asymmetric units. Gratifingly, both compounds bound to LecA in similar fashion to Tolcapone in binding pose II (Figure 2-C), which was determined to be the dominant pose in solution by STD-NMR (Figure 3). Consequently, the key interactions of calcium complexation and H bonding to Asp100 and Asn107 for the catechol moiety are the same as discussed above. A notable difference is the observed presence of the usually conserved water molecule, which was absent for binding pose II in the crystal structure of Tolcapone. As hypothesized before, the ketone of both compounds is in the correct position to form a water-mediated hydrogen bond to the backbone of Pro51 and Gln53. It thereby acts as a mimetic of the OH6 of galactose. The high torsion of phenyl rings in **33** allows for improved hydrophobic interactions with the side chain of Gln53 (Figure 6-A), in line with the entropic contributions determined by ITC. However, the distance of the carbonyl to the Nτ of His50 is increased (3.8 Å) compared to the distance observed for Tolcapone (3.3 Å). In contrast to this, the distance is also 3.3 Å in the case of **15** (Figure 6-B). Here, the angle of the ester residue varied between the observed monomers with the alkyl chain aligning with the protein surface.
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 6** Crystal structures of LecA complexed with (A) Catechol **33** (pdb code 9I7Z) and (B) Catechol **15** (pdb code 9I80). Dashed lines indicated hydrogen bonding distance or metal complexation.</figcaption></figure>
Conclusion and outlook

In our efforts to find potent non-carbohydrate glycomimetic lectin inhibitors, we investigated commercially available nitro-catechols for inhibition of LecA. A substantial increase in affinity of the Parkinson's drug Tolcapone over the initial screening hits was determined by direct (SPR) and competitive (FP) binding assays, while other drugs of this class proved inactive. Tolcapone was then crystallised with LecA to reveal its interactions at atomic level. With the aid of STD NMR spectroscopy, the dominant binding pose in solution could be identified. Important interactions of Tolcapone with LecA include calcium complexation through the catechol moiety, multiple direct and indirect hydrogen bonds to Asp100, Asn107, Pro51 and Gln53 as well as hydrophobic contacts established by the tolyl residue. The structure-activity relationship of (acylated) catechols was investigated by initial screening a Roche library of 3267 compounds in our competitive LecA inhibition assay. 48 potent derivatives were selected for extended dose-response analysis yielding several compounds with double-digit IC₅₀ values. Notably, these non-carbohydrate catechol glycomimetics now showed the same affinity range as conventional carbohydrate-based LecA inhibitors. Isothermal microcalorimetry revealed that the binding of catechols is driven by both enthalpy and entropy, which is in contrast to the enthalpy-driven binding of *p*NPGal. While competitive ¹⁹F NMR spectroscopy highlighted the competitive and reversible binding of a fluorine-bearing nitro-catechol, crystal structures obtained with two Tolcapone derivatives unambiguously revealed their binding modes and key interactions with LecA.

In this work, we demonstrated that non-carbohydrate fragments with weak lectin binding activity can be optimized into potent non-carbohydrate glycomimetics. In contrast to glycosides, these molecules occupy a more druglike chemical space - with several of the tested analogs being actual approved drugs. Finally, the structure-activity relationship paired with structural data for LecA binding of catechols will guide future optimisation to generate a lead structure to fight antimicrobial resistance.
**Acknowledgements**

Authors are grateful to Larissa Valeska Thiele (F. Hoffmann-La Roche) for compound logistics, Dr. Caroline Gramberg (F. Hoffmann-La Roche) for assistance with NMR signal assignment and to Prof. Dr. Christoph Rademacher (University of Vienna) and Hugo Scherer (HIPS) for advise on STD-NMR. We acknowledge synchrotron SOLEIL (Saint Aubin, France) for access and technical support on beamline PROXIMA1 (Proposal 20191314). We thank Emilie Gillon (CERMAV) for providing recombinant LecA for SPR and X-ray crystallography. The work was supported by the ANR/DFG French-German project (ANR-AAPG-2017). The authors acknowledge financial support from Agence Nationale de la Recherche to A.I. (grant no. ANR-17-CE11-0048) and from Deutsche Forschungsgemeinschaft to A.T. (grant no. Ti756/5-1). A.I. further acknowledges Glyco@Alps (ANR-15-IDEX-0002) and Labex Arcane/CBH-EUR-GS (ANR-17-EURE-0003). Support from EU COST Action CA21145 EureStop to A.T. and A.I. is kindly acknowledged.

**Conflict of interest**

B.K., C.L. and U.G. are full employees of F. Hoffmann-La Roche. The authors declare no further competing financial interest.

**Author Information**

Steffen Leusmann 0000-0003-4342-5749

Eike Siebs 0000-0002-7349-9872

Annabelle Varrot 0000-0001-6667-8162

Sakonwan Kuhaudomlarp 0000-0003-4415-3781

Anne Imberty [0000-0001-6825-9527](https://www.cnb.ugent.be/en/imberty/)

Bernd Kuhn 0000-0002-4301-562X

Christian Lerner

Uwe Grether 0000-0002-3164-9270

Alexander Titz [0000-0001-7408-5084](https://www.cnb.ugent.be/en/titz/)
**Supporting Information**

Experimental details, protocols on assays and diverse figures can be found in the electronic
supporting information. The data that support the findings of this study are available in the
supplementary material of this article.

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