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Allosteric inhibition of a mammalian lectin Jonas Aretz, Upendra R. Anumala, Felix F. Fuchsberger, Narges Molavi, Nandor Ziebart, Hengxi Zhang, Marc Nazaré, and Christoph Rademacher J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08644 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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Journal of the American Chemical Society

Allosteric inhibition of a mammalian lectin Jonas Aretz†,‡,⊥, Upendra R. Anumala§,⊥, Felix F. Fuchsberger†, Narges Molavi†,§, Nandor Ziebart§, Hengxi Zhang†, Marc Nazaré§,∥,,⊥,*, Christoph Rademacher†,‡,⊥,* †

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14424 Potsdam, Germany.

‡

Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany.

§

Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125 Berlin, Germany. ∥ Berlin

Institute of Health (BIH), Anna-Louisa-Karsch-Strasse 2, 10178 Berlin, Germany

ABSTRACT: Glycan-binding proteins are key components of central physiological and cellular processes such as self/nonself recognition, cellular tissue homing, and protein homeostasis. Herein, C-type lectins are a diverse protein family that play important roles in the immune system, rendering them attractive drug targets. To evaluate C-type lectin receptors as target proteins for small molecule effectors, chemical probes are required which are, however, still lacking. To overcome the supposedly poor druggability of C-type lectin receptors and to identify starting points for chemical probe development, we screened murine Langerin using 1H and 19F NMR against a library of 871 drug-like fragments. Subsequently, hits were validated by surface plasmon resonance and enzyme-linked lectin assay. Using structure-activity relationship studies and chemical synthesis, we identified thiazolopyrimidine derivatives with double-digit micromolar activity that displayed Langerin selectivity. Based on 1H-15N HSQC NMR and competitive binding and inhibition experiments, we demonstrate that thiazolopyrimidines allosterically inhibit Langerin. To the best of our knowledge, this is the first report of drug-like allosteric inhibitors of a mammalian lectin.

as well as cryptic sites in GBPs13, it appears to be a suitable method to discover drug-like ligands for CLRs.

Introduction Carbohydrates cover every living cell and are fundamental determinants of health and disease. In mammals, there are three major families of glycan-binding proteins (GBPs): Siglecs, Galectins, and C-type lectin receptors (CLRs). The latter comprises several members expressed on cells of the innate immune system that recognize carbohydrate structures to differentiate between “self” and “non-self” antigens1. Hence, those CLRs promote the transition of innate to adaptive immune response, which is involved in a variety of immune-related diseases, such as autoimmunity, cancer, and infections2. However, only a handful of chemical probes or drugs have been developed against this target class3-4. The lack of drug-like ligands may be owed to the low druggability associated with the rather hydrophilic and solvent exposed orthosteric binding sites of mammalian GBPs lacking discrete binding pockets and hot spots, which are also the origin for the low endogenous affinity for monomeric substrates5-8. In case of CLRs, interaction with carbohydrate ligands is mediated via a Ca2+ cofactor exposed by the primary recognition site. On the other hand, CLRs are at least moderately druggable according to experimental assessments9-10, which is most likely mediated by druggable secondary sites11. As the screening of drug-like fragments was successfully applied to identify ligands for challenging targets such as protein-protein interactions12

High-quality chemical probes are powerful research tools and have accelerated biomedical research on several drug targets such as bromodomains14. A CLR with unique biology is Langerin, which is selectively expressed on Langerhans cells (LCs) in human and mouse and additional dendritic cell (DC) subsets in mouse. In both species, its primary function has been associated with the recognition and internalization of pathogens to trigger a response of these innate immune cells. However, certain pathogens have developed mechanisms to evade intracellular processing and make use of such uptake by CLRs to infect their hosts. In case of Langerin this has been reported for Yersinia pestis15 and Influenza A virus16. Due to its selective expression on LCs, Langerin might serve as a receptor for targeted delivery approaches in LC-associated diseases such as Langerhans cell histiocytosis (LCH)17 or skin graft-versus-host disease18. Furthermore, besides DEC205 and CLEC9A, Langerin appears as one of the most promising CLRs for targeted delivery of DC-based vaccines19. This is owed to the slow release of endosomal cargo after Langerin-dependent uptake, which promotes efficient antigen cross-presentation19. To foster our insight into Langerin biology we aimed to develop tool compounds for the murine homolog to allow research in relevant animal models. For this purpose, we performed a fragment-based screening by NMR, followed by a hit-validation cascade using surface plasmon reso-

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nance (SPR) and an enzyme-linked lectin assay (ELLA). Here, we identified three scaffolds that were further explored and validated by saturation transfer difference (STD) NMR epitope mapping, chemical microarray, and initial structure-activity relationship (SAR) studies. Finally, we selected thiazolopyrimidine-5-one derivatives for an in-depth SAR study applying chemical synthesis and fragment growing. Among these derivatives, we identified compounds that exhibited double-digit micromolar inhibition of carbohydrate-protein interactions of Langerin that were highly specific versus the closely related receptors DC-SIGN and human Langerin. By applying derivatized beads and surfaces in flow cytometry and SPR, as well as soluble ligands in 1H-15N HSQC NMR, we demonstrate that these compounds are the first drug-like, allosteric inhibitors of CLR function.

assumption of a unique binding pose20 (Fig. 1 D – F, S3S5). Furthermore, chemical fragment arrays were employed to study compounds from the SAR (Fig. 1 G, Tab. S2). Compounds with significantly higher fluorescent signals compared to DMSO controls were considered as actives (Dunnett’s test, p < 0.001). While for the isoindolene and thiophene series 56% and 63%, respectively, remained active, 93% of the thiazolopyrimidine derivatives were still active on the microarray. Thus, immobilizing the thiazolopyrimidine core by a covalently attached linker did not impair the binding of Langerin in most cases. In combination with the high confirmation rate, this feature indicated a good chemical variability for further fragment growing and led to the prioritization of this scaffold.

Results

At this stage of our hit validation, thiazolopyrimidine-5one derivative 1 consistently passed four validation assays (Fig. 1 B – G). To further rule out potential false positive mechanisms, all hit compounds were analyzed chemoinformatically for promiscuity in badapple and for similarity to known aggregators using Aggregator Advisor21-22 besides the controls that were already performed before the initial and counter-screenings (Fig. 2 A - C). Compounds 1, 4, and 5 were not similar to any known aggregators and the thiazolopyrimidine scaffold was not promiscuous in activity assays, which is in line with published data23. To identify potentially reactive compounds, the hydrolytic stability in aqueous buffers at room temperature and physiological pH (7.4) was monitored for several days (Fig. 2 E). In this assay, 1, 4, and 31 were stable over several weeks whereas 5, which carries a 7-chloromethyl group, displayed a half-life of 370 ± 90 h under these conditions (Fig. 2 E and S6). The chloromethyl group is most likely hydrolyzed, but its half-life exceeded the incubation time of every assay performed in this study. Still, a possible covalent background reaction with the protein cannot be excluded, thus the results for 5 in the inhibition assay have to be interpreted with caution. As reactive compounds are at risk to covalently modify and inhibit proteins non-specifically, compounds 1, 5, 12, 30, 31, 33, 177, and 178 were tested against the related CLRs DC-SIGN and the homolog (hLangerin) using the mannan ELLA (Fig. 2 D). None of these fragments were active against a related CLR (no significant reduction of signal, Dunnett’s test, p>0.05). Hence, we excluded assay artifacts being responsible for their activity against murine Langerin. Remarkably, all these compounds were fully selective for the murine over the human homolog, hinting towards a specific inhibition mechanism via an allosteric or cryptic site24. To exclude aggregation as origin of false positives, STD NMR in absence of protein, IC50 determinations in presence and absence of detergent and with reduced protein concentrations, as well as a NMR aggregation assay were performed (Fig. 2 F-H)21, 25. There were no signals in the STD NMR spectra of 1, 4, and 31 in absence of protein. Furthermore, the IC50 values of 1 and 4 were

Fragment screening against murine Langerin and hit validation identify thiazolopyrimidines inhibitors

Langerin extracellular domain was screened against a library of 660 fragments (Fig. S1) using STD and T2filtered 1H NMR in mixtures of 20 fragments (Fig. 1 A and S2 A). Compounds giving rise to STD NMR signals in absence of protein were identified as potential false positives and not pursued further. Next, binding ligands were identified by the addition of protein, followed by the addition of the cofactor Ca2+, which revealed 67 hits (10.2% hit rate) being increased or decreased in signal intensity (Fig. 1 A, Tab. S1). Eleven additional hits (3.9%, Tab. S1) were added to this set from a screening of 281 fragments with 19F NMR and T2 filtered 19F NMR (Fig. S2 B and C)10. These two libraries include 871 fragments that were evaluated (Fig. S1) with 78 hits (9.0%) being identified in total by two NMR screening methods under the influence of the cofactor Ca2+. To validate and prioritize hits from the screening, two orthogonal counter-screens were performed: a SPR binding assay and a mannan ELLA inhibition assay were performed (Fig. 1 B, C). Using SPR, 53 of 78 screening hits (68%) could be confirmed, from which three compounds (6%) abrogated binding of Langerin to mannan (Tab. S1). SPR and ELLA were applied to estimate binding affinities and allowed ranking the hits according to apparent Kd and ligand efficiency (LE). Considering compounds with moderate affinity below 1 mM and/or good LE above 0.3 kcal mol-1 HA-1, three scaffold series were identified for further studies (Fig. 1 E). To further prioritize scaffolds for a fragment growing approach, compounds from these three ligand series were subjected to in initial SAR studies, by STD NMR epitope mapping, and on a chemical microarray as described earlier9 (Fig 1 D – G, S3 – 5, Tab. S2). A SAR as well as a differentiated binding epitope in STD NMR correlate with a specific binding pose and interaction pattern in a protein pocket and additionally provide a rational to further guide fragment growing. For all three series, a differentiated epitope and a SAR were observed leading to the

Thiazolopyrimidine derivatives are true and selective Langerin inhibitors


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neither affected by detergent nor by protein concentra-

tion.

Figure 1. Examples from fragment screening and follow-up screening against murine Langerin. (A) Exemplary spectra from STD NMR screening in the absence of protein (black), after addition of 10 µM Langerin extracellular domain (orange), and after CaCl2 addition to a final concentration of 25 mM (blue). Expansions from the spectrum display proton peaks (black circles) of compounds (i) that lead to false positive signals in the absence of protein, (ii) that bind to unrelated 2+ pockets, and (iii) that are in competition with the cofactor Ca , i.e. their peak intensity and presumably affinity is reduced in the 2+ presence of Ca . Compounds were screened in mixtures of 20 at 200 µM each with a saturation time of tsat = 4 s. (B) Exemplary SPR sensorgrams of different concentrations of 79 from the validation-screening are shown (left). A one-site-binding model was used for fitting the data (right). SPR sensorgrams are representative of three independent measurements in presence of 2 mM CaCl2. (C) IC50 value of 5 determined by mannan ELLA (left) in comparison to the IC50 value of mannose (right). Error bars represent standard deviation from three independent experiments. (D) STD NMR binding epitope of compound 1 that was identified in the screening. Epitopes were estimated from a STD NMR spectrum recorded with a saturation time of tsat = 1 s at 1 mM compound and 25 µM protein concentration. (E) Three potentially suitable scaffold series with moderate affinity below 1 mM -1 -1 and/or good ligand efficiency (LE) above 0.3 kcal mol HA were identified in the screening and counter-screening. (F) Initial SAR of the thiazolopyrimidine scaffold (blue). Affinities were estimated by SPR and IC50 values by mannan ELLA. Kd,app values were used to calculate LE. For some compounds inhibition in mannan ELLA could not be detected (n.d.) in the tested concen-



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tration range. (G) Microarray data for compounds with thiazolopyrimidine (blue), isoindolene (green), and thiophene (orange) scaffold.

Figure 2. Examples of control experiments that were performed to identify and exclude potential false positives. 1

(A) All compounds in the library were tested for their identity and solubility in aqueous buffers using H NMR. Compounds that were used for NMR screenings were tested in their respective screening mixture for stability at room temperature for 24 h. For compounds that were included in SPR studies the solubility in running buffer was further determined by recording absorption spectra at the tested concentrations and only soluble samples were injected. (B) All compounds were analyzed chemoinformatically for reactivity and PAINS-substructures. Hits were further analyzed for promiscuity with badapple and for similarity to 21-22, 26 reported aggregators with aggregator advisor . (C) Compounds with suspicious SPR sensorgrams such as negative response, slow kinetics that do not reach saturation or super-stoichiometric binding, were flagged as false positives and not followed further. (D) Compounds were tested against the closely related proteins hLangerin and DC-SIGN using the same assay setup in the mannan ELLA as for mLangerin. DMSO was applied as negative and EDTA as positive control. (E) Stability at room temperature 1 in aqueous buffer was monitored for several days and determined by H NMR. (F) Compounds that showed saturation in a STD NMR experiment without protein were flagged as potential false positives. (G) In an NMR aggregation assay, a dilution series of 1 a compound was measured in aqueous buffer using H NMR and the spectra were analyzed for irregularities such as non-linear


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increase in signal integral with rising concentrations, chemical shifts or different peak pattern . (H) IC50 values were determined 21 in the presence of different concentrations of detergent and at different protein concentrations to identify aggregators .

In another NMR aggregation assay, different compound concentrations were measured in aqueous buffer in the presence and absence of detergent, analyzing the spectra for non-linear correlations between signal integral and concentration, chemical shift changes, or changes in the peak patterns25. In this assay 1, 4, 31, 35, and 177 were tested (Fig. 1 F, 2 G). The spectra of 4, 31, and 35 displayed no irregularities with increasing concentration so aggregation can be excluded. The results from the NMR aggregation assay for 1 and 177 were ambiguous; while the signal integrals correlated linearly to concentration and the

spectra were identical after detergent addition, concentration-dependent chemical shift changes were observed for some protons. In summary, compounds with a thiazolopyrimidine-5-one scaffold were tested in orthogonal assays for potential false positive behavior passing these assays for the most part. Together with their specific inhibition of Langerin, an underlying unspecific mechanism is unlikely, rendering thiazolopyrimidines a promising chemotype for further exploration in an extended SAR study.

Scheme 1. Synthetic approaches for the generation of a library of thiazolopyrimidine analogs by diversifying the 6and 7-position of the core scaffold. A) 7-Aminomethyl thiazolopyrimidines 13-22, 24-25, 28; B) 7-Hydroxy-6-carboxamide thiazolopyrimidines 87, 82, 93; C) 6Carboxamide thiazolopyrimidines 26, 37, 53, 76, 111-113, 115, 117, 145, 147-153, 155, 156; D) 7-Aminomethyl thienopyrimidines 168, 176; and E) N-Azetidinyl-6-carboxamide thiazolopyrimidines 137-138, 140-141, 135-136 and N-Piperidinyl-6-carboxamide thia1 2 zolopyrimidines 125-134, 139, 142, 124, 203, 114, 118-119, 120-122, 144. Reagents and conditions: (a) HNR R , ethanol, reflux or alkyl 3 amine, potassium carbonate, DMF or acetonitrile, reflux with R = H, Me; (b) H2NR, DABAL-Me3, THF, microwave heating, 1 2 130°C, 30 min; (c) alkyl- or aryl amine, HATU, DIPEA, dichloromethane, r.t.; (d) HNR R , ethanol, 50°C or reflux; (e) tert-butyl 4aminopiperidine-1-carboxylate or tert-butyl 3-aminoazetidine-1-carboxylate ; HATU, DIPEA, dichloromethane, r.t.; (f) trifluoroacetic acid, dichloromethane, r.t.; (g) alkylisocyanate, triethylamine, dimethylformamide; (h) carboxylic acid, HATU, DIPEA,



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dichloromethane, r.t.; (i) arylboronic acid, copper (II) acetate, DIPEA, dichloromethane, r.t.; (j) sulfonylchloride, triethylamine, dimethylformamide, 16 h, r.t.

Chemical synthesis of a focused library of thiazolopyrimidine analogs

results. Thus, we dismissed a further exploration of these directions.

The strategy for the expansion of the thiazolopyrimidine fragment hit was guided by the initial SAR results and observations from STD NMR, showing that modifications at the 6- and 7-position of the thiazolopyrimidine scaffold were beneficial (Fig. 1 F). Using the 7chloromethyl or the 6-carboxy functionality to attach amines, derivatives were synthesized using N-alkylation or N-acylation (Scheme 1a-c). Simple methylene amine derivatives were synthesized by nucleophilic substitution of the amine with chloro derivative 5 or 6 in ethanol, optionally in the presence of a base such as potassium carbonate and heating. The amide derivatives were synthesized either by classical activation of the carboxylic acid 4 and 4a or 4b using HATU or by Lewis acidmediated activation of the methyl ester 29 by DABALMe3 and reaction with the corresponding amine.

Derivatives of the 7-position are based on scaffolds 5 (Fig. 3, Tab. S4), 164, and 166 (Tab. S3), which display a high LE and good chemical tractability (Scheme 1). In all cases, the chloromethyl group was the most efficient (GECH2-Cl in 5 = 2.1 kcal mol-1 HA-1) and the only substituent that led to considerable inhibition. For scaffold 5 and 166, pyrrolidine enhanced the affinity (GEPyrrolidine in 13 = 1.2 kcal mol-1 HA-1), whereas a decrease was observed for 164 (GEPyrrolidine in 165 = -1.1 kcal mol-1 HA-1). While larger hydrophobic moieties were only tested for scaffold 5, their efficiency was low (GE = 0.1 - 0.4 kcal mol-1 HA-1). Hydrophilic interactions in the 7-position were detected e.g. in 14 compared to 15 in about five atoms distance (ΔΔG0 = 3.6 kcal mol-1). On the other hand, hydrogen bonds are directional interactions and very challenging to implement without structural information. In total, 27 analogs of the 7-position were synthesized and tested with SPR and in an inhibition assay. None of these compounds inhibited Langerin binding to mannan, while only the small hydrophobic residues enhanced affinity. Thus, we decided to solely derivatize this position with small moieties.

To further explore a possible extension of the initial structure and increase the Fsp3 content of the ligand, a linear but flexible bridging spacer was incorporated using 4-amino piperidine as well as a 3-amino azetidine in a further set of analogs. These piperidine and azetidine derivatives were synthesized by a two-step procedure. The Boc-protected 4-amino-piperidine or 3-aminoazetidine derivative was coupled in the presence of HATU followed by TFA mediated cleavage of the Boc group. The resulting free amines 4c and 32 were further converted to sulfonamide derivatives by reaction with sulfonyl chlorides, urea derivatives by using isocyanates, and substituted aryl amine derivatives under Chan-Lam (LIT) conditions by reaction with boronic acids. SAR study of thiazolopyrimidine derivatives

To enhance the LE of the thiazolopyrimidine scaffold, we performed a rescaffolding using commercially available analogs, which were analyzed by SPR and mannan ELLA (Tab. S3). While these results are presented and discussed in detail in the Supporting Information, we observed that most changes to the original thiazolopyrimidine scaffold reduced the LE or induced a change in the binding mode. Nevertheless, a thienopyrimidine-4-one scaffold 164, which could be used for modifications in the 7-position (Tab. S5), was identified to enhance LE from 0.31 to 0.38 kcal mol-1 HA-1. For the 2- and 3-position, commercially available analogs of 4 and 5 with hydrophobic moieties were tested to create matched pairs for 7-chloromethyl- and 6carboxylic acid-substituted thiazolopyrimidine (Fig. 3, Tab. S4). In case of 7-chloromethyl, small hydrophobic substituents were beneficial (GECH3 in 6 = 3.0 kcal mol-1 HA1 ), while for 6-carboxylic acid small as well as larger hydrophobic moieties decreased affinity (GE = -0.1 to -1.2 kcal mol-1 HA-1). A similar trend was observed in the IC50 values, with the exception that 179 led to false positive

Finally, analogs of the 6-position were derived from the building blocks 4 and 29 (Fig. 3, Tab. S4), which introduced an additional 7-hydroxyl group. As the 3-methyl group was beneficial for compounds carrying a substituent in 7-position, 3-methyl-7-hydroxylthiazolopyrimidines were included in this SAR study (Tab. S3, S4). Ligand 4 displayed a high LE, micromolar affinity, and micromolar IC50 value while the 6-position could be derivatized via amide coupling (Scheme 1). Inhibitory activity and affinity were only maintained by scaffold-proximal hydrophilic groups. While a 6-methyl group also displayed a high GE (2.6 kcal mol-1 HA-1), this substitution abrogated inhibition. This effect could originate in detrimental changes of the binding mode as less saturation transfer on the hydrogen in 3-position was observed in STD NMR epitope mapping (Fig. S7-10). Furthermore, aromatic substituents close to the scaffold displayed lower GEs than moieties that can form hydrogen bonds (carboxylic acid 4 and amide 31 > pyrazole in 1 > aryl in 33 > ester, Fig. 1 F). This was also true for the building block 29 (Scheme 1) that was neither able to bind nor inhibit, which could be restored upon derivatization with a hydrogen bond donor or acceptor in 6-position. As a directed interaction of a protein ligand hydrogen bond may increase specificity24 an amide linker was chosen to further explore the 6-position. Here, compounds with secondary amines as part of the linker outperformed derivatives with tertiary amines, strengthening the hypothesis of an essential hydrogen bond donor or acceptor close to the scaffold in 6-position. Attaching aliphatic groups to the linker decreased affinity except for cyclopentane (GE-


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= -0.1 kcal mol-1 HA-1, GEEthyl in 50 = -2.1 kcal mol HA ). With larger substituents in 6-position and a 7hydroxyl group, the 3-methyl decreased affinity (GE = -1.0 to -4.4 kcal mol-1 HA-1), so this series was not followed further. While aromatic groups coupled directly to the Cyclopentane in 37 -1 -1

amide linker led to a complete loss of binding and inhibitory potential, aromatic groups in a certain distance to the scaffold displayed the highest GEs measured in this position (GEBenzyl = 1.0 kcal in 55

Figure 3. Key compounds representing major features of the established SAR of the thiazolopyrimidine scaffold. Thiazolopyrimidine analogs were modified in the (A) 7-position and (B) the 2-, 3-, and 6-position. Apparent Kd values were estimated by SPR using Langerin immobilized on a CM7 sensor chip and IC50 values were determined by an ELLA probing binding of Langerin to mannan-coated 96 well maxisorb plates. Some compounds had no detectable signals in the tested concentration range (n.d.).

mol-1 HA-1, GEBenzyl in 127 = 1.0 kcal mol-1 HA-1). Finally, inhibition was observed for benzyl-derivatives with a methylacetamide linker (55 - 57), while 44, 91, and 143 decreased the IC50 values in the double digit micromolar range. Overall, 83 derivatives varying the 6-position at the thiazolopyrimidine-5-one, 36 modifications at the 7hydroxylthiazolopyrimidine- 5-one, and 19 analogs of the 6-position of the 3-methyl-7-hydroxylthiazolopyrimidine-5-one were tested for activity and inhibition with SPR and mannan ELLA. As a trend, several of the attempted variations on the 3-methyl-7hydroxyl-thiazolopyrimidine-5-one showed decreased

potency. For the remaining two scaffolds, compounds 44, 91, and 92, bound Langerin and inhibited its binding to mannan, respectively, with micromolar potency thus rendering the 6-position of the thiazolopyrimidine-5-one scaffold the most promising for further ligand development. Thiazolopyrimidines are potent allosteric inhibitors of Langerin function

Throughout the SAR study, activity cliffs in the mannan ELLA IC50 data were observed, which were absent in the binding data obtained by SPR (Fig. 4 A, Tab. 1, S3-S5). In addition, the SAR of the 3- and 7-position was non-



additive and visible changes of the STD NMR binding epitopes between several matched pairs were observed (Fig. 4 B, C, S7-10). Hence, compounds either switch from

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an active to an inactive binding site upon derivatization or the binding mode of the thiazolopyrimidine scaffold is little conserved,

Figure 4. Biophysical characterization of thiazolopyrimidines interacting with Langerin. (A - C) Examples from the SAR. (A) Activity cliffs were observed in several mannan ELLA IC50 values which were absent in binding data from SPR. (B) Changes in binding modes were observed in STD NMR epitope mapping. (C) A non-additive SAR was observed for the 3- and 7-position. (D) Exemplary histogram plot of Langerin binding to 4-conjugated DynaBeads in a flow cy1 15 tometric assay in the presence (grey) and absence (blue) of an inhibitor. (E) Overlaid H- N HSQC NMR spectra from a titration with mannose (green) and 91 (blue). As displayed in the expansions from the spectra, certain peaks are affected (i) only by man-


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nose, (ii) only by 91, and (iii) by mannose and 91 along with the shifting of peaks with different vectors. The altered CSP pattern of 91 compared to that of mannose indicates a secondary binding site. F) Competition experiments using 91 (red), mannose (orange), and EDTA (green) in an IC50 assay using 4-conjugated DynaBeads. (G) Exemplary SPR sensorgrams of Langerin bind-1 ing to a 4-derivatized PEGylated matrix on a C1 sensorchip and (H) competition experiments using 6.25 µg mL mannan (blue), 30 mM mannose (orange), and 12.5 mM EDTA (green). (I) STD NMR spectra of 500 µM 91 in the presence of 10 µM Langerin 2+ (left) adding 20 mM mannose (middle) and 10 mM EDTA (right). (J) Determination of Ca affinity in the presence (red, Kd,Ca = 1 15 179 ± 17 µM) and absence (black, Kd,Ca = 177 ± 18 µM) of 91 using H- N HSQC NMR. (K) Determination of EC50 values of Langerin binding to mannan in an ELLA in the presence of 1% DMSO (black squares, EC50 = 3.9 ± 1.1 nM), 30 mM mannose (red circle, EC50 = 6.3 ± 1.8 nM), 1000 µM 4 (grey triangle, EC50 = 53.6 ± 4.0 nM), 250 µM 44 (reversed grey triangle, EC50 = 40.9 ± 1.5 nM), and 250 µM 91 (grey diamond, EC50 = 31.2 ± 3.0 nM).

leading to active and inactive binding poses in the same pocket. To exclude one of these hypotheses, we established an inhibition assay using thiazolopyrimidinederivatized beads (Fig. 4 D). As most tested thiazolopyrimidine-derivatives displayed inhibition in this assay format regardless their substitution (Tab. S4), they most likely interact with the same pocket. Strikingly, thiazolopyrimidines substituted at the 6-position with an amide linker while carrying a 7-OH group lost their ability to compete 4 except for 92. As 92 was the most potent derivative with a 7-OH group that was also active in an orthogonal thiazolopyrimidine competition assay (Fig. S11), we conclude that the 7-OH substitution decreases the ability to outcompete and displace 4 from its binding pocket. Consequently, thiazolopyrimidines likely change their binding pose in the same pocket upon addition of certain substitutions, leading to biologically active or inactive conformations. We were intrigued to find out more about the nature of the binding pocket that can accommodate thiazolopyrimidine-derivatives. During the earlier quality control of thiazolopyrimidines we observed full selectivity in the mannan ELLA for murine over human Langerin and DCSIGN (Fig. 2 D). This also holds true for the most active inhibitors of murine Langerin function (44, 91, Fig. S12). Most importantly, monovalent primary site ligands are rather promiscuous for CLRs with Kd values of mannose for human and murine Langerin and DC-SIGN being 5.6 mM, 7.0 mM, and 3.5 mM, respectively27-28. High specificity as well as less conserved binding modes were reported for secondary site binders and allosteric inhibitors in nonGBPs earlier29-30 which is in agreement with the nonadditive SAR and results from STD NMR epitope mapping. Hence, thiazolopyrimidine-derivatives most likely interact with a secondary site and inhibit Langerin allosterically. To further strengthen our hypothesis of allosteric inhibition of Langerin receptor function, we titrated the primary site ligand mannose as well as 91 to 15N-labeled Langerin CRD and recorded 1H-15N HSQC NMR spectra. Here, 91 and mannose perturbed different resonances (Fig. 4 E), which was also observed for 4 (Fig. S13). In contrast, the ligands 14 and 168, however, only showed the appearance of two new peaks rather than CSPs (Fig. S13). Thus, mannose and thiazolopyrimidine-derivatives likely interact with different pockets, i.e., mannose bind-

ing to the primary site and thiazolopyrimidines to an allosteric secondary site. Next, we focused our investigations onto the potential allosteric mechanism. For this purpose, we tested the primary site inhibitors mannose, mannan, and EDTA for competition in a flow cytometric IC50 assay (Fig. 4 D) using thiazolopyrimidine-derivatized beads and an SPR assay using a thiazolopyrimidine 4-derivatized PEG surface (Fig. 4 F - H). The addition of EDTA and mannose inhibited binding of Langerin to the thiazolopyrimidine scaffold. Thus, the presence of Ca2+ is essential for thiazolopyrimidine recognition, while mannose decreases the affinity of thiazolopyrimidines for their binding site, which was also observed for 91 in a STD NMR competition experiment (Fig. 4 I). We hypothesized that thiazolopyrimidine inhibitors being affected by the Ca2+ concentration, their inhibitory effect may be caused by reducing the Ca2+ affinity and consequently would translate into a reduced affinity for carbohydrates. Hence, we determined the affinity of Langerin for Ca2+ in the presence and absence of 91 via 1H-15N HSQC NMR titrations (Fig. 4 J). The affinity was identical in both cases (Kd,Ca,DMSO = 176 ± 18 µM and Kd,Ca,91 = 179 ± 17 µM) and in the same range as the Ca2+ affinity reported for the human homolog27. On the other hand, 4, 44, and 91 reduced the affinity of Langerin towards mannan in an ELLA about ten-fold (Fig. 4 K, EC50,DMSO = 3.9 ± 1.1 nM, EC50,4 = 53.6 ± 0.4 nM, EC50,44 = 40.9 ± 1.3 nM, EC50,91 = 31.2 ± 3.0), which is a typical behavior of an allosteric inhibitor. Overall, the presence of thiazolopyrimidines decreases the affinity of Langerin towards carbohydrates but not for Ca2+.

Discussion In this study, we performed a fragment screening against murine Langerin followed by orthogonal validation experiments (Fig. 1, 2) and established a SAR (Scheme 1, Fig. 3, Tab. S3-5). As a result, we were able to develop inhibitors with double digit micromolar activity based on a thiazolopyrimidine-5-one scaffold. Interestingly, these inhibitors likely interact with a secondary site, leading to an allosteric modulation of the target receptor function (Fig. 4). With these data in hand, we propose a mechanistic model for the allosteric inhibition of thiazolopyrimidine series (Fig. 5). Binding of the cofactor Ca2+ was unaffected by our inhibitors (Fig. 4 I), while the affinity of thia-



zolopyrimidines was reduced in presence of carbohydrates and Ca2+ (Fig. 4 F, H). Conversely, carbohydrate binding was reduced in the presence of thiazolopyrimidines (Fig. 4 J). Thus, in our model the allosteric site is formed upon Ca2+ binding and is able to perturb the primary carbohydrate site, but does not affect Ca2+ interaction itself. Vice versa, the primary carbohydrate site can modulate the interaction of the inhibitors in the allosteric site (Fig. 5).

Figure 5. Proposed allosteric inhibition ‘switch’ mechanism of thiazolopyrimidines binding to murine Langerin. 2+

(a) Binding of the cofactor Ca induces the formation of the thiazolopyrimidine binding site. (b) Carbohydrate recognition by the conventional recognition site allosterically blocks thiazolopyrimidine binding and (c) Thiazolopyrimidine binding allosterically blocks carbohydrate recognition.

Secondary binding sites exist in several protein families. An analysis of 24 crystallography-based fragment screenings revealed that 67% of the analyzed targets harbored secondary sites in various protein families including chaperones, translation factors, DNA ligases, Kelch-like proteins, ATPases, GTPases, kinases, and proteases. Interestingly, these secondary binding sites were evolutionary conserved and displayed physico-chemical properties comparable to primary sites31. For CLRs, all recent reports applying fragment screening and including structural information on the target site led to the discovery of fragments interacting with a secondary site11, 32-33. Interestingly, this notion can also be expanded to fragment-based campaigns against other GBPs13, 34. Nevertheless, these reports have in common that the targeted secondary sites are close to the primary site and the inhibitory effect was caused by either a steric clash11, 13 or by linking the fragments to a primary site carbohydrate ligand32-34. Here, we report the discovery of drug-like allosteric inhibitors for another CLR and GBP, hence we propose that targeting secondary sites in this challenging target class5-8 might be the method of choice for future drug discovery campaigns. The discovery of an allosteric site in CLRs is in line with our previous reports on an evolutionary conserved allosteric network regulating Ca2+ recognition in human Langerin27, which is also activated upon interaction with carbohydrates that do not bind to the primary site35. This

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could indicate that the existence of secondary sites is common in CLRs and that they are able to communicate via an allosteric network with the primary site. In human Langerin residues were identified as hubs in the allosteric network, which mediated the information transition27. Interestingly, hot spots in murine Langerin that were predicted to accommodate drug-like molecules via organic solvent mapping in FTMap are closely located to regions that undergo major changes upon titration of carbohydrates in human Langerin (Fig. S14)35. Possibly, the thiazolopyrimidines interact with a hub residue that is located in a secondary site. Taking into account our insights into secondary sites identified for the related CLR DC-SIGN and a recent structure of E-selectin in complex with a glycomimetic inhibitor, we propose that such hub residues could be located close to the long loop structure11, 36. As allosteric networks are evolutionary conserved among members of a protein family, they are most likely an inherited feature of at least some CLRs27. This assumption is further supported by reports on allosteric regulation of members of the galectin family. Here, allosteric inhibition was reported for natural products binding to galectin-337, a fragment binding to galectin-738, and peptide mimetics binding to galectin-139, as well as allosteric activation by therapeutic peptides interacting with galectin-1, 2, 7, 8N, 8S, and 9N40. Thus, the identification of the first drug-like allosteric inhibitor of a CLR promises the possibility to also develop allosteric inhibitors against other members of this largest family of mammalian lectins. Whether drug-like allosteric or secondary site CLR inhibitors have in vivo applications is still to be proven. The development of chemical probes is a first step14 on the way to new treatments applying such inhibitors5. The pan-selectin inhibitor GMI-1070 (Rivipansel), which is a glycomimetic and currently in phase 3 clinical trials to treat sickle cell crisis, displayed 4 µM activity in a sialyl Lewis a ELLA against E-selectin4. While the potency of thiazolopyrimidines is still about ten-fold lower and has to be improved further to serve as chemical probes for murine Langerin, this compound series already offers a clear selectivity profile against related CLRs (Fig. 2 D, S12), as well as several inactive negative controls with the same chemotype (Fig. 3, Tab. S4). As the most active thiazolopyrimidine analogs 44 and 91 are still comparably small with 317 and 347 Da, this series represents a suitable starting point for developing the first high-quality chemical probe of a CLR to test possible applications for allosteric CLR inhibitors.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Detailed experimental methods; supplementary Figures S1 – S14 and Tables S1 – S5; additional results and discussion.


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AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected]

Present Addresses J. Aretz: Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany

Author Contributions All authors have given approval to the final version of the manuscript. ⊥These authors contributed equally.

ACKNOWLEDGMENT We thank the Max Planck Society and the German Research Foundation (DFG, RA1944/2-1) for financial support. This work has been supported by iNEXT, grant number 653706, funded by the Horizon 2020 programme of the European Commission. Olaf Niemeyer is acknowledged for NMR technical support and Elena Shanina for her help with protein expression. We further thank Prof. Dr. Peter H. Seeberger for support and helpful discussions.

ABBREVIATIONS APC, antigen presenting cell; CLR, C-type lectin receptor; CSP, chemical shift perturbation; DETC, dendritic epidermal T cells; ELLA, enzyme-linked lectin assay; GBP, glycanbinding protein; GE, group efficiency; HSQC, heteronuclear single quantum coherence; LC, Langerhans cell; LE, ligand efficiency; SAR, structure-activity relationship; SPR, surface plasmon resonance; STD, saturation transfer difference.

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Allosteric inhibition of a mammalian lectin - Journal of the American

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