Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to

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Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to Enzymes of the Human ST8Sia Family Gesa Volkers, Christian Lizak, Jürgen Niesser, Frederico I. Rosell, Johannes Preidl, Vinayaga S. Gnanapragassam, Ruediger Horstkorte, Jörg Rademann, and Natalie C.J. Strynadka ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00478 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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ACS Chemical Biology

Table 1: Half m axim al effective dose (EC50 a ) of fluorescent NeuAc5-CM P m im etics with respect to hum an ST8Sia fam ily enzym es. Probe

hST8SiaII hST8SiaIII hST8SiaIV EC50 [nM ] EC50 [nM ] EC50 [nM ] ( R )-1- m 44.8 ± 7.2 30.7 ± 4.7 29.5 ± 1.2 ( S )-1- m 42.4 ± 3.1 22.7 ± 1.1 17.2 ± 0.3 ( R )-1- p 40.1 ± 9.8 25.1 ± 4.6 24.7 ± 0.5 ( S )-1- p 13.1 ± 2.7 3.8 ± 0.8 6.4 ± 0.1 ( R )-1-G- m 27.5 ± 5.0 10.0 ± 1.3 23.0 ± 1.3 ( S )-1-G- m 34.5 ± 11.0 26.6 ± 5.5 11.5 ± 0.4 GABA-L-Pal 24.4 ± 4.4 6.0 ± 1.0 13.2 ±0.2 a EC50 are identical to Kd values if the fluorescence intensities of the bound ligand (IB) are equal to those of the free ligand in solution (IF), i.e., the ratio Q = IB/IF is close to 1.

Table 2. Displacem ent properties (IC50) of CDP and 1- p Ac . Probe CDP 1- p Ac 1- p Ac + 1% DM SO

hST8SiaII IC50 [μM ] 44.6 ± 12.9 43.8 ± 2.8 23.1 ± 2.1

hST8SiaIII IC50 [μM ] 0.62 ± 0.02 0.62 ± 0.07 0.78 ± 0.05

ACS Paragon Plus Environment

hST8SiaIV IC50 [μM ] 13.1 ± 0.6 32.0 ± 4.3 48.2 ± 4.1

1

a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b

c

d


meta or para Page 2 of 25

*

CMP-Neu5Ac R = methyl

fluorescein

glycine

*(R) or (S)

Compound 1 DP2

ST8SiaIV

ST8SiaIII

ST8SiaII

ST8SiaIV

ST8SiaIII

ST8SiaII

ST8SiaII ST8SiaIII

ST8SiaIV

Enzyme (S)-1-m diSia


(S)-1-m oligoSia polySia

a

Page 3 of 25 1 Trp322 2 3 4 Tyr336 5 6 7 8 9 10 His337 11 12 13 14 15 16 Asn167 17 18 19 Asn190 20 21 22 23 24 25 26 27 Trp350 28 29 30 31Donor analogue 32CMP-3FNeu5Ac 33 Donor mimetic 34 (S)-1-mAc 35 36 37 38 39 40 41 42 43 44 Asn167 45 Asn190 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

b


350-353

Gln351

Trp322

Trp350

His354 Ser300 (S)-1-mAc

His354 Ser300

His337

Thr301 Gly302

(S)-1-mAc

Asn167 Asn190

Thr301 Gly302

d

c

e

Glu352

Tyr336

His354

O8’

Acceptor Sia-6S-LacNAc

f


Loop 348-354

Donor mimetic (S)-1-mAc CDP complex

1 2 3 4 5 6 7 8 9 10 11 12 13 14

0

10

50 100 µM Polysialic acid

b

10


50

100

µM MAA SNA

NCAM

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c

1.20

0

10

50

100

*

**

1.00 Slope (1/hr)

a

0

0.80 0.60 0.40 0.20 0.00

GAPDH

µM (S)-1-G-m ACS Paragon Plus Environment

* p< 0.0012 ** p< 0.0001

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Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to Enzymes of the Human ST8Sia Family Gesa Volkers†,‡, Christian Lizak†,‡, Jürgen Niesser†‡, Frederico I. Rosell†‡, Johannes Preidl§, Vinayaga S. Gnanapragassam║, Ruediger Horstkorte║, Jörg Rademann§, Natalie C.J. Strynadka†,‡ † Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada. ‡ Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada. §

Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße

2+4, 14195 Berlin, Germany. ║

Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystr. 1,

D-06114 Halle/S., Germany.


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Introduction Polysialic acid (polySia), the α-2,8-linked homopolymer of sialic acid, is a posttranslational modification of glycoconjugates, localized on the surface of a wide variety of eukaryotic cells. PolySia often serves as a modulator of cellular interactions and a key regulator of axon growth and maturation, progenitor cell migration and differentiation in the developing mammalian brain.1 The repulsive properties of polySia are derived from the hydration shell it attracts due to its hydrodynamic radius and extensive electronegative charge.2 The polysialyltransferases (polyST) ST8SiaII and IV synthesize polySia on terminally sialylated complex N-glycans protruding from a variety of protein acceptor substrates using CMP-sialic acid (CMP-Neu5Ac) as the donor substrate. PolySTs are highly expressed in developing neural tissue in embryos and newborns with distinct preference for the glycosylated protein acceptors they act on, the most well characterized of which are the neural and synaptic cell adhesion molecules NCAM, SynCAM-1 and neuropilin-2.3–6 The overexpression of polySTs in adults, primarily ST8SiaII, is almost exclusively linked to cancer metastasis with a poor clinical prognosis.7,8 Polysialylation on cell surfaces is a driving factor for metastasis in small cell lung cancer, glioblastoma and neuroblastoma and conveys chemoresistance to tumors under hypoxic conditions.9–13 Concordantly, the development of a drug against polySTs could be highly beneficial for treatment of metastatic cancer, but highthroughput screening for polyST inhibitors has been challenging, partially due to the inaccessibility of purified enzymes as well as the lack of molecular tools and probes.14 Recently, we established the expression and purification of the two human polySTs, ST8SiaII and IV and of a third member, ST8SiaIII. We identified a new sulfated acceptor sugar for ST8SiaIII (Sia-6S-LacNAc, corresponding to terminally sialylated keratin sulfate) but its acceptor protein specificity in vivo is so far unknown. Subsequently, we described the first X-ray crystallographic structures of ST8SiaIII, in the apo-enzyme form, and in complex with donor analogs as well as a ternary complex with an inert donor and acceptor sugar Sia-6S-LacNAc.15 These studies


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established several central molecular principles for substrate binding and catalysis for the mammalian ST8Sia family. Existing sialyltransferase inhibitors are in most cases derived from cytidine analogs in combination with transition state mimics as summarized by Wang and coworkers.16 Often their IC50 or Ki values do not exceed the low micromolar range with affinities in the same order of magnitude, and they lack specificity. Al-Saraireh and coworkers previously proposed the druggability of ST8SiaII and showed that CMP is a competitive inhibitor of polySTs with a Ki in the micromolar range combined with low cellular toxicity in vitro.17 Fukuda et al. demonstrated that C5 or OH-2’ methylated CMP-sialic acid analogs inhibit all polySTs and to a different extent members of the mono-sialyltransferase family.18 We have recently developed a novel class of fluorescent compounds with KD values in the tens of nanomolar range for two mammalian mono-sialyltransferases (rat ST3GalI and human ST6GalI) as well as two prokaryotic mono-sialyltransferases.19 Their characteristic molecular structure 1 (Figure 1, panel a, compounds 1) is derived from the CMP-Neu5Ac donor scaffold, where the natural 2-phosphorylated sialic acid Neu5Ac is replaced by amino-phenyl-hydroxymethyl phosphonic acid with the phosphonic acid residue mimicking the C1 carboxylate.19 In the variants characterized, the aromatic amine occupies the meta- or para-position (m, p) and is either acylated with carboxyfluorescein with or without glycine (G) as an additional spacer, or acetylated without carboxyfluorescein (1-Ac). Asymmetric carbon 1 is either R- or S-configured. Presence of the fluorophore label and the high affinity binding would render these CMP-Neu5Ac mimetics a useful tool for the development of high-affinity inhibitors and probes for the human ST8Sia family. Additionally, their lipophilicity is likely to be sufficient to permeate membranes and reach the Golgi-localized enzymes, and their molecular scaffold may be the starting point for further inhibitor development.19 In this study we use fluorescence polarization assays to determine the binding affinities of these CMP-Neu5Ac mimetics for human poly- and oligoSTs. We demonstrate their use as screening


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tools in displacement assays with the known sialyltransferase inhibitor CDP and with a nonfluorescent racemate.20 X-ray crystallographic structure determination of ST8SiaIII in complex with one of the CMP-Neu5Ac mimetics shows two unique binding modes and demonstrates their transferability for testing of all other members of the human polysialyltransferase family.

RESULTS AND DISCUSSION Affinity of CMP-Neu5Ac Mimetics for ST8SiaII-IV. To probe the binding of the fluorescent CMP-Neu5Ac mimetics to human poly- and oligoSTs, 10 nM of each fluorescent compound was titrated against serial dilutions of recombinantly expressed and purified enzyme (Supplementary Figure 1). We determined the half maximal effective dose (EC50) of the protein-ligand complexes for recombinant polysialyltransferases ST8SiaII and IV, as well as the oligosialyltransferase ST8SiaIII by fluorescence polarization measurements. For ST8SiaII all tested compounds showed EC50s in the tens of nanomolar range, with (S)-1-p exhibiting the lowest EC50 of 13.1 ± 2.7 nM (Table 1, Supplementary Figure 2). We observed the same trend for ST8SiaIII and IV where (S)-1-p again exhibited the lowest EC50’s with 3.8 ± 0.8 nM and 6.4 ± 0.1 nM, respectively (Table 1, Supplementary Figures 3 and 4). The EC50 obtained here for hST8SiaII-IV are in the same range as for the monosialyltransferases rST3GalI and hST6GalI measured by Preidl and coworkers, demonstrating that the high binding affinity makes these compounds potent tools for assay development for this interesting class of enzymes.19 The differences in EC50’s between all compounds tested here were marginal (within 2-3x) which highlights their transferability, but also the challenge in developing donor-based inhibitors with specificity for the active site of a given mono-, oligo- or polyST. The attachment of other fluorophores could be exploited. The total change in fluorescent polarization (∆mP) for all compounds was higher than 220 (Supplementary Table 1) at a probe concentration of 10 nM, demonstrating the reproducibility and robustness of the assay while using low amounts of material.


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Next, we determined the half maximal inhibitory concentration (IC50) of CDP and the racemate 1-pAc (the non fluorescent core scaffold of all compounds tested here, Supplementary Figure 5) using a fluorescence polarization displacement assay, where enzymes bound to compound (S)-1G-m were titrated with serial dilutions of CDP or 1-pAc, respectively. Both CDP and 1-pAc exhibited IC50’s in the low micromolar range for ST8SiaII and ST8SiaIV (Table 2, Supplementary Figures 2 and 4). In the same experiments carried out with the oligosialyltransferase ST8SiaIII and (S)-1-G-m, both CDP and 1-pAc showed a 100-fold lower IC50 compared to polySTs (ST8SiaII and ST8SiaIV), namely 0.64 ± 0.06 µM and 0.69 ± 0.08 µM, respectively (Table 2, Supplementary Figure 3). In comparison to the displacement data published by Preidl and coworkers, we found higher IC50 of CDP and 1-pAc for ST8SiaIII (but not ST8SiaII or IV) compared to the monosialyltransferases ST3GalI and ST6GalI, but only little difference in IC50 between the compounds CDP and 1-pAc (Table 2).19 In the presence of 1% DMSO the values for the IC50 were nearly identical, demonstrating that these probes behave well under buffer conditions for high-throughput compound screening purposes (Table 2, Supplementary Figures 2–4). Although the data indicate that the development of a specific ligand or inhibitor for enzymes of the ST8Sia class is a challenging task, small differences in apparent affinity of the CMP-mimetics do exist. For example (S)-1-p is showing the lowest EC50 for hST8SiaII, III and IV and (R)-1-m the highest EC50 for all three enzymes. The trend of higher affinity from (R)- to (S)-configuration in the chiral center and meta- to para- substitution on the arylamide seems to hold true for the compounds with directly attached fluorophore, but not for compounds (R)-1-G-m and (S)-1-G-m that have a glycine spacer between fluorophore and aryl group. One could argue that the glycine spacer adds more flexibility to the arylamide-fluorophore, which makes a contribution of the aromatic substitution position to binding affinity less relevant. In fact it is well known that there is some tolerability in the sialyltransferase active site for binding CMP-donors or inhibitors with


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differences in the sugar portion, suggesting that the attachment of the fluorophore itself is not a crucial factor for the binding properties of this compound class.21 Inhibition of Enzyme Activity. To test the influence of the CMP mimetics on the activity of human ST8SiaII, III, and IV, we carried out high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) analysis of in vitro sialylation reaction products of hST8SiaII–IV in the presence and absence of (S)-1-m. In the absence of (S)-1-m and under the conditions of the assay using CMP-Neu5Ac as donor and Neu5Ac-2,8-Neu5Ac as acceptor, anion-exchange chromatography coupled with HPLC of the reaction product of ST8SiaII showed that it contained polySia chains up to ~18 Neu5Ac units long (Figure 1, panel b, Supplementary Figure 6). In contrast, the HPLC elution profile of ST8SiaIV reaction products revealed longer chains with up to ~25 units while ST8SiaIII catalyzed the synthesis of ~7–8 units, resembling oligoSia (Figure 1, panel b) which is consistent with previous HPLC analyses by other groups22. In the presence of 10 µM (S)-1-m, HPLC analysis of the reaction product showed that the yields of oligo- and polySia chains were lower for ST8SiaII/IV and to a lesser extent for ST8SiaIII (Figure 1, panel c), but enzymatic activity was not completely abolished. Interestingly, the inhibition patterns observed in the product distribution were slightly different for the three enzymes. Whereas inhibition of ST8SiaII appeared most effective for a product length of ~9 to 19 sialic acid units (i.e., the difference profiles shown on the right side of Figure 1, panel c), (S)-1-m exerted its inhibitory influence on ST8SiaIV activity at a shorter chain length. We hypothesize that these differences in inhibition of certain product lengths of oligo- or polySia could be due to differences in the release and recapture of acceptor chains that may be rate-limiting at certain chain lengths. Furthermore the acceptors used in this study likely have lower affinity compared to naturally occurring glycan acceptors of the ST8Sia family, which are covalent posttranslational modifications of their respective protein acceptors where binding of both the acceptor protein and glycan exert avidity effects on the catalytic activity.


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In the TLC of the reaction products of ST8SiaII-IV using CMP-Neu5Ac donor and a fluorescently labeled boron dipyrromethene (BODIPY)-disialyllactose acceptor in the absence of (S)-1-m, we observed that ST8SiaII and ST8SiaIV synthesized an oligo- to polysialic acid chain product, whereas the oligosialyltransferase ST8SiaIII showed synthesis of shorter oligosialic acid chain products (Figure 1, panel d, lanes 3, 9, and 5). This is consistent with the analysis of reaction products by HPLC. In the presence of 100 µM (S)-1-m the activity of ST8SiaII and ST8SiaIV was almost completely diminished as observed by TLC, with only small amounts of oligosialic acid synthesized, while ST8SiaIII showed nearly complete inhibition with no oligosialic acid products visible (Figure 1, panel d, lanes 4, 10, and 6).

Structure of hST8SiaIII crystallized with (S)-1-m. We determined the structure of human ST8SiaIII co-crystallized with (S)-1-m to 2.15 Å resolution (Supplementary Table 2, Figure 2). ST8SiaIII is a dimer in solution and we observe one copy of the ligand in the active site of each monomer comprising the biological dimer of ST8SiaIII (Figure 2, panels a and b). The nucleotide, aryl-phosphonate and amide group at the meta-position of (S)-1-m are well ordered in the electron density map of both active sites (Supplementary Figure 7, panels a and b), with Bfactors comparable to surrounding amino acids (~20–50 Å2). The fluorescein fluorophore is disordered in both active sites of the dimer and could not be modeled, likely because it is able to rotate and points outward into the solvent, making no apparent interactions with the protein. Thus, the ligand modeled here corresponds to (S)-1-mAc which is the core structure in (S) configuration common to all fluorescent CMP-Neu5Ac mimics described in this study. In the following description of its binding mode we will refer to the refined ligand as (S)-1-mAc.19 It is not unusual to observe partially disordered ligands, backbone atoms, or side chains in sialyltransferase or glycosyltransferase structures, especially in the highly dynamic active site loop region, and this has been observed before for ST8SiaIII.15,23 In both monomers of the dimer the CMP portion of (S)-1-mAc shows similar interactions with protein backbone as in the


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previously published nucleotide complexes of ST8SiaIII bound to CDP or CTP (PDB 5BO6 and 5BO7).15 The cytidine interacts via hydrogen bonds with the protein backbone of residues Trp322 and Tyr336. The ribose is hydrogen bonded with the side chain hydroxyl group of Ser300 and backbone amide of Thr301–Gly302 while the alpha-phosphate interacts with side chains of Asn167 and Asn190 in both monomers (Figure 2, panels a and b, all residues conserved in hST8SiaII–IV, see alignment in Supplementary Figure 8). All compounds discussed in this study have an arylamide group bound to an alpha-phosphonate via a benzylic carbon position, a hydroxymethyl group. This hydroxymethyl group is positioned similarly to the anomeric C2-atom of the natural donor CMP-Neu5Ac but in the compounds of this study they are bound to a second phosphorus atom of the inhibitor, referred to as terminal phosphonate. In the ST8SiaIII complex with (S)-1-mAc the terminal phosphonate is positioned similarly as its negatively charged counterpart of the natural donor CMP-Neu5Ac, namely the C1 carboxylate characteristic for neuraminic acid sugars and, importantly, interacts with the imidazole side chain of His354. This is demonstrated by the overlap of the ST8SiaIII:(S)-1-mAc complex with our previously published ternary complex of ST8SiaIII (PDB 5BO915, r.m.s.d. of 0.62 Å over 554 common Cαresidues, Figure 2, panel c). This conserved histidine is the key catalytic base of poly- and oligoSTs and activates the C8-hydroxyl group of the acceptor sialic acid.24 In previously obtained ST8SiaIII structures, His354 is either disordered alongside with the active site loop comprised of residues 341–354 or fully ordered in only one of the monomers of the dimer, with varying degrees of disorder in the active site loop of the other monomer. (S)-1-mAc cannot be hydrolyzed because there is no anomeric carbon that is able to form a stabilized planar oxocarbenium ion as in the native neuraminic acid donor. His354 in the (S)-1-mAc complex is likely blocked from its catalytic activity and it may be why it is ordered in both active sites of the dimer. Further differences exist in the active site loop conformations between the (S)-1-mAc and previously obtained CDP/CTP complexes. The backbone of the active site loop alongside with the side chain of His354 of ST8SiaIII is clearly defined in the electron density of both monomers of the (S)-1-


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mAc complex, but with different conformations and varying degree of disorder for some of the other side chains in either monomer (Figure 2, panel d and e). Residues 348-352 of monomer A (but not of monomer B) fold into a helical turn which is another example of the highly dynamic active site that is characteristic for glycosyltransferases. In the CDP complex of ST8SiaIII (PDB 5BO6) for example, the side chains of Lys349 and Trp350 of the monomer A active site loop bind the beta-phosphate of CDP and His354 does not interact with CDP (Figure 2, panel f). Compared to the closed lid conformation in the CDP complex the lid conformation in the (S)-1mAc complex could be described as half-open. A stronger engineered interaction of the donor mimetics with the active site loop, leading to a defined, closed lid conformation, might offer an opportunity to engineer the donor mimetics described here further to be more powerful inhibitors, and at the same time provide an opportunity to increase selectivity between oligo- and polySTs due to their difference in the amino acid sequences of the active site lid.

The differential conformation of the active site loop also translates into two slightly different binding modes of (S)-1-mAc in the homodimer. The backbone carbonyl group of Trp350 in monomer A makes a hydrogen bond with the meta-amide group of (S)-1-mAc (distance 2.9 Å) while the side chains of residues 349–351 are disordered in the electron density. The back of the hydrophobic aryl ring of (S)-1-mAc packs against the side chain of Leu356 of ST8SiaIII. In ST8SiaII and IV this position is a methionine ((Trp350 is absent from ST8SiaII/IV, see Supplementary Figure 8), suggesting that the carbon-carbon van-der-Waals interaction would be maintained. One side of the aryl ring of (S)-1-mAc is packed against His354 in a pi-stacking manner, and the opposite side of the aryl ring faces Tyr336 (conserved in ST8SiaII and IV) in a face-to-edge fashion (Figure 2, panel a). In monomer B the binding mode of (S)-1-mAc is different. Rather than the backbone carbonyl group, the side chain of Trp350 packs against the aryl ring in a pi-pi stacking interaction with a face-to-face displaced geometry (Figure 2, panel b). On the opposite side of the aryl ring the


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imidazole side chain of His354 binds the terminal phosphonate. The amide bond of (S)-1-mAc in monomer B is satisfied by a weak hydrogen bond with the hydroxyl group of Ser353 (distance 3.3 Å). The proximal and terminal phosphonate in both monomers interacts further with the side chains of Asn167 and Asn190 and His337 (all conserved in the ST8Sia family) as well as Ser300 (Thr294 in ST8SiaII, Ser297 in ST8SiaIV). Trp350 is present in hST8SiaIII but in ST8SiaII and IV it is a tyrosine (Tyr330 and Tyr315, respectively). The Trp350 side chain interaction with the aryl ring in monomer B of hST8SiaIII (Figure 2, panel b) is absent in monomer A and is compensated for by the well-conserved Tyr336 of monomer A of ST8SiaIII (Figure 2, panel a), which demonstrates that the ST8SiaIII complex of monomer A is also a good representative for the pi-pi stacking binding mode of these compounds in ST8SiaII and IV. Sialyltransferases transfer sialic acid to glycoconjugates through a trigonal planar oxocarbenium ion-like intermediate in the transition state by partially dissociating the CMP moiety and nucleophilic attack at the anomeric carbon.25 The affinity of this intermediate is considerably higher, and the use of this concept has been established for sialyltransferase inhibitors.16,26 The compounds tested here mimic this intermediate geometry as our crystal structure shows. A phosphonate compared to a carboxylate has a higher negative charge due to being a bivalent acid and likely exhibits greater affinity to sialyltransferases, this is exemplified by the competitive inhibitors CDP and CTP that harbor two and three negative charges.27 Our crystal structure of ST8SiaIII with one of the compounds, (S)-1-mAc, shows that in both monomers of the biological dimer, the aryl ring of the compound is bound with strong pi-stacking interactions, but facilitated by different residues. In monomer A we observe Tyr336 as the main contributor to the aromatic interaction, whereas in monomer B we observe that the side chain of Trp350 is ordered and interacts with the aryl ring. In both monomers the backbone of the active site lid is involved in binding the CMP-Neu5Ac but with different loop conformations. Most residues involved in binding in ST8SiaIII are conserved in the human polySTs, with the exception of Trp350. This


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supports the underlying findings that the compounds studied here work well for all human ST8Sia members, as the interaction is mediated by aromatic amino acids in the active site loop. Altogether, the non-hydrolysable nature of the compounds tested here and their trigonal-planar transition state geometry as well as the aromatic pi-stacking interactions contribute significantly to the high affinity of this compound class to the donor-binding pocket of ST8SiaIII and likely also the polySTs, based on the conserved active site features.28

Effect of CMP-Neu5Ac Mimetics on NCAM. The effect of the CMP-mimetics on the polysialylation of NCAM was analyzed by Western blotting and in cell adhesion assays using Kelly cells, a neuroblastoma cell line (Figure 3). Kelly cells strongly express ST8SiaII and to a lesser extent ST8SiaIV, and it is to our knowledge unknown whether they also express ST8SiaIII.29,30 In the presence of

Structural Basis for Binding of Fluorescent CMP-Neu5Ac Mimetics to

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