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A High-Throughput “FP-Tag” Assay for the Identification of Glycosyltransferase Inhibitors Zhizeng Gao, Olga G. Ovchinnikova, Bo-Shun Huang, Feng Liu, David E. Williams, Raymond Andersen, Todd L. Lowary, Chris Whitfield, and Stephen G. Withers J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b10940 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Journal of the American Chemical Society

A High-Throughput “FP-Tag” Assay for the Identification of Glycosyltransferase Inhibitors Zhizeng Gao1†*, Olga G. Ovchinnikova2, Bo-Shun Huang3, Feng Liu1, David E Williams1, Raymond J Andersen1, Todd L Lowary3, Chris Whitfield2 and Stephen G Withers1,4* 1 2 3

Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1 Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2G2

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Department of Biochemistry and Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada Supporting Information Placeholder

ABSTRACT: Bacterial capsular polysaccharides are im-

portant virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing -3-deoxy-Dmanno-oct-2-ulosonic acid (-Kdo). The -Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive anti-virulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for -Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This “FP-tag” assay should be readily adaptable to highthroughput screens of other glycosyltransferases.

Glycans play vital roles in biology, mediating a number of cellular processes through their locations on cell surfaces or extracellular proteins.1,2 Assembly of these glycans is accomplished by glycosyltransferases (GTs) that use activated donors to transfer a monosaccharide to the specific acceptor for that enzyme. Since a number of human diseases are related to inappropriate glycosylation, their inhibition is an attractive therapeutic approach.3–5 Microorganisms also frequently possess a glycan coat, thus the assembly of capsular polysaccharides (CPS) and lipopolysaccharides (LPS), in Gram-negative bacteria, both offer targets for therapeutics.6–8 High-throughput screening for inhibitors of these GTs is therefore an attractive approach to new therapeutics. However, design of efficient screens for GTs has proved challenging as the reaction catalyzed by these enzymes is not accompanied by a useful change in UV/Vis absorbance or fluorescence. Perhaps the most common approach to the design of plate-based GT screens is one in which transfer of the glycosyl moiety to an acceptor attached to the plate surface

or beads is detected, in a stopped fashion, using a fluorescent donor analogue or a fluorescence-tagged antibody or lectin. Since this involves at least one washing step to remove unbound fluorophore, automation is needed if a larger scale screen is performed. Another limitation is that many GTs do not easily transfer to immobilised ligands. Approaches not requiring a washing step include coupled assays where the reaction product is quantified9 or in which “in situ” synthesis of the NDP sugar donor occurs with release of a chromophore.10,11 An alternative is the detection of loss of substrate as a consequence of attachment of the sugar moiety, as in the protease protection assay.12 The classic coupled assay of Gosselin et al 13 has not proved generally useful due to challenges in detection of NADH oxidation in such assays,14,15 although the luminescence version has proved useful in secondary screens. 16 Approaches using mass spectrometry 17 are not accessible as truly high-throughput approaches to most labs. The need for washing steps can be avoided by use of fluorescence polarization (FP) in which plane-polarized light is used to excite a fluorophore. If that fluorophore is immobilized, its emitted light will retain the initial polarization, while if it tumbles in solution it will emit light in different planes from that of the initial excitation. Thus attachment of a fluorophore to a slowly tumbling macromolecule is readily detected through its FP signal. A substrate displacement FP assay has been used 18–20 in which fluorescence-tagged sugar-nucleotide analogs are displaced by the inhibitor from the GT of interest, thereby decreasing the FP signal (Figure 1A). However this approach requires high affinity fluorescent substrate analogs, is only likely to identify molecules that bind in the donor site and uses large amounts of enzyme.

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The inner core of E. coli LPS has a di- or tri-Kdo structure incorporating an -(14) linkage(s) connecting the glycan backbone to the lipid A moiety23 while the so-called “group 2” CPS produced by extra-intestinal pathogenic Escherichia coli (ExPEC), Neisseria meningitidis, Haemophilus influenzae and others contains several Kdo sugars with alternating -(24) and -(27) linkages8,24,25 connecting the repeat unit CPS polysaccharide to phosphatidylglycerol. Because the Kdo glycosyltransferases involved in both LPS and CPS assembly use CMP--Kdo as donor, the different stereochemistries of these linkages dictate that the GTs involved in their assembly must employ different mechanisms, one being a retaining GT and the other inverting. Accordingly, the retaining -Kdo transferase shares no significant homology with the inverting -Kdo transferases or indeed with any known GTs, thus forming a separate GT family.26 The importance of the CPS to bacterial virulence, the absence of Kdo in mammals, and the unique nature of the -Kdo transferase enzyme class combine to make this a good candidate for the development of therapeutics that specifically target bacterial capsule formation. Figure 1. A) Walker’s substrate displacement assay.18–20 Fluorescencetagged sugar-nucleotide analogs are initially bound to the GT, which results in a high FP signal. Once the ligand is displaced by a more potent binder, the FP signal decreases. B) Paulson’s catalytic FP assay.21 Fluorescencetagged sugar-nucleotide analogs are transferred to fetuin by sialyltransferases or fucosyltransferases, generating a high FP signal. The addition of inhibitors lowers the FP signal. C) This work: the FP tag assay. Fluorescence-tagged sugar-nucleotide analogs are transferred to a biotin conjugate by the target GT, and a high FP signal is generated when streptavidin is added. The addition of inhibitors lowers the FP signal.

The Paulson group developed a “catalytic” FP-based assay for sialyl- and fucosyltransferases21 in which the transfer of fluorescence-tagged sugars to asialo-fetuin or fetuin (ca. 50 kDa), generates a strong FP signal (Figure 1B). This assay requires much less enzyme and can identify inhibitors that target both the donor and acceptor binding sites. However it only works for GTs that transfer to proteins and the cognate protein acceptors are not always known or available in quantity. We aimed to develop a generalizable version of the Paulson assay based upon GT-catalyzed transfer to a biotinylated acceptor that could subsequently be bound to streptavidin (52 kDa) as a surrogate protein, thereby allowing use of FP detection in a simple assay with no washing steps (Figure 1C). This “FP-Tag”assay could also be performed using a fluorescent acceptor and a biotinylated donor. Specifically, we wanted to develop an assay for a recently discovered GT that is involved in the assembly of some bacterial CPS. Many bacteria coat their surfaces with a CPS to serve as a defensive structure against other microorganisms and against host defence mechanisms. Additionally, Gramnegative bacteria assemble a layer of LPS on their surface.8,22 Both LPS and a well distributed class of CPS incorporate Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) as a key component of their structure, but the glycosidic linkage formed is different in the two cases (Figure 2).

Figure 2. Kdo linkages in E. coli CPS (left) and LPS (right).

The enzyme responsible for extension of the -Kdo oligosaccharide is KpsC.24,26 Establishment of a high throughput screening assay for E. coli KpsC is significantly more challenging than for most GTs. There are three major obstacles. (i) KpsC has two closely (sequence) related catalytic domains that form the alternating (24) and -(27)-Kdo linkages. The full-length enzyme generates products with heterogeneous chain lengths, making comparison of activities from differentially inhibited reactions difficult.27 To avoid such complications a truncated KpsC construct containing only the N-domain was employed to transfer a single residue; this also has the advantage of better solubility and expression yield. This protein catalyzes a single Kdo addition, forming the -(24) linkage.27 (ii) the short (~30 min) halflife of the donor sugar nucleotide, CMP--Kdo, makes it impractical to directly use this donor substrate in screens.28 Thus, in situ generation of CMP--Kdo by a CMP--Kdo synthetase, KdsB,24 is needed. (iii) KpsC Ndomain requires a -(27)-linked di-Kdo acceptor, which is a synthetically challenging molecule but this was resolved (Scheme S1). We initially planned to synthesize a derivative of Kdo bearing a fluorophore at C-8 and monitor its addition to biotinylated acceptor by KdsB/KpsC using FP (Figure S1A). Unfortunately, the system proved incapable of its transfer. However, 8-N3-Kdo29 was accepted well by both


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KdsB and the N-domain of KpsC; thus, click additions were instead performed after the enzymatic reaction. The terminal 8-N3-Kdo residue did not serve as an acceptor for further chain extension, precluding investigation of the dual domains of full-length KpsC (Figure S2). With the sugar donor established, we moved to evaluating the relative merits of biotinylated vs BODIPY-modified (27)-linked di-Kdo acceptors (1, Figure 3A). The transfer of 8-N3-Kdo to both acceptors occurred at comparable rates, as was demonstrated by LC-MS analysis (Figure S3). However an assay format using fluorophore-labeled acceptor has two advantages over biotin-labeled acceptor: (i) The final step in both strategies (Figure S1B,C) is the addition of streptavidin and BODIPY- or biotin-alkyne. To push the click reaction to completion, an excess of alkyne is needed but excess BODIPY-alkyne would decrease the overall FP reading since the FP is the ratio between streptavidin-bounded BODIPY and the free BODIPY. In contrast, an excess of biotin-alkyne in the reaction would not compromise the FP signal intensity. (ii) The fluorophore labeled acceptor provides convenient detection options, so the same compound can be used for both the “FP-tag” HTS assay and secondary TLC- and/or HPLC-based assays used for validation.27 Thus, the final assay format included transfer of 8-N3-Kdo to acceptor 1 (Figure 3A). We employed the copper-free dibenzocyclooctyne (DBCO)-mediated click chemistry in the “FP-tag” assay, as shown in Figure 3A, because fewer operations are needed than for copper-catalysed reactions. To confirm that binding of the BODIPY fluorophore to streptavidin generates an FP signal, we directly monitored binding of the product of the Kdo-transferase reaction, 2 (1.0 µM) to the Streptavidin–Biotin–DBCO (SBD conjugate, 1:4) by FP. As seen in Figure 3B, the FP signal increases as the click reaction proceeds, reaching a plateau at ~ 5 h when using higher (2.4 – 3.6 µM) concentrations of SBD, and 15 h using 1.2 µM. A final FP reading of ~ 260 mP, corresponding to a S/N ratio of almost 10, indicates that the assay is robust and easily adaptable to HTS campaigns.

Figure 3. A) Step 1. Enzymatic step adding 8-N3-Kdo to 1. Step 2. The SBD “FP tag” is “clicked” onto the transfer product. B) Time courses for the “click” reaction of 2 (1.0 µM) and 1.2, 2.4 and 3.6 µM Streptavidin–Biotin– DBCO (1:4) monitored by FP. C) Time course for the KpsC reaction monitored by FP.


Encouraged by these results, we explored optimal conditions for a screen. Continuous monitoring of this process with all reagents in place proved not to be feasible, as the binding of the biotinylated ligand to SA and the click reaction with 8-N3-Kdo yields the very large streptavidin-bound complex, which cannot be processed by KdsB or KpsC. A stopped assay was thus developed in which aliquots of the reaction mixture were drawn at different time points and mixed with excess Kdo (to quench the reaction as it is a much better KdsB substrate than 8N3-Kdo) and the SBD conjugate. After 12 h of incubation to let the click reaction go to completion, the FP signals of all aliquots were measured. As seen in Figure 3C the rate of the glycosylation reaction is proportional to the KpsC concentration and reaction proceeds to completion within 3 h when using 60 ng/µL KpsC. The utility of the stopped assay was then assessed by measuring the IC50 of the substrate analog cytidine diphosphate: 0.8 mM (Figure S4).

Figure 4. A) Marine sponge screening results. B) IC50 plot of aeroplysinin1 and verongiaquinol: relative activity vs concentration (M).

To evaluate the performance of this “FP tag” assay in a real high-throughput assay, a collection of about 1000 marine sponge extracts that has proved fruitful previously was screened in a 384-well format against KpsC. The assay was robust, yielding a very solid Z'-factor30 of approx. 0.9, and identified several hits (Figure 4A). Extensive fractionation of the marine sponge extracts, guided by the “FP-tagged” assay, identified two known natural products, aeroplysinin-1,31,32 and verongiaquinol33, as useful lead inhibitors, with IC50 values of 7.9 and 3.3 µM, respectively (Figure 4B). A secondary TLC-based assay (Figure S5) confirmed that the observed inhibition was due to slower reaction of KpsC/KdsB, while use of a KdsB assay confirmed that KpsC was the inhibitory target and not KdsB. It has been suggested that reagent costs of less than 50 cents USD/well are necessary for a large screen to be economically viable.34 Beyond the acceptor and donor sugar,



which will be needed for any assay and for which only very small quantities are used, the most expensive reagent employed in this approach is streptavidin for which about 2 mg are needed per plate. Bulk streptavidin can be purchased at 1 g for about $3500, thus the cost per well is less than 2 cents. The low cost and robust S/N ratio suggest that it is feasible to screen very large compound collections with this assay. In summary, the “FP tag” HTS assay we have developed should be useful for a wide range of GTs, or indeed for a range of enzymatic reactions in which two small molecules are coupled. The assay works particularly well for enzymes that can accommodate a donor sugar modified by biotin or a fluorophore, since reaction can then either be monitored directly or after a single addition of streptavidin. Indeed, many GTs of interest, such as sialyl, fucosyl21 and HexNAc transferases,35 have such relaxed substrate specificity. However, as demonstrated here, the screen can also be performed using an azidosugar and derivatised with biotin or a fluorophore after the enzymatic reaction is complete. In this respect we note that a number of GTs have been shown to transfer azide-modified derivatives of the parent monosaccharides.36 Our pilot screen of marine sponge extract collections proceeded with an excellent Z'-factor (0.9) and identified two low M inhibitors, demonstrating the practical applicability of this approach. We envision that this “FP tag” assay will find broad application in the search for potent GT inhibitors, as well as in directed evolution of these enzymes.

We thank GlycoNet, the Canadian Network of Centres of Excellence in glycoscience for financial support: doi: 10.13039/501100009056. TLL and CW are recipients of Canada Research Chairs.

ASSOCIATED CONTENT

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ACKNOWLEDGMENT We thank Dr. Tom Pfeifer, CDRD, Vancouver for helpful discussions.

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Supporting Information:

Synthetic procedures and characterisation of products; experimental procedures for the FP Tag assay and IC50 measurements; procedures for isolation of aeroplysinin-1 and verongiaquinol (3,5-dibromoverongiaquinol) from the sponge tissue.

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AUTHOR INFORMATION Corresponding Authors

10.

Zhizeng Gao: [email protected] Stephen G Withers: [email protected]

11.

Present Addresses

School of Marine Sciences, Sun Yat-sen University, Zhuhai, Guangdong, China, 518082 ORCID:

12.

Stephen G Withers 0000 0002-6722-5001

Todd Lowary 0000-0002-8331-8211

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Chris Whitfield 0000-0002-5267-7027 Funding Sources

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