Systematic Tuning of Fluoro-galectin-3 Interactions Provides

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Systematic tuning of fluoro-galectin-3 interactions provides thiodigalactoside derivatives with single digit nM affinity and high selectivity Kristoffer Peterson, rohit kumar, Olof Stenström, Priya Verma, Prashant R Verma, Maria Håkansson, Barbro Kahl-Knutsson, Fredrik Zetterberg, Hakon Leffler, Mikael Akke, Derek Thomas Logan, and Ulf J. Nilsson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01626 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Journal of Medicinal Chemistry

Systematic tuning of fluoro-galectin-3 interactions provides thiodigalactoside derivatives with single digit nM affinity and high selectivity

Kristoffer Peterson,a Rohit Kumar,b Olof Stenström,c Priya Verma,a Prashant R. Verma,a Maria Håkansson,d Barbro Kahl-Knutsson,e Fredrik Zetterberg,f Hakon Leffler,e Mikael Akke,c Derek T. Logan,b,d Ulf J. Nilssona*

a

Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Box

124, SE-221 00, Lund, Sweden b

Biochemistry and Structural Biology, Center for Molecular Protein Science,

Department of Chemistry, Lund University, Box 124, SE-221 00 Lund, Sweden c

Biophysical Chemistry, Center for Molecular Protein Science, Department of

Chemistry, Lund University, Box 124, SE-221 00 Lund, Sweden d

SARomics Biostructures AB, Medicon Village, SE-223 63 Lund, Sweden

e

Department of Laboratory Medicine, Section MIG, Lund University BMC-C1228b,

Klinikgatan 28, 221 84 Lund, Sweden f

Galecto Biotech AB, Sahlgrenska Science Park, Medicinaregatan 8 A, SE-413 46

Gothenburg, Sweden

ACS Paragon Plus Environment

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ABSTRACT Symmetrical

and

asymmetrical

fluorinated

phenyltriazolyl-thiodigalactoside

derivatives have been synthesized and evaluated as inhibitors of galectin-1 and galectin-3. Systematic tuning of the phenyltriazolyl-thiodigalactosides’ fluorointeractions with galectin-3 led to the discovery of inhibitors with exceptional affinities (Kd down to 1-2 nM) in symmetrically substituted thiodigalactosides, as well as unsurpassed combination of high affinity (Kd 7.5 nM) and selectivity (46-fold) over

galectin-1

for

asymmetrical

thiodigalactosides

carrying

one

trifluorphenyltriazole and one coumaryl moiety. Studies of the inhibitor-galectincomplexes with isothermal titration calorimetry and X-ray crystallography revealed the importance of fluoro-amide interaction for affinity and for selectivity. Finally, the high affinity of the discovered inhibitors required two competitive titration assay tools to be developed: a new high affinity fluorescent probe for competitive fluorescent polarization and a competitive ligand optimal for analyzing high affinity galectin-3 inhibitors with competitive isothermal titration calorimetry.

KEYWORDS Thiodigalactoside, triazole, coumarin, selectivity, galectin-3, galectin-1, ITC, X-ray


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Introduction Galectins are a family of glycan-binding proteins known for their ability to cross-link glycoconjugates to form lattices. They are defined by their affinity for β-Dgalactopyranoside moieties and their possession of a conserved carbohydraterecognition domain (CRD).1 Prototype galectins (1, 2, 5, 7, 10, 11, 13, 14 and 15) exists as monomers with one CRD, which dimerize at higher concentrations. Galectins (4, 6, 8, 9, and 12) are of the tandem-repeat type with two different CRDs joined by a linker polypeptide, and galectin-3 is a chimera-type protein with a single CRD and a non-lectin N-terminal domain that promotes oligomerization.2 Galectin lattices have been proposed to influence the behavior and activities of cross-linked glycoconjugates by regulating their cellular trafficking, localization, residence times and molecular interactions. Interactions of galectin-3, the most extensively studied family member, with glycoconjugates control cell properties and functions, cell adhesion, and have modulatory effects on the immune system3, tumor growth and metastasis,4 which suggests that galectin-3 CRD inhibitors can be used as therapeutic agents. This has been validated by several ex vivo5,6 and in vivo7,8 studies. Selective small molecule galectin-3 inhibitors are not only valuable as research tools but also as drug candidates. Small molecule derivatives often involve lactose-based derivatives,7,9–12 1- and 3-substitutions of galactose13–17 or 3,3’-disubstitution of thiodigalactoside. Other strategies involve multivalent inhibitors.18–22 The highest affinity small molecule galectin-3 inhibitors achieve low nM affinity by 3-benzamido,23,24 3-O-coumarylmethyl,25,26 4-amido-1,2,3-triazolyl,27 or 4-aryl-1,2,3-triazolyl28 substitutions at the 3- and 3’-positions of thiodigalactoside. Recently, X-ray structural analysis28 of the two galectin-3 CRD (galectin-3C) complexes with bis-[4-(3-


3


fluorophenyl)-1,2,3-triazol-1-yl]

or

Page 4 of 54

bis-[4-(4-fluorophenyl)-1,2,3-triazol-1-yl]

thiodigalactosides 1 and 2 (Figure 1) revealed two types of important interactions, namely guanidine-arene interactions, involving R144 and R186 and the aryls of 1 or 2, and orthogonal multipolar fluorine-amide interactions29 involving the backbone amides of R144 and I145 and the fluorine atom of 1, or the backbone amide of S237 with the fluorine atom of 2. This observation suggests that fluorine-amide interactions are attractive design motifs towards discovery of high-affinity ligands of lectins that have inherently low affinity for natural saccharide fragments. Herein, we report thiogalactoside and thiodigalactoside derivatives carrying one or two mono- to tri-fluorinated 3-(4-aryl-1,2,3-triazol-1-yl) moieties at thiodigalactoside C3 and C3´ towards the discovery of galectin-3 ligands with improved selectivity and affinity. Additionally, an in-depth analysis of competitive fluorescence polarizationand competitive isothermal titration calorimetry assays used for evaluating galectin-3 ligands with very high affinity is reported.


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Figure 1. A) The 3- and 4-fluorophenyl thiodigalactoside derivatives 1 and 2 with high affinity for galectin-3.28 B) and C) Galectin-3C in complexes with compounds 1 and 2. The R144 side chain atop the mono-fluorophenyl is highlighted for clarity. Orthogonal multipolar fluorine interactions between compound 1 and the carbonyl groups of R144 and I145 and between compound 2 and the carbonyl group of S237 are depicted with dashed lines.

Results and discussion Synthesis and galectin-3 affinities of 3-(4-aryl-1,2,3-triazol-1-yl)-thiogalactosides Deprotection and acetylation of azide 330 to azide 4 allowed the synthesis of a library of 1,4-disubstituted triazoles 5-16 at the C3-galactose position by a 1,3-dipolar cycloaddition and subsequent deacetylation (Scheme 1).


5


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Scheme 1. Synthesis of triazoles 5-16. Reagents and conditions: (a) i. AcOH (90%), 90 oC, 4 h; ii. Ac2O, pyridine, rt; (b) i. alkyne, CuI, DIPEA, MeCN, 50 oC; ii. NaOMe, MeOH, rt.

As thiodigalactosides 1 and 2 showed high affinities for both galectin-1 and galectin3, we evaluated the inhibition potencies of triazoles 5-16, together with reference compound p-methylphenyl 1-thio-β-D-galactopyranoside 17 (Table 1), towards galectin-1 and galectin-3 using a previously described competitive fluorescence polarization assay.31,32 All monogalactoside triazoles 5-16 bound galectin-1 and galectin-3 better than the unsubstituted thiogalactoside 17, and all phenyltriazoles except 6 showed better galectin-3 binding than the unsubstituted phenyltriazole 5. The galectin-3 affinities revealed that meta- and para-fluorination (7-8), meta- and parachlorination (13-14) or para-bromination (16) increase binding. Additional


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fluorination (9-11) or chlorination (15) further increased binding, with the trifluorophenyltriazole 11 showing remarkably high affinity, for a monosaccharide ligand, towards galectin-3. These results provided a basis for selecting phenyltriazolyl structures to implement and investigate on a thiodigalactoside scaffold and triazolylthiodigalactoside analogs (19-21) of the three best monogalactosides (9-11) were selected for synthesis.

Table 1. Kd (µM) values for monogalactoside triazoles 5-16 and reference 17 determined by competitive fluorescence polarization.

Galectin-1 Galectin-1

Galectin-3 /Galectin-3

5

220 ± 6

88 ± 3

2.5

6

190 ± 13

92 ± 5

2.1

7

180 ± 7

22 ± 0.7

8.2

8

140 ± 6

31 ± 1.3

4.5

9

530 ± 40

8.8 ± 0.3

60

10

290 ± 9

15 ± 0.3

19

11

180 ± 12

5.2 ± 0.3

35

12

170 ± 2

23 ± 1.0

7.4

13

100 ± 2

44 ± 1.1

2.3

14

680 ± 80

19 ± 1.0

36

15

120 ± 8

14 ± 1.3

8.6

16

300 ± 20

44 ± 3.9

6.8

17

1100 ± 110 230 ± 30

4.7


7


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Synthesis and galectin-3 affinities of symmetrical bis-3-(4-aryl-1,2,3-triazol-1-yl)thiodigalactosides Three

symmetrical

tetra-

or

hexafluorinated

bis-3-(4-aryl-1,2,3-triazol-1-yl)-

thiodigalactosides 19-21 were synthesized in order to compare with the known monofluorinated phenyl groups of 1-2. Compounds 19-21 were obtained in good yields from the known diazide 18,33 following similar conditions to those used for the synthesis of monogalactoside triazoles 5-16 (Scheme 2).

Scheme 2. Synthesis of triazoles 19-21. Reagents and conditions: (a) i. alkyne, CuI, DIPEA, DMF, 50 o

C; ii. NaOMe, MeOH, CH2Cl2, rt.

Evaluation of potentially very high affinity inhibitors, such as 19-21, in competitive fluorescence polarization assays31 requires that the competitive fluorescent probe molecule also possesses high affinity for galectin-3, in order to allow lower experimental concentrations of the probe molecule and the protein in the range of, or ideally lower than, the anticipated dissociation constants for the inhibitors. Hence, having discovered that the derivatives 9-11 were indeed potent mono-galactoside inhibitors compared to the corresponding unsubstituted galactoside 17, we designed


8

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and synthesized fluorescent probe molecules 25-26 based on thiodigalactoside equipped with the affinity-enhancing mono- and trifluorophenyltriazole moieties discovered on the monogalactosides 7 and 11, respectively (Scheme 3).

Scheme 3. Synthesis of fluorescent probes 25-26. Reagents and conditions: (a) 3,4,5trifluorophenylacetylene, CuI, DIPEA, DMF, 50 oC, 1 h; (b) i. 3-fluorophenylacetylene, CuI, DIPEA, DMF, rt, 40 min; ii. NaOMe, MeOH, rt; (c) NaOMe, MeOH, CH2Cl2, rt; (d) 5-FAM alkyne, CuI, DIPEA, DMF, 50 oC, 24 h.

Indeed, high affinity towards galectin-3 was observed for both fluorescent probes 2526 while affinities were significantly lower for galectin-1 (Table 2, Figure S1-S2 in the Supporting Information). Furthermore, a 3-fold higher affinity and 4-fold better galectin-3-selectivity of the trifluoride 26 over the monofluoride 25 confirms the importance of the multiple fluoro substituents for galectin-3 affinity and selectivity, thus reflecting the differences seen between the monosaccharides 7 and 11. The high affinity of 26 for galectin-3 allowed for competitive fluorescence polarization


9


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experiments at lower concentrations of both the probe 26 (4.0 nM) and galectin-3 (10.0 nM), resulting in improved assay sensitivity and accuracy. A blocking protein (100 nM BSA) was required to prevent perturbing absorption losses at such low concentrations. These assay conditions were used for evaluation of the symmetrical thiodigalactosides 1-228 and 19-21 as inhibitors of galectin-3 (Table 2). For comparison, we also included as reference compounds unsubstituted thiodigalactoside 27 and the more galectin-3 selective bis-coumaryl thiodigalactoside 2826, and bisbutylaminocarbonyltriazolyl thiodigalactoside 2927 (Figure 2). It is important to note that the improved 26-based assay yielded galectin-3 affinities in the same range as previously determined for compounds 1-228, 2826, and 2927. All compounds except 19 bound galectin-1 with double-digit nM affinity or worse, thus it was decided that a previously reported fluorescent probe28 was sufficient to evaluate galectin-1 affinity of 1-2, 19-21, and 29.

Figure 2. Reference compounds 27-29.


10

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Table 2. Kd (nM) values for symmetrical thiodigalacosides 1-2, 19-21 and references 27-29 determined by competitive fluorescence polarization and for probes 25-26 with direct fluorescence polarization titration.

Galectin-

Galectin-

Galectin-1

1

3

/Galectin-3

1

1228

2.3 ± 0.2

5.2

2

2728

4.0 ± 0.6

6.8

19

Systematic Tuning of Fluoro-galectin-3 Interactions Provides

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