Design, synthesis and evaluation of carbamate-linked uridyl-based inhibitors of human ST6Gal I
Graphical abstract
Introduction
Sialic acid (N-acetylneuraminic acid, Neu5Ac) is one of the human body’s most important sugars next to glucose.1 These negatively charged nine-carbon α-keto aldonic acids are located at the terminal end of glycan chains on cell surface and secreted molecules, where they play essential roles in cellular biology.2, 3 These functions are mainly related to cellular and molecular recognition events, such as activation or inhibition of intracellular and intramolecular interactions, cell-cell recognition, receptor binding, protein-lectin interactions, protein targeting, cell adhesion, and immune responses.4, 5, 6, 7 The biosynthesis of sialic acid containing glycoconjugates in humans is mediated by 20 sialyltransferases (STs) anchored within the Golgi apparatus’ membrane with the catalytic domain present within the lumen.4, 5 Sialylation catalysed by STs uses the sugar nucleotide donor CMP-Neu5Ac and an oligosaccharide or glycoconjugate terminated by a galactose (Gal), N-acetylgalactosamine (GalNAc), or another sialic acid residue as the acceptor.4, 8
Upregulation of ST activity and the resulting modified cell surface sialylation is strongly associated with cancer, with hypersialylation of 30–50% observed in several cancers.9 This has been directly correlated with an increased metastatic potential of tumours, facilitation of apoptotic avoidance mechanisms and poor patient prognosis.10, 11 Hypersialylation as a result of ST upregulation is also linked to chemo-resistance and thus reduced treatment efficacy in ovarian,12, 13, 14 colorectal,15 and cervical cancer,16 hepatocellular carcinoma,17 pancreatic ductal adenocarcinoma18 and myeloid leukaemia.19 Exposure to radiation induces an increased expression of human ST6Gal I (hST6Gal I) and reduces radiation-induced cell death in colon cancer.20 Thus, the critical role of STs in tumour growth, progression and resistance to both radio- and chemotherapy demonstrates the importance of developing small molecule modulators of this family of enzymes as tools to help understand a potential new anti-cancer drug target.
A number of ST inhibitors have been reported ranging from those isolated from natural sources (e.g. lithocholic acid and soyasaponin) and high throughput screening to those specifically designed to mimic ST substrates as reviewed recently.9 Of these, analogues mimicking the proposed oxocarbenium ion-like transition state of the donor (Fig. 1A) pioneered by RR Schmidt, exhibit the highest affinity to STs.21, 22 The most potent are those incorporating a 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) moiety, where the C2–C3 double bond mimics the planar anomeric carbon; and an extra carbon between the anomeric carbon and CMP leaving group mimics the elongated distance in the transition state.21, 23 Of the various modifications, substituting the glycerol side chain of the Neu5Ac2en moiety with a phenoxy group gave derivative 1 with a Ki value of 29 nM against rat liver ST6Gal in a HPLC-based assay (Fig. 1B).24 More synthetically accessible derivatives that replace the Neu5Ac2en with an aryl moiety have been developed.21, 22, 25 For example, the (R)-isomer of the 3-phenoxy derivative 2 (Fig. 1B) is a potent inhibitor of both rat liver ST6Gals (Ki = 70 nM)21 and recombinant human ST6Gal I (Ki = 19 nM),26 along with cyclopentyl26 (3) and amide27 (4) derivatives with Ki values of 28 nM and 16 nM respectively against hST6Gal I, also in a HPLC-based assay. It has also been demonstrated that the size of the aryl substituent does not impact binding to human ST6Gal I, with fluorescein-labelled derivatives such as (R)-5 and (S)-5, exhibiting Kd values of 22 nM and 9 nM, respectively, against human ST6Gal I.28 These fluorescein-labelled derivatives have since been reported as important high-throughput in vivo screening tools to assist in the development of inhibitors of human oligo- and polysialyltransferases.29
The charged phosphodiester linkage present in the majority of known ST inhibitors (e.g. Fig. 1B) is considered essential for activity.21, 22, 25, 30 Yet it may also lead to low bioavailability in vivo and loss of activity due to cleavage by phosphatases or instability in the ST active site.31, 32 The charged nature of the linker may also lead to poor cellular permeability, although recent reports of phosphodiester derivatives bearing a fluorescent probe (such as 5, Fig. 1B) have suggested these charged compounds may be able to cross cellular membrane via a vesicular uptake mechanism.28 In addition to the potential pharmacokinetic issues, the synthesis of the phosphodiester-linked inhibitors utilises a capricious condensation of an α-hydroxyphosphonate and an highly air-sensitive cytidine phosphitamide.21, 26, 27
To improve synthetic accessibility to ST inhibitors, we have replaced the phosphodiester linker with an uncharged carbamate, which can be synthesised using a wide range of alkoxy carbonylating agents.33 Structurally, the carbamate functionality is related to an amide-ester hybrid and in general displays very good chemical and proteolytic stability. Their capability to permeate cell membranes has resulted in carbamates being widely utilised as peptide bond isosteres,33 which would also provide advantages in this case. More specifically a carbamate moiety would have similar hydrogen bonding capabilities to a phosphodiester group and maintain the three-atom distance between the nucleoside (Fig. 2) and the Neu5Ac mimic observed in reported phosphodiester-linked ST inhibitors such as 1–4 (Fig. 1B).
We have demonstrated previously using docking and molecular dynamics (MD) simulations that carbamate- and 1,2,3-triazole-linked derivatives have comparable interactions to their phosphodiester-linked counterparts with the hST6Gal I active site (PDB ID: 4JS2).34, 35 Using free energy perturbation (FEP) calculations we have shown that these compounds can successfully mimic the charged phosphodiester linkage with a comparable binding affinity through an enthalpy-entropy compensation.36 We have also explored replacing the cytidine moiety with uridine, which has the advantage of requiring less protecting groups enabling inhibitors to be produced more readily and in fewer steps. In addition, as CMP-Neu5Ac is the common natural donor for all ST subtypes, the replacement of the cytidine moiety with uridine could be a hitherto unexplored route to selectivity.
Herein, the phosphodiester linker of classical ST inhibitors has been replaced with a carbamate, along with uridine in place of cytidine and the effects investigated both computationally and experimentally. Additionally, the difference of diastereomers and various substituents of the aryl sialic acid mimic, particularly at the 3-position, were also explored. The target compounds were prepared in 7 steps from two protected building blocks: the 5′-amino-5′-deoxynucleoside and an α-hydroxyphosphonate. The inhibitory activity of the newly synthesised compounds was then evaluated against recombinant hST6Gal I in a luminescence microplate assay, along with cellular toxicity assessment in a pancreatic cancer cell line.
Section snippets
Free energy calculations
Building on previous computational studies demonstrating the feasibility of using carbamates and 1,2,3-triazoles as phosphodiester isosteres,34, 35, 36, 37 we have performed FEP calculations, which are one of the most rigorous calculation methods available to compare the relative binding affinity of cytidine- and uridine-based carbamate inhibitors with hST6Gal I (PDB ID: 4JS2).38, 39 Alchemical transformation of the cytidine-based inhibitor (R)-6 to the uridine-based inhibitor (R)-7 in complex
Conclusions
Herein, we examined the replacement of the phosphodiester linker and cytidine of classical ST inhibitors26, 27 with a carbamate linker and uridine respectively. Additionally, the difference of diastereomers and various substituents of the aryl sialic acid mimic were also explored. FEP calculations demonstrated that for binding to hST6Gal I, uridine was a beneficial alternative to cytidine. Thus, 26 carbamate-linked compounds were synthesised by coupling various α-hydroxyphosphonates with
Free energy calculation
The FEP calculation describing the alchemical transformation40, 41 of the cytidine-based inhibitor (R)-6 to the uridine based inhibitor (R)-7 was prepared using VMD version 1.9.2.57 and carried out using the NAMD 2.10 package.58 The change in the binding free energy with the perturbation from cytidine to uridine (Fig. 5) can be expressed as:
In the FEP method, one introduces a hybrid Hamiltonian, (λ)=(1 – λ) + λ, where H0 represents the Hamiltonian
Associated content
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We wish to acknowledge the Australian Government as H.Y. is the recipient of an Australian Research Council Future Fellowship (Project number FT110100034) and for an Australian Government Research Training Program Award scholarship for A.M. We also wish to acknowledge Phil Clingan, Maxine Stewart and the Illawarra Cancer Carers for financial support, including funding for a PhD scholarship for R.S. and C.D. matched by the University of Wollongong. This research was in part supported under the
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