Design, synthesis and evaluation of tool compounds for studying endosulfatases

The extracellular endo-6-O-sulfatases (Sulfs) catalyse the hydrolysis of the glucosamaine 6-O-sulfate groups within heparan sulfate (HS). By controlling the sulfation status of HS, the endosulfatases modulate the bioavailability of various signalling ligands that use HS chains to interact with their cell-surface receptors and thus play an important role in the regulation of these pathways. GlcNS-6-sulfamate derivatives have been reported as weak inhibitors of the Sulfs; however, more potent inhibitors of these enzymes are required to probe the biological relevance of endosulfatases in disease models. This work describes the synthesis of tool compounds for targeting endosulfatases for enzymatic and in-cell experiments, which can be categorised into inhibitors, target engagement probes and targeted protein degradation.
Firstly, a small library of putative Sulf inhibitors, based on GlcNS-6-sulfamate monosaccharide were prepared, however when these compounds were evaluated as inhibitors of Sulf-2 only very weak inhibition was observed even at high concentration (<30% at 500 μM). In an attempt to increase the potency of these analogues, the size of the HS-fragment was increased to the disaccharide IdoA(2S)-GlcNS(6Sulfamate). This was synthesised in 15-steps in 3% overall yield. When this disaccharide was evaluated for Sulf2 inhibition at 1 mM, nearly complete inhibition of the enzyme was observed. The IC50 value of this compound was determined to be 42.6 μM ± 18.3.
A multivalent display approach was utilised to improve potency further via altering the presentation of the disaccharide inhibitor to Sulf-2. A glycopolymer was designed with a polynorbornene backbone to allow for maximal control over chain length and low polydispersity. Polymerisation of an NHS-activated norbornene carboxylate monomer through ring-opening metathesis polymerization chemistry followed by an amide coupling reaction to install the disaccharide inhibitor was found to be superior to polymerisation of a sulphated-disaccharide functionalised norbornene monomer. The glycopolymer with the highest disaccharide loading (20%) was evaluated as a Sulf-2 inhibitor, using the effective molarity of the disaccharide to investigate the multivalent effect and it was found to be approximately 5-fold more potent than the parent disaccharide, (IC50 = 9 μM ± 4.75).
Small molecule target engagement probes were designed and synthesised using two photoaffinity techniques for proximity labelling. First, the disaccharide IdoA(2S)-GlcNS(6Sulfamate) was functionalised with a diazirine and a terminal alkyne, using traditional PAL probe design. However, it was anticipated that traditional photoaffinity labelling of Sulf-2 in the extracellular matrix would be challenging, a second approach by the MacMillan group was utilised, which takes advantage of catalytic diazirine activation to achieve greater than single stoichiometric labelling. To this end, the IdoA(2S)-GlcNS(6Sulfamate) was tethered to a cell-impermeable Ir-photocatalyst via a CuAAC reaction. The target engagement probes were investigated in MCF-7 cells, that are reported to express high levels of HSulf-2, and pleasingly the disaccharide-based probes labelled various extracellular proteins. These proteins were successfully off-competed by a high concentration of the ‘parent’ inhibitor. Identification of these proteins by LC-MS/MS is on-going.
Finally, a probe was designed for targeted protein degradation using LYTAC technology. It was envisioned that the HSulf-2 inhibiting glycopolymer could be end-functionalised with an azide group. It was planned that this would be joined to the poly(M6P-ala) glycopeptide reported by the Bertozzi group via a CuAAC reaction. Synthesis of both endlabelled polymers is on-going and is high priority for future work.