Discovery, affinity maturation and multimerization of small molecule ligands against human tyrosinase and tyrosinase-related protein 1

Abstract

Human tyrosinase (hTYR) and tyrosinase-related protein 1 (hTYRP1) are closely-related enzymes involved in the synthesis of melanin, which are selectively expressed in melanocytes and, in a pathological context, in melanoma lesions. We used a previously described tyrosinase inhibitor (Thiamidol™) and DNA-encoded library technology for the discovery of novel hTYR and hTYRP1 ligands, that could be used as vehicles for melanoma targeting. Performing de novo selections with DNA-encoded libraries, we discovered novel ligands capable of binding to both hTYR and hTYRP1. More potent ligands were obtained by multimerizing Thiamidol™ moieties, leading to homotetrameric structures that avidly bound to melanoma cells, as revealed by flow cytometry. These findings suggest that melanoma lesions may, in the future, be targeted not only by monoclonal antibody reagents but also by small organic ligands.

Fig. 1. Human tyrosinase (hTYR) and tyrosinase-related protein 1 (hTYRP1) are melanoma associated antigens that present favorable ligandability properties. a) Tyrosine is converted in eumelanin through the interaction with metal ions placed within the active site (depicted in red); b) chromatographic elution profiles of purified intra melanosomal human hTYR and hTYRP1 proteins. In the insets, SDS-PAGE gels showing the purity of the eluted fractions in the peak volume. The first lane contains the MW marker, confirming the expected size of the proteins (PDB: 5M8P).

Fig. 2. A known hTYR inhibitor (Thiamidol™) was modified with a derivatizable handle for later modifications. The new Thiamidol™ alkyne derivative retains the inhibitory capacity of the parent compound in presence of human hTYR.

Fig. 3. Discovery of novel binding fragments for hTYR and hTYRP1 through DNA-encoded library (GB-DEL). a) A single-pharmacophore DEL, based on a racemic glutamic acid scaffold, was screened in presence of immobilized hTYR and hTYRP1 proteins; b–d) bi- and tri-dimensional plots of the resulting selections fingerprints after incubation with streptavidin coated beads, hTYRP1 and hTYR are reported. Selections against hTYR and hTYRP1 displayed A26/B389 as the most enriched combination of building blocks; e) the chemical structures of the enriched fragments are reported, whose binding affinities were evaluated through fluorescence polarization measurements in presence of hTYRP1 [Fig. S1†].

Fig. 4. ESAC-based affinity maturation of Thiamidol™ ligand. a) The single-stranded GB-DEL library was paired with a complementary DNA-fragment displaying a Thiamidol™ derivative and screened in presence of hTYR and hTYRP1; b and c) decoding plots of the selection experiment delineate fragments particularly enriched upon incubation with hTYRP1 and hTYR; d) DNA-linked fragments were paired to fluoresceinated LNA-scaffolds presenting a Thiamidol™ derivative. Binding affinities towards hTYRP1 were measured through fluorescence polarization.

Fig. 5. Thiamidol™ alkyne was conjugated to multimeric scaffolds in order to achieve ligands with different binding avidities. The peptidic linker composed by the triad Asp–Arg–Asp was conjugated to fluorescein and to Thiamidol™ derivative through three scaffolds with different valences (i.e., monomeric, dimeric and tetrameric). Binding affinities of monomer, dimer and tetramer were evaluated through ELISA assay on hTYR and hTYRP1 coated microtiter wells.

Fig. 6. Binding properties of Thiamidol™ based fluorescein-labelled ligands were evaluated in presence of B16F10 melanoma cells. Cells were incubated with a fixed concentration of ligands (1 μM) and the residual binding was detected by flow-cytometry. An anti-fluorescein antibody labelled with Cy5-dye was employed to enhance signal intensity and reduce autofluorescence background.