X-ray structures of human furin in complex with competitive inhibitors.

Furin inhibitors are promising therapeutics for the treatment of cancer and numerous infections caused by bacteria and viruses, including the highly lethal Bacillus anthracis or the pandemic influenza virus. Development and improvement of inhibitors for pharmacological use require a detailed knowledge of the protease's substrate and inhibitor binding properties. Here we present a novel preparation of human furin and the first crystal structures of this enzyme in complex with noncovalent inhibitors. We show the inhibitor exchange by soaking, allowing the investigation of additional inhibitors and substrate analogues. Thus, our work provides a basis for the rational design of furin inhibitors.

Furin is a member of the pro-hormone/pro-protein convertase family (PCs) of subtilisin-like endoproteinases.[1] PCs are required for activation and maturation of many secreted proteins. Target proteins include peptide hormones, growth factors, matrix metalloproteases, blood clotting factors, regulators of the cholesterol metabolism, bacterial toxins, and viral capsid proteins.[2,3] Therefore furin and other PCs are intensively investigated as pharmacological targets for the treatment of many diseases, e.g., atherosclerosis, hypercholesterolaemia, and cancer, as well as viral and bacterial infections.[4] Proteolysis by furin is highly specific and occurs C-terminal to a multibasic recognition motive. The extended substrate binding site gives rise to diverging specificities, strongly favoring arginine at P1 and basic amino acid side chains at P2, P4, and/or P6, whereby R-[X]-(R/K)-R↓ is the most common recognition sequence.

Up to now several compound classes have been identified as promising starting points for drug development. In addition to small molecules and peptide based inhibitors,[5] also camelid VHH-antibodies were found to selectively inhibit furin.[6] It was shown that furin inhibitors are indeed suitable to prevent the growth and invasiveness of tumors (e.g., refs (7 and 8)), the replication of viruses (e.g., refs (9 and 10)), or the toxicity of bacterial toxins (e.g., refs (11 and 12)). For their broad pharmacological application, next generation compounds require, however, improvements of their stability, selectivity, bioavailability, and/or pharmacokinetics.[5]

Structure-guided drug design provides the possibility for rational modification and directed development of enhanced inhibitors. This approach requires an in-depth structural understanding of furin–inhibitor complexes. So far, structures of mouse furin[13] and of its yeast homologue kexin[14] are available only in complex with covalently attached peptides. The mouse furin structure showed the interaction with a prototypical R-V-K-R↓ recognition motive. Investigation of other furin substrate analogues or inhibitors by exchange of the initially co-crystallized compound, however, was not possible.

Peptidomimetic compounds based on a phenylacetyl-Arg-Val-Arg-4-(amidomethyl)benzamidine (Phac-RVR-4-Amba) core structure (15) belong to the strongest noncovalent inhibitors available so far. Upon variation of the P5 position, dramatic changes of the Ki values were observed that cannot be explained by the known recognition motive. The Ki improved by approximately 2 orders of magnitude after addition of basic substituents, e.g., by modification of the Phac-moiety at P5 by a m- or p-guanidinomethyl group.[15]

Here we describe a novel preparation of human furin and two crystal structures of this enzyme in complex with competitive, noncovalent inhibitors. The tight binding observed for the inhibitor complexes is accompanied by a very strong increase of the structural stability in thermal denaturation experiments. The structures explain the different affinities of the inhibitors and the related specificity of the protease for substrates with Arg/Lys residues at the P5 position.

Methods

The coding sequence of human furin was inserted into the plasmid pHLsec[28] and expressed by transient transfection of human embryonic kidney cells. The protein was purified in a three-step chromatography scheme, employing metal affinity chromatography, inhibitor based affinity chromatography,[17] and size exclusion chromatography. Finally a ∼300-fold enrichment of human furin was observed, corresponding to a specific activity of 57 ± 1 u. One unit corresponds to 1 μmol AMC (h × mg)−1 released from the peptide pGlu-Arg-Thr-Lys-Arg-AMC (200 μM) at 37 °C in 100 mM Hepes, pH 7.0, 5 mM CaCl2, 0.5% (v/v) TritonX-100. Details of the expression, preparation, kinetic analyses, and thermal denaturation assays are described in Supporting Information.

For crystallization furin was concentrated to 140–150 μM (∼7.5 mg mL–1), and I1 was added to a final concentration of 290 μM. Crystals were grown at 30 °C in 50 mM Tris, pH 8.5, 2.8 M sodium formate and 0.015 mM Cymal-7. For the structural investigation of the complex of furin with I2, crystals were soaked in crystallization solution supplemented with 3 mM of I2. Diffraction data were collected at 100 K at the BESSY-II beamline 14.1 of the Helmholtz-Zentrum Berlin (HZB)[29] and processed with XDS (v.03/2013[30]). Model building was carried out in COOT (v.0.6.2[31]). CNS (v.1.3[32]) was used for refinement of the structures of furin in complex with I1 and I2 up to 2.3 and 2.7 Å resolution, respectively.