Designing photoaffinity tool compounds for the investigation of the DENV NS2B-NS3 protease allosteric binding pocket.

Dengue virus (DENV) infection still lacks specific antiviral therapy, making the NS2B-NS3 protease an attractive target for drug development. However, allosteric inhibitors that bind to a site other than the active site still need to be better understood. In this study, we designed and synthesised tool compounds for photoaffinity labelling (PAL) to investigate the binding site of allosteric inhibitors on the DENV protease. These tool compounds contained an affinity moiety, a photoreactive group, and a reporter tag for detection. Upon irradiation, the photoreactive group formed a covalent bond with the protease, allowing for binding site identification. SDS-PAGE-based assays confirmed the qualitative binding of the designed inhibitors to the allosteric pocket, and pull-down experiments validated the interaction. Tryptic protein digestion following liquid chromatography/mass spectrometry analysis further supported the binding of the inhibitor to the proposed pocket revealing photo-attachment to an NS3 loop close to the C-terminus. These results enhance our understanding of allosteric inhibitors and their mechanism of action against the DENV protease. The developed tool compounds and PAL are potent tools for future drug discovery efforts and investigations targeting the DENV protease.

SAR of novel benzothiazoles targeting an allosteric pocket of DENV and ZIKV NS2B/NS3 proteases.

In recent years, dengue virus (DENV) and Zika virus (ZIKV), both mosquito-borne members of the Flaviviridae family, have emerged as intercontinental health issues since their vectors have spread from their tropical origins to temperate climate zones due to climate change and increasing globalization. DENV and ZIKV are positive-sense, single-stranded RNA viruses, whose genomes consist of three structural (capsid, membrane precursor, envelope) and seven non-structural (NS) proteins, all of which are initially expressed as a single precursor polyprotein. For virus maturation, the polyprotein processing is accomplished by host proteases and the viral NS2B/NS3 protease complex, whose inhibitors have been shown to be effective antiviral agents with loss of viral pathogenicity. In this work, we elucidate new structure-activity relationships of benzo[d]thiazole-based allosteric NS2B/NS3 inhibitors. We developed a new series of Y-shaped inhibitors, which, with its larger hydrophobic contact surface, should bind to previously unaddressed regions of the allosteric NS2B/NS3 binding pocket. By scaffold-hopping, we varied the benzo[d]thiazole core and identified benzofuran as a new lead scaffold shifting the selectivity of initially ZIKV-targeting inhibitors to higher activities towards the DENV protease. In addition, we were able to increase the ligand efficiency from 0.27 to 0.41 by subsequent inhibitor truncation and identified N-(5,6-dihydroxybenzo[d]thiazol-2-yl)-4-iodobenzamide as a novel sub-micromolar NS2B/NS3 inhibitor. Utilizing cell-based assays, we could prove the antiviral activity in cellulo. Overall, we report new series of sub-micromolar allosteric DENV and ZIKV inhibitors with good efficacy profile in terms of cytotoxicity and protease inhibition selectivity.

Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.

Proline-Based Allosteric Inhibitors of Zika and Dengue Virus NS2B/NS3 Proteases.

The NS2B/NS3 serine proteases of the Zika and Dengue flaviviruses are attractive targets for the development of antiviral drugs. We report the synthesis and evaluation of a new, proline-based compound class that displays allosteric inhibition of both proteases. The structural features relevant for protease binding and inhibition were determined to establish them as new lead compounds for flaviviral inhibitors. Based on our structure-activity relationship studies, the molecules were further optimized, leading to inhibitors with submicromolar IC50 values and improved lipophilic ligand efficiency. The allosteric binding site in the proteases was probed using mutagenesis and covalent modification of the obtained cysteine mutants with maleimides, followed by computational elucidation of the possible binding modes. In infected cells, antiviral activity against Dengue virus serotype 2 using prodrugs of the inhibitors was observed. In summary, a novel inhibitor scaffold targeting an allosteric site shared between flaviviral NS2B/NS3 proteases is presented whose efficacy is demonstrated in vitro and in cellulo.