N-sulfonyl peptide-hybrids as a new class of dengue virus protease inhibitors.

Dengue virus (DENV) from the Flaviviridae family causes an epidemic disease that seriously threatens human life. The viral serine protease NS2B-NS3 is a promising target for drug development against DENV and other flaviviruses. We here report the design, synthesis, and in-vitro characterization of potent peptidic inhibitors of DENV protease with a sulfonyl moiety as N-terminal cap, thereby creating sulfonamide-peptide hybrids. The in-vitro target affinities of some synthesized compounds were in the nanomolar range, with the most promising derivative reaching a Ki value of 78 nM against DENV-2 protease. The synthesized compounds did not have relevant off-target activity nor cytotoxicity. The metabolic stability of compounds against rat liver microsomes and pancreatic enzymes was remarkable. In general, the integration of sulfonamide moieties at the N-terminus of peptidic inhibitors proved to be a promising and attractive strategy for further drug development against DENV infections.

Antibody-drug conjugates: What drives their progress?

Antibody-drug conjugates (ADCs) are on the brink of widespread use for the targeted treatment of cancer. ADCs manage the toxicity of drugs with unacceptable narrow therapeutic windows by guiding highly toxic compounds to the target cells, thereby sparing healthy cells. In this review, we describe approved ADCs and discuss their modes of action, together with medicinal chemical aspects, to evaluate the potential for improvement and to combat tumor-acquired resistance. A recent research focus has centered on the stimulation of immune responses to induce immunogenic cell death and the influence on the tumor microenvironment to enhance bystander effects.

Discovery of potent benzoxaborole inhibitors against SARS-CoV-2 main and dengue virus proteases.

The RNA viruses SARS-CoV-2 and dengue pose a major threat to human health worldwide and their proteases (Mpro; NS2B/NS3) are considered as promising targets for drug development. We present the synthesis and biological evaluation of novel benzoxaborole inhibitors of these two proteases. The most active compound achieves single-digit micromolar activity against SARS-CoV-2 Mpro in a biochemical assay. The most active substance against dengue NS2B/NS3 protease has submicromolar activity in cells (EC50 0.54 µM) and inhibits DENV-2 replication in cell culture. Most benzoxaboroles had no relevant cytotoxicity or significant off-target inhibition. Furthermore, the class demonstrated passive membrane penetration and stability against the evaluated proteases. This compound class may contribute to the development of antiviral agents with activity against DENV or SARS-CoV-2.

The spectrum between substrates and inhibitors: Pinpointing the binding mode of dengue protease ligands with modulated basicity and hydrophobicity.

Peptides can be inhibitors and substrates of proteases. The present study describes the inhibitor- vs. substrate-like properties of peptidic ligands of dengue protease which were designed to provide insight into their binding modes. Of particular interest was the localization of the cleavable peptide bond and the placement of hydrophobic elements in the binding site. The findings provide clues for the design of covalent inhibitors in which electrophilic functional groups bind to the catalytic serine, and in addition for the development of inhibitors that are less basic than the natural substrate and therefore have an improved pharmacokinetic profile. We observed a tendency of basic elements to favor a substrate-like binding mode, whereas hydrophobic elements decrease or eliminate enzymatic cleavage. This indicates a necessity to include basic elements which closely mimic the natural substrates into covalent inhibitors, posing a challenge from the chemical and pharmacokinetic perspective. However, hydrophobic elements may offer opportunities to develop non-covalent inhibitors with a favorable ADME profile and potentially improved target-binding kinetics.

Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

Beyond Basicity: Discovery of Nonbasic DENV-2 Protease Inhibitors with Potent Activity in Cell Culture.

The viral serine protease NS2B-NS3 is one of the promising targets for drug discovery against dengue virus and other flaviviruses. The molecular recognition preferences of the protease favor basic, positively charged moieties as substrates and inhibitors, which leads to pharmacokinetic liabilities and off-target interactions with host proteases such as thrombin. We here present the results of efforts that were aimed specifically at the discovery and development of noncharged, small-molecular inhibitors of the flaviviral proteases. A key factor in the discovery of these compounds was a cellular reporter gene assay for the dengue protease, the DENV2proHeLa system. Extensive structure-activity relationship explorations resulted in novel benzamide derivatives with submicromolar activities in viral replication assays (EC50 0.24 µM), selectivity against off-target proteases, and negligible cytotoxicity. This structural class has increased drug-likeness compared to most of the previously published active-site-directed flaviviral protease inhibitors and includes promising candidates for further preclinical development.

A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays.

Dengue and West Nile virus are rapidly spreading global pathogens for which no specific therapeutic treatments are available. One of the promising targets for drug discovery against dengue and other flaviviruses is the viral serine protease NS2B-NS3. We present the design, synthesis, and in vitro and cellular characterization of a novel chemotype of potent small-molecule non-peptidic dengue protease inhibitors derived from 4-benzyloxyphenylglycine. A newly developed luciferase-based DENV-2 protease reporter system in HeLa cells (DENV2proHeLa) was employed to determine the activity of the compounds in a cellular environment. Specificity and selectivity of the DENV2proHeLa system were confirmed by viral titer reduction assays. The compounds reach low micromolar to upper nanomolar inhibitory potency in cell-based assays, are selective against other serine proteases, and do not show relevant cytotoxicity. An extensive structure-activity relationship study provides a perspective for further drug development against flaviviral infections.