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

Abstract

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.

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

“…With compound 29 2-fold improvement could be achieved whereas compound 33 had a 4-fold lower EC 50 value. Compound 33 is therefore one of the most active DENV-2 protease inhibitors in cells and is in the same activity range as recently published inhibitors [ 27 , 28 ]. The substituents and structural variations of the 3-(benzyloxy) derivatives also had a major influence on the cytotoxicity of the compounds.…”

“…In the biochemical assay of DENV-2 and WNV protease, no relevant or only low activity could be observed for the compounds ( Table S1 ). However, based on our previous experience with phenylglycine derivatives [ 27 , 28 ], we nevertheless proceeded to test these compounds in the cellular DENV-2 protease reporter assay (DENV2proHeLa). This cellular reporter assay provides the full-length NS3 protein together with the NS2 cofactor in a natural environment without the need of an artificial linker, which is expected to influence protease conformations and dynamics [ 32 ] and therefore affects target-inhibitor recognition.…”

“…Initial screenings were performed in biochemical assays. In addition, for the DENV protease, we carried out a cellular assay (DENV2proHeLa) that had a crucial role in the discovery of potent monobasic and non-basic inhibitors of DENV and WNV proteases [ 27 , 28 ].…”

Repurposing of investigational cancer drugs: Early phase discovery of dengue virus NS2B/NS3 protease inhibitors

“…[7][8][9] Thus, taking into account the pivotal role played in the viral replication, the DENV NS2B/NS3 protease has been extensively exploited as a drug target for the discovery and development of direct-acting antivirals against dengue. [10,11] Even though several peptidic or peptidomimetic compounds containing reactive "warheads," [12][13][14][15][16][17][18] and noncovalent small organic molecules [19][20][21][22][23][24][25][26] have been identified as inhibitors of DENV NS2B/NS3 protease, no inhibitor has reached clinical development as a specific antidengue drug. This may be due to unfavorable drug metabolism and pharmacokinetic (DMPK) properties and/or lower target affinity of known inhibitors.…”

“…Even though several peptidic or peptidomimetic compounds containing reactive “warheads,” [ 12–18 ] and noncovalent small organic molecules [ 19–26 ] have been identified as inhibitors of DENV NS2B/NS3 protease, no inhibitor has reached clinical development as a specific anti‐dengue drug. This may be due to unfavorable drug metabolism and pharmacokinetic (DMPK) properties and/or lower target affinity of known inhibitors.…”