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.
Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, αfluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (K i = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo [b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC 50 values (0.14/0.80 μM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.
Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with drugresistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active-site cysteine. A broad series of derivatives were synthesized to derive structure-activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single-digit micromolar K i values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase-mediated adherence of S. aureus cells. a 100 % � 0.5 % 93 % � 5.6 % n.i. 7 a 98 % � 0.1 % 14 % � 8.9 % 18 % � 1.1 % 7 f 60 % � 0.5 % n.i. 12 % � 3.1 % 7 g 18 % � 3.9 % n.i. 17 % � 7.7 % 7 h 69 % � 1.7 % 23 % � 11 % 13 % � 8.0 % 12 a 90 % � 0.5 % n.i. 15 % � 5.7 % n.i. = no inhibition at 20 μM compound concentration. All results include the mean value and standard deviations from triplicate measurements.
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