The global prevalence of hepatitis C virus (HCV) infection and the serious consequences associated with the chronic state of the disease have become a worldwide health problem. A combination therapy ...comprising Interferon-alpha and Ribavirin represents the current standard treatment for chronic HCV infection, although it has demonstrated limited success and causes serious side effects. Promising alternative approaches toward the control of HCV infection include the development of small molecule inhibitors of viral enzymes interfering with the essential steps in the life cycle of the virus. In this review we will focus on inhibitors of the HCV-encoded NS5B RNA-dependent RNA polymerase (NS5B RdRp) which is essential for viral replication and has been recognized as a prime target for therapeutic intervention.
Kinetics of the PROTAC‐induced protein degradation were modelled using the equilibrium approximation, accounting for the protein recovery rate with a time lag. The simulated kinetic curves resemble ...what is experimentally observed, and the physical formulas of the half‐maximal degradation concentration (DC50) were derived from them. The equations reveal that DC50 is proportional to the dissociation constant of the ternary complex (Kd) and inversely proportional to the expression level of the E3 ligase and the effective ubiquitylation rate (kub). The predicted relationships were rigorously confirmed by experimental evidences from a matched molecular pair analysis using a set of published PROTACs.
The equations derived from kinetic modelling of PROTAC‐induced protein degradation reveal the dependency of DC50 on the dissociation constant of the ternary complex, the expression level of the E3 ligase, and the effective ubiquitylation rate.
The hepatitis C virus (HCV) polymerase is required for replication of the viral genome and is a key target for therapeutic
intervention against HCV. We have determined the crystal structures of the ...HCV polymerase complexed with two indole-based
allosteric inhibitors at 2.3- and 2.4-Ã resolution. The structures show that these inhibitors bind to a site on the surface
of the thumb domain. A cyclohexyl and phenyl ring substituents, bridged by an indole moiety, fill two closely spaced pockets,
whereas a carboxylate substituent forms a salt bridge with an exposed arginine side chain. Interestingly, in the apoenzyme,
the inhibitor binding site is occupied by a small α-helix at the tip of the N-terminal loop that connects the fingers and
thumb domains. Thus, these molecules inhibit the enzyme by preventing formation of intramolecular contacts between these two
domains and consequently precluding their coordinated movements during RNA synthesis. Our structures identify a novel mechanism
by which a new class of allosteric inhibitors inhibits the HCV polymerase and open the way to the development of novel antiviral
agents against this clinically relevant human pathogen.
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Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified ...through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.
The evolutionary path is reported to conformationally constrained indole inhibitors of HCV NS5B-polymerase. Biochemical and cell-based potency was achieved, coupled with attractive DMPK ...properties—leading ultimately to the identification of a pre-clinical candidate with an excellent predicted human pharmacokinetic profile.
We report the evolutionary path from an open-chain series to conformationally constrained tetracyclic indole inhibitors of HCV NS5B-polymerase, where the C2 aromatic is tethered to the indole nitrogen. SAR studies led to the discovery of zwitterionic compounds endowed with good intrinsic enzyme affinity and cell-based potency, as well as superior DMPK profiles to their acyclic counterparts, and ultimately to the identification of a pre-clinical candidate with an excellent predicted human pharmacokinetic profile.
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. Compounds that block replication of subgenomic HCV RNA in liver ...cells are of interest because of their demonstrated antiviral effect in the clinic. In followup to our recent report that indole-N-acetamides (e.g., 1) are potent allosteric inhibitors of the HCV NS5B polymerase enzyme, we describe here their optimization as cell-based inhibitors. The crystal structure of 1 bound to NS5B was a guide in the design of a two-dimensional compound array that highlighted that formally zwitterionic inhibitors have strong intracellular potency and that pregnane X receptor (PXR) activation (an undesired off-target activity) is linked to a structural feature of the inhibitor. Optimized analogues devoid of PXR activation (e.g., 55, EC50 = 127 nM) retain strong cell-based efficacy under high serum conditions and show acceptable pharmacokinetics parameters in rat and dog.
Aromatic and heteroaromatic amines (ArNH2) are activated by cytochrome P450 monooxygenases, primarily CYP1A2, into reactive N-arylhydroxylamines that can lead to covalent adducts with DNA ...nucleobases. Hereby, we give hands-on mechanism-based guidelines to design mutagenicity-free ArNH2. The mechanism of N-hydroxylation of ArNH2 by CYP1A2 is investigated by density functional theory (DFT) calculations. Two putative pathways are considered, the radicaloid route that goes via the classical ferryl-oxo oxidant and an alternative anionic pathway through Fenton-like oxidation by ferriheme-bound H2O2. Results suggest that bioactivation of ArNH2 follows the anionic pathway. We demonstrate that H-bonding and/or geometric fit of ArNH2 to CYP1A2 as well as feasibility of both proton abstraction by the ferriheme-peroxo base and heterolytic cleavage of arylhydroxylamines render molecules mutagenic. Mutagenicity of ArNH2 can be removed by structural alterations that disrupt geometric and/or electrostatic fit to CYP1A2, decrease the acidity of the NH2 group, destabilize arylnitrenium ions, or disrupt their pre-covalent transition states with guanine.
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