As a part of our systematic study of foldamer structural elements, we analyze and quantify the conformational behavior of two model compounds based on a frequently used class of aromatic oligoamide building blocks. Combining computational and NMR approaches, we investigate ortho-fluoro- and ortho-chloro-N-methylbenzamide. Our results indicate that the -F substituent in an ortho position can be used to fine-tune the rigidity of the oligomer backbone. It provides a measurably attenuated but still considerably strong hydrogen bond (H-bond) to the peptide group proton when compared to the -OCH3 substituent in the same position. On the other hand, the ortho-Cl substituent does not impose significant restrictions on the flexibility of the backbone. Its effect on the final shape of an oligomer is likely governed by its size rather than by noncovalent intramolecular interactions. Furthermore, the effect of solvent on the conformational preferences of these building blocks has been quantified. The number of intramolecularly H-bonded conformations decreases significantly when going from nonprotic to protic environments. This study will facilitate rational design of novel arylamide foldamers.

The RNA-dependent RNA polymerase from the Hepatitis C Virus (gene product NS5B) is a validated drug target because of its critical role in genome replication. There are at least four distinct allosteric sites on the polymerase to which several small molecule inhibitors bind. In addition, numerous crystal structures have been solved with different allosteric inhibitors bound to the polymerase. However, the molecular mechanisms by which these small molecules inhibit the enzyme have not been fully elucidated. There is evidence that allosteric inhibitors alter the intrinsic motions and distribution of conformations sampled by the enzyme. In this study we use molecular dynamics simulations to understand the structural and dynamic changes that result when inhibitors are bound at three different allosteric binding sites on the enzyme. We observe that ligand binding at each site alters the structure and dynamics of NS5B in a distinct manner. Nonetheless, our studies also highlight commonalities in the mechanisms of action of the different inhibitors. Each inhibitor alters the conformational states sampled by the enzyme, either by rigidifying the enzyme and preventing transitions between functional conformational states or by destabilizing the enzyme and preventing functionally relevant conformations from being adequately sampled. By illuminating the molecular mechanisms of allosteric inhibition, these studies delineate the intrinsic functional properties of the enzyme and pave the way for designing novel and more effective polymerase inhibitors. This information may also be important to understand how allosteric regulation occurs in related viral polymerases and other enzymes.

The hepatitis C virus (HCV) infects close to 200 million people globally, resulting in a significant need for effective HCV therapies. The HCV polymerase (gene product NS5B) is a valuable target for therapeutics because of its role in replicating the viral genome. Various studies have identified inhibitors for this enzyme, including non-nucleoside inhibitors (NNIs) that bind distal to the enzyme active site. Recently, it has been shown that simultaneously challenging the enzyme with two NNIs results in enhanced inhibition relative to that observed after challenge with individual inhibitors, suggesting that employing multiple NNIs might be the basis of more effective therapeutics. Nevertheless, the molecular mechanisms responsible for this enhanced inhibition remain unclear. We employ molecular dynamics simulations to determine the origin of enhanced inhibition when two NNIs bind to NS5B. Our results suggest that nonoverlapping NNI sites are compatible with simultaneous binding of dual NNIs. We observe that both inhibitors act in concert to induce novel enzyme conformations and dynamics, allowing us to identify molecular mechanisms underlying enhanced inhibition of NS5B. This knowledge will be useful in optimizing combinations of NNIs to target NS5B, helping to prevent the acquisition of viral resistance that remains a significant barrier to the development of HCV therapeutics.

Hepatitis C virus (HCV) is a global health challenge, affecting approximately 200 million people worldwide. In this study we developed SAR models with advanced machine learning classifiers Random Forest and k Nearest Neighbor Simulated Annealing for 679 small molecules with measured inhibition activity for NS5B genotype 1b. The activity was expressed as a binary value (active/inactive), where actives were considered molecules with IC50 ≤ 0.95 μM. We applied our SAR models to various drug-like databases and identified novel chemical scaffolds for NS5B inhibitors. Subsequent in vitro antiviral assays suggested a new activity for an existing prodrug, Candesartan cilexetil, which is currently used to treat hypertension and heart failure but has not been previously tested for anti-HCV activity. We also identified NS5B inhibitors with two novel non-nucleoside chemical motifs.

Recently discovered information alters the interpretation of our paper published previously in Biophysical Journal. In the study, we used molecular dynamics (MD) simulations to understand the mechanisms that govern allosteric inhibition of the RNA polymerase from hepatitis C virus (HCV). Our findings revealed that allosteric inhibitors alter the conformations adopted by the enzyme in a manner that allows their inhibitory effects to be readily understood. Their primary modes of action were found to be: 1) restricting enzyme fluctuations to nonfunctional regions of conformational space, 2) destabilizing the enzyme to the extent that it is unable to stably adopt functional conformations, and 3) blocking RNA template access to the enzyme's active site.