Structure-Based Design of Highly Potent HIV-1 Protease Inhibitors Containing New Tricyclic Ring P2-Ligands: Design, Synthesis, Biological, and X-ray Structural Studies.

We describe here design, synthesis, and biological evaluation of a series of highly potent HIV-1 protease inhibitors containing stereochemically defined and unprecedented tricyclic furanofuran derivatives as P2 ligands in combination with a variety of sulfonamide derivatives as P2' ligands. These inhibitors were designed to enhance the ligand-backbone binding and van der Waals interactions in the protease active site. A number of inhibitors containing the new P2 ligand, an aminobenzothiazole as the P2' ligand and a difluorophenylmethyl as the P1 ligand, displayed very potent enzyme inhibitory potency and also showed excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The tricyclic P2 ligand has been synthesized efficiently in an optically active form using enzymatic desymmetrization of meso-1,2-(dihydroxymethyl)cyclohex-4-ene as the key step. We determined high-resolution X-ray structures of inhibitor-bound HIV-1 protease. These structures revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insights into the binding properties of these new inhibitors.

X-ray structure and inhibition of the feline infectious peritonitis virus 3C-like protease: Structural implications for drug design.

Feline infectious peritonitis (FIP) is a deadly disease that effects both domestic and wild cats and is caused by a mutation in feline coronavirus (FCoV) that allows the virus to replicate in macrophages. Currently, there are no treatments or vaccines available for the treatment of FIP even though it kills approximately 5% of cats in multi-cat households per year. In an effort to develop small molecule drugs targeting FIP for the treatment of cats, we screened a small set of designed peptidomimetic inhibitors for inhibition of FIPV-3CL(pro), identifying two compounds with low to sub-micromolar inhibition, compound 6 (IC50=0.59±0.06 µM) and compound 7 (IC50=1.3±0.1 µM). We determined the first X-ray crystal structure of FIPV-3CL(pro) in complex with the best inhibitor identified, compound 6, to a resolution of 2.10 Å to better understand the structural basis for inhibitor specificity. Our study provides important insights into the structural requirements for the inhibition of FIPV-3CL(pro) by peptidomimetic inhibitors and expands the current structural knowledge of coronaviral 3CL(pro) architecture.

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the ß-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL(pro) from other ß-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL(pro) is less efficient at processing a peptide substrate due to MERS-CoV 3CL(pro) being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL(pro) enzymes are tightly associated dimers. Analytical ultracentrifugation studies support that MERS-CoV 3CL(pro) is a weakly associated dimer (Kd ∼52 µm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL(pro) undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL(pro) from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL(pro) dimerization. Activation of MERS-CoV 3CL(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL(pro) inhibitors as antiviral agents.

Coronaviruses resistant to a 3C-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance.

Viral protease inhibitors are remarkably effective at blocking the replication of viruses such as human immunodeficiency virus and hepatitis C virus, but they inevitably lead to the selection of inhibitor-resistant mutants, which may contribute to ongoing disease. Protease inhibitors blocking the replication of coronavirus (CoV), including the causative agents of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), provide a promising foundation for the development of anticoronaviral therapeutics. However, the selection and consequences of inhibitor-resistant CoVs are unknown. In this study, we exploited the model coronavirus, mouse hepatitis virus (MHV), to investigate the genotype and phenotype of MHV quasispecies selected for resistance to a broad-spectrum CoV 3C-like protease (3CLpro) inhibitor. Clonal sequencing identified single or double mutations within the 3CLpro coding sequence of inhibitor-resistant virus. Using reverse genetics to generate isogenic viruses with mutant 3CLpros, we found that viruses encoding double-mutant 3CLpros are fully resistant to the inhibitor and exhibit a significant delay in proteolytic processing of the viral replicase polyprotein. The inhibitor-resistant viruses also exhibited postponed and reduced production of infectious virus particles. Biochemical analysis verified double-mutant 3CLpro enzyme as impaired for protease activity and exhibiting reduced sensitivity to the inhibitor and revealed a delayed kinetics of inhibitor hydrolysis and activity restoration. Furthermore, the inhibitor-resistant virus was shown to be highly attenuated in mice. Our study provides the first insight into the pathogenicity and mechanism of 3CLpro inhibitor-resistant CoV mutants, revealing a low genetic barrier but high fitness cost of resistance. Importance: RNA viruses are infamous for their ability to evolve in response to selective pressure, such as the presence of antiviral drugs. For coronaviruses such as the causative agent of Middle East respiratory syndrome (MERS), protease inhibitors have been developed and shown to block virus replication, but the consequences of selection of inhibitor-resistant mutants have not been studied. Here, we report the low genetic barrier and relatively high deleterious consequences of CoV resistance to a 3CLpro protease inhibitor in a coronavirus model system, mouse hepatitis virus (MHV). We found that although mutations that confer resistance arise quickly, the resistant viruses replicate slowly and do not cause lethal disease in mice. Overall, our study provides the first analysis of the low barrier but high cost of resistance to a CoV 3CLpro inhibitor, which will facilitate the further development of protease inhibitors as anti-coronavirus therapeutics.

BACE1 (ß-secretase) inhibitors for the treatment of Alzheimer's disease.

BACE1 (ß-secretase, memapsin 2, Asp2) has emerged as a promising target for the treatment of Alzheimer's disease. BACE1 is an aspartic protease which functions in the first step of the pathway leading to the production and deposition of amyloid-ß peptide (Aß). Its gene deletion showed only mild phenotypes. BACE1 inhibition has direct implications in the Alzheimer's disease pathology without largely affecting viability. However, inhibiting BACE1 selectively in vivo has presented many challenges to medicinal chemists. Since its identification in 2000, inhibitors covering many different structural classes have been designed and developed. These inhibitors can be largely classified as either peptidomimetic or non-peptidic inhibitors. Progress in these fields resulted in inhibitors that contain many targeted drug-like characteristics. In this review, we describe structure-based design strategies and evolution of a wide range of BACE1 inhibitors including compounds that have been shown to reduce brain Aß, rescue the cognitive decline in transgenic AD mice and inhibitor drug candidates that are currently in clinical trials.