Structure-Guided Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic, Cell-Based, and In Vivo Studies

Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of anti-norovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection

Structure-Based Exploration and Exploitation of the S4 Subsite of Norovirus 3CL Protease in the Design of Potent and Permeable Inhibitors

Human noroviruses are the primary cause of epidemic and sporadic acute gastroenteritis. The worldwide high morbidity and mortality associated with norovirus infections, particularly among the elderly, immunocompromised patients and children, constitute a serious public health concern. There are currently no approved human vaccines or norovirus-specific small-molecule therapeutics or prophylactics. Norovirus 3CL protease has recently emerged as a potential therapeutic target for the development of anti-norovirus agents. We hypothesized that the S4 subsite of the enzyme may provide an effective means of designing potent and cell permeable inhibitors of the enzyme. We report herein the structure-guided exploration and exploitation of the S4 subsite of norovirus 3CL protease in the design and synthesis of effective inhibitors of the protease

Oxadiazole-Based Cell Permeable Macrocyclic Transition State Inhibitors of Norovirus 2CL Protease

Human noroviruses are the primary causative agents of acute gastroenteritis and a pressing public health burden worldwide. There are currently no vaccines or small molecule therapeutics available for the treatment or prophylaxis of norovirus infections. Norovirus 3CL protease plays a vital role in viral replication by generating structural and nonstructural proteins via the cleavage of the viral polyprotein. Thus, molecules that inhibit the viral protease may have potential therapeutic value. We describe herein the structure-based design, synthesis, and in vitro and cell-based evaluation of the first class of oxadiazole-based, permeable macrocyclic inhibitors of norovirus 3CL protease

Efficacy of a 3C-like protease inhibitor in treating various forms of acquired feline infectious peritonitis

Objectives The safety and efficacy of the 3C-like protease inhibitor GC376 was tested on a cohort of client-owned cats with various forms of feline infectious peritonitis (FIP). Methods Twenty cats from 3.3-82 months of age (mean 10.4 months) with various forms of FIP were accepted into a field trial. Fourteen cats presented with wet or dry-to-wet FIP and six cats presented with dry FIP. GC376 was administered subcutaneously every 12 h at a dose of 15 mg/kg. Cats with neurologic signs were excluded from the study. Results Nineteen of 20 cats treated with GC376 regained outward health within 2 weeks of initial treatment. However, disease signs recurred 1-7 weeks after primary treatment and relapses and new cases were ultimately treated for a minimum of 12 weeks. Relapses no longer responsive to treatment occurred in 13 of these 19 cats within 1-7 weeks of initial or repeat treatment(s). Severe neurologic disease occurred in 8/13 cats that failed treatment and five cats had recurrences of abdominal lesions. At the time of writing, seven cats were in disease remission. Five kittens aged 3.3-4.4 months with wet FIP were treated for 12 weeks and have been in disease remission after stopping treatment and at the time of writing for 5-14 months (mean 11.2 months). A sixth kitten was in remission for 10 weeks after 12 weeks of treatment, relapsed and is responding to a second round of GC376. The seventh was a 6.8-year-old cat with only mesenteric lymph node involvement that went into remission after three relapses that required progressively longer repeat treatments over a 10 month period. Side effects of treatment included transient stinging upon injection and occasional foci of subcutaneous fibrosis and hair loss. There was retarded development and abnormal eruption of permanent teeth in cats treated before 16-18 weeks of age. Conclusions and relevance GC376 showed promise in treating cats with certain presentations of FIP and has opened the door to targeted antiviral drug therapy

Changes in the viral RNA levels in P15 and P16 before and during antiviral treatment.

<p>(A and B) The viral RNA fold changes in the macrophages from the ascites of P15 (A) and P16 (B) over time are shown. The Ct values from viral RNA real-time qRT-PCR were normalized to β-actin and the 2^-ΔΔCt method was used to calculate the relative change in viral RNA level, compared to the pre-treatment value. (C) The viral RNA level (2^-ΔCt) in the omentum of P15 and P16 which are collected after 4 and 7 days of antiviral treatment, respectively. The bar graph shows the 2^-ΔCt values calculated by normalizing the Ct values from viral RNA real-time qRT-PCR to β-actin. (D) The 2^-ΔCt values for each viral RNA in the macrophages from the ascites of P15 and P16 at pre-treatment and during treatment are listed in the table. N/A, not available.</p

Clinical and laboratory findings in cats challenged with FIPV prior to antiviral treatment.

<p>Clinical and laboratory findings in cats challenged with FIPV prior to antiviral treatment.</p

Activity of GC376 against 3CLpro of various coronaviruses in a fluorescence resonance energy transfer (FRET) assay.

<p>The upper graph shows the percent activity of 3CLpro of FIPV, SARS-CoV, and MERS-CoV in the presence of GC376, determined by a FRET assay. The lower table summarizes the 50% inhibitory concentration (IC₅₀) values of GC376 against 3CLpro of FIPV, SARS-CoV, and MERS-CoV. Asterisks indicate the previously published value [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005531#ppat.1005531.ref028" target="_blank">28</a>].</p