Comparison of <i>in vitro</i> anti-influenza activities of RO5464466 and RO5487624.

a<p>: All numbers (EC₅₀, CC₅₀, and IC₅₀) are means of three independent experiments and shown in micromolar concentrations;</p><p>Some data from A/Weiss/43 have been shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#pone.0029120-Tang1" target="_blank">[29]</a>.</p>b<p>: MDCK cells were used;</p>c<p>: + means nearly 100% protection of BHA from trypsin degradation in the presence of compounds (10 µM).</p

RO5464466 inhibited HA-mediated hemolysis of chicken erythrocytes.

<p>After mixing RO5464466 with virus-containing allantoic fluid, suspension of freshly prepared chicken erythrocytes was added. The mixture was then acidified with different pH from 4.85 to 6. The suspension was incubated at 37°C for 30 minutes. The final concentration of RO5464466 was 10 µM. After a brief spin, supernatants containing released hemoglobin were transferred to a second plate for measuring OD₅₄₀. As a control, suspensions of erythrocytes alone were acidified (pH control) similarly to measure low pH-caused hemolysis. In the DMSO control group, every step was the same as described in RO5464466 group except the testing compound was omitted. All conditions were tested in duplicated wells. *, results significantly different (RO5464466 treated samples vs. DMSO controls) by Student's t test (<i>P</i><0.05).</p

SDS-PAGE of trypsin sensitivity assay showing RO5464466 protected BHA from trypsin digestion in pH-dependent and dose-dependent manner.

<p>(A) 6 µg BHA was incubated with compounds of different concentrations for 15 minute at 31°C prior to acidification to pH 5.0 with 0.25 M citrate (pH 4.2). The mixture was neutralized to a final pH of 7.5 and treated with 2 µg of trypsin for 30 minutes at 37°C. The extent of trypsin cleavage on BHA was analyzed on a 10% SDS-PAGE gel. MW was shown on the right in thousands. Untreated BHA, trypsin alone, BHA trypsin digestion without a prior acidification step were used as controls and included in this figure. (B) Dose-dependent protection of BHA by RO5464466. (C) pH-dependent protection of BHA by RO5464466. After incubated with 10 µM of RO5464466, BHA was acidified to different final pH shown on the top of the gel before neutralization and trypsin digestion. As a negative control, DMSO-treated BHA was adjusted to two pH values (5.0 and 5.2) before neutralization and trypsin digestion. (D) RO5464466 protected BHA from trypsin digestion not by directly inhibiting trypsin enzymatic activity. RO5464466 was added into the reaction either before or after acidification of BHA.</p

<i>In vivo</i> efficacy of RO5487624.

<p>40 LD₅₀ of A/FM/1/47 (H1N1) were used for viral challenge in all groups by intranasal inoculation. The first dosage of all treatments was given either 1 h before (A) or 3 h after (B) virus challenge (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a> section). Mice in untreated control group were orally administrated with PBS twice a day for 7 days. Animals (n = 10 in each group) was monitored for 14 days starting from virus challenge. Data was analyzed with Chi-square for mortality rate, * <i>P</i><0.05; ** <i>P</i><0.001, and with Kaplan-Meier for mean survival day, # <i>P</i><0.05; ## <i>P</i><0.001, respectively.</p

Pharmacokinetic analysis of RO5487624.

<p>Mean plasma concentration-time profiles of RO5487624 after an oral dose (30 mg/kg, ◊, 100 mg/kg □, and 200 mg/kg, Δ) to CD-1 mice. Vertical lines are standard deviation of each group (n = 3). Concentration corresponding to RO5487624's EC₉₀ after protein binding adjustment is marked by a dotted line.</p

RO5464466 blocked the production of progeny virus and inhibited an established influenza virus infection.

<p>(A) Decrease of progeny virus production in cells treated with RO5464466 and other reference compounds. 12-well plates were seeded with MDCK cells at a density of 1.5×10⁵ cells per well. On the next day, serially diluted compounds and 150 pfu virus were added per well. 48 hours later, treatment was stopped and viral titer in cell monolayer was measured in TCID₅₀. Virus yield was shown in a log scale, a mean of two wells. (B) Dynamics of virus yield reduction in the presence of inhibitors. In this experiment, all treatments began at the same time when virus was added into culture wells but were stopped at different time points of 24, 48, and 72 hours post-infection, respectively. (C) Inhibition of plaque formation by RO5464466. Details of seeding MDCK cells into 6-well plates and infecting monolayers with influenza A/Weiss/43 (H1N1) were described in the plaque reduction assay in the section of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a>. Instead of using 3 ml of agarose-containing overlay for each well, 2.5 ml of overlay was used. At each time point (0, 12, 24, 48 hours post-infection), 2.5 ml of culture medium that contained compound of 2-fold final concentration was added on the top of the overlay. At 72 hours post-infection, cell monolayers were fixed and stained to show plaques. Representative data of three independent experiments was shown.</p

Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure–activity relationship (SAR) and protease selectivity are also discussed

Design and Synthesis of Orally Bioavailable 4‑Methyl Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors

Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal capsid formation have been an attractive approach to block the replication of HBV viruses. We carried out multidimensional structural optimizations based on the heteroaryldihydropyrimidine (HAP) analogue Bay41-4109 (<b>1</b>) and identified a novel series of HBV capsid inhibitors that demonstrated promising cellular selectivity indexes, metabolic stabilities, and in vitro safety profiles. Herein we disclose the design, synthesis, structure–activity relationship (SAR), cocrystal structure in complex with HBV capsid proteins and in vivo pharmacological study of the 4-methyl HAP analogues. In particular, the (2<i>S</i>,4<i>S</i>)-4,4-difluoroproline substituted analogue <b>34a</b> demonstrated high oral bioavailability and liver exposure and achieved over 2 log viral load reduction in a hydrodynamic injected (HDI) HBV mouse model

Design and Synthesis of Orally Bioavailable 4‑Methyl Heteroaryldihydropyrimidine Based Hepatitis B Virus (HBV) Capsid Inhibitors

Targeting the capsid protein of hepatitis B virus (HBV) and thus interrupting normal capsid formation have been an attractive approach to block the replication of HBV viruses. We carried out multidimensional structural optimizations based on the heteroaryldihydropyrimidine (HAP) analogue Bay41-4109 (<b>1</b>) and identified a novel series of HBV capsid inhibitors that demonstrated promising cellular selectivity indexes, metabolic stabilities, and in vitro safety profiles. Herein we disclose the design, synthesis, structure–activity relationship (SAR), cocrystal structure in complex with HBV capsid proteins and in vivo pharmacological study of the 4-methyl HAP analogues. In particular, the (2<i>S</i>,4<i>S</i>)-4,4-difluoroproline substituted analogue <b>34a</b> demonstrated high oral bioavailability and liver exposure and achieved over 2 log viral load reduction in a hydrodynamic injected (HDI) HBV mouse model

Discovery and Pre-Clinical Characterization of Third-Generation 4‑H Heteroaryldihydropyrimidine (HAP) Analogues as Hepatitis B Virus (HBV) Capsid Inhibitors

Described herein are the discovery and structure–activity relationship (SAR) studies of the third-generation 4-H heteroaryldihydropyrimidines (4-H HAPs) featuring the introduction of a C6 carboxyl group as novel HBV capsid inhibitors. This new series of 4-H HAPs showed improved anti-HBV activity and better drug-like properties compared to the first- and second-generation 4-H HAPs. X-ray crystallographic study of analogue <b>12</b> (HAP_R01) with Cp149 Y132A mutant hexamer clearly elucidated the role of C6 carboxyl group played for the increased binding affinity, which formed strong hydrogen bonding interactions with capsid protein and coordinated waters. The representative analogue <b>10</b> (HAP_R10) was extensively characterized in vitro (ADMET) and in vivo (mouse PK and PD) and subsequently selected for further development as oral anti-HBV infection agent