Incorporation of [³H]-ETV into HBV nucleocapsid DNA in culture.

<p>[³H]-ETV was added to cultures of HepG2 cells transfected with an HBV expression construct, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>. HBV nucleocapsids were isolated from cell lysates, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>, and radiolabeled HBV DNA was quantified through scintillation counting. The levels of nucleocapsid-associated [³H] from cells grown in [³H]-ETV are presented as percent wildtype control values ± standard deviation. Yields of HBV nucleocapsid DNA were standardized according to real-time PCR quantification of HBV DNA within isolated nucleocapsids, as detailed under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#s4" target="_blank">Materials and Methods</a>. Similar results were obtained by standardizing total HBV nucleocapsid DNA levels with nucleocapsids from parallel cultures metabolically labeled with [³H]-thymidine (data not shown). WT, wildtype nucleocapsids; LVDr, M204V+L180M substituted HBV nucleocapsids, LVDr+M250V, M204V+L180M+M250V substituted nucleocapsids; LVDr+T184G+S202I, M204V+L180M+T184G+S202I substituted nucleocapsids. HBVs were tested independently 3 to 4 times, except the LVDr+M250V, which was tested twice.</p

The T184-S202-204 hydrogen bonding network stabilizes the YMDD loop.

<p>The YMDD loop is shown with residues M204V+L180M and the H-bonding between residues T184, S202 and M204V are shown as dotted white lines.</p

Analysis of ETVr through strand-specific DNA synthesis in culture.

<p>Cell culture ETV EC₅₀ determinations were made for wildtype and various resistant HBVs. The levels of HBV DNA synthesized in the presence of ETV was determined by HBV-specific probe hybridization to HBV nucleocapsid DNA from the cultures. The probes used were the typical double-stranded DNA probe, or strand-specific riboprobes which hybridized to a single HBV DNA strand. Comparison of strand-specific EC₅₀ versus double stranded EC₅₀ for wildtype polymerase (WT) or LVDr M204V+L180M HBV, or the LVDr substitutions with ETVr substitutions (+T184, +S202, +M250) as indicated. Values at 4000 nM indicate that 50% inhibition was not observed at the highest ETV concentration tested.</p

Molecular homology model of resistant HBV RTs.

<p>The ETV-TP binding pocket of HBV RT. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Langley1" target="_blank">[3]</a>. (A) HBV RT with LVDr substitutions, M204V+L180M, (B) HBV RT with LVDr + S202G. HBV RT, ETV-TP and primer-template DNAs are labeled. The residues lining the pocket are orange, changes from LVDr are red, from ETVr are blue, and the M250, S202, and T184 residue positions (panel B) are pink. Panel A was essential reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Langley1" target="_blank">[3]</a> with permission.</p

Phenotypic ETV Resistance of HBV Clones.

a<p>EC₅₀ and IC₅₀ values represent the mean ± standard deviation (SD) from at least 3 independent experiments.</p>b<p>Wildtype values from a single isolate tested in parallel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Tenney1" target="_blank">[5]</a>, the average ETV EC₅₀ for a panel of 76 wildtype isolates  = 3.4±2.0 nM.</p>c<p>LVDr values from a single isolate tested in parallel <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Tenney1" target="_blank">[5]</a>, the average ETV EC₅₀ for a panel of 15 LVDr isolates  = 31.1±17.5 nM.</p>d<p>Values averaged from 2 independent isolates.</p>e<p>Values obtained from a single patient isolate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009195#pone.0009195-Tenney1" target="_blank">[5]</a>.</p

Kinetic Parameters for HBV Polymerases.

a<p>Values are the mean ± standard deviation from at least three independent experiments.</p>b<p>Fold change from wildtype value.</p

Discovery of the Human Immunodeficiency Virus Type 1 (HIV-1) Attachment Inhibitor Temsavir and Its Phosphonooxymethyl Prodrug Fostemsavir

The optimization of the 4-methoxy-6-azaindole series of HIV-1 attachment inhibitors (AIs) that originated with <b>1</b> to deliver temsavir (<b>3</b>, BMS-626529) is described. The most beneficial increases in potency and pharmacokinetic (PK) properties were attained by incorporating N-linked, sp²-hybridized heteroaryl rings at the 7-position of the heterocyclic nucleus. Compounds that adhered to a coplanarity model afforded targeted antiviral potency, leading to the identification of <b>3</b> with characteristics that provided for targeted exposure and PK properties in three preclinical species. However, the physical properties of <b>3</b> limited plasma exposure at higher doses, both in preclinical studies and in clinical trials as the result of dissolution- and/or solubility-limited absorption, a deficiency addressed by the preparation of the phosphono­oxymethyl prodrug <b>4</b> (BMS-663068, fostemsavir). An extended-release formulation of <b>4</b> is currently in phase III clinical trials where it has shown promise as part of a drug combination therapy in highly treatment-experienced HIV-1 infected patients

Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1) Attachment 6. Preclinical and Human Pharmacokinetic Profiling of BMS-663749, a Phosphonooxymethyl Prodrug of the HIV-1 Attachment Inhibitor 2-(4-Benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1<i>H</i>-pyrrolo[2,3-<i>c</i>]pyridin-3-yl)-2-oxoethanone (BMS-488043)

BMS-663749, a phosphonooxymethyl prodrug <b>4</b> of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1<i>H</i>-pyrrolo­[2,3-<i>c</i>]­pyridin-3-yl)-2-oxoethanone (BMS-488043) (<b>2</b>) was prepared and profiled in a variety of preclinical in vitro and in vivo models designed to assess its ability to deliver parent drug following oral administration. The data showed that prodrug <b>4</b> had excellent potential to significantly reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies in normal healthy subjects confirmed the potential of <b>4</b>, revealing that the prodrug significantly increased both the AUC and <i>C</i>_max of <b>2</b> compared to a solid capsule formulation containing the parent drug upon dose escalation. These data provided guidance for further efforts to obtain an effective HIV-1 attachment inhibitor

Inhibitors of Human Immunodeficiency Virus Type 1 (HIV-1) Attachment. 12. Structure–Activity Relationships Associated with 4‑Fluoro-6-azaindole Derivatives Leading to the Identification of 1‑(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]triazol-1-yl‑1<i>H</i>‑pyrrolo[2,3‑<i>c</i>]pyridin-3-yl)ethane-1,2-dione (BMS-585248)

A series of highly potent HIV-1 attachment inhibitors with 4-fluoro-6-azaindole core heterocycles that target the viral envelope protein gp120 has been prepared. Substitution in the 7-position of the azaindole core with amides (<b>12a</b>,<b>b</b>), C-linked heterocycles (<b>12c</b>–<b>l</b>), and N-linked heterocycles (<b>12m</b>–<b>u</b>) provided compounds with subnanomolar potency in a pseudotype infectivity assay and good pharmacokinetic profiles in vivo. A predictive model was developed from the initial SAR in which the potency of the analogues correlated with the ability of the substituent in the 7-position of the azaindole to adopt a coplanar conformation by either forming internal hydrogen bonds or avoiding repulsive substitution patterns. 1-(4-Benzoylpiperazin-1-yl)-2-(4-fluoro-7-[1,2,3]­triazol-1-yl-1<i>H</i>-pyrrolo­[2,3-<i>c</i>]­pyridin-3-yl)­ethane-1,2-dione (BMS-585248, <b>12m</b>) exhibited much improved in vitro potency and pharmacokinetic properties than the previous clinical candidate BMS-488043 (<b>1</b>). The predicted low clearance in humans, modest protein binding, and good potency in the presence of 40% human serum for <b>12m</b> led to its selection for human clinical studies

Discovery and Early Clinical Evaluation of BMS-605339, a Potent and Orally Efficacious Tripeptidic Acylsulfonamide NS3 Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

The discovery of BMS-605339 (<b>35</b>), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropyl­acylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1′ site of the protease. The identification of <b>35</b> was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound <b>35</b> which demonstrated antiviral activity in HCV-infected patients