Discovery of Potent, Orally Bioavailable Inhibitors of Human Cytomegalovirus.

A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 μM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle.

Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling.

Development of a high-throughput human cytomegalovirus quantitative PCR cell-based assay.

This report describes the development and optimization of a quantitative real-time PCR assay for evaluating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assay measures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalization of CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability.

Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells.

Discovery of novel small-molecule HIV-1 replication inhibitors that stabilize capsid complexes.

The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex.

Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs.

Nucleotide competing reverse transcriptase inhibitors: discovery of a series of non-basic benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

A HTS screen led to the identification of a benzofurano[3,2-d]pyrimidin-2-one core structure which upon further optimization resulted in 1 as a potent HIV-1 nucleotide competing reverse transcriptase inhibitor (NcRTI). Investigation of the SAR at N-1 allowed significant improvements in potency and when combined with the incorporation of heterocycles at C-8 resulted in potent analogues not requiring a basic amine to achieve antiviral activity. Additional modifications at N-1 resulted in 33 which demonstrated excellent antiviral potency and improved physicochemical properties.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency.

ARF1 and ARF6 are dispensable for Crk-dependent epithelial-mesenchymal-like transitions.

Background: Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes.

Crk associates with a multimolecular Paxillin/GIT2/beta-PIX complex and promotes Rac-dependent relocalization of Paxillin to focal contacts.

We have previously demonstrated that the CrkII and CrkL adapter proteins are required for the spreading of epithelial colonies and the breakdown of adherens junctions in response to hepatocyte growth factor. When overexpressed, CrkII and CrkL promote lamellipodia formation, cell spreading, and the loss of epithelial adherens junctions in the absence of hepatocyte growth factor. The exact mechanism by which Crk proteins elicit these changes is unclear.

Crk synergizes with epidermal growth factor for epithelial invasion and morphogenesis and is required for the met morphogenic program.

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process.

Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes.