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. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 µM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

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. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid.

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 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. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.

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. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

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. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells.

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. We show that the overexpression of CrkII or CrkL, but not Src homology 2 or amino-terminal Src homology 3 domain mutant Crk proteins, promotes the relocalization of Paxillin to focal contacts throughout the cell and within lamellipodia in a Rac-dependent manner. In stable cell lines overexpressing CrkII, enhanced lamellipodia formation and cell spreading correlate with an increased association of CrkII with Paxillin, GIT2 (an ARF-GAP) and beta-PIX (a Rac1 exchange factor). Mutants of Paxillin that fail to associate with Crk or GIT2, or do not target to focal adhesions inhibit Crk-dependent cell spreading and lamellipodia formation. We conclude from these studies that the association of Crk with Paxillin is important for the spreading of epithelial colonies, by influencing the recruitment of Paxillin to focal complexes and promoting the enhanced assembly of Paxillin/GIT2/beta-PIX complexes.

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. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.

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. Overexpression of Crk adapter proteins in MDCK cells promotes spreading and loss of adherens junctions, events regulated by HGF. We recently demonstrated that the overexpression of CrkII promotes the formation of a multi-molecular complex containing CrkII, Paxillin and GIT-2, an ARF-GAP. MATERIALS AND METHODS: To determine the possible role of ARF1 and ARF6 in Crk- and HGF-dependent cell spreading, dominant negative mutants of ARF1 or ARF6 were microinjected into MDCK cells. RESULTS: We report that MDCK cell lines overexpressing CrkII display reduced ARF6 but not ARF1 activity. While both ARF1 and ARF6 are required for the spreading of MDCK cells stimulated with HGF, ARF1 and ARF6 activity is dispensable for the spreading of cells microinjected with Crk. CONCLUSION: We propose that Crk adapter proteins may act downstream of ARF1 and ARF6 to promote cell spreading or rely on different pathways to enhance cell spreading.

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. The assay throughput was further enhanced by eliminating several wash steps and by lysing the cells directly in the presence of cell culture media, both of which had no impact on the assay metrics. The assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR) assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratory strains and clinical isolates.

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. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection.

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. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.