Discovery of the Potent and Selective ATR Inhibitor Camonsertib (RP-3500).

ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties.

Small Molecule Inhibition of an Exopolysaccharide Modification Enzyme is a Viable Strategy To Block Pseudomonas aeruginosa Pel Biofilm Formation.

Biosynthesis of the Pel exopolysaccharide in Pseudomonas aeruginosa requires all seven genes of the operon. The periplasmic modification enzyme PelA contains a C-terminal deacetylase domain that is necessary for Pel-dependent biofilm formation. Herein, we show that extracellular Pel is not produced by a P.

Identification of novel bifunctional HIV-1 reverse transcriptase inhibitors.

Objectives: The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles.

Methods: Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells.

Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin.

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector.

Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage.

Clostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding.

Derivatives of mesoxalic acid block translocation of HIV-1 reverse transcriptase.

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation.

Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore.

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore.

Impact of primer-induced conformational dynamics of HIV-1 reverse transcriptase on polymerase translocation and inhibition.

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity.

A high-throughput continuous assay for screening and characterization of inhibitors of HIV reverse-transcriptase DNA polymerase activity.

The authors have devised a continuous fluorescence-based assay to measure HIV reverse transcriptase (RT) polymerase activity for both high-throughput screening (HTS) and mechanistic characterization of inhibitors. The designed substrate is composed of a recessed DNA primer annealed to a DNA template that is labeled at the 5'-terminus with a donor fluorophore (AlexaFluor 488). RT-catalyzed incorporation of an acceptor-labeled deoxyuridine (dUTP-AlexaFluor 555) at the 3'-terminus of the fully extended DNA primer juxtaposes donor and acceptor fluorophores, resulting in robust fluorescence resonance energy transfer that can be monitored kinetically in real time.

Characterization of a stable, hypertensive rat model suitable for the consecutive evaluation of human renin inhibitors.

Introduction: The hypertensive double-transgenic (dTG) rat strain, expressing human renin and angiotensinogen, develops severe hypertension and organ damage and 50% of individuals die by 7 weeks of age. Here, we characterise a variation of this model in which animals present stable hypertension.

Materials And Methods: The effect of renin-angiotensin system blockers on blood pressure was determined with adult dTG rats treated with enalapril from 3 to 12 weeks of age.

Identification of transforming growth factor beta1-driven genetic programs of acute lung fibrosis.

Lung fibrosis is characterized by excessive accumulation of extracellular matrix components leading to progressive airflow limitation. Distinct profibrotic pathways converge on the activation of transforming growth factor-beta (TGF-beta), a central growth factor implicated in most fibroproliferative diseases. Recently, enforced expression of bioactive human TGF-beta1 (hTGF-beta1) in lungs of transgenic mice was shown to recapitulate several key pathophysiologies observed in fibrotic disorders of the lung, including cellular inflammation, tissue fibrosis, and myofibroblast hyperplasia.

Raloxifene and ICI182,780 increase estrogen receptor-alpha association with a nuclear compartment via overlapping sets of hydrophobic amino acids in activation function 2 helix 12.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-alpha (ERalpha) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERalpha in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERalpha remained nuclear in HepG2 cells treated with either compound.

Tamoxifen and raloxifene differ in their functional interactions with aspartate 351 of estrogen receptor alpha.

The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ERalpha], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hERalpha on the activity profiles of tamoxifen derivatives.

Opposite effects of histone deacetylase inhibitors on glucocorticoid and estrogen signaling in human endometrial Ishikawa cells.

Histone deacetylase inhibitors (HDACi), which have emerged as a new class of anticancer agents, act by modulating expression of genes controlling apoptosis or cell proliferation. Here, we compared the effect of HDACi on transcriptional activation by estrogen or glucocorticoid receptors (ER and GR, respectively), two members of the steroid receptor family with cell growth regulatory properties. Like other transcription factors, steroid receptors modulate histone acetylation on target promoters.

Regulation of Rho signaling pathways in interleukin-2-stimulated human T-lymphocytes.

Rho GTPases are key regulators of many cellular functions, including cytoskeleton organization which is important for cell morphology and mobility, gene expression, cell cycle progression, and cytokinesis. In addition, it has recently been recognized that Rho GTPase activity is required for development of the immune system, as well as for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen-specific populations and provides a model to study cell cycle entry and cell cycle progression.