Design and synthesis of a series of bioavailable fatty acid synthase (FASN) KR domain inhibitors for cancer therapy.

We designed and synthesized a new series of fatty acid synthase (FASN) inhibitors with potential utility for the treatment of cancer. Extensive SAR studies led to highly active FASN inhibitors with good cellular activity and oral bioavailability, exemplified by compound 34. Compound 34 is a potent inhibitor of human FASN (IC50 = 28 nM) that effectively inhibits proliferation of A2780 ovarian cells (IC50 = 13 nM) in lipid-reduced serum (LRS). This cellular activity can be rescued by addition of palmitate, consistent with an on-target effect. Compound 34 is also active in many other cell types, including PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM), and is highly bioavailable (F 61%) with good exposure after oral administration. In a pharmacodynamics study in H460 lung xenograft-bearing mice, oral treatment with compound 34 results in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate, fully consistent with the desired target engagement.

A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.

Automated quality control tool for high-content imaging data by building 2D prediction intervals on reference biosignatures.

Recent advances in automated microscopy and image analysis enables quantitative profiling of cellular phenotypes (Cell Painting). It paves the way for studying the broad effects of chemical perturbations on biological systems at large scale during lead optimization. Comparison of perturbation biosignatures with biosignatures of annotated compounds can inform on both on- and off-target effects. When building databases with phenotypic profiles of thousands of compounds, it is vital to control the quality of Cell Painting assays over time. A tool for this to our knowledge does not yet exist within the imaging community. In this paper, we introduce an automated tool to assess the quality of Cell Painting assays by quantifying the reproducibility of biosignatures of annotated reference compounds. The tool learns the biosignature of those treatments from a historical dataset, and subsequently, it builds a two-dimensional probabilistic quality control (QC) limit. The limit will then be used to detect aberrations in new Cell Painting experiments. The tool is illustrated using simulated data and further demonstrated on Cell Painting data of the A549 cell line. In general, the tool provides a sensitive, detailed and easy-to-interpret mechanism to validate the quality of Cell Painting assays.

Development of a cellular high-content, immunofluorescent HBV core assay to identify novel capsid assembly modulators that induce the formation of aberrant HBV core structures.

Hepatitis B Virus (HBV) core protein has multiple functions in the viral life cycle and is an attractive target for new anti-viral therapies. Capsid assembly modulators (CAMs) target the core protein and induce the formation of either morphologically normal (CAM-N) or aberrant structures (CAM-A), both devoid of genomic material. To date a diverse family of CAM-N chemotypes has been identified, but in contrast, described CAM-As are based on the heteroaryldihydropyrimidine (HAP) scaffold. We used the HBV-inducible HepG2.117 cell line with immunofluorescent labeling of HBV core to develop and validate a cellular high-content image-based assay where aggregated core structures are identified using image analysis spot texture features. Treatment with HAPs led to a dose- and time-dependent formation of aggregated core appearing as dot-like structures in the cytoplasm and nucleus. By combining a biochemical and cellular screening approach, a compound was identified as a novel non-HAP scaffold able to induce dose-dependent formation of aberrant core structures, which was confirmed by electron microscopy and native gel electrophoresis. This compound displayed anti-HBV activity in HepG2.117 cells, providing proof-of-concept for our screening approach. We believe our combined biochemical and cellular high-content screening method will aid in expanding the range of CAM-A chemotypes.

A small-molecule fusion inhibitor of influenza virus is orally active in mice.

Recent characterization of broadly neutralizing antibodies (bnAbs) against influenza virus identified the conserved hemagglutinin (HA) stem as a target for development of universal vaccines and therapeutics. Although several stem bnAbs are being evaluated in clinical trials, antibodies are generally unsuited for oral delivery. Guided by structural knowledge of the interactions and mechanism of anti-stem bnAb CR6261, we selected and optimized small molecules that mimic the bnAb functionality. Our lead compound neutralizes influenza A group 1 viruses by inhibiting HA-mediated fusion in vitro, protects mice against lethal and sublethal influenza challenge after oral administration, and effectively neutralizes virus infection in reconstituted three-dimensional cell culture of fully differentiated human bronchial epithelial cells. Cocrystal structures with H1 and H5 HAs reveal that the lead compound recapitulates the bnAb hotspot interactions.

A Prospective Virtual Screening Study: Enriching Hit Rates and Designing Focus Libraries To Find Inhibitors of PI3Kδ and PI3Kγ.

Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.

Design, synthesis, and tripeptidyl peptidase II inhibitory activity of a novel series of (S)-2,3-dihydro-2-(4-alkyl-1H-imidazol-2-yl)-1H-indoles.

Butabindide, 1, was previously reported as a potent inhibitor (IC50 = 7 nM) of the serine protease enzyme tripeptidyl peptidase II (TPPII), an endogenous protease that degrades cholecystokinin-8 (CCK-8). We found that 1 has some inherent chemical instability, yielding diketopiperazine 2 fairly readily under mimicked physiological conditions. We therefore prepared imidazoles 3, which are void of 1's inherent instability, and have found that our novel analogues maintained comparable TPPII inhibitory activity (e.g.,for 3c, IC50 = 4 nM) as 1.

Synthesis and biological evaluation of imidazol-2-one and 2-cyanoiminoimidazole derivatives: novel series of PDE4 inhibitors.

This communication describes the synthesis and in vitro PDE4 inhibitory activity of a novel series of imidazol-2-one and 2-cyanoiminoimidazole derivatives. The compounds described were also tested in in vivo models to evaluate their anti-inflammatory activity after topical administration as well as their gastro-intestinal side effects. Several compounds proved to be potent PDE4 inhibitors and some 2-cyanoiminoimidazoles showed less pronounced gastro-intestinal side effects than reference compounds but maintained anti-inflammatory activity after topical administration.