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Recent advances with deep neural networks have shown the feasibility of acquiring brightfield images with transmitted light and applying in-silico labeling to predict fluorescent images. We have developed a novel in-silico labeling method based on a generative adversarial network and outperforms the state-of-the-art Unet method in generating realistic fluorescent images and quantitatively recapitulating real staining signals, as demonstrated in a complex co-culture myelination assay. Furthermore, we have performed the assay in live mode with multiple kinetic points, applied in-silico labeling to predict fluorescent images from brightfield and quantified the kinetic phenotypic changes. Thus, the proposed approach provides a potential tool to study the kinetics of cellular phenotypic changes with brightfield imaging.

FGF19 signaling through the FGFR4/β-klotho receptor complex has been shown to be a key driver of growth and survival in a subset of hepatocellular carcinomas, making selective FGFR4 inhibition an attractive treatment opportunity. A kinome-wide sequence alignment highlighted a poorly conserved cysteine residue within the FGFR4 ATP-binding site at position 552, two positions beyond the gate-keeper residue. Several strategies for targeting this cysteine to identify FGFR4 selective inhibitor starting points are summarized which made use of both rational and unbiased screening approaches. The optimization of a 2-formylquinoline amide hit series is described in which the aldehyde makes a hemithioacetal reversible-covalent interaction with cysteine 552. Key challenges addressed during the optimization are improving the FGFR4 potency, metabolic stability, and solubility leading ultimately to the highly selective first-in-class clinical candidate roblitinib.

[Image: see text] As part of a project to identify FGFR4 selective inhibitors, scaffold morphing of a 2-formylquinoline amide hit identified series of 2-formylpyridine ureas (2-FPUs) with improved potency and physicochemical properties. In particular, tetrahydronaphthyridine urea analogues with cellular activities below 30 nM have been identified. Consistent with the hypothesized reversible-covalent mechanism of inhibition, the 2-FPUs exhibited slow binding kinetics, and the aldehyde, as the putative electrophile, could be demonstrated to be a key structural element for activity.

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.

Patients with International Union Against Cancer (UICC) stage IIb and III colon cancer and stage II and III rectal cancer may receive adjuvant chemotherapy with 5-fluorouracil (5-FU). High levels of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been associated with resistance to 5-FU in advanced colorectal cancer. The aim of this study was to investigate the association of TS and DPD mRNA levels with recurrence-free survival in patients with colorectal cancer who are receiving adjuvant 5-FU-based chemotherapy. TS and DPD mRNA quantitation was retrospectively performed in primary colorectal cancer specimens from patients receiving adjuvant 5-FU using a reverse transcription- polymerase chain reaction technique. The median TS mRNA level in patients with a recurrence (n = 142) was 0.68, and in patients without a recurrence (n = 206) the median level was 0.80 (P0.01). Patients with a recurrence who had a low TS level (TSor = 0.9; n = 102) had a median recurrence-free survival of 18 months (range 3.0 to 54 months), and those with a high TS level (TS0.9; n = 40) had a median recurrence-free survival of 11 months (range 1.7 to 53 months; P = 0.0024). There was no difference in the median recurrence-free survival of patients with low and high DPD mRNA levels. The TS mRNA level may be a useful marker to predict the time to recurrence in patients with colorectal cancer who are receiving adjuvant 5-FU treatment.