Anti-Cancer Activity Profiling of Chemotherapeutic Agents in 3D Co-Cultures of Pancreatic Tumor Spheroids with Cancer-Associated Fibroblasts and Macrophages

Activated pancreatic stellate cells (aPSCs) and M2 macrophages modulate tumor progression and therapeutic efficacy in pancreatic ductal adenocarcinoma (PDAC) via epithelial-mesenchymal transition (EMT). Here, our aim was to analyze the anti-invasion effects of anti-cancer agents where EMT-inducing cancer-stroma interaction occurs under three-dimensional (3D) culture conditions. We used microfluidic channel chips to co-culture pancreatic tumor spheroids (TSs) with aPSCs and THP-1-derived M2 macrophages (M2 THP-1 cells) embedded in type I collagen. Under stromal cell co-culture conditions, PANC-1 TSs displayed elevated expression of EMT-related proteins and increased invasion and migration. When PANC-1 TSs were exposed to gemcitabine, 5-fluorouracil, oxaliplatin, or paclitaxel, 30–50% cells were found unaffected, with no significant changes in the dose-response profiles under stromal cell co-culture conditions. This indicated intrinsic resistance to these drugs and no further induction of drug resistance by stromal cells. Paclitaxel had a significant anti-invasion effect; in contrast, oxaliplatin did not show such effect despite its specific cytotoxicity in M2 THP-1 cells. Overall, our findings demonstrate that the TS-stroma co-culture model of PDAC is useful for activity profiling of anti-cancer agents against cancer and stromal cells, and analyzing the relationship between anti-stromal activity and anti-invasion effects.

Phosphocatalytic Kinome Activity Profiling of Apoptotic and Ferroptotic Agents in Multiple Myeloma Cells

Through phosphorylation of their substrate proteins, protein kinases are crucial for transducing cellular signals and orchestrating biological processes, including cell death and survival. Recent studies have revealed that kinases are involved in ferroptosis, an iron-dependent mode of cell death associated with toxic lipid peroxidation. Given that ferroptosis is being explored as an alternative strategy to eliminate apoptosis-resistant tumor cells, further characterization of ferroptosis-dependent kinase changes might aid in identifying novel druggable targets for protein kinase inhibitors in the context of cancer treatment. To this end, we performed a phosphopeptidome based kinase activity profiling of glucocorticoid-resistant multiple myeloma cells treated with either the apoptosis inducer staurosporine (STS) or ferroptosis inducer RSL3 and compared their kinome activity signatures. Our data demonstrate that both cell death mechanisms inhibit the activity of kinases classified into the CMGC and AGC families, with STS showing a broader spectrum of serine/threonine kinase inhibition. In contrast, RSL3 targets a significant number of tyrosine kinases, including key players of the B-cell receptor signaling pathway. Remarkably, additional kinase profiling of the anti-cancer agent withaferin A revealed considerable overlap with ferroptosis and apoptosis kinome activity, explaining why withaferin A can induce mixed ferroptotic and apoptotic cell death features. Altogether, we show that apoptotic and ferroptotic cell death induce different kinase signaling changes and that kinome profiling might become a valid approach to identify cell death chemosensitization modalities of novel anti-cancer agents.

Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3’,5’-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), Cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

Analogs of TIQ-A as inhibitors of human mono-ADP-ribosylating PARPs

The scaffold of TIQ-A, a previously known inhibitor of human poly-ADP-ribosyltransferase PARP1, was utilized to develop inhibitors against human mono-ADP-ribosyltransferases through structure-guided design and activity profiling. By supplementing the TIQ-A scaffold with small structural changes, based on a PARP10 inhibitor OUL35, selectivity changed from poly-ADP-ribosyltransferases towards mono-ADP-ribosyltransferases. Binding modes of analogs were experimentally verified by determining complex crystal structures with mono-ADP-ribosyltransferase PARP15 and with poly-ADP-ribosyltransferase TNKS2. The best analogs of the study achieved 10-20-fold selectivity towards mono-ADP-ribosyltransferases PARP10 and PARP15 while maintaining micromolar potencies. The work demonstrates a route to differentiate compound selectivity between mono- and poly-ribosyltransferases of the human ARTD family.

Selective DNA Gyrase Inhibitors: Multi-Target In Silico Profiling with 3D-Pharmacophores

(1) Background: DNA gyrase is an important target for the development of novel antibiotics. Although ATP-competitive DNA gyrase (GyrB) inhibitors are a well-studied class of antibacterial agents, there is currently no representative used in therapy, largely due to unwanted off-target activities. Selectivity of GyrB inhibitors against closely related human ATP-binding enzymes should be evaluated early in development to avoid off-target binding to homologous binding domains. (2) Methods: To address this challenge, we developed selective 3D-pharmacophore models for GyrB, human topoisomerase IIα (TopoII), and the Hsp90 N-terminal domain (NTD) to be used in in silico activity profiling paradigms to identify molecules selective for GyrB over TopoII and Hsp90, as starting points for hit expansion and lead optimization. (3) Results: The models were used to profile highly active GyrB, TopoII, and Hsp90 inhibitors. Selected compounds were tested in in vitro assays. GyrB inhibitors 1 and 2 were inactive against TopoII and Hsp90, while 3 and 4, potent Hsp90 inhibitors, displayed no inhibition of GyrB and TopoII, and TopoII inhibitors 5 and 6 were inactive at GyrB and Hsp90. (4) Conclusions: The results provide a proof of concept for the use of target activity profiling methods to identify selective starting points for hit and lead identification.

Laying the Foundation for Pregnancy Physical Activity Profiling: A Framework for Providing Tailored Physical Activity Advice and Guidance to Pregnant Women

The aim of this study was to examine the predictive utility of the theory of planned behaviour (TPB) in explaining pregnant women’s physical activity (PA) intentions and behaviour and to scrutinise the role of past behaviour within this context. Pregnant women (n = 89) completed the pregnancy physical activity questionnaire (PPAQ) and newly developed TPB questionnaire on two separate occasions during their pregnancy. Analyses were carried out in relation to three scenarios. Firstly, when considering the original TPB, intention emerged as the strongest determinant of pregnant women’s PA behaviour. Secondly, controlling for past behaviour attenuated the influence of intention and perceived behavioural control on behaviour, with neither of the original variables providing a unique influence. Finally, the addition of past behaviour added significantly to the prediction of intention with the model as a whole, explaining 85% of the variance in pregnant women’s PA intention, and with past behaviour uniquely contributing 44.8% of the variance. Pregnancy physical activity profiling based on intention and behaviour status is subsequently introduced as a novel and practical framework. This provides healthcare professionals with the opportunity and structure to provide tailored advice and guidance to pregnant women, thereby facilitating engagement with PA throughout motherhood.