Fuertes-Garcia, L. | Search bioRxiv

bioRxiv2026

Targeting the CDK4-Cyclin D Complex: A New Generation of Selective Kinase Inhibitors for Cancer Therapy

Glioblastoma (GBM) is an aggressive malignancy with limited therapeutic options and poor survival. Cyclin-dependent kinase 4 (CDK4) is overexpressed in GBM and promotes tumor progression through canonical cell-cycle regulation and extranuclear functions related to metabolism, invasion, and tumor microenvironment modulation. Current ATP-competitive CDK4/6 inhibitors show limited selectivity, potentially causing off-target effects and resistance. We developed a strategy to identify novel CDK4 inhibitors targeting the CDK4-Cyclin D1 protein-protein interaction, a key determinant of CDK4 activation. Virtual screening of approximately 950,000 compounds, combining molecular docking and molecular dynamics simulations, identified candidate molecules that were subsequently evaluated using proximity ligation assays, in vitro kinase activity assays, phosphokinase profiling, and functional analyses in patient-derived GBM cell lines and additional cancer models. Diophenic emerged as the lead candidate, demonstrating consistent antiproliferative activity across multiple GBM and epithelial cancer models. The compound selectively disrupted the CDK4-Cyclin D1 interaction while sparing the closely related CDK6 complex, and inhibited CDK4 kinase activity in vitro. Functional studies showed that diophenic induced substantial cell death in GBM cells, significantly reduced invasiveness, and exhibited distinct effects on cell migration compared with CDK4/6 inhibitors. Furthermore, phosphokinase profiling revealed that diophenic modulates a more restricted signaling network than ATP-competitive inhibitors, supporting greater functional selectivity and reduced off-target activity. Aiming the CDK4-Cyclin D1 interface represents a promising alternative to ATP-competitive CDK4/6 inhibition, which target a highly conserved site across the protein kinase superfamily. This approach provides a proof of concept for developing next-generation CDK4-selective therapeutics. Further studies should evaluate its translational potential in GBM.

cancer biology↗

bioRxiv2025

Temperature changes are signaled in cyanobacteria through the PipX interaction network

Cyanobacteria perform oxygenic photosynthesis and have evolved sophisticated mechanisms to adapt their metabolism to challenging environmental changes. Despite their ecological and biotechnological importance, many regulatory proteins are still uncharacterised, and their signalling networks are poorly studied in comparison to other bacterial phyla. Two small proteins, PipX, unique to cyanobacteria, and PII, widespread in bacteria and plants, are the hubs of a protein interaction network involved in carbon/nitrogen homeostasis, energy sensing, translational regulation and growth. Here we exploit the NanoBiT complementation system to demonstrate in real time that temperature affects PipX interactions with its best studied partners: the signal transduction protein PII, the global transcriptional regulator NtcA, and the ribosome-assembly GTPase EngA. While heat shock increased PipX-PII complex formation and impaired PipX-EngA and PipX-NtcA interactions, cold shock resulted in a decrease of all three complexes. Far-UV circular dichroism spectra of isolated PipX suggested the involvement of its C-terminal -helix in the common response to cold shock. However, during longer term acclimatization, each type of complex responded distinctively after up- or downshifts in temperature and PipX-PII and PipX-NtcA interactions were influenced in opposite ways. Altogether the results indicate that PipX is a thermometer of low temperatures, bringing new light to the study of environmental signaling in cyanobacteria. Our results also illustrate the enormous potential of the NanoBiT complementation system to fuel understanding of the mechanisms allowing cyanobacteria to initially respond and/or acclimatize to environmental factors. IMPORTANCECyanobacteria are a group of organisms of great ecological and biotechnological importance but relatively little understood in terms of the regulatory components and molecular mechanisms that make them so unique. PipX is a small protein exclusive to cyanobacteria that functions by binding to other regulators in response to intracellular metabolic signals. We used a bioluminescence reporter system to show that temperature shifts significantly alter the relative affinity of PipX for its well-known partners. By showing the impact of a highly relevant environmentally factor such as temperature on the regulatory details of a protein interaction network and implicating PipX in the response to cold shock this work paves the way for significant advancements in both basic and applied research of cyanobacteria.

molecular biology↗