Kinases and Phosphatases

Reversible protein phosphorylation is an important regulatory mechanism in cellular signalling and disease, regulated by the opposing actions of kinases and phosphatases. Modern computer methods predict kinase–substrate or phosphatase–substrate interactions in isolation and lack specificity for biological conditions, neglecting triadic regulation. We present SPINET-KSP, a multi-modal LLM–Graph foundation model engineered for the prediction of kinase–substrate–phosphatase (KSP) triads with contextual awareness. SPINET-KSP integrates high-confidence interactomes (SIGNOR, BioGRID, STRING), structural contacts obtained from AlphaFold3, ESM-3 sequence embeddings, and a 512-dimensional cell-state manifold with 1612 quantitative phosphoproteomic conditions. A heterogeneous KSP graph is examined utilising a cross-attention Graphormer with Reversible Triad Attention to mimic kinase–phosphatase antagonism. SPINET-KSP, pre-trained on 3.41 million validated phospho-sites utilising masked phosphorylation modelling and contrastive cell-state learning, achieves an AUROC of 0.852 for kinase-family classification (sensitivity 0.821, specificity 0.834, MCC 0.655) and a Pearson correlation coefficient of 0.712 for phospho-occupancy prediction. In distinct 2025 mass spectrometry datasets, it identifies 72% of acknowledged cancer-resistance triads within the top 10 rankings and uncovers 247 supplementary triads validated using orthogonal proteomics. SPINET-KSP is the first foundational model for simulating context-dependent reversible phosphorylation, enabling the targeting of dysregulated kinase-phosphatase pathways in diseases.

Trypanosoma cruzi is the protozoan parasite responsible for Chagas disease, a neglected tropical disease caused by trypanosomatids. Its success as pathogen relies on remarkable metabolic adaptability, stress tolerance, and complex interactions with mammalian hosts. Among the proteins contributing to these processes, nucleoside diphosphate kinases (NDPKs) and arginine kinase (AK) have emerged as central enzymes for parasite metabolism. NDPKs, beyond their canonical role in nucleotide homeostasis, are implicated in DNA repair and oxidative stress responses and are also secreted enzymes. AK, on the other hand, serves as a unique energy-buffering system absent in mammals, supporting parasite growth and adaptation to oxidative and metabolic stresses, including modulation of host immunity. Both enzymes display distinct subcellular localizations all along the parasite and through the life cycle, linking them to multiple roles important for parasite biology and survival. Recent studies have highlighted the impact of interfering these enzymes with several compounds on the viability of the organisms, suggesting new avenues to explore them as drug targets. This review provides a general overview of NDPKs and AK in T. cruzi, aiming to underline their relevance to a broader context of trypanosomatids. Their study not only broadens our understanding of parasite biology but also opens perspectives for applied research, including therapeutic alternatives for Chagas and related diseases.

CK2α and CK2α’, two paralogous members of the human kinome, are catalytic subunits of protein kinase CK2. Together with the regulatory subunit CK2β, they form heterotetrameric holoenzymes. CK2 is the subject of efforts to develop effective and selective inhibitors. For this, secondary binding sites remote from the canonical ATP/GTP cavity are critical. A crystallographic fragment screening with CK2α’ crystals and an established molecular fragment collection was performed to identify new ligands at known or novel sites. It resulted in fourteen CK2α’/fragment structures. Five fragments were found at the CK2β interface of CK2α’ and three fragments at the established αD pocket, which exhibits subtle differences between CK2α and CK2α’; comparative co-crystallisations with CK2α showed that one of them binds to the αD pocket of CK2α’ exclusively. No fragments bound at the substrate-binding region of CK2α’, but a CK2α’ structure with dp10, a decameric section of the substrate-competitive inhibitor heparin, and the indenoindole-type ATP-competitive inhibitor 4w was determined. A comparison with a published CK2α/dp10 structure revealed features consistent with reports about substrate specificity differences between the isoenzymes: dp10 binds to CK2α’ and CK2α with opposite strand orientations, and the local conformations of the isoenzymes in the helix αD region are significantly different.