A fluorescence-based sensor for calibrated measurement of protein kinase stability in live cells.

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

Explanatory review on pyrimidine/fused pyrimidine derivatives as anticancer agents targeting Src kinase.

The pyrimidine and fused pyrimidine ring systems play vital roles to inhibit the c-Src kinase. The Src kinase is made of different domains but the kinase domain is responsible for inhibition of Src kinase. In which the kinase domain is the main domain that is made of several amino acids. The Src kinase is inhibited by its inhibitors when it is activated by phosphorylation. Although dysregulation of Src kinase caused cancer in the late nineteenth century, medicinal chemists have not explored it extensively; therefore it is still regarded as a cult pathway. There are numerous FDA-approved drugs on the market, yet novel anticancer drugs are still in demand. Existing medications have adverse effects and drug resistance owing to rapid protein mutation. In this review, we discussed the activation process of Src kinase, chemistry of pyrimidine ring and its different synthetic routes, as well as the recent development in c-Src kinase inhibitors containing pyrimidine and their biological activity, SAR, and selectivity. The c-Src binding pocket has been predicted in detail to discover the vital amino acids which will interact with inhibitors. The potent derivatives were docked to discover the binding pattern. The derivative 2 established three hydrogen bonds with the amino acid residues Thr341 and Gln278 and had the greatest binding energy of -13.0 kcal/mol. The top docked molecules were further studied for ADMET studies. The derivative 1, 2, and 43 did not show any violation of Lipinski's rule. All derivatives used for the prediction of toxicity showed toxicity.

Medicinal Chemistry Perspectives on Recent Advances in Src Kinase Inhibitors as a Potential Target for the Development of Anticancer Agents: Biological Profile, Selectivity, Structure-Activity Relationship.

The physiological Src proto-oncogene is a protein tyrosine kinase receptor that served as the essential signaling pathway in different types of cancer. Src kinase receptor is divided into different domains: a unique domain, an SH3 domain, an SH2 domain, a protein tyrosine kinase domain, and a regulatory tail, which runs from the N-terminus to the C-terminus. Src kinase inhibitors bind in the kinase domain and are activated by phosphorylation. The etiology of cancer involved various signaling pathways and Src signaling pathways are also involved in those clusters. Although the dysregulation of Src kinase resulted in cancer being discovered in the late 19th century it is still considered a cult pathway because it is not much explored by different medicinal chemists and oncologists. The Src kinase regulated through different kinase pathways (MAPK, PI3K/Akt/mTOR, JAK/STAT3, Hippo kinase, PEAK1, and Rho/ROCK pathways) and proceeded downstream signaling to conduct cell proliferation, angiogenesis, migration, invasion, and metastasis of cancer cells. There are numerous FDA-approved drugs flooded the market but still, there is a huge demand for the creation of novel anticancer drugs. As the existing drugs are accompanied by several adverse effects and drug resistance due to rapid mutation in proteins. In this review, we have elaborated about the structure and activation of Src kinase, as well as the development of Src kinase inhibitors. Our group also provided a comprehensive overview of Src inhibitors throughout the last two decades, including their biological activity, structure-activity relationship, and Src kinase selectivity. The Src binding pocket has been investigated in detail to better comprehend the interaction of Src inhibitors with amino acid residues. We have strengthened the literature with our contribution in terms of molecular docking and ADMET studies of top compounds. We hope that the current analysis will be a useful resource for researchers and provide glimpse of direction toward the design and development of more specific, selective, and potent Src kinase inhibitors.

Structural basis of constitutive c-Src kinase activity due to R175L and W118A mutations.

Cellular Src (c-Src) belongs to a non-receptor membrane-associated tyrosine kinase family that plays essential roles in cellular processes. Growing evidence suggests that R175L and W118A mutations in SH2/SH3 domains of c-Src functionally inactivate these domains leading to constitutive activation of kinase domain (KD). Here we modeled c-SrcR175L, c-SrcW118A and c-SrcW118A+R175L structures by inducing phosphorylation at Y416 or Y527, respectively to characterize the comparative dynamics in the active versus inactive states through molecular dynamics simulation assay. We observed more conformational readjustments in c-Srcopen than its close variants. In particular, C-terminal tail residues of c-SrcW118A-open and c-SrcW118A+R175L-open demonstrate significantly higher transitions. The cross-correlation analysis revealed an anticorrelation behavior in the motion of KD with respect to SH2, SH3 and the linker region of SrcW118A+R175L-open, while in c-SrcWT-open, SH2 and SH3 domains were anticorrelated, while KD and C-terminal tail motions were correlated. Due to these conformational differences, c-Src open forms exhibited lower interaction between pY527 and SH2 domain. Through detailed structural analysis, we observed a uniform myristate binding cavity in c-SrcWT-open, while the myristoyl pockets of mutant forms were deformed. We propose that constitutive activation of mutant Src forms may presumably be achieved by the prolonged membrane binding due to unusual conformations of C-terminal and myristoyl switch residues that may result in a higher dephosphorylation rate at pY527 in the myristoylated c-Src. Thus, our study establishes novel clues to decipher the constitutive activation status of c-Src in response to known mutations that may help in devising novel therapeutic strategies for cancer metastasis treatment.Communicated by Ramaswamy H. Sarma.

Csk αC Helix: A Computational Analysis of an Essential Region for Conformational Transitions.

Conformational changes are an essential feature for the function of some dynamic proteins. Understanding the mechanism of such motions may allow us to identify important properties, which may be directly related to the regulatory function of a protein. Also, this knowledge may be employed for a rational design of drugs that can shift the balance between active and inactive conformations, as well as affect the kinetics of the activation process. Here, the conformational changes in carboxyl-terminal Src kinase, the major catalytic repressor to the Src family of kinases, was investigated, and it was proposed as a functionally related hypothesis. A Cα Structure-Based Model (Cα-SBM) was applied to provide a description of the overall conformational landscape and further analysis complemented by detailed molecular dynamics simulations. As a first approach to Cα-SBM simulations, reversible transitions between active (closed) and inactive (open) forms were modeled as fluctuations between these two energetic basins. It was found that, in addition to the interdomain Carboxyl-terminal SRC Kinase (Csk) correlated motions, a conformational change in the αC helix is required for a complete conformational transition. The result reveals this as an important region of transition control and domain coordination. Restrictions in the αC helix region of the Csk protein were performed, and the analyses showed a direct correlation with the global conformational changes, with this location being propitious for future studies of ligands. Also, the Src Homology 3 (SH3) and SH3 plus Src Homology 2 (SH2) domains were excluded for a direct comparison with experimental results previously published. Simulations where the SH3 was deleted presented a reduction of the transitions during the simulations, while the SH3-SH2 deletion vanishes the Csk transitions, corroborating the experimental results mentioned and linking the conformational changes with the catalytic functionality of Csk. The study was complemented by the introduction of a known kinase inhibitor close to the Csk αC helix region where its consequences for the kinetic behavior and domain displacement of Csk were verified through detailed molecular dynamics. The findings describe the mechanisms involving the Csk αC helix for the transitions and also support the dynamic correlation between SH3 and SH2 domains against the Csk lobes and how local energetic restrictions or interactions in the Csk αC helix can play an important role for long-range motions. The results also allow speculation if the Csk activity is restricted to one specific conformation or a consequence of a state transition, this point being a target for future studies. However, the αC helix is revealed as a potential region for rational drug design.

ATP-site inhibitors induce unique conformations of the acute myeloid leukemia-associated Src-family kinase, Fgr.

The Src-family kinase Fgr is expressed primarily in myeloid hematopoietic cells and contributes to myeloid leukemia. Here, we present X-ray crystal structures of Fgr bound to the ATP-site inhibitors A-419259 and TL02-59, which show promise as anti-leukemic agents. A-419259 induces a closed Fgr conformation, with the SH3 and SH2 domains engaging the SH2-kinase linker and C-terminal tail, respectively. In the Fgr:A-419259 complex, the activation loop of one monomer inserts into the active site of the other, providing a snapshot of trans-autophosphorylation. By contrast, TL02-59 binding induced SH2 domain displacement from the C-terminal tail and SH3 domain release from the linker. Solution studies using HDX MS were consistent with the crystal structures, with A-419259 reducing and TL02-59 enhancing solvent exposure of the SH3 domain. These structures demonstrate that allosteric connections between the kinase and regulatory domains of Src-family kinases are regulated by the ligand bound to the active site.

Lipid chain-driven interaction of a lipidated Src-family kinase Lyn with the bilayer membrane.

N-Myristoylation is a process of ubiquitous protein modification, which promotes the interaction of lipidated proteins on cell surfaces, in conjunction with reversible S-palmitoylation. We report the cooperative lipid-lipid interaction of two acyl chains of proteins, which increases the protein-membrane interaction and facilitates selective targeting of membranes containing anionic lipids. Lyn is a member of the Src family kinases distributed on the membrane surface by N-myristoyl and neighbouring S-palmitoyl chain anchors at the unique N-terminus domain. We prepared N-terminal short segments of lipidated Lyn to investigate the behaviour of each acyl chain in the lipid composition-dependent membrane interaction by solid-state nuclear magnetic resonance (NMR) analysis. Solid-state 31P-NMR studies revealed that S-palmitoylation of N-myristoylated Lyn peptides increased the interaction between peptides and phospholipid head groups, particularly with the anionic phosphatidylserine-containing bilayers. The solid-state 2H-NMR of Lyn peptides with a perdeutero N-myristoyl chain indicated an increase (0.6-0.8 Å) in the extent of the N-myristoyl chain in the presence of nearby S-palmitoyl chains, probably through the interaction via the acyl chains. The cooperative hydrocarbon chain interaction of the two acyl chains of Lyn increased membrane binding by extending the hydrocarbon chains deeper into the membrane interior, thereby promoting the peptide-membrane surface interaction between the cationic peptide side chains and the anionic lipid head groups. This lipid-driven mechanism by S-palmitoylation promotes the partition of the lipidated proteins to the cytoplasmic surface of the cell membranes and may be involved in recruiting Lyn at the signalling domains rich in anionic lipids.

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations.

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often "cryptic" without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein-ligand systems.

A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Structural study of ponatinib in inhibiting SRC kinase.

Ponatinib is a multi-target tyrosine kinase inhibitor that targets ABL, SRC, FGFR, and so on. It was designed to overcome the resistance of BCR-ABL mutation to imatinib, especially the gatekeeper mutation ABLT315I. The molecular mechanism by which ponatinib overcomes mutations of BCR-ABL and some other targets has been explained, but little information is known about the characteristics of ponatinib binding to SRC. Here, we showed that ponatinib inhibited wild type SRC kinase but failed to inhibit SRC gatekeeper mutants in both biochemical and cellular assays. We determined the crystal structure of ponatinib in complex with the SRC kinase domain. In addition, by structural analysis, we provided a possible explanation for why ponatinib showed different effects on SRC and other kinases with gatekeeper mutations. The resistance mechanism of SRC gatekeeper mutations to ponatinib may provide meaningful information for designing inhibitors against SRC family kinases in the future.

A Model for the Signal Initiation Complex Between Arrestin-3 and the Src Family Kinase Fgr.

Arrestins regulate a wide range of signaling events, most notably when bound to active G protein-coupled receptors (GPCRs). Among the known effectors recruited by GPCR-bound arrestins are Src family kinases, which regulate cellular growth and proliferation. Here, we focus on arrestin-3 interactions with Fgr kinase, a member of the Src family. Previous reports demonstrated that Fgr exhibits high constitutive activity, but can be further activated by both arrestin-dependent and arrestin-independent pathways. We report that arrestin-3 modulates Fgr activity with a hallmark bell-shaped concentration-dependence, consistent with a role as a signaling scaffold. We further demonstrate using NMR spectroscopy that a polyproline motif within arrestin-3 interacts directly with the SH3 domain of Fgr. To provide a framework for this interaction, we determined the crystal structure of the Fgr SH3 domain at 1.9 Å resolution and developed a model for the GPCR-arrestin-3-Fgr complex that is supported by mutagenesis. This model suggests that Fgr interacts with arrestin-3 at multiple sites and is consistent with the locations of disease-associated Fgr mutations. Collectively, these studies provide a structural framework for arrestin-dependent activation of Fgr.

Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways.

Studies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) identified Src as the target of matrine. Cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DARTS) provided solid evidences that matrine directly bound to Src. Bioinformatics prediction and pull-down experiment demonstrated that Src kinase domain was required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419 in Src kinase domain. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3, and PI3K/Akt signaling pathways via targeting Src. Collectively, matrine targeted Src, inhibited its kinase activity, and down-regulated its downstream MAPK/ERK, JAK2/STAT3, and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

A Conformation Selective Mode of Inhibiting SRC Improves Drug Efficacy and Tolerability.

Despite the approval of several multikinase inhibitors that target SRC and the overwhelming evidence of the role of SRC in the progression and resistance mechanisms of many solid malignancies, inhibition of its kinase activity has thus far failed to improve patient outcomes. Here we report the small molecule eCF506 locks SRC in its native inactive conformation, thereby inhibiting both enzymatic and scaffolding functions that prevent phosphorylation and complex formation with its partner FAK. This mechanism of action resulted in highly potent and selective pathway inhibition in culture and in vivo. Treatment with eCF506 resulted in increased antitumor efficacy and tolerability in syngeneic murine cancer models, demonstrating significant therapeutic advantages over existing SRC/ABL inhibitors. Therefore, this mode of inhibiting SRC could lead to improved treatment of SRC-associated disorders. SIGNIFICANCE: Small molecule-mediated inhibition of SRC impairing both catalytic and scaffolding functions confers increased anticancer properties and tolerability compared with other SRC/ABL inhibitors.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

Reduced efficacy of a Src kinase inhibitor in crowded protein solution.

The inside of a cell is highly crowded with proteins and other biomolecules. How proteins express their specific functions together with many off-target proteins in crowded cellular environments is largely unknown. Here, we investigate an inhibitor binding with c-Src kinase using atomistic molecular dynamics (MD) simulations in dilute as well as crowded protein solution. The populations of the inhibitor, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), in bulk solution and on the surface of c-Src kinase are reduced as the concentration of crowder bovine serum albumins (BSAs) increases. This observation is consistent with the reduced PP1 inhibitor efficacy in experimental c-Src kinase assays in addition with BSAs. The crowded environment changes the major binding pathway of PP1 toward c-Src kinase compared to that in dilute solution. This change is explained based on the population shift mechanism of local conformations near the inhibitor binding site in c-Src kinase.

Src family kinases, adaptor proteins and the actin cytoskeleton in epithelial-to-mesenchymal transition.

Over a century of scientific inquiry since the discovery of v-SRC but still no final judgement on SRC function. However, a significant body of work has defined Src family kinases as key players in tumor progression, invasion and metastasis in human cancer. With the ever-growing evidence supporting the role of epithelial-mesenchymal transition (EMT) in invasion and metastasis, so does our understanding of the role SFKs play in mediating these processes. Here we describe some key mechanisms through which Src family kinases play critical role in epithelial homeostasis and how their function is essential for the propagation of invasive signals. Video abstract.

Novel Roles of SH2 and SH3 Domains in Lipid Binding.

Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.

The impact of oncogenic mutations of the viral Src kinase on the structure and stability of the SH3 domain.

Src kinase belongs to the family of Src-related nonreceptor tyrosine kinases. Because of its physiological role in cell growth and proliferation, its activity is strictly controlled by several mechanisms. Nevertheless, in viral Src kinase (v-Src) some of these mechanisms fail, and its uncontrolled activity is responsible for the occurrence of cancer. Here, the crystal structures of three SH3-domain mutants of v-Src were determined to unveil the effects of these oncogenic mutations in this regulatory domain. Mutations in the n-Src and distal loops have a low impact on the overall structure of the domain and its capacity to form intertwined dimers. However, mutations in the RT loop compromise the stability of the domain and make the protein very prone to aggregation. Additionally, these mutations prevent the formation of intertwined dimers. The results show a synergistic effect between mutations in the RT loop and those in the n-Src and distal loops. Analysis of the structures of the v-Src SH3-domain mutants and the closed inactive conformation of cellular Src kinase (c-Src) point to a loss of the interactions that are required to establish the compact inactive form of the kinase. Nevertheless, an analysis of structures of the c-Src SH3 domain complexed with class I and II peptides points to minor changes in the interactions between the v-Src SH3 domain and these peptides. In this way, the structures reported here indicate that mutations in the RT loop might impair the kinase regulation mechanism without affecting the recognition of short proline-rich motifs in the target proteins of the kinase, thus explaining the oncogenic behaviour of the protein.

Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells.

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

Tuning Local Hydration Enables a Deeper Understanding of Protein-Ligand Binding: The PP1-Src Kinase Case.

Water plays a key role in biomolecular recognition and binding. Despite the development of several computational and experimental approaches, it is still challenging to comprehensively characterize water-mediated effects on the binding process. Here, we investigate how water affects the binding of Src kinase to one of its inhibitors, PP1. Src kinase is a target for treating several diseases, including cancer. We use biased molecular dynamics simulations, where the hydration of predetermined regions is tuned at will. This computational technique efficiently accelerates the SRC-PP1 binding simulation and allows us to identify several key and yet unexplored aspects of the solvent's role. This study provides a further perspective on the binding phenomenon, which may advance the current drug design approaches for the development of new kinase inhibitors.