Insight into the mechanism of molecular recognition between human Integrin-Linked Kinase and Cpd22 and its implication at atomic level

Pseudokinases have received increasing attention over the past decade because of their role in different physiological phenomena. Although pseudokinases lack several active-site residues, thereby hindering their catalytic activity, recent discoveries have shown that these proteins can play a role in intracellular signaling thanks to their non-catalytic functions. Integrin-linked kinase (ILK) was discovered more than two decades ago and was subsequently validated as a promising target for neoplastic diseases. Since then, only a few small-molecule inhibitors have been described, with the V-shaped pyrazole Cpd22 being the most interesting and characterized. However, little is known about its detailed mechanism of action at atomic level. In this study, using a combination of computational chemistry methods including PELE calculations, docking, molecular dynamics and experimental surface plasmon resonance, we were able to prove the direct binding of this molecule to ILK, thus providing the basis of its molecular recognition by the protein and the effect over its architecture. Our breakthroughs show that Cpd22 binding stabilizes the ILK domain by binding to the pseudo-active site in a similar way to the ATP, possibly modulating its scaffolding properties as pseudokinase. Moreover, our results explain the experimental observations obtained during Cpd22 development, thus paving the way to the development of new chemical probes and potential drugs

A Computer-Driven Scaffold-Hopping Approach Generating New PTP1B Inhibitors from the Pyrrolo[1,2-a]quinoxaline Core

Protein tyrosine phosphatase 1B (PTP1B) is a very promising target for the treatment of metabolic disorders such as type II diabetes mellitus. Although it was validated as a promising target for this disease more than 30 years ago, as yet there is no drug in advanced clinical trials, and its biochemical mechanism and functions are still being studied. In the present study, based on our experience generating PTP1B inhibitors, we have developed and implemented a scaffold-hopping approach to vary the pyrrole ring of the pyrrolo[1,2-a]quinoxaline core, supported by extensive computational techniques aimed to explain the molecular interaction with PTP1B. Using a combination of docking, molecular dynamics and end-point free-energy calculations, we have rationally designed a hypothesis for new PTP1B inhibitors, supporting their recognition mechanism at a molecular level. After the design phase, we were able to easily synthesize proposed candidates and their evaluation against PTP1B was found to be in good concordance with our predictions. Moreover, the best candidates exhibited glucose uptake increments in cellulo model, thus confirming their utility for PTP1B inhibition and validating this approach for inhibitors design and molecules thus obtained

Integrin Linked Kinase (ILK) Downregulation as an Early Event During the Development of Metabolic Alterations in a Short-Term High Fat Diet Mice Model.

Background/Aims: Diabetes type 2, metabolic syndrome or non-alcoholic fatty liver disease are insulin resistance-related metabolic disorders, which lack a better prognosis before their full establishment. We studied the importance of the intracellular scaffold protein integrin linked kinaes (ILK) as a key modulator in the initial pathogenesis and the early progression of those insulin resistance- related disorders. Methods: Adult mice with a global transgenic downregulation of ILK expression (cKD-ILK) and littermates without that depletion (CT) were fed with either standard (STD) or high fat (HFD) diets during 2 and 6 weeks. Weights, blood glucose and other systemic biochemical parameters were determined in animals under fasting conditions and after glucose or pyruvate intraperitoneal injections to test their tolerance. In RNA or proteins extracted from insulin-sensitive tissues, we determined by reverse transcription?quantitative PCR and western blot the expression of ILK, metabolites transporters and other metabolism and inflammatory markers. Glucose uptake capacity was studied in freshly isolated tissues. Results: HFD feeding was able to early and progressively increase glycaemia, insulinemia, circulating glycerol, body weight gain, liver-mediated gluconeogenesis along this time lapse, but cKD-ILK have all these systemic misbalances exacerbated compared to CT in the same HFD time lapse. Interestingly, the tisular expression of ILK in HFD-fed CT was dramatically downregulated in white adipose tissue (WAT), skeletal muscle and liver at the same extent of the original ILK downregulation of cKD-ILK. We previously published that basal STD-fed cKD-ILK compared to basal STD-CT have different expression of glucose transporters GLUT4 in WAT and skeletal muscle. In the same STD-fed cKD-ILK, we observed here the increased expressions of hepatic GLUT2 and WAT pro-inflammatory cytokines TNF-? and MCP-1. The administration of HFD exacerbated the expression changes in cKD-ILK of these and other markers related to the imbalanced metabolism observed, such as WAT lipolysis (HSL), hepatic gluconeogenesis (PCK-1) and glycerol transport (AQP9). Conclusion: ILK expression may be taken as a predictive determinant of metabolic disorders establishment, because its downregulation seems to correlate with the early imbalance of glucose and glycerol transport and the subsequent loss of systemic homeostasis of these metabolites.Instituto de Salud Carlos III-ISCIIIComunidad de MadridFondo Europeo de Desarrollo Regional-FEDERInstituto Ramon y Cajal de Investigación Sanitária-IRYCISFundación Renal Iñigo Álvarez de Toledo-FRIA

Tripeptides as Integrin-Linked Kinase Modulating Agents Based on a Protein-Protein Interaction with alfa-Parvin

Integrin-linked kinase (ILK) has emerged as a controversial pseudokinase protein that plays a crucial role in the signaling process initiated by integrin-mediated signaling. However, ILK also exhibits a scaffolding protein function inside cells, controlling cytoskeletal dynamics, and has been related to non-neoplastic diseases such as chronic kidney disease (CKD). Although this protein always acts as a heterotrimeric complex bound to PINCH and parvin adaptor proteins, the role of parvin proteins is currently not well understood. Using in silico approaches for the design, we have generated and prepared a set of new tripeptides mimicking an alpha-parvin segment. These derivatives exhibit activity in phenotypic assays in an ILK-dependent manner without altering kinase activity, thus allowing the generation of new chemical probes and drug candidates with interesting ILK-modulating activities

The Integrin Beta1 Modulator Tirofiban Prevents Adipogenesis and Obesity by the Overexpression of Integrin-Linked Kinase: a Pre-Clinical Approach In Vitro and In Vivo

de Frutos, S., Griera, M., Hatem-Vaquero, M. et al. The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo. Cell Biosci 12, 10 (2022)Background: Obesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrin?1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scafold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fght obesity have brought long-term cardiovascular side efects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofban (TF), stated in preclinical studies as a cardiovascular protector. Methods: Fully diferentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specifc INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specifc dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-infammatory cytokines (MCP1, IL6), adipogenesis (PPAR?, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite trans porters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3? (GSK3?) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT. Results: TF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not afected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3?. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF efect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3? were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF efects on the TG content and the transcriptional expression of PPAR? and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-infammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic efect of in vivo TF administration. Conclusions: ILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increas ing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagu lant and cardiovascular protective advantages.Instituto de Salud Carlos IIIComunidad de MadridFondo Europeo de Desarrollo Regional-FEDE

H- ras deletion protects against angiotensin II-induced arterial hypertension and cardiac remodeling through protein kinase G-Ibeta pathway activation

15 p.Ras proteins regulate cell survival, growth, differentiation, blood pressure, and fibrosis in some organs.We have demonstrated that H-rasgene deletion produces mice hypotensionviaa soluble guanylate cyclase-proteinkinase G (PKG)–dependent mechanism. In this study, we analyzed the consequences of H-rasdeletiononcardiacremodeling induced by continuous angiotensin II (AngII) infusion and the molecular mechanisms implied. Leftventricular posterior wall thickness and mass and cardiomyocyte cross-sectional area were similar between AngII-treated H-Ras knockout (H-ras2/2) and control wild-type (H-ras+/+) mice, as were extracellular matrix proteinexpression. Increased cardiac PKG-Ibprotein expression in H-ras2/2mice suggests the involvement of this proteinin heart protection.Ex vivoexperiments on cardiac explants could support this mechanism, as PKG blockadeblunted protection against AngII-induced cardiac hypertrophy and fibrosis markers in H-ras2/2mice. Geneticmodulation studies in cardiomyocytes and cardiac and embryonic fibroblasts revealed that the lack of H-Ras down-regulates the B-RAF/MEK/ERK pathway, which induces the glycogen synthase kinase-3b-dependent activation ofthe transcription factor, cAMP response element-binding protein, which is responsible for PKG-Iboverexpressionin H-ras2/2mouse embryonic fibroblasts. This study demonstrates that H-rasdeletion protects against AngII-induced cardiac remodeling, possiblyviaa mechanism in which PKG-Iboverexpression could play a partial role, andpoints to H-Ras and/or downstream proteins as potential therapeutic targets in cardiovascular disease.Instituto de Salud Carlos IIIUniversidad de AlcaláFundación SenefroFEDE

Uraemic toxins impair skeletal muscle regeneration by inhibiting myoblast proliferation, reducing myogenic differentiation, and promoting muscular fibrosis

17 p.Uraemic toxins increase in serum parallel to a decline in the glomerular filtration rate and the development of sarcopenia in patients with chronic kidney disease (CKD). This study analyses the role of uraemic toxins in sarcopenia at different stages of CKD, evaluating changes in the muscular regeneration process. Cultured C2C12 cells were incubated with a combination of indoxyl sulphate and p-cresol at high doses (100 mu g/mL) or low doses (25 mu g/mL and 10 mu g/mL) resembling late or early CKD stages, respectively. Cell proliferation (analysed by scratch assays and flow cytometry) was inhibited only by high doses of uraemic toxins, which inactivated the cdc2-cyclin B complex, inhibiting mitosis and inducing apoptosis (analysed by annexin V staining). By contrast, low doses of uraemic toxins did not affect proliferation, but reduced myogenic differentiation, primed with 2% horse serum, by inhibiting myogenin expression and promoting fibro-adipogenic differentiation. Finally, to assess the in vivo relevance of these results, studies were performed in gastrocnemii from uraemic rats, which showed higher collagen expression and lower myosin heavy chain expression than those from healthy rats. In conclusion, uraemic toxins impair the skeletal muscular regeneration process, even at low concentrations, suggesting that sarcopenia can progress from the early stages of CKD.Instituto de Salud Carlos II

Endothelin-1 induces cellular senescence and fibrosis in cultured myoblasts. A potential mechanism of aging-related sarcopenia

Endothelial dysfunction, with increased endothelin-1 (ET-1) synthesis, and sarcopenia, characterized by the loss of muscular mass and strength, are two aging-related conditions. However, a relationship between them has not been already established. The aim of this study was to determine whether ET-1 induces senescence and fibrosis in cultured murine myoblasts, which could be involved in the development of sarcopenia related to aging. For this purpose, myoblasts were incubated with ET-1 to assess cellular senescence, analyzed by senescence associated beta-galactosidase activity and p16 expression; and fibrosis, assessed by fibronectin expression. ET-1 induced myoblast senescence and fibrosis through ETA receptor. The use of antioxidants and several antagonists revealed that ET-1 effect on senescence and fibrosis depended on ROS production and activation of PI3K-AKT-GSK pathway. To stress the in vivo relevance of these results, circulating ET-1, muscular strength, muscular fibrosis and p16 expression were measured in male C57Bl6 mice from 5-18-24-months-old. Old mice shown high levels of ET-1 correlated with muscular fibrosis, muscular p16 expression and loss of muscle strength. In conclusion, ET-1 promotes fibrosis and senescence in cultured myoblasts, similar results were found in old mice, suggesting a potential role for ET-1 in the development of sarcopenia related to aging

Hyperphosphatemia Promotes Senescence of Myoblasts by Impairing Autophagy Through Ilk Overexpression, A Possible Mechanism Involved in Sarcopenia

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated beta-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53