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c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P 31 VHP 34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein-protein interactions (PPIs) have emerged in the past years as significant pharmacological targets in the development of new therapeutics due to their key roles in determining pathological pathways. Herein, we present fragments on energy surfaces, a simple and general design strategy that integrates the analysis of the dynamic and energetic signatures of proteins to unveil the substructures involved in PPIs, with docking, selection, and combination of drug-like fragments to generate new PPI inhibitor candidates. Specifically, structural representatives of the target protein are used as inputs for the blind physics-based prediction of potential protein interaction surfaces using the matrix of low coupling energy decomposition method. The predicted interaction surfaces are subdivided into overlapping windows that are used as templates to direct the docking and combination of fragments representative of moieties typically found in active drugs. This protocol is then applied and validated using structurally diverse, important PPI targets as test systems. We demonstrate that our approach facilitates the exploration of the molecular diversity space of potential ligands, with no requirement of prior information on the location and properties of interaction surfaces or on the structures of potential lead compounds. Importantly, the hit molecules that emerge from our ab initio design share high chemical similarity with experimentally tested active PPI inhibitors. We propose that the protocol we describe here represents a valuable means of generating initial leads against difficult targets for further development and refinement.

Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties. TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential. Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands' fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Computational chemistry has come of age in drug discovery. Indeed, most pharmaceutical development programs rely on computer-based data and results at some point. Herein, we discuss recent applications of advanced simulation techniques to difficult challenges in drug discovery. These entail the characterization of allosteric mechanisms and the identification of allosteric sites or cryptic pockets determined by protein motions, which are not immediately evident in the experimental structure of the target; the study of ligand binding mechanisms and their kinetic profiles; and the evaluation of drug-target affinities. We analyze different approaches to tackle challenging and emerging biological targets. Finally, we discuss the possible perspectives of future application of computation in drug discovery., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of α-adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, α-adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that α-adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2+/+ but not in sik2-/- mice. Thus LVH triggered by HS or the presence of a genetic variant of α-adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

The Milan hypertensive strain of rats (MHS) develops hypertension as a consequence of the increased tubular Na(+) reabsorption sustained by enhanced expression and activity of the renal tubular Na-K-ATPase. To verify whether the Na-K-2Cl cotransporter (NKCC2) is involved in the maintenance of hypertension in MHS rats, we have analysed the phosphorylation state and the activation of NKCC2 in Milan rats. Western blotting and immunofluorescence experiments were performed using specific antibodies against the regulatory phospho-threonines in the NKCC2 N terminus (R5 antibody). The phosphorylation levels of NKCC2 were significantly increased in the kidney of MHS rats. Moreover, the administration of furosemide in vivo decreased the blood pressure and increased the urine output and natriuresis in MHS rats demonstrating the actual involvement of NKCC2 activity in the pathogenesis of hypertension in this strain of rats. The up-regulation of NKCC2 activity is most probably mediated by a STE20/SPS1-related proline/alanine-rich kinase (SPAK) phosphorylation at serine-325 since it was significantly increased in MHS rats. Interestingly, aldosterone treatment caused an increase in NKCC2 phosphorylation in NKCC2-expressing MDCK cells. In conclusion, we demonstrated an increase in the activity of NKCC2 along the TAL that significantly contributes to the increase in systemic blood pressure in MHS rats. The elevated plasma levels of aldosterone, found in MHS rats, may influence Na(+) balance through a SPAK-dependent regulation of NKCC2 accounting for the maintenance of the hypertensive state in MHS rats.

Background: Sodium-sensitive hypertension is caused by renal tubular dysfunction, leading to increased retention of sodium and water. Previous findings have suggested that single-nucleotide polymorphisms of the cytoskeletal protein, α-adducin, are associated with increased membrane expression of the Na/K pump and abnormal renal sodium transport in Milan hypertensive strain (MHS) rats and in humans. However, the possible contribution of renal aquaporins (AQPs) to water retention remains undefined in MHS rats., Methods: Kidneys from MHS rats were analyzed and compared with those from age-matched Milan normotensive strain (MNS) animals by quantitative-PCR, immunoblotting, and immunoperoxidase. Endocytosis assay was performed on renal cells stably expressing AQP4 and co-transfected either with wild-type normotensive (NT) or with mutated hypertensive (HT) α-adducin., Results: Semiquantitative immunoblotting revealed that AQP1 abundance was significantly decreased only in HT MHS whereas AQP2 was reduced in both young pre-HT and adult-HT animals. On the other hand, AQP4 was dramatically upregulated in MHS regardless of the age. These results were confirmed by immunoperoxidase microscopy. Endocytosis assays clearly showed that the expression of mutated adducin strongly reduced the rate of constitutive AQP4 endocytosis, thereby increasing its abundance at the plasma membrane., Conclusions: We provide here the first evidence that AQP1, AQP2, and AQP4 are dysregulated in the kidneys of MHS animals. In particular, we provide evidence that α-adducin mutations may be responsible for AQP4 upregulation. The downregulation of AQP1 and AQP2 and the upregulation of AQP4 may be relevant for the onset and maintenance of salt-sensitive hypertension.

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.