Abstract-Apelin peptides mediate beneficial effects on the cardiovascular system and are being targeted as potential new drugs. However, apelin peptides have extremely short biological half-lives, and improved understanding of apelin peptide metabolism may lead to the discovery of biologically stable analogues with therapeutic potential. We examined the ability of angiotensin-converting enzyme 2 (ACE2) to cleave and inactivate pyr-apelin 13 and apelin 17, the dominant apelin peptides. Computer-assisted modeling shows a conserved binding of pyr-apelin 13 and apelin 17 to the ACE2 catalytic site. In ACE2 knockout mice, hypotensive action of pyr-apelin 13 and apelin 17 was potentiated, with a corresponding greater elevation in plasma apelin levels. Similarly, pharmacological inhibition of ACE2 potentiated the vasodepressor action of apelin peptides. Biochemical analysis confirmed that recombinant human ACE2 can cleave pyr-apelin 13 and apelin 17 efficiently, and apelin peptides are degraded slower in ACE2-deficient plasma. The biological relevance of ACE2-mediated proteolytic processing of apelin peptides was further supported by the reduced potency of pyr-apelin 12 and apelin 16 on the activation of signaling pathways and nitric oxide production from endothelial cells. Importantly, although pyr-apelin 13 and apelin 17 rescued contractile function in a myocardial ischemia-reperfusion model, ACE2 cleavage products, pyr-apelin 12 and 16, were devoid of these cardioprotective effects. We designed and synthesized active apelin analogues that were resistant to ACE2-mediated degradation, thereby confirming that stable apelin analogues can be designed as potential drugs. We conclude that ACE2 represents a major negative regulator of apelin action in the vasculature and heart. residue and the enzymatic processes involved in its removal from the native apelin peptide remains poorly defined. Using loss-of-function and gain-of-function strategies, we here define a critical role of angiotensin-converting enzyme 2 (ACE2) in the proteolytic cleavage of the C-terminal phenylalanine residue in pyr-apelin 13 and apelin 17. Importantly, this degradative site can be modified to produce relatively stable apelin analogues as potential therapeutic agents. Methods In Silico Modeling of Apelin Peptide Binding to ACE2We selected the structure of human apo-ACE2 (PDB ID: 1R42) mainly because of its high resolution, appropriate R-factor, and errorless electron density map. 16 This structure was equilibrated in constant pressure-temperature condition (NVT, NPT) in the Groningen Machine for Chemical Simulations (GROMACS). 17 We performed knowledgebased docking using our understanding from ACE-substrate bound complex structure. For this purpose, we have used the Angiotensin II (Ang II)-bound ACE complex (PDB ID: 4APH) 18 and MLN-4760 inhibitor bound ACE2 complex (PDB ID: 1R4L). 16,[18][19][20] We modeled the ACE2-Ang II, ACE2-pyr-apelin 13, and ACE2-apelin 17 complexes using these 2 above mentioned reference structures as a template. We built th...

Epithelial–mesenchymal transition (EMT) is a key step in transdifferentiation process in solid cancer development. Forthcoming evidence suggest that the stratified program transforms polarized, immotile epithelial cells to migratory mesenchymal cells associated with enhancement of breast cancer stemness, metastasis, and drug resistance. It involves primarily several signaling pathways, such as transforming growth factor‐β (TGF‐β), cadherin, notch, plasminogen activator protein inhibitor, urokinase plasminogen activator, and WNT/beta catenin pathways. However, current understanding on the crosstalk of multisignaling pathways and assemblies of key transcription factors remain to be explored. In this review, we focus on the crosstalk of signal transduction pathways linked to the current therapeutic and drug development strategies. We have also performed the computational modeling on indepth the structure and conformational dynamic studies of regulatory proteins and analyze molecular interactions with their associate factors to understand the complicated process of EMT in breast cancer progression and metastasis. Electrostatic potential surfaces have been analyzed that help in optimization of electrostatic interactions between the protein and its ligand. Therefore, understanding the biological implications underlying the EMT process through molecular biology with biocomputation and structural biology approaches will enable the development of new therapeutic strategies to sensitize tumors to conventional therapy and suppress their metastatic phenotype.

cNXL104 is a novel β-lactamase inhibitor with a non-lactam structural scaffold. Our kinetic and mass spectrometric analysis demonstrates that NXL104 quantitatively inhibits BlaC, the only chromosomally encoded β-lactamase from Mycobacterium tuberculosis, by forming a carbamyl adduct with the enzyme. The inhibition efficiency (k2/K) of NXL104 was shown to be more than 100-fold lower than that of clavulanate, a classical β-lactamase inhibitor, probably caused by the bulky rings of NXL104. However, the decarbamylation rate constant (k3) was determined to be close to zero. The BlaC-NXL104 adduct remained stable for at least 48 hours, while the hydrolysis of the BlaC-clavulanate adduct was observed after two days. The three-dimensional crystal structure of the BlaC-NXL104 carbamyl adduct was determined at a resolution of 2.3 A. Interestingly, the sulfate group of NXL104 occupies the position of a phosphate ion in the structure of the BlaC-clavulanate adduct, and is hydrogen bonded to residues Ser128, Thr 237 and Thr239. Favorable interactions are also seen in the electrostatic potential map. We propose that these additional interactions, as well as the intrinsic stability of carbamyl linkage, contribute to the extraordinary stability of the BlaC-NXL104 adduct.

Biomechanical stress and cytoskeletal remodeling are key determinants of cellular homeostasis and tissue responses to mechanical stimuli and injury. Here we document the increased activity of gelsolin, an actin filament severing and capping protein, in failing human hearts. Deletion of gelsolin prevents biomechanical stress-induced adverse cytoskeletal remodeling and heart failure in mice. We show that phosphatidylinositol (3,4,5)-triphosphate (PIP3) lipid suppresses gelsolin actin-severing and capping activities. Accordingly, loss of PI3Kα, the key PIP3-producing enzyme in the heart, increases gelsolin-mediated actin-severing activities in the myocardium in vivo, resulting in dilated cardiomyopathy in response to pressure-overload. Mechanical stretching of adult PI3Kα-deficient cardiomyocytes disrupts the actin cytoskeleton, which is prevented by reconstituting cells with PIP3. The actin severing and capping activities of recombinant gelsolin are effectively suppressed by PIP3. Our data identify the role of gelsolin-driven cytoskeletal remodeling in heart failure in which PI3Kα/PIP3 act as negative regulators of gelsolin activity.

l-nucleoside analogs represent an important class of small molecules for treating both viral infections and cancers. These pro-drugs achieve pharmacological activity only after enzyme-catalyzed conversion to their tri-phosphorylated forms. Herein, we report the crystal structures of human deoxycytidine kinase (dCK) in complex with the l-nucleosides (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC)—an approved anti-human immunodeficiency virus (HIV) agent—and troxacitabine (TRO)—an experimental anti-neoplastic agent. The first step in activating these agents is catalyzed by dCK. Our studies reveal how dCK, which normally catalyzes phosphorylation of the natural d-nucleosides, can efficiently phosphorylate substrates with non-physiologic chirality. The capability of dCK to phosphorylate both d- and l-nucleosides and nucleoside analogs derives from structural properties of both the enzyme and the substrates themselves. First, the nucleoside-binding site tolerates substrates with different chiral configurations by maintaining virtually all of the protein-ligand interactions responsible for productive substrate positioning. Second, the pseudo-symmetry of nucleosides and nucleoside analogs in combination with their conformational flexibility allows the l- and d-enantiomeric forms to adopt similar shapes when bound to the enzyme. This is the first analysis of the structural basis for activation of l-nucleoside analogs, providing further impetus for discovery and clinical development of new agents in this molecular class.