Georgios A. Spyroulias

Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE)

Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS.

Mechanism of Cu(A) assembly.

Copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. Here we show that a new periplasmic protein (PCu(A)C) selectively inserts Cu(I) ions into subunit II of Thermus thermophilus ba(3) oxidase to generate a native Cu(A) site. The purported metallochaperone Sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the Cu(A) cysteine ligands.

Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress

Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with H(2)O(2) reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with H(2)O(2) significantly increased HMR-1766 responses. Similar results were obtained with SIN-1, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, H(2)O(2) inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the NH(2)-terminus of the beta(1)-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production.

Pyrrolo[2,3-a]carbazoles as potential cyclin dependent kinase 1 (CDK1) Inhibitors. Synthesis, biological evaluation, and binding mode through docking simulations.

Pyrrolo[2,3- a]carbazole derivatives were synthesized, and their effects on CDK1/cyclinB activity were evaluated. The most potent and efficacious inhibitor was found to be ethyl 9-chloro-1H-pyrrolo[2,3-alpha]carbazole-2-carboxylate (1e), exhibiting an IC50 in the low micromolar range and leading to 90% at higher concentrations. Using a computational model for CDK1-1e, binding we have observed that 1e exhibited two likely binding modes in the ATP-binding cleft that involve interactions with Lys130, Thr14, and Asp146 of the enzyme.

Study of the interaction between the amyloid beta peptide (1–40) and antioxidant compounds by nuclear magnetic resonance spectroscopy

Amyloid beta peptide (Abeta) aggregation leads to the senile plaque formation, a process that is strongly influenced by oxidative stress and is considered as the molecular basis of various neurodegenerative diseases, such as Alzheimers disease (AD). Endogenous antioxidants or dietary derived compounds may down-regulate this process. In this study, the interaction of two antioxidants, oleuropein (OE) and melatonin (M), with Abeta is monitored through nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. The concerted application of these two analytical techniques provides new experimental evidence and residue-specific insights into the interacting Abeta peptide amino acids that are implicated in this process. Both antioxidant compounds interact in a similar way with the peptide and cause chemical shift variations. The most pronounced resonance changes have been observed for the 1H-15N signals of N-terminal region and Leu17-Phe20 residues, as monitored by NMR titration studies.

Structural features of angiotensin-I converting enzyme catalytic sites: conformational studies in solution, homology models and comparison with other zinc metallopeptidases.

Angiotensin-I Converting Enzyme (ACE) is a Zinc Metallopeptidase of which the three-dimensional structure was unknown until recently, when the X-ray structure of testis isoform (C-terminal domain of somatic) was determined. ACE plays an important role in the regulation of blood pressure due to its action in the frame of the Renin-Angiotensin System. Efforts for the specific inhibition of the catalytic function of this enzyme have been made on the basis of the X-ray structures of other enzymes with analogous efficacy in the hydrolytic cleavage of peptide substrate terminal fragments. Angiotensin-I Converting Enzyme bears the sequence and topology characteristics of the well-known gluzincins, a sub-family of zincins metallopeptidases and these similarities are exploited in order to reveal common structural elements among these enzymes. 3D homology models are also built using the X-ray structure of Thermolysin as template and peptide models that represent the amino acid sequence of the ACEs two catalytic, zinc-containing sites are designed and synthesized. Conformational analysis of the zinc-free and zinc-bound peptides through high resolution 1H NMR Spectroscopy provides new insights into the solution structure of ACE catalytic centers. Structural properties of these peptides could provide valuable information towards the design and preparation of new potent ACE inhibitors.

Conformational Properties of HIV-1 gp120/V3 Immunogenic Domains

Infection of target host cells by the human immunodeficiency virus-1 (HIV-1) is a multi-step process involving a series of conformational changes in the viral gp120 and gp41 proteins. Gp120 binding to the main cell receptor, CD4, on the surface of cells expressing this molecule, and interaction with the cell chemokine receptors CCR5 and CXCR4, are among the key events for HIV-1 infection. These steps are crucial for the virus and offer potential therapeutic targets. For this reason, understanding the structure and the physicochemical characteristics of the gp120 in relation to these interactions has drawn much attention. This review article focuses on the biologically important V3 region of the gp120 and summarizes the functional role, the sequence variation and the conformational features of V3 peptides, which are important for co-receptor selectivity, specificity and interaction. Synthetic V3 peptides have been extensively studied by NMR spectroscopy and X-ray crystallography, in solution or in solid state, in their free or bound form, and valuable information was generated with the aim to be exploited in the design of new, effective inhibitors of HIV-1 infection. The features of the potential gp120 interacting sites on the two chemokine co-receptors, CCR5 and CXCR4, are also discussed, and co-receptor blocking molecules under clinical trial are also reported.

Current Inhibition Concepts of Zinc Metallopeptidases Involved in Blood Pressure Regulation

Certain Zinc Metallopeptidases, such as Angiotensin Converting Enzyme (ACE), Neutral Endopeptidase (NEP) and Endothelin Converting Enzyme (ECE), play a key role in vascular homeostasis through their proteolytic activity in various vasoactive peptides. Effective inhibitors for these enzymes were until recently designed in the absence of the X-ray structure of these enzymes, and a variety of ACE inhibitors are commercially available. A new class of promising compounds, namely vasopeptidase inhibitors, have emerged and they represent a new concept in hypertension and cardiovascular disease therapeutics. They contemporarily inhibit the catalytic function of more than one of the above enzymes and they are undergoing extensive clinical trials exhibiting increased efficacy in hypertension treatment and higher risk for side effects such as angioedema when compared to ACE inhibitors. The determination of the substrate-free and substrate- loaded X-ray models of NEP and ACE provides valuable insight of the structure determinants in enzyme - substrate interaction and it is believed that new more selective inhibitors could be afforded through structure- based drug design process. Selectivity towards target enzyme even in simultaneous inhibition could modify the breakdown of vasodilator and vasoconstrictor peptides and could therefore modulate the balance of the risk-benefit profile.

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin‐releasing hormone peptide analogs

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

A Computational Approach to the Study of the Binding Mode of Dual ACE/NEP Inhibitors

Combined blockade of the renin-angiotensin-aldosterone system (RAAS) is an attractive therapeutic strategy for the treatment of cardiovascular diseases. Vasopeptidase inhibitors are a group of compounds capable of inhibiting more than one enzyme, which leads to potentiation of natriuretic peptide actions and suppression of the RAAS. In this study, molecular modeling has been used to elucidate key structural features that govern the binding and/or selectivity of a single compound toward the zinc catalytic sites of the N- and C-domains of the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (NEP). Eleven dual inhibitors were categorized in three classes, according to their zinc binding groups. Analysis of their docked conformers revealed the molecular environment of the catalytic sites and the specific interactions between the inhibitors and amino acid residues that are important for selectivity and cooperativity. In addition, inhibitors were predicted to bind to the C-domain of the ACE with greater affinity than the N-domain, with an average difference in the free energy of binding approximately 2-3 kcal mol(-1). Residues that were identified to actively participate in the binding and stabilizing of the enzyme-inhibitor complexes were analyzed in a consensus way for both the ACE and the NEP. These atomic-level insights into enzyme-ligand binding can be used to drive new structure-based drug design processes in the quest for more selective and effective vasopeptidase inhibitors.