A multicenter cross-sectional study of the quality of life and iron chelation treatment satisfaction of patients with transfusion-dependent ß-thalassemia, in routine care settings in Western Greece.

PURPOSE: To evaluate health-related quality of life (HRQoL) and satisfaction with iron chelation therapy (ICT) of patients with transfusion-dependent ß-thalassemia (TDT) managed under routine care conditions. PATIENTS AND METHODS: This was an observational, multicenter, cross-sectional study conducted in three hospital-based Thalassemia Units of Western Greece. Patients confidentially completed the 36-item short-form (SF-36) and the "satisfaction with ICT" (SICT) instruments to assess HRQoL and ICT satisfaction respectively. RESULTS: One hundred and thirty-one adult TDT patients [74 female, median (IQR) age: 41 (36-47) years] were enrolled. Eighty patients (61.1%) were receiving parenteral ICT, with or without oral chelators (Group I), whereas 51 (38.9%) were only receiving oral ICT (Group II). The median SF-36 physical component summary and mental component summary scores were 76.3 and 75.7 among Group I, and 76.9 and 74.5 among Group II patients, not differing between the two groups. In their majority, Group I (84.6%) and Group II (92.9%) patients reported preferring oral ICT. Moreover, Group I patients reported greater perceived ICT effectiveness (median SICT score: 4.3 versus 4.2; p = 0.039), whereas patients receiving deferasirox-containing ICT reported higher treatment acceptance (median SICT score: 4.0 versus 3.6, p = 0.038) and greater satisfaction with the burden of their ICT (median SICT score: 4.4 versus 3.9, p = 0.033). CONCLUSION: TDT patients prefer to receive oral ICT and are more satisfied of the burden of deferasirox-containing ICT, even though those receiving parenteral ICT are more satisfied by the effectiveness of their treatment. No differences in HRQoL were not noted between patients receiving parenteral versus oral ICT.

The Isoxazole Ring and Its N-Oxide: A Privileged Core Structure in Neuropsychiatric Therapeutics.

Mental disorders are neuropsychiatric conditions that are marked by unusual or irregular thinking, feelings, or behavior, and lead to distress and/or impaired functions. Major psychiatric conditions are depression, anxiety, and psychoses of various types. Their etiopathogeneses, of a primary or secondary origin, are associated with genetic and environmental factors. They are commonly treated with psychoactive drugs (also known as psychotropics), which target serotonin, dopamine, norepinephrine, glutamate, and nuclear receptors (NRs), including retinoic acid receptor-related orphan receptors (RORs) and other receptors in the central nervous system (CNS). Herein we present a diverse array of isoxazole derivatives, among which are some prominent marketed drugs. Some of the derivatives and forms, including N-oxides, are under either (pre)clinical evaluation or patent protection as new generation of psychotropics, and a few have effective blood-brain barrier (BBB) permeability. Various drug-like isoxazol(in)es and their structural features and efficiency, modified through scaffold hopping, are described and discussed in the context of treating neuropsychiatric conditions.

H-Bond: Τhe Chemistry-Biology H-Bridge.

H-bonding, as a non covalent stabilizing interaction of diverse nature, has a central role in the structure, function and dynamics of chemical and biological processes, pivotal to molecular recognition and eventually to drug design. Types of conventional and non conventional (H-H, dihydrogen, H- π, CH- π, anti- , proton coordination and H-S) H-bonding interactions are discussed as well as features emerging from their interplay, such as cooperativity (σ- and π-) effects and allostery. Its utility in many applications is described. Catalysis, proton and electron transfer processes in various materials or supramolecular architectures of preorganized hosts for guest binding, are front-line technology. The H-bond-related concept of proton transfer (PT) addresses energy issues or deciphering the mechanism of many natural and synthetic processes. PT is also of paramount importance in the functions of cells and is assisted by large complex proteins embedded in membranes. Both intermolecular and intramolecular PT in H-bonded systems has received attention, theoretically and experimentally, using prototype molecules. It is found in rearrangement reactions, protein functions, and enzyme reactions or across proton channels and pumps. Investigations on the competition between intra- and intermolecular H bonding are discussed. Of particular interest is the H-bond furcation, a common phenomenon in protein-ligand binding. Multiple H-bonding (H-bond furcation) is observed in supramolecular structures.

Structural studies and cytotoxicity assays of "aggregation-prone" IAPP(8-16) and its non-amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin.

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic ß-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8-20, 20-29, and 30-37. A smaller fragment of the 8-20 region, spanning residues 8-16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8-16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8-16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8-16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8-16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8-16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling.

NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.

Zn(II)/pyridyloxime complexes as potential reactivators of OP-inhibited acetylcholinesterase: in vitro and docking simulation studies.

In order to investigate the ability of metal complexes to act as reactivators of organophosphorus compounds (OP)-inhibited acetylcholinesterase (AChE), we have synthesized and crystallographically characterized three novel mononuclear Zn(II) complexes formulated as [ZnCl2{(4-py)CHNOH}2] (1), [ZnBr2{(4-py)CHNOH}2] (2) and [Zn(O2CMe)2{(4-py)CHNOH}2]∙2MeCN (3∙2MeCN), where (4-py)CHNOH is 4-pyridinealdoxime. Their reactivation potency was tested in vitro with a slight modification of the Ellman's method using Electric eel acetylcholinesterase and the insecticide paraoxon (diethyl 4-nitrophenyl phosphate) as inhibitor. The activity of the already reported complex [Zn2(O2CPh)2{(4-py)CHNOH}2]·2MeCN (4·2MeCN) and of the clinically used drug obidoxime 1,1'-[oxybis(methylene)]bis{4-[(E)- (hydroxyimino)methyl]pyridinium} was also examined. The results of the in vitro experiments demonstrate moderate reactivation of the metal complexes compared to the drug obidoxime. On the other hand, it is clearly shown that the metal complex is the responsible molecular entity for the observed activity, as the reactivation efficacy of the organic ligand (4-pyridinealdoxime) is found to be inconsequential. Docking simulation studies were performed in the light of predicted complex-enzyme interactions using the paraoxon-inhibited enzyme along with the four Zn(II) complexes and obidoxime as a reference reactivator. The results showed that the three mononuclear metal complexes possess the required characteristics to be accommodated into the active site of AChE, while the entrance of the dinuclear Zn(II) compound is unsuccessful. An interesting outcome of docking simulations is the fact that the mononuclear compounds accommodate into the active site of AChE in a similar mode as obidoxime.

Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.

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.

Synthesis and Biological Evaluation of New CRH Analogues.

A series of 7 new human/rat Corticotropin Releasing Hormone (h/r-CRH) analogues were synthesized. The induced alterations include substitution of Phe at position 12 with D-Phe, Leu at positions 14 and 15 with Aib and Met at positions 21 and 38 with Cys(Et) and Cys(Pr). The analogues were tested regarding their binding affinity to the CRH-1 receptor and their activity which is represented by means of percentage of maximum response in comparison to the native molecule. The results indicated that the introduction of Aib, or Cys derivatives although altering the secondary structure of the molecule, did not hinder receptor recognition and binding.

Synthetic peptides as structural maquettes of Angiotensin-I converting enzyme catalytic sites.

The rational design of synthetic peptides is proposed as an efficient strategy for the structural investigation of crucial protein domains difficult to be produced. Only after half a century since the function of ACE was first reported, was its crystal structure solved. The main obstacle to be overcome for the determination of the high resolution structure was the crystallization of the highly hydrophobic transmembrane domain. Following our previous work, synthetic peptides and Zinc(II) metal ions are used to build structural maquettes of the two Zn-catalytic active sites of the ACE somatic isoform. Structural investigations of the synthetic peptides, representing the two different somatic isoform active sites, through circular dichroism and NMR experiments are reported.

Cytogenetic and antineoplastic effects of modified steroidal alkylators.

INTRODUCTION: The aim of this study was to design new potentially antineoplastic agents by combining nitrogen mustard with steroidal skeleton, in an effort to improve specificity and simultaneously to reduce systemic toxicity. The steroidal part is aimed to act as a biological platform enabling the alkylating moiety to approach its site of action by altering its physicochemical properties. MATERIALS AND METHODS: The compounds tested have, as alkylating agents, either p-N,N-bis(2-chloroethyl)aminophenyl-butyrate or p-N,N-bis(2-chloroethyl)aminophenyl-acetate esterified with a modified steroidal nucleus. The four newly synthesized compounds were compared on a molar basis, regarding their ability to induce sister chromatid exchanges and modify proliferation rate indices in cultured human lymphocytes. Life span of BDF1 mice inoculated with L1210 leukemia was also estimated (antileukemic activity). RESULTS: A compound having p-N,N-bis(2-chloroethyl)aminophenyl-acetate as the alkylator and two ketone groups in the steroidal part demonstrated the highest statistically significant enhancement of sister chromatid exchanges and suppression of proliferation rate indices, and also caused significant antineoplastic activity. The other compounds proved less active. CONCLUSION: These results suggest that cytogenetic and antileukemic activity of alkylating steroidal esters depends on the configuration of the whole molecule and the appropriate combination of the alkylator with the steroidal molecule.

Folding in solution of the C-catalytic protein fragment of angiotensin-converting enzyme.

Angiotensin-converting enzyme (ACE) is a key molecule of the renin-angiotensin-aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala(959) to Ser(1066) region (ACE_C) that corresponds to the C-catalytic domain of human somatic angiotensin-I-converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1-trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.

Synthesis, in vivo antileukemic evaluation and comparative study of novel 5alpha-7-keto steroidal esters of chlorambucil and its active metabolite.

Recent structure-antileukemic activity studies showed that the steroidal part of complex molecules containing DNA alkylators does not play only the role of the "biological carrier". New such compounds designed to possess an allylic 7-ketone showed enhanced antileukemic potency compared with derivatives with a simple steroidal skeleton. In order to investigate whether the enhancement of the antileukemic potency is attributed to the introduction of the 7-ketone or to the Delta5-7-keto conjugated steroidal system we decided to reduce the Delta5 double bond. The 5alpha-7-keto-steroidal skeletons synthesized were tethered to chlorambucil and phenyl acetic acid's nitrogen mustard and studied against leukemia P338 in vivo. The reduction of the double bond had a negative impact on the antileukemic potency since the comparative study of the novel derivatives showed that a series of very potent Delta 5-7-keto-steroidal esters were converted by this modification to compounds with marginally accepted activity.

Rational design, synthesis, and in vivo evaluation of the antileukemic activity of six new alkylating steroidal esters.

The synthesis and the in vivo evaluation against leukemias P388 and L1210 of six new alkylating steroidal esters are described. The esteric derivatives incorporating the 17beta-acetamido-B-lactamic steroidal skeleton exhibited increased antileukemic activity and lower toxicity, compared to the 17beta-acetamido-7-keto analogs. Among the 17beta-acetamido-B-lactamic steroidal esters, the most potent compound afforded four out of six cures in leukemia P388 and was measured to be almost non-toxic, producing significant low levels of toxicity.

Synthesis and sst(2) binding profiles of new [Tyr(3)]octreotate analogs.

One of the main objectives of our current work is the development of new somatostatin analogs that would retain the general characteristics of [Tyr(3)]octreotate (Tate) while showing potential for clinical application. In this respect, study of their interaction with the sst(2) is crucial in providing preliminary structure-activity relationships data. In the present work we report on the synthesis and the preliminary biological evaluation of a total of 15 new structurally modified [Tyr(3)]octreotate analogs. The binding affinities were determined during competition binding assays in sst(2)-positive rat acinar pancreatic AR4-2J cell membranes using [(125)I-Tyr(3)]octreotide as the radioligand.

Expression, purification, and physicochemical characterization of the N-terminal active site of human angiotensin-I converting enzyme.

We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, ACE-N) of human somatic angiotensin-I converting enzyme in Escherichia coli. This construct represents the N-catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8-M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE-N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE-N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X-ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy.

On the formation of 4-[N,N-bis(2-chloroethyl)amino]phenyl acetic acid esters of hecogenin and aza-homo-hecogenin and their antileukemic activity.

The p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid esters of hecogenin and aza-homo-hecogenin have been prepared and their antineoplastic activity was evaluated against two basic drug screening systems in rodents, P388 lymphocytic and L1210 lymphoid murine leukemias. Among the compounds tested, the p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid ester of aza-homo-hecogenin was appeared to possess a significant higher antileukemic effect. These results support that the alkylating esters of hecogenin produce important antitumor activity as well as, indicate that the aza-homo-hecogenin ester exhibits significantly higher activity due to lactam group (-NHCO-) modification.

Synthesis and cytogenetic studies of structure--biological activity relationship of esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers.

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.

Solid-phase synthesis and conformational properties of angiotensin converting enzyme catalytic-site peptides: the basis for a structural study on the enzyme-substrate interaction.

The solution NMR conformational properties of two angiotensin converting enzyme (ACE) Zn catalytic-site 36-residue peptides, with the general sequence HEMGHX23EAIGDX3, synthesized through solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, is reported. The 1H resonance assignment of Zn-bound peptides is presented and the characteristic features of the NMR solution models of the two ACE Zn(II)-bound peptides are reported. The solid-state and solution structures of the ACE C-domain catalytic site are compared while biologically important structural similarities and differences of the N- and C-terminal catalytic sites are discussed. Additionally, the structural features of the ACE substrate, the angiotensin I (AI) decapeptide, are studied using NMR spectroscopy, in order to set the structural basis for the ACE-substrate interaction in solution.

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 ACE's 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.