Search

We report here the synthesis and preliminary pharmacological characterization of a novel Neurokinin A receptor antagonist. This molecule contains a dehydroalanine residue. It displays a high conformational rigidity and possesses very high activity. Its pharmacological properties as a neurokinin A receptor antagonist were assessed in in vitro experiments on rat vas deferens and were compared to those of Neuronorm and MEN10627.

Abstract 2871 Introduction. The addition of Rituximab to standard CHOP chemotherapy has improved outcome of DLBCL; however, 30–40% of patients do not response or relapse after induction treatment. The use of Lenalidomide, as single-agent in relapsed or refractory aggressive NHL has been encouraging, with an overall response rate (ORR) of 19% for DLBCL. Preclinical evidence suggests that Lenalidomide may be synergistic with Rituximab. However the safety and efficacy of Lenalidomide combined with R-CHOP is not yet fully evaluated. Aims. On these basis, the IIL is running a prospective multicenter dose finding phase I-II pilot trial to evaluate efficacy and safety of Lenalidomide treatment plus R-CHOP21 (LR-CHOP21) for elderly patients with untreated DLBCL (registered at http://www.clinicaltrials.gov, NCT00907348). The primary endpoint for the phase I part of the study was: definition of Dose Limiting Toxicity (DLT) considered as the maximum dose inducing any grade ≥3 non-hematologic toxicity, or a >15 days delay of planned cycle date. The primary endpoint for the phase II part of the study was: complete response (CR) and ORR. Herein we report the results of phase I. Patients and Methods. Inclusion Criteria were: age 60–80; histologically proven CD20+ DLBCL; no prior chemotherapy and no prior malignancies in the last 3 years; Ann Arbor stage II, III, IV; International Prognostic Index (IPI) at low-intermediate, intermediate-high or high risk score. Treatment consisted of 6 courses of R-CHOP21 in association with Lenalidomide for days 1–14 at the established dose level (5, 10, 15, 20 mg), with Peg-Filgrastim support and prophylactic sc. low-molecular weight heparin. Phase I of the study was planned to define the Maximum Tolerated Dose (MTD) that is the dose that achieves a DLT in 33% or less patients evaluated after the first three courses of LR-CHOP21. The study was designed with the Continual Reassessment Method, a Bayesian “memory design” that begins with a subjective prediction of the dose-response relationship and uses, as dose allocation rule of the sequentially incoming patients, the re-estimated probability of toxicity based on the results obtained for the patients already observed. Four doses of Lenalidomide were tested: 5, 10, 15 and 20 mg. By decision of the steering committee of the study, a dose of 10 mg was administered to the first cohort of 3 patients; in the light of the observed toxicity the probability model assigned the next cohort to the dose with the higher probability to met the required MTD. At the end of each cohort, the dose level associated with an updated DLT probability closest to 33% was recommended to be administered to the next patient cohort. Results. In phase I, from May 2008 to February 2010, 21 patients were enrolled. Median age was 69 years (61-78), stage II/III/IV were 4/4/13 respectively, B symptoms in 11, PS 2 in 8, bone marrow involvement in 6, abnormal LDH in 8, intermediate-high or high IPI score in 15. Patient allocation by Lenalidomide dose was: none patients received 5 mg/day, nine patients received 10 mg/day, nine patients 15 mg/day and three patients 20 mg/day on days 1–14 of each LR-CHOP21 courses. The flow of the dose allocation and the observed DLTs are shown in figure 1. DLTs in the first three courses were recorded in seven patients (see figure 1 for details). These data, according to the continual reassessment method, determined Lenalidomide 15 mg as MTD in association to R-CHOP21. One-hundred-fifteen courses of LR-CHOP21 were performed in the whole series of 21 patients. Overall hematological toxicity was moderate: grade III or IV thrombocytopenia occurred in 10%, anemia in 4% and neutropenia in 28% of the courses. Extra-hematological toxicities were mild: only one patient with grade IV (increase of CPK), two patients with grade III cardiac, three with grade III neurological toxicities, and three patients with grade III infections (two pneumonias and one febrile neutropenia with diarrhea). At the end of six LR-CHOP21 courses, 15/21 patients achieved CR, 1/21 partial remission and 5/21 were not responsive. Conclusions. Lenalidomide 15 mg on days 1–14 in association with R-CHOP21 is the MTD for LR-CHOP. This schedule is safe and well tolerated in a population of elderly DLBCL patients. Preliminary efficacy results are promising. The ongoing phase II part of the LR-CHOP21 trial aims at evaluating the efficacy of 15 mg of Lenalidomide in association with R-CHOP21. Disclosure: Vitolo: Roche Italy: advisory committee; Celgene Italy: advisory committee; Janssen-Cilag: lecture-fee. Off Label Use: The use of Lenalidomide is off-label in untreated DLBCL.

The design, synthesis, and metal-binding properties of DF3, a new de novo designed di-iron protein model are described (‘‘DF’’ represents due ferri, Italian for ‘‘two iron,’’ ‘‘di-iron’’). DF3 is the latest member of the DF family of synthetic proteins. They consist of helix–loop– helix hairpins, designed to dimerize and form an antiparallel four-helix bundle that encompasses a metal-binding site similar to those of non-heme carboxylate-bridged di-iron proteins. Unlike previous DF proteins, DF3 is highly soluble in water (up to 3 mM) and forms stable complexes with several metal ions (Zn, Co, and Mn), with the desired secondary structure and the expected stoichiometry of two ions per protein. UV–vis studies of Co(II) and Fe(III) complexes confirm a metal-binding environment similar to previous di- Co(II)- and di-Fe(III)-DF proteins, including the presence of a l-oxo-di-Fe(III) unit. Interestingly, UV–vis, EPR, and resonance Raman studies suggest the interaction of a tyrosine adjacent to the di-Fe(III) center. The design of DF3 was aimed at increasing the accessibility of small molecules to the active site of the four-helix bundle. Indeed, binding of azide to the di-Fe(III) site demonstrates a more accessible metal site compared with previous DFs. In fact, fitting of the binding curve to the Hill equation allows us to quantify a 150% accessibility enhancement, with respect to DF2. All these results represent a significant step towards the development of a functional synthetic DF metalloprotein.

In this paper we explore the use of fluorescently labeled cytochrome c peroxidase (CcP) from baker's yeast for monitoring nitric oxide (NO) down to the sub-micromolar level, by means of a FRET (Forster Resonance Energy Transfer) mechanism. The binding affinity constant ( K d ) for the NO binding to CcP was determined to be 10 ± 1.5 µM. The rate of NO dissociation from the CcP ( k off ) and the second order rate constant for the NO association ( k on ) were found to be 0.22 ± 0.08 min − 1 and 0.024 ± 0.002 µM − 1 min − 1 respectively. The immobilization of fluorescently labeled CcP into a polymeric matrix for use in a solid state NO sensing device was also explored. The results provide proof-of-principle that labeled CcP can be successfully implemented in a fast, simple, quantitative and sensitive NO sensing device.

Summary. Background: The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser88-Arg-Ser-Arg-Tyr92 internal sequence. Objective: To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser88-Arg-Ser-Arg-Tyr92 sequence. Methods and Results: In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser88-Arg-Ser-Arg-Tyr92 chemotactic sequence, the maximal effect being reached at 10 nmol L−1 SRSRY peptide (SRSRY). This effect is mediated by the αvβ3 vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2. Conclusions: Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser88-Arg-Ser-Arg-Tyr92 chemotactic sequence.

The conformational analysis of [Aib 5,6 -D-Ala 8 ]cyclolinopeptide A, in the solid state and solution, has been carried out by X-ray diffraction and NMR spectroscopy. The structure of the orthorhombic form, obtained from methanol-water mixture [a=29.92 (3) A b=19.85 (3) A, c=9.90 (1) A, space group P2 1 2 1 2 1 Z=4], shows the presence of five in N-H...O=C hydrogen bonds, with formation of one C 17 ring structure, one α-turn (C 13 ), one γ-turn (C 7 ), and two β-turns (C 10 , one of type III and one of type I)

The trans-[Cl2-Pt-(H-Aib-OH)2] and trans-[Pt-(H-Aib-O−)2] complexes have been synthesized and characterized in solution and in the solid state. Their crystal and molecular structures have been determined by X-ray diffraction analyses. The complexes show a distorted planar coordination around the heavy atom. The organic moiety in the two complexes shows different conformations: in trans-[Pt-(H-Aib-O−)2] the amino acid residue, acting as a bidentate, is forced to assume an unusual conformation. On the other hand, in trans-[Cl2-Pt-(H-Aib-OH)2], in which the amino acid acts as a monodentate ligand, the α-aminoisobutyric acid assumes an extended conformation, which in general for α-monosubstituted amino acid residues coordinated to the platinum should be the preferred conformation. In both crystal structures hydrogen bonds are formed between the donor and acceptor groups with further stabilization deriving from van der Waals interactions between hydrophobic moieties.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and solution conformational characterization of the cyclic tetrapeptide cyclo-(L-Pro-beta-Ala-L-Pro-beta-Ala). This peptide was synthesized by classical solution methods and the cyclization of the free tetrapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide (DCCI). The compound crystallizes in the orthorombic space group P2(1)2(1)2(1) from ethyl acetate. All peptide bonds are trans. The molecular conformation is stabilized by two intramolecular hydrogen bonds between the CO and NH groups of the two beta-alanine residues. These hydrogen bonds take place in a C7 structure in which both proline residues occupy the 2 position of an inverse gamma-turn. The two beta-alanine residues have a typical folded conformation (around the C alpha-C beta bond) observed in other cyclic peptides containing this residue. A detailed 1H-nmr analysis in CD3CN solution has been carried out. The molecule assumes a twofold symmetry in solution with a molecular conformation consistent with that observed in the solid state.

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser88-Arg-Ser-Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH2 (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe–dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an αv integrin–dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/αv association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein–expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy. [Mol Cancer Ther 2009;8(9):2708–17]

This paper reports the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

A synthetic, terminally blocked homohexapeptide fromα-aminoisobutyric acid has been examined by single-crystal X-ray diffraction and the structure refined toR = 0.047. The compound is folded into almost two turns of a 310-helix, stabilized by four consecutive intramolecular N—H⋯·O=C H-bonds. An asymmetric covalent geometry for theα-aminoisobutyric acid residues, known to be responsible for the onset of the 310-helix in homopeptides from this Cα,α-dialkylatedα-aminoacid on the basis of a theoretrical conformational analysis, has been experimentally found also in this structure.

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser 88 -Arg-Ser-Arg-Tyr 92 (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH 2 ) shares the same binding site with SRSRY and competes with N -formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N -formyl-peptide receptor (FPR). pERERY-NH 2 is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERY-NH 2 is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.

The solid state conformational analysis of the ionophoric homodetic bicyclic cyclo(Glu-Leu-Pro-Gly-Lys-Leu-Pro-Gly)cyclo(1γ-5ϵ)Gly (BCP3) has been carried out by x-ray diffraction. It crystallizes with 4.5 molecules of water per peptide molecule in the monoclinic system, space group P21, with a = 11.425 A, b = 16.616 A, c = 13.931 A, β = 109.24°, and Z = 2. The structure has been determined by direct methods and refined to an R factor of 0.061 for 2448 observed reflections. The structure characterized by all trans peptide bonds is stabilized by three intramolecular hydrogen bonds: a type II β-turn, a mixed type I-type III β-turn, and a pseudo γ-turn, which involves the side chain CO and the main-chain NH groups of the Glu1 residue. The resulting globular molecule presents a rather hydrophilic surface with most of the CO groups available to hydration of the solvent molecules, which are linked through hydrogen bonds of the NH … O or OH … O types in a complicated H-bonding scheme. The conformation observed in the solid state is rather different from the conformation proposed in solution for both the free and the Ca2+-complexed BCP3 molecule.

The solid state structure of the Zn-complex of clathridine (1), a genuine metabolite of the sponge Clathrina clathrus, was established by X-ray diffraction analysis. In addition, the conformation of 1 in CDCl3 solution was investigated by NOE difference NMR experiments.

Background. A randomised trial has been performed among several GITMO/IIL italian centers, comparing Rituximab-supplemented High-Dose Sequential Chemotherapy (R-HDS) and CHOP-R in high-risk FL Patients (pts) and Methods. Eligibility was based on age-adjusted IPI >=2 (125 pts) or on the IIL score >=3 (11 pts). 136 pts were randomized (68/arm). Main clinical features were: median age 51 yrs. (22–59), stage III–IV 98%, elevated LDH 59%, bulky disease 56%, B symptoms 47%, extranodal disease other than bone marrow (BM) 31%, PS >1 (ECOG) 60%. Both R-HDS and CHOP-R have been already described (Ladetto et al ASH 2005, Rambaldi et al Blood 2000). Cross-over was allowed for pts failing CHOP-R. Centralized minimal residual disease (MRD) analysis with the bcl-2/IgH was performed on BM samples. Analysis was intention to treat. Results. Early toxic deaths were 5 (2 in CHOP-R, 3 in R-HDS); CR rates were 61% with CHOP-R and 85% with R-HDS (p Conclusions: In high-risk FL: a) first-line R-HDS ensures better EFS and PFS and superior molecular outcome than CHOP-R; b) pts undergoing salvage R-HDS have an excellent clinical outcome; c) the long-term OS is markedly improved compared to the pre-rituximab era, with no difference between CHOP-R and R-HDS; d) both the high efficacy as salvage treatment and the potential side effects suggest that the ideal positioning of R-HDS-like treatments is beyond first line treatment, for relapsed or refractory disease; e) a PCR-negative status is the most important prognostic factor regardless of treatment received and pts with persistent PCR-positivity might potentially be considered for specific experimental intervention.

significant accumulation of the NuRD complex at MYC TSS after IMiDs exposure. Importantly, CHD4 silencing attenuated lenalidomide induced MYC transcriptional repression and cell death. In summary, our studies suggest that IKZF1 drives MYC expression in MM by regulating the activity of the IGH 3’ enhancer and preventing the repositioning of the NURD complex to its promoter. 5. Multiple Myeloma Therapy in Newly Diagnosed Patients excluding Transplantation

HIV initiates its infectious cycle by docking to CD4 and a chemokine receptor, most commonly CCR5. RANTES, a natural CCR5 ligand, is a potent inhibitor of HIV-1. Despite the lack of structural information on the RANTES-CCR5 complex, determinants of HIV blockade were previously identified within the RANTES N-loop and beta1-strand regions. A prototype N-loop/beta1-strand peptide, named R11-29, contains two terminal hydrophobic stretches separated by a central hydrophilic region. Here, the role of the terminal hydrophobic clusters was investigated by means of amino acid substitutions or deletions. Most hydrophobic residues in these clusters were shown to be fundamental for the anti-HIV activity. However, increasing the hydrophobicity of the two clusters using non-natural amino acids did not significantly improve the potency of the peptides. These results may provide instrumental knowledge for the rational design of RANTES-derivative molecules with increased anti-HIV activity.

OBJECTIVES Aim of this prospective study was to evaluate the risk of invasive fungal infection (IFI) in patients (pts) with acute promyelocytic leukemia (APL) and to compare APL pts with patients affected by non promyelocytic acute myeloid leukemia (npAML) in order to evaluate factors potentially linked to IFI in these two subsets of acute myeloid leukemia. PATIENTS AND METHODS From January 2010 to April 2012 all pts with newly diagnosed AML were registered in 33 Italian participating centers. A minimum follow up of 90 days after 1st induction chemotherapy was requested for all pts. A prolonged follow up until June 2014 was made only for APL. Data were collected about age, gender, AML subtype, treatment and also about post chemotherapy risk factors for IFI (duration of neutropenia, mucosal damages, vomiting, diarrhea, presence of medical devices), antifungal prophylaxis, onset of IFI, level of certainty (possible/probable/proven), and antifungal treatment. Only for APL the survey was prolonged for at least 3 months in order to analyze if these pts have an IFI risk during other than first induction phases. RESULTS 1,192 consecutive newly diagnosed adult AML pts (npAML:1,086/APL:106) were enrolled in the study. Among npAML pts, those receiving low dose chemotherapy and/or palliative treatment were excluded from the analysis; in the remaining 881 pts 214 cases (24%) of IFI were recorded. Considering APL, 3 pts were excluded from the analysis due to early death (1 pt) or bad performance status (2 pts). The remaining 103 pts received APL treatment according to local protocols: all trans retinoic acid (ATRA) plus chemotherapy (90 pts) or ATRA plus arsenic trioxide (ATO)(13 pts). Only 8 (8%) APL pts developed an IFI after the induction phase: 1 proven, 3 probable and 4 possible IFI. All cases were caused by molds. All APL were followed for a median follow up of 36 months (range 3-54). During this time only 2 other cases of IFI were observed: 1 possible IFI during consolidation at 16 weeks from APL diagnosis and 1 probable aspergillosis in a rare case of APL relapse at 132 weeks from APL diagnosis. All the IFI occurred in pts treated with ATRA plus chemotherapy. IFI was fatal in only 1 case (cerebral aspergillosis), all the other pts recovered after antifungal treatment. A comparison between npAML and APL was made in order to analyze the risk of IFI within 90 days after induction treatment among these 2 groups of patients (see table). A significantly lower number of overall IFI and systemic antifungal treatment was observed in the APL group, in spite of the fact that systemic anti mold prophylaxis was significantly less frequently utilized. | | || | | APL | npAML | p | | Number of pts | 103 | 881 | | | Mean age | 51 | 55 | 0.01 | | m/f | 50/53 | 448/433 | N.S. | | Performance status (WHO) 0-1 >1 | . 76 27 | . 284 597 | .

Calmodulin (CaM) is an acidic protein of 148 amino acid residues that plays a central role in cellular regulation by relating the levels of cellular calcium to the activities of regulatory proteins, such as kinases, phosphatases, etc. Recent studies with synthetic models and natural peptides have shown that CaM recognizes positively charged, amphiphilic peptides [1]. There is no exact sequence homology in the CaM binding proteins, but they frequently contain segments rich in basic residues (Arg, Lys, His) and are capable of adopting, when they bind to CaM, an amphiphilic conformation. These observations demonstrated that this structural feature is important for binding, and that hydrophobic and electrostatic interactions both play a relevant role. We report here our approach for the design of an dimer covalently linked in a parallel or antiparallel orientation that is able to specifically interact with only one of the target enzymes recognized by CaM and form three stranded coiled coils. The target sequence that we chose for binding was the CaM binding domain of calcineurin. This important enzyme is the target of immunosuppressive drugs, such as cyclosporin A and FK506. A peptide that specifically binds to calcineurin could be expected to be either an immune-stimulant or an immune-suppressant, depending on whether it activates or inhibits activation of the enzyme.

The properties of mimochrome II, a hemoprotein model compound, were evaluated in relation to the peptide chain composition and length and its importance in controlling heme properties.

We previously identified the major structural determinants of CCR5 binding and HIV blockade in RANTES, describing linear RANTES-derived peptides with biological activity in the low micromolar range (Nardese et al., 2001). To deepen our understanding of RANTES structure-function relations and obtain more potent antiviral peptido-mimetics, we have extensively mutagenized the prototypic peptide, R11-29. This presents two clusters of hydrophobic residues at its termini, (corresponding to RANTES N-loop and b1-strand) connected by a positivelycharged linker. Single or multiple alanine substitutions within the Nor C-terminal hydrophobic clusters resulted in a dramatic loss of antiviral activity, whereas deletion of selected residues within the hydrophilic linker had no major functional consequences. Based on RANTES 3D structure, we designed a series of modified peptides, resulting in a progressive increase in specific antiviral activity. These peptides also displayed anti-inflammatory properties blocking RANTES-elicited lymphocyte chemotaxis. Through serial passages in culture in the presence of increasing concentrations of the most effective antiviral peptides, we have derived variants of the R5 HIV-1 isolate BaL resistant to the peptide inhibitory activity. Complete sequencing of the envelope genes from such variants is currently underway. Our results provide new insights into the structure of the receptor-binding region of RANTES and identify new antiviral peptides that may be instrumental in the development of effective HIV-1 entry inhibitors. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

Peptide-based models have an enormous impact for the development of metalloprotein models, as they seem appropriate candidates to mimic both the structural characteristics and reactivity of the natural systems. Through the de novo design of four-helix bundles, we developed the DF (Due Ferri) family of artificial proteins, as models of di-iron and di-manganese metalloproteins. The goal of our research is to elucidate how the electrostatic environment, polarity and solvent accessibility of the metal-binding site, influence the functional properties of di-iron proteins. The first two subsets of the DF protein family, DF1 and DF2, consist of two non-covalently associated helix-loop-helix motifs, which bind the di-metal cofactor near the center of the structure. The DF2 subset was designed to improve the properties of DF1: DF2 and DF2t have several changes in their sequences to improve solubility and metal ion access, as well as a change in the loop connecting the two helices. In order to evaluate how these changes affect the overall structure of the model proteins, we solved the NMR structures of the di-Zn(II) complexes of DF2 and DF2t, and compared these structures with those recently obtained from X-ray crystallography. Further, we examined the thermodynamic consequences associated with the mutations, by measuring the stability of DF2t in the presence of different metal ions, and comparing the results with the data already obtained for DF2. Taken together, analysis of all the data showed the importance of the turn conformation in the design and stability of four-helix bundle.

The objective of this study was to make a contribution to the debate on the cause-effect relationship between low-dose exposure to benzene and onset of hemopathies. We report the case of a coke plant worker suffering from aplastic anemia. Before being hired at the coke plant, he underwent a medical examination including a blood cell count: no disease or abnormalities of the blood crasis were found. For 3 years the patient was then exposed to gas containing-as measured in environmental investigation carried out at the coke plant-concentrations of benzene lower than TLV-TWA ACGIH (measured values 21-109 micrograms/m3). In the absence from the patient's history of any exposure to other myelotoxic agents and of any earlier pathology causing aplastic anemia, we assume there is a relationship between exposure to low levels of benzene and onset of the disease. However, it is very important to consider that exposure to low levels of benzene could promote myelotoxic reactions when the working environment contains other substances that may act synergistically or compete for the same metabolism sites, or when carcinogenic substances are present in the working environment.

Protein design provides an attractive approach to test the essential features required for folding and function. Previously, we described the design and structural characterization in solution of mimochromes, a series of miniaturized metalloproteins, patterned after the F-helix of the hemoglobin beta-chain. Mimochromes consist of two medium-sized helical peptides, covalently linked to the deuteroporphyrin. CD and NMR characterization of the prototype, mimochrome I, revealed that the overall structure conforms well to the design. However, formation of Delta and Lambda diastereomers was observed. To overcome the problem of diastereomer formation, we re-designed mimochrome I, by engineering intramolecular, interchain interactions. The resulting model was mimochrome IV: the solution structural characterization showed the presence of the Lambda isomer as a unique form. To examine the extent to which the stereochemical stability and uniqueness of mimochrome IV was retained in the solid state, the crystal structure of Co(III)-mimochrome IV was solved by X-ray diffraction, and compared to the solution structure of the same derivative. Co(III)-mimochrome IV structures, both in solution and in the solid state, are characterized by the following common features: a bis-His axial coordination, a Lambda configuration around the metal ion, and a predominant helical conformation of the peptide chains. However, in the crystal structure, intrachain Glu1-Arg9 ion pairs are preferred over the designed, and experimentally found in solution, interchain interactions. This ion pairing switch may be related to strong packing interactions.

De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein ( K diss = 0.001 fM), but the dissociation constant increases to 0.6 nM (ΔΔ G = 5.4 kcal/mol monomer) as the active-site cavity is increased to accommodate small molecules.

University of Napoli Federico II, Department of Chemistry One of the most challenging topics in hemoprotein chemistry is to identify and to rationalise the structural requirements for specific defined functions (dioxygen activation/transport/storage, electron transfer etc.). Recently, mimochrome-IV a synthetic based-peptide model of hemoproteins was designed [Chem.Rev. (2001) 101, 3165]. The molecule is composed by two identical nine-peptide chains covalently linked to the propionic groups of a deuteroporphyrin-IX through the epsilon-amino function of Lys. Each peptide sequence bears a His residue in the central position that may act as axial ligand in metal coordination. With the aim to study the metal environment of this system we have undertaken the X-ray diffraction experiments of the mimochrome-IV cobalt(III) derivative. After several unsuccessful attempts to solve the phase problem by molecular replacement and the direct methods, the presence of a cobalt atom in the structure suggested to perform an MAD experiment. Four MAD data sets, collected at Elettra, were used to solve the XRD structure of the lambda isomer of Co(III)-mimochrome-IV. The structure consists of two antiparallel peptide chains in -helix conformation, sandwiching the porphyrin plane with a pseudo C2 symmetry. This is the first crystal structure of an artificial heme protein of the mimochrome series and represents the starting point to address the future design directions to reproduce in artificial molecules the subtle mechanisms that control the heme functions, thus ensuring the selectivity of the natural systems. Furthermore, at best of our knowledge this result represents the first structure solved using MAD technique on the cobalt absorption edge. Keywords: MINIATURIZED PROTEIN HEMOPROTEIN MAD Acta Cryst. (2002). A58 (Supplement), C277 ENGINEERING GREEN FLUORESCENT PROTEIN TO A REDOX SWITCH A. Henriksen

8566 Background: Implementing strategies for Hodgkin lymphoma (HL) patients failing stem cell transplantation (SCT) remains a crucial need. We investigated for the first time safety and efficacy of a combination of lenalidomide and bendamustine (LEBEN), two agents synergistically framing different targets on both neoplastic and microenvironment tumor-supporting cells of HL. Methods: A weekly schedule of bendamustine (60 mg/m2; d 1, 8, 15) plus continuous daily lenalidomide (10, 15, 20 or 25 mg), in a 28-day cycle, was tested in a Bayesian phase 1/2 study (Thall & Cook, Biometrics 2004) defining the optimal lenalidomide dosage through the best efficacy/toxicity trade-off. Response (CR or PR at PET/CT) and toxicity (CTCv3.0 grade >3 lasting >2 weeks) endpoints were assessed at 2 cycles (day +56). Non-progressing patients received additional 4 courses. Results: Seventeen chemorefractory patients [SCT failures n=12 (single auto SCT, n=4; tandem n=3; tandem auto/allo, n=5 ); unresponsive to pre-SCT salvage, n=...

The miniaturization process applied to rubredoxins generated a class of peptide-based metalloprotein models, named METP ( m iniaturized e lectron t ransfer p rotein). The crystal structure of Desulfovibrio vulgaris rubredoxin was selected as a template for the construction of a tetrahedral (S γ -Cys) 4 iron-binding site. Analysis of the structure showed that a sphere of 17 Å in diameter, centered on the metal, circumscribes two unconnected approximately C 2 symmetry related β-hairpins, each containing the -Cys-(Aaa) 2 -Cys- sequence. These observations provided a starting point for the design of an undecapeptide, which self assembles in the presence of tetrahedrally coordinating metal ions. The METP peptide was synthesized in good yield by standard methodologies. Successful assembly of the METP peptide with Co(II), Zn(II), Fe(II/III), in the expected 2:1 stoichiometry, was proven by UV-visible and circular dichroism spectroscopies. UV-visible analysis of the metal complexes indicated the four Cys ligands tetrahedrally arrange around the metal ion, as designed. Circular dichroism measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. NMR characterization of the Zn(II)-METP complex fully supported the structure of the designed model. These results prove that METP reproduces the main features of rubredoxin.

EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time. Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106.4 Å. A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 Å resolution, using a single frozen crystal.

We report here the synthesis and preliminary pharmacological characterization of a novel water soluble Neurokinin A receptor antagonist named Neuronorm. The synthesis was achieved in high yield by a combination of classical peptide synthesis methodologies, in both solution and solid phase. The pharmacological properties as neurokinin A receptor antagonist were assessed in in vitro experiments on rat vas deferens and guinea pig trachea, and were compared to those of MEN10627.

In this paper we have reported a preliminary analysis of the functional and structural properties of a rationally designed DNA-binding peptide. Finger 4 is able to bind a DNA target sequence. More remarkably, the peptide sequence shows a high helical content, thus demonstrating the effectiveness of the Aib residue in inducing helical structures even in highly charged, water-solubles peptides.

The X-ray crystal structure of the human alpha-thrombin-hirunorm IV complex has been determined at 2.5 A resolution, and refined to an R-factor of 0.173. The structure reveals an inhibitor binding mode distinctive of a true hirudin mimetic, which justifies the high inhibitory potency and the selectivity of hirunorm IV. This novel inhibitor, composed of 26 amino acids, interacts through the N-terminal end with the alpha-thrombin active site in a nonsubstrate mode, and binds specifically to the fibrinogen recognition exosite through the C-terminal end. The backbone of the N-terminal tripeptide Chg1"-Arg2"-2Na13" (Chg, cyclohexyl-glycine; 2Na1, beta-(2-naphthyl)-alanine) forms a parallel beta-strand to the thrombin main-chain segment Ser214-Gly216. The Chg1" side chain occupies the S2 site, Arg2" penetrates into the S1 specificity site, while the 2Na13" side chain occupies the aryl binding site. The Arg2" side chain enters the S1 specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2" side chain establishes the typical ion pair with the carboxylate group of Asp189.

A synthetic, terminally blocked homodecapeptide from the C alpha, alpha-dimethylated glycyl residue alpha-aminoisobutyric acid has been analyzed by single-crystal X-ray diffraction and the structure refined to R = 0.073. The compound crystallizes as a perfect 3(10) helix, stabilized by eight consecutive intramolecular N-H . . . O = C hydrogen bonds. This is the first observation at atomic resolution of a regular polypeptide 3(10) helix as long as three complete turns.

We describe here the solution 1H NMR analysis, restrained and unrestrained molecular dynamic simulations of the bicyclic peptide cyclo(Met1-asp2-Trp3-Phe4-dap5-Leu6)cyclo(2β-5β) (MEN10701) (dap: (2R)-2,3-diaminopropionic acid). This compound is an analogue of cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)cyclo(2β-5β) (MEN10627) (Dap: (2S)-2,3-diaminopropionic acid), which is the most potent and selective, peptide-based NK2 receptor antagonist known to date. MEN10701 differs from MEN10627 for the d chirality of the Asp2 and Dap5 residues; it was designed to better understand the role of the lactame bridge in determining the shape of the molecule and to elucidate whether its position, above or below the plane containing the pharmacophores (Met1, Trp3, Phe4, and Leu6 side chains), could modulate the biological response. Despite our expectations, the uncoercible bicyclic structure of MEN10627 is dramatically coerced into a novel conformation, by the replacement of the lactame bridge forming units (Asp2 and Dap5) with residues of opposite chirality. The overall shape of MEN10701 is also quite unique because of its compactness. It is ellipsoidal instead of being rectangle-like, and the structure is stabilized by two intramolecular hydrogen bonds encompassing two type I‘ β-turns. This structure can be added to the repertoire of rigid β-turn scaffolds for the design of bioactive molecules, which require turned motifs to elicit potency and specificity.

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z–X1–Afc2–Y3–OMe (peptide a: X = Y = Gly; peptide b: X = Aib, Cα,α-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a–c). In contrast, Afc was found in a folded conformation, in the 310-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib. © 2000 John Wiley & Sons, Inc. Biopoly 53: 150–160, 2000

FeIII mimochrome I is the prototype of a new class of hemoprotein models characterized by a covalent helix–heme–helix sandwich. It contains deuterohemin bound through two propionyl groups to two identical N- and C-terminal protected α-helical nonapeptides, each of which bears a His residue (a potential axial lig-and of the iron ion) in the central position. In order to understand better the three-dimensional structure of FeIII mimochrome I and its correlation with spectral properties, we have characterized the fully diamagnetic parent compound CoIII mimochrome I by UV/visible, CD, and NMR spectroscopy, coupled with conformational energy calculations. CoIII mimochrome I is a highly water-soluble compound present in solution as two isomers, which slowly interconvert only at very low pH values. These isomers were isolated and separately characterized. Their UV/visible spectral properties are very similar, while their CD spectral properties differ markedly in both the far UV and Soret regions. The isomers were identified by 1H NMR spectroscopy as diastereomers of the δ and δ type. This is the first example of an accurate three-dimensional structure determination in solution of a hemoprotein mimetic that allows a straightforward correlation between structure and spectral properties.

Introduction The role of bone marrow (BM) involvement as prognostic factor in untreated young patients with diffuse large B-cell lymphoma at poor prognosis is still a matter of debate. Recent data showed an adverse prognostic role of BM involvement in DLBCL including patients both at low and high IPI score (Sehn L et al, J Clin Oncol 2011). On this basis, FIL analyzed the impact of BM involvement in the prospective randomized phase III trial DLCL04 (Vitolo U et al, Blood, ASH annual Meeting 2012) that included only young patients at high-risk age-adjusted IPI (aa-IPI) score 2 or 3. Patients and Methods Inclusion criteria were: age 18-65; untreated DLBCL or follicular grade IIIb; aa-IPI score 2 or 3. Patients were stratified according to aa-IPI and randomized at diagnosis to receive: R-CHOP14 x 8 cycles; R-MegaCHOP14 (1200 mg/sqm Cyclophosphamide, 70 mg/sqm Doxorubicin, standard dose Vincristine and Prednisone) x 6; R-CHOP14 x 4 + R-HDC (Rituximab + High Dose Cytarabine + Mitoxantrone + Dexamethasone) followed by BEAM and ASCT; R-MegaCHOP14 x 4 + R-HDC + BEAM and ASCT. BM biopsy and aspirate were mandatory at diagnosis; at the end of treatment, BM assessment was mandatory only in case of positivity at baseline. BM involvement was defined as concordant if marrow was involved by large B-cell and discordant if involved by small B-cells. Flow cytometry, immunohistochemistry, and/or molecular studies were utilized to confirm a clonal B-cell population. Results From June 2005 to September 2010, 399 patients were randomized to receive: 199 R-HDC+ASCT and 200 R-dose-dense chemotherapy without ASCT. All patients were evaluable for analysis. BM involvement was reported in 84 patients (21%): 39 (20%) in the R-HDC+ASCT group and 45 (22%) in the R-dose-dense chemotherapy group. Pattern of involvement was: concordant in 63 patients, discordant in 14 and not specified in 7 patients. Patients with BM involvement (BM positive: 84) compared to those without BM involvement (BM negative: 315) were significantly older (median age 53 years vs 47 years, p1 43% vs 45%, bulky 25% vs 33%, extranodal sites > 1 26% vs 33%, LDH higher than normal value 93% vs 89%). With a median follow-up of 49 months, 3-year PFS for the whole series of 399 patients enrolled in the trial was: 67% (95% CI: 62-72). Three-year PFS was significantly worse in BM positive vs. BM negative: 46% (95% CI:35-56%) vs. 73% (95% CI:67-77%) p Conclusions Bone marrow involvement, namely concordant pattern, is a strong adverse predictor of outcome in young patients with untreated DLBCL at poor prognosis treated with R-chemotherapy regardless of intensification with HDC+ASCT. New treatment approaches are needed for these high-risk patients at poor prognosis. Disclosures: Vitolo: Roche: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau.

Introduction The standard treatment for elderly untreated diffuse large B-cell lymphoma (DLBCL) is RCHOP21, however up to 40% of patients experienced failures. Lenalidomide showed activity in heavily pretreated DLBCL and in vivo and in vitro data demonstrated a synergism with rituximab. In the phase I trial REAL07 (Chiappella et al, Haematol 2013), FIL demonstrated that the association of LRCHOP21 was feasible in elderly untreated DLBCL and identified 15 mg lenalidomide from day 1 to day 14 as the maximum tolerated dose in combination with RCHOP21. Patients and methods. The phase II trial REAL07 was designed based on Simon's two stage design to demonstrate an improvement of overall response rate (ORR) of 15% in LRCHOP21 compared to 70% of standard RCHOP21. Secondary endpoints were progression-free survival (PFS), overall survival (OS), event-free survival (EFS) and to correlate outcome with cell of origin (COO) profile. Response was evaluated according to 2007 Cheson criteria. Inclusion criteria were: age 60-80 FIT at the comprehensive geriatric assessment; untreated CD20+ DLBCL; Ann Arbor stage II/III/IV; international prognostic index (IPI) at low-intermediate/intermediate-high/high (LI/IH/H) risk. Treatment plan was: RCHOP21 plus 15 mg lenalidomide from day 1 to 14 for 6 courses. All cases were centrally reviewed by expert pathologist; COO profile analysis was conducted with immunohistochemistry according to Hans' algorithm and with gene expression profile (DASL assay). Results. From April 2010 to May 2011, 49 patients were enrolled. Clinical characteristics were: median age 69 years (range 61-80); stage III/IV 43 (88%), IPI IH/H 30 (61%). At the end of 6 LRCHOP21, ORR was 92%. Complete remissions (CR) were 42 (86%) and partial remission 3 (6%); 3 patients (6%) did not respond and one (2%) died for homicide. At a median follow-up of 28 months, 2-year OS was 92% (95% CI: 79-97), 2-year PFS was 80% (95% CI: 64-89) and 2-years EFS was 70% (95% CI: 55-81); 2-year PFS for IPI LI was 89% (95% CI: 62-97) and for IPI IH 76% (95% CI: 47-90) and for IPI H 72% (95% CI: 36-90). Hematological and extra-hematological toxicities were mild, with no grade IV extra-hematological events and no toxic deaths during treatment. Of the 294 planned courses of LRCHOP21, 277 (94%) were administered; median dose of lenalidomide delivered was 1185 mg (94% of the planned dose); at least 90% of the planned dose of each drug was administered in 91% of the RCHOP21 courses. Median interval time between RCHOP21 courses was 21 days (range 19-48). All 49 cases underwent central pathology review and diagnosis of DLBCL was confirmed. Regarding COO analysis, tissue block or stained slides were collected in 40/49 (82%), of which 32 were adequate for analysis. At the time of this abstract, COO analysis was reported according to immunohistochemistry data; DASL analysis is ongoing. Clinical characteristics between germinal center (GCB, 16 patients) and non-GCB (16 patients) were superimposable, excepted for a majority of H IPI risk in non-GCB group (p 0.067). ORR for GCB and non-GCB were 88% (CR 81%) and 88% (CR 88%), respectively. At a median follow-up of 28 months, 2-year PFS was 71% (95% CI: 40-88) in GCB-group and 2-years PFS was 81% (95% CI: 51-93) in non-GCB-group (Figure 1). Conclusions. In conclusion, LRCHOP21 is effective, also in poor risk patients, namely in non-GCB subgroup. These encouraging data warrant a future phase III randomized trial comparing LRCHOP21 vs. RCHOP21 in untreated non-GCB DLBCL. Disclosures: Off Label Use: lenalidomide in first line DLBCL is off lable. drug provided free by Celgene. Vitolo:Roche: Speakers Bureau; Celgene: Speakers Bureau; Takeda: Speakers Bureau.

Background Elderly multiple myeloma (MM) patients are an heterogeneous population. Aging is associated with an increased frequency of co-morbidities, frailty and disability, with negative impact on treatment tolerance and outcome. A simple and reliable scoring system, based on geriatric assessment, has been developed to predict survival and used also to predict the risk of severe toxicities or treatment discontinuation in elderly newly diagnosed MM patients treated with lenalidomide-, bortezomib- or carfilzomib-based induction regimens. Methods Patients with newly diagnosed MM, ineligible for high-dose therapy and autologous stem cell transplantation due to age (≥65 years) or coexisting co-morbidities, enrolled in 3 prospective multicenter trials, were included in the analysis. Up-front dose reductions were performed according to patients age (full doses for patients ≤75 years and reduced for patients >75 years). Details on treatment regimens and results of these studies have previously been reported (Gay F et al EHA 2013, Larocca A et al EHA 2013, Bringhen S et al EHA 2013). At diagnosis, a geriatric assessment had been performed, to assess co-morbidities, cognitive and physical conditions. Results 869 patients were included in the analysis: 659 enrolled in the lenalidomide-based, 152 in the bortezomib-based and 58 in the carfilzomib-based trial. Median age was 74 years, and 44% of patients were older than 75 years. Median follow-up was 18 months. In univariable analysis, the risk of death was higher in patients aged 75-80 (Hazard Ratio, HR 1.37, p=0.11), and in patients older than 80 years (HR 2.75, p In a Cox's model, including ISS, gender and performance status, the HR compared to the fit category was 1.4 (p=0.18) and 2.9 (p Conclusions The use of a simple scoring system, based on geriatric assessment, allows the identification of groups of patients with different survival and risk of severe toxicities. Disclosures: Larocca: Celgene: Honoraria; Janssen-Cilag: Honoraria. Bringhen:Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Merck Sharpe & Dohme: Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy. Gay:Celgene: Honoraria; Janssen-Cilag: Honoraria; Celgene: Membership on an entity’s Board of Directors or advisory committees; Byotest: Membership on an entity’s Board of Directors or advisory committees. Boccadoro:Celgene: Research Funding; Janssen-Cilag: Research Funding; Celgene: Consultancy; Janssen-Cilag: Consultancy; Celgene: Membership on an entity’s Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity’s Board of Directors or advisory committees. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria.

Introduction The role of interim-PET (i-PET), after two cycles of chemotherapy, in advanced stage Hodgkin's lymphoma is well defined; the same role in diffuse large B cell lymphoma (DLBCL) is still controversial. The Fondazione Italiana Linfomi planned a prospective randomized phase III trial, addressed to young poor prognosis DLBCL patients at the diagnosis, with a 2x2 factorial design aimed at investigating the possible benefit of intensification with R-HDC+ASCT after R-dose-dense chemotherapy delivered at two different level of dose (R-CHOP14/R-MegaCHOP14). During this study an ancillary study was designed to define the prognostic impact of i-PET on progression free survival (PFS) after two cycles of immunochemotherapy in all treatments arms. Patients and methods As described by Vitolo et al (oral communication, abs 688 ASH 2012), patients were stratified according to aa-IPI and randomized at diagnosis to receive: R-CHOP14 x 8 cycles; R-MegaCHOP14 (1200 mg/sqm Cyclophosphamide, 70 mg/sqm Doxorubicin, standard dose Vincristine and Prednisone) x 6; R-CHOP14 x 4 + R-HDC (Rituximab + High Dose Cytarabine + Mitoxantrone + Dexamethasone) followed by BEAM and ASCT; R-MegaCHOP14 x 4 + R-HDC + BEAM and ASCT. G-CSF support was mandatory. Seventeen centers agreed to participate in the study and enrolled patients. All evaluable patients had performed basal PET and i-PET. A visual dichotomous criteria, according to Deauville Criteria (First Consensus Conference, 2009), was used to define the i-PET result. The final response was identified according to 2007 Cheson response criteria. Results From June 2005 to September 2010, 399 patients were randomized in the overall DLCL04 study and 130 were enrolled for the ancillary PET study , 69 in the R-HDC+ASCT and 61 in R dose dense therapy respectively. The clinical characteristics, as in the overall DLCL04 study, were well balanced among the four arms of therapy excluding a selection bias in the group of patients studied with i-PET. The i-PET result was positive in 74 patients and negative in 56 patients, no differences were observed between negative and positive i-PET results according to clinical characteristics and group of treatment. In particular, in R-dose dense arm 27 were negative and 34 positive, in R-HDC+ASCT 29 were negative and 40 positive, according to immunochemotherapy scheme in R-CHOP14 29 were negative and 40 were positive, in MegaCHOP14 27 were negative and 34 were positive. With a median follow-up of 36 months, 3-year PFS and 3-year Overall Survival (OS) rates for the whole series were: 64% (95% CI:59-69) and 79% (95% CI:74-83) respectively. No differences in PFS was reported according to i-PET result. In particular 3-year PFS were 87%(95% CI:75-94) in i-PET negative patients and 76% (95% CI: 65-85) in i-PET positive patients respectively (p: 0.09 The 3-year PFS according to I- PET results in the different treatment arm were: in the R-HDT+ASCT group i-PET negative or i-PET positive patients had a 3-year PFS of 83% (95% CI:63-92) and 79% (95% CI:63-89) respectively; in the R-dose dense i-PET negative or i-PET positive patients had a 3-year PFS of 92% (95% CI:73-98) and 72% (95% CI:54-85) with a p value near the significance (0.07). According to OS, i-PET negative and i-PET positive patients had a 3-year OS of 89%(95% CI:76-95) and 83%(95% CI:72-90) respectively, p=0.219. Conclusions In our multicenter prospective study, addressed to young poor prognosis DLBCL patients at the diagnosis, i-PET, performed after two immunochemotherapy cycles and analyzed with a visual method, is not able to identify two different risk population. To confirm our data, we are planning in the near future a centralized revision of all evaluable PET. In conclusion, our feeling is that the i-PET after two cycles in poor prognosis DLBCL patients is too early to predict a chemorefractory disease and more parameters must be considered to define clinical response in these patients. Disclosures: No relevant conflicts of interest to declare.

We review the reported data on the design, the conformational features and the pharmacological properties of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta). MEN 10,627 possesses a highly constrained structure characterized by two consecutive beta-turns, as confirmed by the almost coincident results of NMR and X-ray analyses. The compound has been efficiently synthesized by solid-phase methodology using either Boc or Fmoc strategies. It is quite stable to metabolic degradation and is endowed with high affinity and selectivity for NK2 receptor expressed in various species. At the hamster NK2 receptor MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist, SR 48,968, while the converse is true for the rabbit NK2 receptor. MEN 10,627 and SR 48,968 show comparable affinities for the human NK2 receptor. MEN 10,627 produces a long lasting inhibition of the response to the selective NK2 receptor agonist [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo after intravenous, intranasal and intraduodenal administration. Therefore different administration routes are possible for this compound that overcomes the usual drawbacks for the application of peptides as drugs.

We describe here the design, synthesis, and activity of a novel class of alpha-thrombin inhibitors named hirunorms. They were rationally designed to interact through their N-terminal end with the alpha-thrombin active site in a nonsubstrate mode and to specifically bind the fibrinogen recognition exosite. An appropriate spacer that is able to properly orient the N-terminal end in the active site was also selected. This spacer allowed the size of the inhibitors to be reduced to about one-third of the amino acid residues in the hirudin sequence. Hirunorms specifically inhibit the amidolytic action of human alpha-thrombin toward a small chromogenic substrate. The most active compounds of the series, hirunorms IV and V, inhibit alpha-thrombin catalyzed hydrolysis of Tos-Gly-Pro-Arg-p-nitroanilide with K(i) = 0.09 and K(i) = 0.21 nM, respectively. Comparison of the anticoagulant properties of hirunorms, natural hirudin from the European leech Hirudo medicinalis, and the synthetic analog hirulog-1 revealed that hirunorm IV is about 10-fold and 3-fold more active, on a molar base, than hirudin and hirulog-1 in increasing the aPTT, PT, and TT of normal human plasma. The peculiar structure of hirunorms makes them stable to the amidolytic action of thrombin without the introduction of any peptide bond modification. These molecules display long-lasting activity in human plasma, due to the presence of several unnatural amino acids in susceptible positions. Hirunorms are potential candidates for injectable anticoagulants, due to their potency, specificity of action, long-lasting activity, and safety profiles.

Irregular protein secondary structures are believed to be important structural domains involved in molecular recognition processes between proteins, in interactions between peptide substrates and receptors, and in protein folding. In these respects tight turns are being studied in detail. They also represent template structures for the design of new molecules such as drugs, pesticides, or antigens. Isolated α-turns, not participating in α-helical structures, have received little attention due to the overwhelming presence of other types of tight turns in peptide and protein structures. The growing number of protein X-ray structures allowed us to undertake a systematic search into the Protein Data Bank of this uncharacterized protein secondary structure. A classification of isolated α-turns into different types, based on conformational similarity, is reported here. A preliminary analysis on the occurrence of some particular amino acids in certain positions of the turned structure is also presented. © 1996 John Wiley & Sons, Inc.

Gliadin amino acid sequence(s) responsible for toxicity in susceptible individuals have not been fully elucidated. Previous in vitro studies have suggested the presence of active sequences in the NH(2)-terminal part of the A-gliadin molecule. In this paper the in vitro activity of A-gliadin synthetic peptides 31-55, 31-43, and 44-55 has been investigated.Organ culture of jejunal mucosa from untreated and treated coeliac patients was used. In the first system enterocyte height was used as a measure of peptide toxicity; in the second system evidence of activated mucosal cell-mediated immune response was sought.Peptides 31-55 and 31-43 were active on untreated coeliac mucosa at a concentration of 0.5 mg/ml and peptide 44-55 only at a concentration of 3 mg/ml. In in vitro-cultured treated coeliac mucosa peptides 31-55 and 31-43 at 1 mg/ml and peptide 44-55 at 3 mg/ml were able to induce enhanced epithelial expression of HLA-DR and 4F2 molecules and the appearance of CD25 positive cells.Our results suggest that 31-43 and 44-55 A-gliadin peptides are both active, even if to different extents. In vitro systems remain essential tools to screen material to be subsequently tested in vivo.