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The lack of a randomized trial comparing carfilzomib (K) versus elotuzumab (Elo) associated with lenalidomide and dexamethasone (Rd) prompted us to assess the relative usefulness of one triplet over the other. Five independent retrospective cohorts of 883 relapsed/refractory multiple myeloma (RRMM) patients, including 300 EloRd and 583 KRd cases, outside clinical trials, entered this non-randomized comparison. KRd cohort accounted for a higher incidence of younger patients, cases with ≥3 lines of therapy, already exposed to lenalidomide, International Staging System (ISS) stage III, and abnormal lactic dehydrogenase (LDH) level compared with EloRd cohort. Moreover, cytogenetic risk categories, detected in roughly one-third of cases, were equally distributed between the two therapy arms. The probability of CR+VGPR response was significantly higher in KRd (n = 314, 53.9%) than in EloRd patients (n = 111, 37.0%). Likewise, the cumulative incidence function of CR+VGPR, taking into account the competitive risk of death, was significantly higher in KRd arm patients than those in the EloRd arm (p = .003). Moreover, KRd treatment significantly reduced the progression or death risk by 46% in an adjusted multivariate analysis (HR: 0.54, 95% CI 0.42-0.69, p .0001). Finally, in an adjusted illness-progression/death model, the effect of KRd versus EloRd was of higher magnitude among those who achieved CR+VGPR (-39% hazard ratio reduction, p = .02) than among those who achieved VGPR (-29% hazard ratio reduction, p = .007). With limitations characteristic to any retrospective analysis, this current clinical practice study's overall results demonstrated potential benefits of KRd therapy compared with EloRd. This observation may help the daily clinical practice.

Carfilzomib, lenalidomide, and dexamethasone (KRd) have been approved for the treatment of relapsed and refractory multiple myeloma (RRMM) based on ASPIRE clinical trial. However, its effectiveness and safety profile in real clinical practice should be further assessed. We retrospectively evaluated 130 consecutive RRMM patients treated with KRd between December 2015 and August 2018, in 9 Hematology Departments of Rete Ematologica Pugliese (REP). The overall response rate (ORR) was 79%, with 37% complete response (CR). Treatment with KRd led to an improvement in response regardless of age, refractory disease, and number and type of previous therapies. After a median follow-up of 18 months, median PFS was 24 months and 2y-PFS was 54%. PFS was longer in patients achieving a very good partial response (VGPR) with median PFS of 32.4 months. The relapses after prior autologous transplant (ASCT) positively impact median PFS. Several baseline disease characteristics, such as III ISS scoring or elevated LDH, and prior exposure to lenalidomide were found to negatively impact PFS. Primary refractory or relapsed myeloma patients have been treated with KRd as bridge to ASCT with a great benefit. Thirty-four (83%) reached at least a partial response after KRd and 21 (61%) performed ASCT. In transplanted patients, median PFS was not reached and 2y-PFS was 100%. The treatment discontinuation rate due to adverse events (AEs) was 18%, most commonly for lenalidomide (11%). Overall, in 10% of patients, a KRd dose reduction was necessary at least once (2.5% for carfilzomib and 8% for lenalidomide). The most frequent AE was neutropenia (44%) and anemia (41%). Infections occurred in 14% of patients. Cardiovascular events occurred in 11% of patients. Elderly patients have tolerated therapy very well, without additional side effects compared to younger patients, except for cardiac impairment. Our analysis confirmed that KRd is effective in RRMM patients. It is well tolerated and applicable to the majority of patients outside clinical trials. A longer PFS was shown in patients achieving VGPR, in those lenalidomide naive and in patients relapsing after previous ASCT. Previous ASCT should not hamper the option for KRd therapy. Accordingly, KRd should be used as bridge regimen to ASCT with remarkable improvement in response and PFS rates. Further clinical studies are needed.

Artificial metalloenzymes as catalysts are promising candidates for their use in different technologies, such as bioremediation, biomass transformation, or biosensing. Despite this, their practical exploitation is still at an early stage. Immobilized natural enzymes have been proposed to enhance their applicability. Immobilization may offer several advantages: (i) catalyst reuse; (ii) easy separation of the enzyme from the reaction medium; (iii) better tolerance to harsh temperature and pH conditions. Here, we report an easy immobilization procedure of an artificial peroxidase on different surfaces, by means of click chemistry. FeMC6*a, a recently developed peroxidase mimic, has been functionalized with a pegylated aza-dibenzocyclooctyne to afford a "clickable" biocatalyst, namely FeMC6*a-PEG4@DBCO, which easily reacts with azide-functionalized molecules and/or nanomaterials to afford functional bioconjugates. The clicked biocatalyst retains its structural and, to some extent, its functional behaviors, thus housing high potential for biotechnological applications.

The R-BAC regimen is considered among standard first-line treatment for elderly fit patients with mantle cell lymphoma (MCL). In the previous R-BAC500 FIL trial, patients with the blastoid variant and/or high Ki67 proliferative index (High Risk - HR-) had a significantly higher risk of progression (2-years PFS of 40%), as compared to classical histologies and low proliferative index (Low Risk -LR-). When treated with R-BAC, LR patients had excellent outcome (median PFS not reached after 7 years), although no maintenance therapy was delivered. For this reason we designed a phase 2 trial that enrolled patients from 35 centers of the Fondazione Italiana Linfomi (FIL). At study entry, patients were centrally reviewed and stratified as "low risk (LR)", or "high risk (HR)", depending on morphology (blastoid versus others), Ki67 expression (≥30% versus others), TP53 mutation/TP53 deletions (present versus not). Patients with any of the three risk factors were classified as HR. The primary endpoint was 2-years progression-free survival (PFS) for the HR patients. Patients had to be aged ≥65 years and fit according to the geriatric CGA assessment, or age ≤64 years if not eliglible to high-dose chemotherapy plus transplantation. Asymptomatic patients with non-nodal disease were excluded. Treatment consisted of 6 cycles of R-BAC (rituximab 375 mg/m2 d 1; bendamustine 70 mg/m2 d 1,2; cytarabine 500 mg/m2 d 1,2,3) for LR patients. HR patients received abbreviated induction with a maximum of 4 R-BAC followed by consolidation (4 months, 800 mg/d), and maintenance (20 months, 400 mg/d) with venetoclax. First patient was included on the 3rd of september, 2018, and last patient on the 20th of july, 2021. Overall, 140 patients were enrolled, of whom 52 were HR (37%). Median age was 72 (range 57-79), and 75% were males. The prevalence of TP53 mutations and deletions in the whole series was 21%, and 13%, respectively; Ki67 was ≥30% in 24%, and the blastoid variant was diagnosed in 9%. Demographic characteristics of HR versus LR patients (127 patients with available data at the present time) are reported in Table 1A. Median follow-up was 9 months (range 0-34). The two groups (HR and LR) had similar age, gender, and MIPI, but differed for LDH, and SUVmax at diagnosis, both being significantly more elevated in the HR group. The VR-BAC trial represents the first prospective study that stratified patients with MCL to different frontline treatments according to centralized on-time evaluation of the risk profile. We have shown that almost 40% of elderly patients with MCL in need of treatment have HR features. Data on tolerance, and tumor response will be presented at the meeting. Figure 1 Figure 1. Disclosures Tisi: GILEAD: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; BWS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Zilioli: Roche, Italfarmaco: Consultancy, Honoraria; MSD, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations; Takeda: Other: travel expenses, accommodation; Gentili, Takeda, Gilead, Servier: Consultancy, Speakers Bureau. Corradini: AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; BMS: Other: Travel and accommodation; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; Sanofi: Consultancy, Honoraria; Incyte: Consultancy; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations. Musuraca: janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Puccini: Takeda: Membership on an entity's Board of Directors or advisory committees. Cavallo: Servier: Speakers Bureau; ROCHE: Membership on an entity's Board of Directors or advisory committees; Gilead: Speakers Bureau. Merli: EUSA Pharma: Other: Travel, Accomodations, Expenses; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses; MSD: Membership on an entity's Board of Directors or advisory committees; Gilead Science: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accomodations, Expenses; Celgene: Other: Travel, Accomodations, Expenses. Ferreri: PletixaPharm: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Beigene: Research Funding; Hutchison Medipharma: Research Funding; ADC Therapeutics: Research Funding; Adienne: Membership on an entity's Board of Directors or advisory committees; Genmab: Research Funding; Amgen: Research Funding; x Incyte: Membership on an entity's Board of Directors or advisory committees; Ospedale San Raffaele srl: Patents & Royalties; Pfizer: Research Funding. Santoro: AstraZeneca: Speakers Bureau; AbbVie: Speakers Bureau; Amgen: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Eli-Lilly: Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Speakers Bureau; Sanofi: Consultancy; Arqule: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Zinzani: KYOWA KIRIN: Other, Speakers Bureau; SERVIER: Other: Advisory board, Speakers Bureau; SANDOZ: Other: Advisory board; TG Therapeutics: Other: Advisory board, Speakers Bureau; ROCHE: Other, Speakers Bureau; BMS: Other: Advisory board, Speakers Bureau; TAKEDA: Other: Advisory board, Speakers Bureau; MSD: Consultancy, Other: Advisory board, Speakers Bureau; NOVARTIS: Consultancy, Other, Speakers Bureau; EUSAPHARMA: Consultancy, Other, Speakers Bureau; Beigene: Other, Speakers Bureau; ADC Therap.: Other; Incyte: Other, Speakers Bureau; JANSSEN-CILAG: Other: Advisory board, Speakers Bureau; GILEAD: Other: Advisory board, Speakers Bureau; CELLTRION: Other: Advisory board, Speakers Bureau; VERASTEM: Consultancy, Other: Advisory board, Speakers Bureau. OffLabel Disclosure: Venetoclax is off-label in Italy in mantle cell lymphoma

Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

Retinitis pigmentosa (RP) is characterized by progressive loss of vision due to photoreceptor degeneration leading to secondary inflammation. The urokinase‐type plasminogen activator (uPA) system contributes to retinal inflammation, but its role in RP is unknown. In the rd10 mouse model of RP, we addressed this question with the use of the peptide UPARANT designed to interact with the uPA system. UPARANT was systemically administered from post‐natal day (PD) 10 to PD30 when its efficacy in RP rescue was investigated using electroretinographic recordings, Western blot and immunocytochemistry. Temporal profile of protein expression in the uPA system was also investigated. UPARANT reduced both Müller cell gliosis and up‐regulated levels of inflammatory markers and exerted major anti‐apoptotic effects without influencing the autophagy cascade. Rescue from retinal cell degeneration was accompanied by improved retinal function. No scotopic phototransduction was rescued in the UPARANT‐treated animals as determined by the kinetic analysis of rod‐mediated a‐waves and confirmed by rod photoreceptor markers. In contrast, the cone photopic b‐wave was recovered and its rescue was confirmed in the whole mounts using cone arrestin antibody. Investigation of the uPA system regulation over RP progression revealed extremely low levels of uPA and its receptor uPAR both of which were recovered by HIF‐1α stabilization indicating that HIF‐1 regulates the expression of the uPA/uPAR gene in the retina. Ameliorative effects of UPARANT were likely to occur through an inhibitory action on up‐regulated activity of the αvβ3 integrin/Rac1 pathway that was suggested as a novel target for the development of therapeutic approaches against RP.

Puncture-induced iris neovascularization (rubeosis iridis; RI) in mice is associated with upregulation of extracellular matrix (ECM) degradation and inflammatory factors. The anti-angiogenic and anti-inflammatory efficacy of UPARANT in reducing RI was determined by noninvasive, in vivo iris vascular densitometry, and confirmed in vitro by quantitative vascular-specific immunostaining. Intravitreal administration of UPARANT successfully and rapidly reduced RI to non-induced control levels. Molecular analysis revealed that UPARANT inhibits formyl peptide receptors through a predominantly anti-inflammatory response, accompanied with a significant reduction in ECM degradation and inflammation markers. Similar results were observed with UPARANT administered systemically by subcutaneous injection. These data suggest that the tetrapeptide UPARANT is an effective anti-angiogenic agent for the treatment of RI, both by local and systemic administrations. The effectiveness of UPARANT in reducing RI in a model independent of the canonical vascular endothelial growth factor (VEGF) proposes an alternative for patients that do not respond to anti-VEGF treatments, which could improve treatment in proliferative ocular diseases. Key messages UPARANT is effective in the treatment of rubeosis iridis, both by local and systemic administrations.UPARANT can reduce VEGF-independent neovascularization. Electronic supplementary material The online version of this article (10.1007/s00109-019-01794-w) contains supplementary material, which is available to authorized users.

Manganese-porphyrins are important tools in catalysis, due to their capability to promote a wide variety of synthetically valuable transformations. Despite their great reactivity, the difficulties to control the reaction selectivity and to protect the catalyst from self-degradation hamper their practical application. Compared to small-molecule porphyrin complexes, metalloenzymes display remarkable features, because the reactivity of the metal center is finely modulated by a complex interplay of interactions within the protein matrix. In the effort to combine the catalytic potential of manganese porphyrins with the unique properties of biological catalysts, artificial metalloenzymes have been reported, mainly by incorporation of manganese-porphyrins into native protein scaffolds. Here we describe the spectroscopic and catalytic properties of Mn-Mimochrome VI*a (Mn-MC6*a), a mini-protein with a manganese deuteroporphyrin active site within a scaffold of two synthetic peptides covalently bound to the porphyrin. Mn-MC6*a is an efficient catalyst endowed with peroxygenase activity. The UV-vis absorption spectrum of Mn-MC6*a resembles that of Mn-reconstituted horseradish peroxidase (Mn-HRP), both in the resting and high-valent oxidized states. Remarkably, Mn-MC6*a shows a higher reactivity compared to Mn-HRP, because higher yields and chemoselectivity were observed in thioether oxidation. Experimental evidences also provided indications on the nature of the high-valent reactive intermediate and on the sulfoxidation mechanism.

Purpose The clinical impact of positron emission tomography (PET) evaluation performed early during first-line therapy in patients with advanced-stage Hodgkin lymphoma, in terms of providing a rationale to shift patients who respond poorly onto a more intensive regimen (PET response-adapted therapy), remains to be confirmed. Patients and Methods The phase II part of the multicenter HD0801 study involved 519 patients with advanced-stage de novo Hodgkin lymphoma who received an initial treatment with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and who underwent an early ifosfamide-containing salvage treatment followed by stem-cell transplantation if they showed a positive PET evaluation after two cycles of chemotherapy (PET2). The primary end point was 2-year progression-free survival calculated for both PET2-negative patients (who completed a full six cycles of ABVD treatment) and PET2-positive patients. Overall survival was a secondary end point. Results In all, 103 of the 512 evaluable patients were PET2 positive. Among them, 81 received the scheduled salvage regimen with transplantation, 15 remained on ABVD (physician’s decision, mostly because of minimally positive PET2), five received an alternative treatment, and two were excluded because of diagnostic error. On intention-to-treat analysis, the 2-year progression-free survival was 76% for PET2-positive patients (regardless of the salvage treatment they received) and 81% for PET2-negative patients. Conclusion Patients with advanced-stage Hodgkin lymphoma for whom treatment was at high risk of failing appear to benefit from early treatment intensification with autologous transplantation, as indicated by the possibility of successful salvage treatment in more than 70% of PET2-positive patients through obtaining the same 2-year progression-free survival as the PET2-negative subgroup.

Advanced oxidation processes represent a viable alternative in water reclamation for potable reuse. Sensing methods of hydrogen peroxide are, therefore, needed to test both process progress and final quality of the produced water. Several bio-based assays have been developed so far, mainly relying on peroxidase enzymes, which have the advantage of being fast, efficient, reusable, and environmentally safe. However, their production/purification and, most of all, batch-to-batch consistency may inherently prevent their standardization. Here, we provide evidence that a synthetic de novo miniaturized designed heme-enzyme, namely Mimochrome VI*a, can be proficiently used in hydrogen peroxide assays. Furthermore, a fast and automated assay has been developed by using a lab-bench microplate reader. Under the best working conditions, the assay showed a linear response in the 10.0–120 μM range, together with a second linearity range between 120 and 500 μM for higher hydrogen peroxide concentrations. The detection limit was 4.6 μM and quantitation limits for the two datasets were 15.5 and 186 μM, respectively. In perspective, Mimochrome VI*a could be used as an active biological sensing unit in different sensor configurations.

The folding of a synthetic mini-hydrogenase is shown to enhance catalyst efficiency and longevity., A synthetic enzyme is reported that electrocatalytically reduces protons to hydrogen (H2) in water near neutral pH under aerobic conditions. Cobalt mimochrome VI*a (CoMC6*a) is a mini-protein with a cobalt deuteroporphyrin active site within a scaffold of two synthetic peptides covalently bound to the porphyrin. Comparison of the activity of CoMC6*a to that of cobalt microperoxidase-11 (CoMP11-Ac), a cobalt porphyrin catalyst with a single “proximal” peptide and no organized secondary structure, reveals that CoMC6*a has significantly enhanced longevity, yielding a turnover number exceeding 230 000, in comparison to 25 000 for CoMP11-Ac. Furthermore, comparison of cyclic voltammograms of CoMC6*a and CoMP11-Ac indicates that the trifluoroethanol-induced folding of CoMC6*a lowers the overpotential for catalytic H2 evolution by up to 100 mV. These results demonstrate that even a minimal polypeptide matrix can enhance longevity and efficiency of a H2-evolution catalyst.

Rational design provides an attractive strategy to tune and control the reactivity of bioinspired catalysts. While there has been considerable progress in the design of heme oxidase mimetics with active site environments of ever-growing complexity and catalytic efficiency, their stability during turnover is still an open challenge. Here we show that the simple incorporation of two 2-amino isobutyric acids into an artificial peptide-based peroxidase resulted in a new catalyst (FeIII-MC6*a), with higher resistance against oxidative damage, and higher catalytic efficiency. A two-fold enhancement of the turnover number, respect to its predecessor, was observed. These results point out the protective role exerted by the peptide matrix and pave the way to the synthesis of robust bioinspired catalysts.

Synthetic proteins represent useful tools for reproducing metalloprotein functions in minimal, well-defined scaffolds. Herein, we describe the rational refinement of function into heme-protein models from the Mimochrome family. Originally designed to mimic the bis-His cytochrome b, the Mimochrome structure was modified to introduce a peroxidase-like activity, by creating a distal cavity on the heme. The success with the first asymmetric system, Mimochrome VI (MC6), gave the opportunity to explore further modifications in order to improve the catalytic activity. Starting from ferric MC6, single amino acid substitutions were introduced in the peptide chains to obtain four compounds, which were screened for peroxidase activity. The detailed structural and functional analysis of the best analogue, Fe(III)-E(2)L(TD)-MC6, indicates that an arginine residue in proximity to the heme-distal site could assist with catalysis by favoring the formation of the intermediate "compound I", thus mimicking R(38) in HRP. This result highlights the potential of using small scaffolds for exploring the main factors that tune the heme-protein activity, and for programming new desired functions.

We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.

Purpose The activation of the urokinase-type plasminogen activator and its receptor system is associated with retinal diseases. Among peptide inhibitors of this system, UPARANT acts by preventing the onset of pathologic signs of neovascular ocular diseases. We investigated whether systemic UPARANT may act in a therapeutic regimen by suppressing the retinal damage that characterizes diabetic retinopathy using a rat model of streptozotocin-induced diabetes. Methods In healthy rats, plasma, eye, and retina concentrations of UPARANT were evaluated by mass spectrometry. In rat models of streptozotocin-induced diabetes, the appearance of diabetic retinopathy was assessed by electroretinogram (ERG). UPARANT was then administered at different dosages and daily regimens. ERG recording, Evans blue perfusion, and real-time PCR were used to evaluate UPARANT efficacy. UPARANT safety was also determined. Results UPARANT was found in plasma, eye, and retina soon after its administration and remained detectable after 24 hours. Between the 4th and the 5th week after diabetes onset, UPARANT at 8 mg/kg (daily for 5 days) was effective in recovering dysfunctional ERG. Three-day treatments at 8 mg/kg or a half dose for 5 days were ineffective. ERG recovery lasted approximately 2 weeks. ERG recovery was accompanied by restored blood-retinal barrier integrity and inhibition of inflammatory and angiogenic responses. UPARANT showed a safety profile. Conclusions These data suggest that targeting the urokinase-type plasminogen activator and its receptor system by systemic UPARANT is a potential therapeutic approach for the treatment of early diabetic retinopathy, thus providing a potential alternative approach to delay disease progression in humans.

The spontaneously diabetic Torii (SDT) rat is of increasing preclinical interest because of its similarities to human type 2 diabetic retinopathy (DR). The system formed by urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is a player in blood-retinal barrier (BRB) breakdown in DR. Here, we investigated whether in SDT rats, preventive administration of UPARANT, an inhibitor of the uPAR pathway, counteracts the retinal impairment in response to chronic hyperglycemia. Electroretinogram (ERG) monitoring was followed over time. Fluorescein-dextran microscopy, CD31 immunohistochemistry, quantitative PCR, ELISA, Evans blue perfusion, and Western blot were also used. UPARANT prevented ERG dysfunction, upregulation of vascular endothelial growth factor and fibroblast growth factor-2, BRB leakage, gliosis, and retinal cell death. The mechanisms underlying UPARANT benefits were studied comparing them with the acute streptozotocin (STZ) model in which UPARANT is known to inhibit DR signs. In SDT rats, but not in the STZ model, UPARANT downregulated the expression of uPAR and its membrane partners. In both models, UPARANT reduced the levels of transcription factors coupled to inflammation or inflammatory factors themselves. These findings may help to establish the uPAR system as putative target for the development of novel drugs that may prevent type 2 DR.

Metalloproteins utilize O-2 as an oxidant, and they often achieve a 4-electron reduction without H2O2 or oxygen radical release. Several proteins have been designed to catalyze one or two-electron oxidative chemistry, but the de novo design of a protein that catalyzes the net 4-electron reduction of O-2 has not been reported yet. We report the construction of a diiron-binding four-helix bundle, made up of two different covalently linked (2) monomers, through click chemistry. Surprisingly, the prototype protein, DF-C1, showed a large divergence in its reactivity from earlier DFs (DF: due ferri, two iron). DFs release the quinone imine and free H2O2 in the oxidation of 4-aminophenol in the presence of O-2, whereas Fe-III-DF-C1 sequesters the quinone imine into the active site, and catalyzes inside the scaffold an oxidative coupling between oxidized and reduced 4-aminophenol. The asymmetry of the scaffold allowed a fine-engineering of the substrate binding pocket, that ensures selectivity.

Bioconjugation is a rapidly expanding field because of the numerous potential applications of bioconjugate materials. We explored the usefulness of branched porphyrins as rigid scaffolds, bearing multiple sites for bioconjugation. To this end, we first selected the tetrakis(p-(aminomethyl) phenyl) porphyrin (TAMPP) macrocycle and developed a straightforward synthetic protocol, able to provide the desired tetraphenylporphyrin, carrying four functional amino groups. The partially protection of the amino groups by tert-butoxy-carbonyl allowed the selective and specific decoration of the porphyrin with different peptide sequences. To explore the utility of the macrocycle as molecular scaffold for bioconjugation, we selected peptide sequences able to function as thrombin inhibitors. In particular, two peptide sequences, named CS3 and ES7, able to interact, respectively, with the thrombin catalytic site and the fibrinogen recognition exosite, were joined onto the porphyrin macrocycle, providing the multisite-directed inhibitor CS3-TAMPP-ES7. This multisite inhibitor and its Mn III complex are able to inhibit α-thrombin-catalyzed hydrolysis of Tos-Gly-Pro-Arg-nitroanilide with inhibition constants in the micromolar range, as well as the hydrolysis of the natural substrate fibrinogen. The inhibitor is resistant against enzymatic degradation by thrombin and is highly selective. The Mn III complex is capable of interacting with clot-bound thrombin and partially inhibits clot growth in the presence of fibrinogen. The results herein reported are very promising, suggesting the potential of the newly developed conjugate as new imaging agents for clot detection. C

Glycosylation is an important PTM in proteins, and it is of paramount importance for the manufacture and efficacy of therapeutic glycoproteins. The elucidation of the glycosylation patterns is greatly hampered by the structural heterogeneity, which involves the oligosaccharide moieties, and sometimes also the protein chain. Several strategies are used to map the glycosylation state of glycoproteins: they generally involve analysis of the glycopeptides obtained upon glycoprotein proteolytic digestion, and/or analysis of the glycans, enzymatic or chemically released from the glycoproteins. These approaches are efficient in determining the total glycan content, as well as the glycan structures; nevertheless, information regarding the whole protein is lost. Recent advances in MS allowed us to directly analyze human follicle stimulating hormone (hFSH). RP-HPLC/IT-TOF MS, coupled to bioinformatic algorithms, was used to determine the glycan content of intact hFSH preparations, either urinary-derived or recombinant. More traditional and complementary analytical methods (oligosaccharide profiling, IEF, and peptide mapping analyses) were employed to compare the obtained results. Our analysis shows a predominance of highly sialylated, highly branched glycans in a urinary hFSH preparation as compared to recombinant hFSH expressed in rodent cell lines. Noteworthy, high-resolution mass spectra may allow to evaluate the glycosylation profile during the manufacturing process of different preparations.

Background: Ibrutinib is a first-in-class oral inhibitor of Bruton Tyrosine Kinase (BTK) approved for the treatment patients with CLL who have received at least one prior therapy (R/R) or Treatment Naïve (TN) with del17p. Ibrutinib has been showed to improve progression free survival (PFS) and overall survival (OS) even in presence of del17p. Moreover there is a good tolerability profile such as to not lead to frequent and permanent interruption of therapy.Methods: In 131 CLL/SLL patients (including previously treated and untreated with del17) from eight different hematologic centers of REP (Rete Ematologica Pugliese) Ibrutinib 420 mg once daily was administrated until disease progression or unacceptable toxicity. PFS, OS and Overall response rate (ORR) were calculated. Kaplan Meier method was used to estimate PFS/OS and differences in survival times between groups were assessed with a log-rank test.Results: Among 131 pts, 9 (6.9%) received Ibrutinib as first line therapy (TN) and 122 (R/R) received prior therapies before Ibrutinib (39.7% BR, 18.3% FCR and other treatments); 90% (1-2) lines of therapy, 8% (3-4) lines and 2% more than 5. Median age of the 131 pts with CLL/SLL was 65 y (range, 28-84) with 50% ≥65 y.ECOG Performance Status was observed in 108 pts (0, 56%; 1, 40%; 2, 4%). FISH analysis was performed in 85/131 pts (65%) and some patients had more than one deletion. Among these, 42% had del17p (TN 100% and R/R 36%) and for the remaining R/R was 16% del13q, 4% del11q, 7% trisomy12 and in the others no abnormalities were found. Unmutated and mutated IGVH was performed in 78/131 patients (60%), 63% and 37% respectively. The ORR was 80% (complete response [CR], 10%) for all-treated pts (TN: 86%, R/R: 77%) and for R/R pts with del17p the ORR was 61%.With a median time on study of 17 months (range, 1-45), median PFS was not reached for all TN or R/R patients and the estimated PFS at 30 months was 66,7% for R/R patients. We exclude from our further analysis 9 TN patients because of the small sample size and the lack of events in a median follow up of 15 months (range, 3-24). In 122 R/R pts, median PFS was 39 months for pts with del17p and median PFS was not reached for pts without abnormality (overall logrank p=0.543). At 30 m the estimated PFS was 65.1% for del17p pts and 80.4% for no abnormality pts . Median OS was not reached for 122 R/R patients and the estimated PFS at 30 months was 72.7%. Median OS was not reached in pts with and without del17p (overall logrank p=0.807) with an estimated OS at 30 months of 81.7% and 90.2% respectively.In 47% of all treated patients adverse events (AEs) with grade ≥3 were neutropenia (16%), hypertension (12%), atrial fibrillation (3%), anemia (3%), diarrhea (2%). With a median follow up of 17 months 80% of pts remain on Ibrutinib treatment.Conclusions: In our real life experience with data from REP, Ibrutinib single agent demonstred to be effective. In R/R patients, with a median time on study of 17 months, the estimated PFS at 30 m was 66.7%. The differences in PFS between patients with and without del17p was not statistically significant (p=0.543) but at 30 months the estimated PFS was lower in del17p pts than in no abnormality pts (65.1% vs 80.4%).The treatment was well tolerated with AEs of grade ≥3 in a low percentage (36%) that did not result in treatment interruptions in the most of cases. Clearly, these results will require longer follow up time to be further confirmed. Disclosures No relevant conflicts of interest to declare.

Many efforts are continuously devoted to the construction of hybrid biomaterials for specific applications, by immobilizing enzymes on different types of surfaces and/or nanomaterials. In addition, advances in computational, molecular and structural biology have led to a variety of strategies for designing and engineering artificial enzymes with defined catalytic properties. Here, we report the conjugation of an artificial heme enzyme (MIMO) with lipoic acid (LA) as a building block for the development of gold-based biomaterials. We show that the artificial MIMO@LA can be successfully conjugated to gold nanoparticles or immobilized onto gold electrode surfaces, displaying quasi-reversible redox properties and peroxidase activity. The results of this work open interesting perspectives toward the development of new totally-synthetic catalytic biomaterials for application in biotechnology and biomedicine, expanding the range of the biomolecular component aside from traditional native enzymes.

The crystal and molecular structure of the fully protected dipeptide Boc-Val-(S)-alpha-MeSer-OMe has been determined by X-ray diffraction techniques. Crystals grown from ethyl acetate/n-pentane mixtures are tetragonal, space group I4(1), with cell parameters at 295 K of a = 15.307(2), c = 18.937(10)angstrom, V = 4437.1 angstrom3, M.W. = 332.40, Z = 8, D(m) = 0.99 g/cm3 and D(x) = 0. 995 g/cm3. The structure was solved by application of direct methods and refined to an R value of 0.028 for 1773 reflections with I greater-than-or-equal 3sigma(I) collected on a CAD-4 diffractometer. Both chiral centers have the (S) configuration. The dipeptide assumes in the solid state an S shape. The urethane moiety is in the cis conformation, while the amide bond is in the common trans conformation. The conformational angles phi1, psi1 of the Val and phi2, and psi2 of the (S)-alphaMeSer fall in the F region of the phi-psi map. The isopropyl side chain of the Val residue has the (t, g-) conformation, while the Ser side chain has a g+ conformation. The hydrogen bond donor groups are all involved in intermolecular H-bond interactions. Along the quaternary axis the dipeptide molecules are linked to each other with the formation of infinite rows.

The synthesis, physical and analytical characterization, and crystal‐state structural analysis by X‐ray diffraction of three analogues of the Nα‐acylated tripeptide amide tail of oxytocin, each containing a cyclic Cα, α‐ disubstituted glycine at position 2, have been performed. The peptides arc Boc‐L‐Pro‐Ac3c‐Gly‐NH2, Z‐L‐Pro‐Ac5c‐Gly‐NH2 and Z‐L‐Pro‐Ac5c‐Gly‐NH2. While the former is folded in a type‐II β‐turn conformation at the ‐L‐Pro‐Ac3c‐ sequence, the two latter tripeptides form two consecutive (type‐II, type‐I′) β‐turns. The Ac5c‐ and Ac6c‐tripeptides are the first examples of such a highly folded structural combination in a position‐2 analogue of the Nα‐acylated ‐L‐Pro‐L‐Leu‐GIy‐NH2 sequence. Copyright © 1993, Wiley Blackwell. All rights reserved

An empirical equation, describing the relationship between the porphyrin methyl hyperfine shifts and the position of the axial ligand(s), has been applied to an artificial heme-protein in order to obtain insight into the active site properties of heme-protein models.

We have investigated the conformational preferences of a newly synthesized C(alpha,alpha) symmetrically disubstituted glycine, namely alpha,alpha-dicyclopropylglycine (Dcp). We report here the crystal structure of a fully protected dipeptide containing Dcp, namely Z-Dcp(1)-Dcp(2)-OCH(3). Both Dcp residues are in a folded conformation. The overall peptide structural organization corresponds to an alpha-pleated sheet conformation, similar to that observed in linear peptides made up of alternating D- and L-residues and in Z-Aib-Aib-OCH(3) (Aib: alpha,alpha-dimethylglycine). These preliminary data suggest that the Dcp could represent an alternative as molecular tool to stabilize folded conformations.

Erythropoietin (Epo) is the crucial cytokine regulator of red blood cell production, and recombinant human erythropoietin (rHuEpo) is widely used in clinical practice for the treatment of anemia, primarily in kidney disease and in cancer. Increasing evidence suggests several biological roles for Epo and its receptor, Epo-R, unrelated to erythropoiesis, including angiogenesis. Epo-R has been found expressed in various non-haematopoietic cells and tissues, and in cancer cells. Here, we detected the expression of Epo-R in bone marrow-derived macrophages (BMMAs) from multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) patients and assessed whether Epo/Epo-R axis plays a role in MM macrophage-mediated angiogenesis. We found that Epo-R is over-expressed in BMMAs from MM patients with active disease compared to MGUS patients. The treatment of BMMAs with rHuEpo significantly increased the expression and secretion of key pro-angiogenic mediators, such as vascular endothelial growth factor, hepatocyte growth factor and monocyte chemotactic protein (MCP-1/CCL-2), through activation of JAK2/STAT5 and PI3 K/Akt pathways. In addition, the conditioned media harvested from rHuEpo-treated BMMAs enhanced bone marrow-derived endothelial cell migration and capillary morphogenesis in vitro, and induced angiogenesis in the chorioallantoic membrane of chick embryos in vivo. Furthermore, we found an increase in the circulating levels of several pro-angiogenic cytokines in serum of MM patients with anemia under treatment with Epo. Our findings highlight the direct effect of rHuEpo on macrophage-mediated production of pro-angiogenic factors, suggesting that Epo/Epo-R pathway may be involved in the regulation of angiogenic response occurring in MM.

We recently reported the rational design, synthetics, and structural characterization of the most potent and selective peptide-based neurokinin A antagonist thus far described: cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)cyclo(2 beta-5 beta). Its bicyclic structure is characterized by a type I and a type II two beta-turn around Trp3-Phe4 and Leu6-Met1, respectively. In order to understand whether the two different beta-turned structures are determined by the bicyclic structure or by the amino acid type at the corner positions, we have synthesized the pseudo-symmetrical analogue cyclo(Phe1-Asp2-Trp3-Phe4-Dap5-Trp6)cyclo(2 beta-5 beta). The structural characterization in the crystal state and in solution, here reported, gives an experimental evidence that the backbone of the bicyclic structure is a rigid scaffold that can be used to build both a type I and type II beta-turn independently from the amino acid composition.

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L-Pro-L-Phe-β-Ala)2. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P21 from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L-Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d6 solutions as a mixture of conformational families. One of these corresponds to a C2 symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ϕ,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.

In the present paper we describe the synthesis, purification, single-crystal x-ray analysis, solution conformational characterization, and conformational energy calculations of the cyclic tetrapeptide cyclo- (β-Ala-L-Pro-β-Ala-L-Val). The 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. The compound crystallizes in the monoclinic space group P21 from ethanol with two independent molecules in the unit cell. All peptide bonds are trans. The nmr molecular conformation in the acetonitrile solution as well as that derived from the molecular dynamic simulation in vacuo is quite different from those observed in the solid state and is very similar to that previously observed for the parent compound cyclo-(β-Ala-L-Pro-β-Ala-L-Pro). © 1993 John Wiley & Sons, Inc.

The complex [Cl(Ph3P)Pt(H-Aib-O)], where Ph3P and H-Aib-O are triphenylphosphine and α-aminoisobutyrate, respectively, has been synthesized and characterized in the solid state by X-ray diffraction analysis. It crystallizes as a monohydrate in the triclinic system, space group P with a=9.020, b=10.011, c=13.748 Å, α=91.74, β=90.29, γ=111.26° and Z=2 molecules per unit cell. The crystal structure has been solved by the Patterson technique and refined to a final R factor of 0.038 for the 3826 observed reflections with I≥3.0σ(I). The Pt(II) atom displays the square planar coordination with the H-Aib-O− amino acid acting as a bidentate ligand. The N atom of the H-Aib-O− residue in trans position with respect to the P atom of the triphenylphosphine moiety and correspondingly the O atom of the carboxylate is trans to the chlorine atom. The observed distortions in the bond angles around the Pt(II) atom as well as the conformation assumed by the triphenylphosphine and the H-Aib-O− residue can be all explained on the basis of the need to release the intramolecular non-bonded interactions between atoms of the various ligands. Three intermolecular hydrogen bonds, involving as donors the cocrystallized water molecule and the two hydrogen atoms of the NH groups and as acceptors the chlorine, the oxygen of the water molecule, and one of the oxygen atoms of the carboxylic acid group, together with van der Waals interactions between hydrophobic groups held the molecules in the crystal state.

Self-aggregation of water-soluble pyridinium-substituted porphyrins with functionalized calixarenes leads to well-defined pH-tunable stoichiometries. Two assemblies with different porphyrin:calixarene ratios that crystallized at pH 2 and 6 have been structurally characterized. The Job plots of Soret band broadenings indicate that these structures are also preserved in solution.

Mimicking enzymes with alternative molecules represents an important objective in synthetic biology, aimed to obtain new chemical entities for specific applications. This objective is hampered by the large size and complexity of enzymes. The manipulation of their structures often leads to a reduction of enzyme activity. Herein, we describe the spectroscopic and functional characterization of Fe(III)-mimochrome VI, a 3.5 kDa synthetic heme-protein model, which displays a peroxidase-like catalytic activity. By the use of hydrogen peroxide, Fe(III)-mimochrome VI efficiently catalyzes the oxidation of several substrates, with a typical Michaelis-Menten mechanism and with several multiple turnovers. The catalytic efficiency of Fe(III)-mimochrome VI in the oxidation of 2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) and guaiacol (k(cat)/K(m)=4417 and 870 mM(-1) s(-1), respectively) is comparable to that of native horseradish peroxidase (HRP, k(cat)/K(m)=5125 and 500 mM(-1) s(-1), respectively). Fe(III)-mimochrome VI also converts phenol to 4- and 2-nitrophenol in the presence of NO(2) (-) and H(2) O(2) in high yields. These results demonstrate that small synthetic peptides can impart high enzyme activities to metal cofactors, and anticipate the possibility of constructing new biocatalysts tailored to specific functions.

Mimochrome VI (MC-VI) is a synthetic heme peptide containing a helix-heme-helix sandwich motif designed to reproduce the catalytic activity of heme oxidases. The thermodynamics of Fe(III) to Fe(II) reduction and the kinetics of the electron-transfer process for MC-VI immobilized through hydrophobic interactions on a gold electrode coated with a nonpolar SAM of decane-1-thiol have been determined through cyclic voltammetry. Immobilization slightly affects the reduction potential of MC-VI, which under these conditions electrocatalytically turns over molecular oxygen. This work sets the premise for the exploitation of totally synthetic mimochrome-modified electrode surfaces for clinical and pharmaceutical biosensing.

The 2,2,8-triisopropyl-4,5,7,8-tetrahydroimidazo[1,2-a]pyrazin-3,6-dione molecule, C15H25N3O2, has a double bond and two partial double bonds in the bicyclic skeleton, with some π-delocalization along C'1—N3—C'2. The conformation parameters of the diisopropyl (Dip) residue reveals that it is in an unusually high energy conformation. The peptide bond between the glycine and valine residues is cis [Cα3—C'3—N1—Cα1 = −7.0 (3)°]. In the crystal, the molecules are held together in the ac plane of the P2/n space group by intermolecular hydrogen bonds formed around a twofold axis by molecules related by symmetry centres.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.