In the dog, the endotheliochorial placenta allows only the 5% to 10% transfer of maternal antibodies to the fetus, but the timing and the factors influencing the immunoglobulin G (IgG) transplacental transport were not fully investigated. The aims of the present study were the following: (1) to assess the presence of both IgG and lysozyme in amniotic and allantoic fluids collected from fully developed and viable newborn puppies born by elective cesarean section at term and possible correlations between amniotic and allantoic IgG and lysozyme levels; (2) to verify possible differences in IgG and lysozyme concentrations between the two fluids; and (3) to detect possible differences in IgG and lysozyme fetal fluid levels in relation to the maternal breed body size and parity, as well as to the neonatal gender. The study, performed on 41 purebred bitches submitted to elective cesarean section at term, enrolled 142 puppies, 74 males and 68 females, born mature, viable, without gross malformations, and with a normal weight. At surgery, a total of 129 amniotic and 84 allantoic samples were collected for IgG and lysozyme analysis. Class G immunoglobulins and lysozyme were detected in both fluids, but IgG concentrations were higher (P < 0.01) in amniotic fluid. Moreover, a significant positive correlation (P < 0.01) between IgG amniotic and allantoic levels, but not for lysozyme, was observed. A significant effect of the maternal parity (P < 0.05), but not of the breed body size, on the amniotic IgG concentrations was found, whereas the newborn gender was not associated to different IgG or lysozyme amniotic or allantoic levels. Given the significant contributions of fetal fluids to fetal and neonatal health, the results reported that the amniotic and allantoic fluids play a role in the immune protection of the fetus/newborn also in canine species. However, additional research is needed to better elucidate both the origin of IgG and lysozyme and the factors influencing the wide interindividual variations.

Defective apoptosis is a hallmark of the progression of B chronic lymphocytic leukaemia (B-CLL). Smac-mimetics have been shown to induce apoptosis in several tumours. We describe the in vitro pro-apoptotic activity and regulation of the molecular pathway induced by new Smac-mimetics in B-CLL. The cytotoxic effect was significantly higher in B-CLL samples than in healthy controls. No significant synergistic effect was observed in combined treatment. In conclusion one of our compounds (Smac66), used as monotherapy and not in combination, is highly active against B-CLL cells thus suggesting a promising therapeutic potential as a new class of antileukemic drugs in haematology.

Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.

OBJECTIVE:Microvesicles (MVs) have been indicated as important mediators of intercellular communication and are emerging as new biomarkers of tissue damage. Our previous data indicate that reactive microglia/macrophages release MVs in vitro. The aim of the study was to evaluate whether MVs are released by microglia/macrophages in vivo and whether their number varies in brain inflammatory conditions, such as multiple sclerosis (MS).METHODS:Electron and fluorescence microscopy and flow cytometry were used to detect myeloid MVs in the cerebrospinal fluid (CSF) of healthy controls, MS patients, and rodents affected by experimental autoimmune encephalomyelitis (EAE), the animal model of MS.RESULTS:Myeloid MVs were detected in CSF of healthy controls. In relapsing and remitting EAE mice, the concentration of myeloid MVs in the CSF was significantly increased and closely associated with disease course. Analysis of MVs in the CSF of 28 relapsing patients and 28 patients with clinical isolated syndrome from 2 independent cohorts revealed higher levels of myeloid MVs than in 13 age-matched controls, indicating a clinical value of MVs as a companion tool to capture disease activity. Myeloid MVs were found to spread inflammatory signals both in vitro and in vivo at the site of administration; mice impaired in MV shedding were protected from EAE, suggesting a pathogenic role for MVs in the disease. Finally, FTY720, the first approved oral MS drug, significantly reduced the amount of MVs in the CSF of EAE-treated mice.INTERPRETATION:These findings identify myeloid MVs as a marker and therapeutic target of brain inflammation.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head–head (8), tail–tail (9) or head–tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2–BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail–tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has a large number of biological effects, including skin, cardiovascular, neurologic diseases, diabetes, infertility, cancers and immunotoxicity. We analysed the in vitro TCDD effects on human CD34+ cells and tested the gene expression modulation by means of microarray analyses before and after TCDD exposure. We identified 257 differentially modulated probe sets, identifying 221 well characterized genes. A large part of these resulted associated to cell adhesion and/or angiogenesis and to transcription regulation. Synaptic transmission and visual perception functions, with the particular involvement of the GABAergic pathway were also significantly modulated. Numerous transcripts involved in cell cycle or cell proliferation, immune response, signal transduction, ion channel activity or calcium ion binding, tissue development and differentiation, female or male fertility or in several metabolic pathways were also affected after dioxin exposure. The transcriptional profile induced by TCDD treatment on human CD34+ cells strikingly reproduces the clinical and biological effects observed in individuals exposed to dioxin and in biological experimental systems. Our data support a role of dioxin in the neoplastic transformation of hemopoietic stem cells and in immune modulation processes after in vivo exposure, as indicated by the epidemiologic data in dioxin accidentally exposed populations, providing a molecular basis for it. In addition, TCDD alters genes associated to glucidic and lipidic metabolisms, to GABAergic transmission or involved in male and female fertility, thus providing a possible explanation of the diabetogenic, dyslipidemic, neurologic and fertility effects induced by TCDD in vivo exposure.

A case is reported of fatal cardiomyopathy in an 8-year-old female German Shepherd after standard chemotherapy with doxorubicin for splenic hemangiosarcoma. The main gross lesion was a moderate bilateral cardiac ventricular dilation with diffusely pale myocardium. Histological analysis revealed severe multifocal vacuolar degeneration of cardiomyocytes, myocytolysis, myofibril loss, myocardial fibrosis, and edema. Myocardial fiber vacuolization and myocytolysis were highly suggestive of doxorubicin cardiotoxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, nowadays considered as suitable candidate for autologous stem therapy with bone marrow (BM). A careful characterization of BM stem cell (SC) compartment is mandatory before its extensive application to clinic. Indeed, widespread systemic involvement has been recently advocated given that non-neuronal neighboring cells actively influence the pathological neuronal loss. We therefore investigated BM samples from 21 ALS patients and reported normal hematopoietic biological properties while an atypical behavior and impaired SC capabilities affected only the mesenchymal compartment. Moreover, by quantitative real-time approach, we observed altered Collagen IV and Metalloproteinase-9 levels in patients’ derived mesenchymal stem cells (MSCs). Widespread metalloproteinase (MMPs) and their tissue inhibitor (TIMPs) alterations were established by multiplex ELISA analysis, demonstrating diffuse enzymatic variations in MSC compartment. Since MMPs act as fundamental effectors of extra-cellular matrix remodeling and stem cell mobilization, their modifications in ALS may influence reparative mechanisms effective in counteracting the pathology. In conclusion, ALS is further confirmed to be a systemic disease, not restricted to the nervous system, but affecting also the BM stromal compartment, even in sporadic cases. Therefore, therapeutic implantation of autologous BM derived SC in ALS patients needs to be carefully reevaluated.

Veterinary RecordVolume 166, Issue 3 p. 83-85 Short Communication Colostrum and serum lysozyme levels in Mediterranean buffaloes (Bubalus bubalis) and in their newborn calves D. Piantedosi DVM, PhD, Corresponding Author D. Piantedosi DVM, PhD [email protected] Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalyE-mail for correspondence: [email protected]Search for more papers by this authorF. Servida DVM, PhD, F. Servida DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorL. Cortese DVM, PhD, L. Cortese DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorM. Puricelli DVM, PhD, M. Puricelli DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorV. Benedetti DVM, V. Benedetti DVM Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorA. Di Loria DVM, PhD, A. Di Loria DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorV. Foglia Manzillo DVM, PhD, V. Foglia Manzillo DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorP. Dall'Ara DVM, PhD, P. Dall'Ara DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorP. Ciaramella DVM, P. Ciaramella DVM Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this author D. Piantedosi DVM, PhD, Corresponding Author D. Piantedosi DVM, PhD [email protected] Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalyE-mail for correspondence: [email protected]Search for more papers by this authorF. Servida DVM, PhD, F. Servida DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorL. Cortese DVM, PhD, L. Cortese DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorM. Puricelli DVM, PhD, M. Puricelli DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorV. Benedetti DVM, V. Benedetti DVM Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorA. Di Loria DVM, PhD, A. Di Loria DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorV. Foglia Manzillo DVM, PhD, V. Foglia Manzillo DVM, PhD Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this authorP. Dall'Ara DVM, PhD, P. Dall'Ara DVM, PhD Microbiology and Immunology Division, Department of Veterinary Animal Pathology and Public Health, University of Milan, Via Celoria 10, 20133 Milan, ItalySearch for more papers by this authorP. Ciaramella DVM, P. Ciaramella DVM Internal Medicine Division, Department of Veterinary Clinical Sciences, University of Naples, ‘Federico II’, Via F. Delpino 1, 80137 Naples, ItalySearch for more papers by this author First published: 16 January 2010 https://doi.org/10.1136/vr.c179Citations: 4 Provenance: not commissioned; externally peer reviewed Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1CIARAMELLA P., DALL'ARA P., CORTESE L., PURICELLI M., PIANTEDOSI D., SERVIDA F., CORONA M. & PERSECHINO A. 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(1986) A genetic association between bovine serum and colostrum lysozyme levels. Animal Genetics 17, 39–45 6LOMBARDI P., AVALLONE L., D'ANGELO A. & BOGIN E. (1996) γ-Glutamyltransferase and serum proteins in buffalo calves following colostral ingestion. European Journal of Clinical Chemistry and Clinical Biochemistry 34, 965–968 7MAGLIONE E., FERRARI A., MAGLIONE D. & ROSATI S. (1988) Sul contenuto di immunoglobuline A nel sangue di vacche immunizzate contro il BVD, nel loro colostro e nel colostro e nel sangue dei loro vitelli. Proceedings of the 20th Annual Conference of the Italian Association for Buiatrics (SIB). Marina di Ravenna, May 6 to 8, 1988. pp 287–297 8MCCLELLAND D. B., MCGRATH J. & SAMSON R. R. (1978) Antimicrobial factors in human milk. Studies of concentration and transfer to the infant during the early stages of lactation. Acta Paediatrica Scandinava Supplementum 271, 1–20 9MULLER G., SCHULTZE F., ERLER W. & JACOB B. (1990) Untersuchungen uber Lysozym beim kalb. 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The clinical and biochemical features of an acute and initial Wegener's granulomatosis case were analysed in a young woman. A multifactorial aspects are evident. A chronic inflammation of the superior respiratory tract has been observed. Staphylococcus aureus has been isolated. An oligoclonal component constituted of high levels of anti-PR3 autoantibodies was detected: initial autoreactive B cell clone activation is probable. The chronological link with postpartum is present: our study excluded foetal microchimerism; the hormonal state can be a trigger factor. Serical IL-17 was negative.

The clinical and biochemical features of an acute and initial Wegener's granulomatosis case were analysed in a young woman. A multifactorial aspects are evident. A chronic inflammation of the superior respiratory tract has been observed. Staphylococcus aureus has been isolated. An oligoclonal component constituted of high levels of anti-PR3 autoantibodies was detected: initial autoreactive B cell clone activation is probable. The chronological link with postpartum is present: our study excluded foetal microchimerism; the hormonal state can be a trigger factor. Serical IL-17 was negative.

The Inhibitor of Apoptosis Proteins (IAPs) are important regulators of programmed cell death. XIAP is the most potent among them and is over-expressed in several hematological malignancies. Its activity is endogenously antagonized by SMAC/DIABLO, and also by small molecules mimicking Smac that can induce apoptosis in tumor cells. Here we describe the activity of 56 newly synthesized Smac-mimetics in human leukemic cell lines and normal CD34(+) progenitor cells. Our compounds bind to XIAP with high affinity, reduce the levels of cIAP1 and are cytotoxic at nanomolar or low micromolar concentrations. Furthermore, the Smac-mimetics synergize with Cytarabine, Etoposide and especially with TRAIL in combination treatments. Apoptosis activation was clearly detectable by the occurrence of sub G(1) apoptotic peak and the accumulation of cleaved PARP, caspase 8 and caspase 3. Interestingly, the down-regulation of XIAP sensitized Jurkat cells to drugs too, confirming the role of this protein in drug-resistance. In conclusion, while being very active in leukemic cells, our Smac-mimetics have modest effects on normal hematopoietic progenitors, suggesting their promising therapeutic potential as a new class of anticancer drugs in onco-hematology, particularly when combined with TRAIL, to overcome the resistance of cancer cells.

Abstract Abstract 1835 Defective apoptosis has a central role both in development and progression of chronic lymphocytic leukemia (CLL). Indeed in this hematological malignancy the expression of inhibitor of apoptosis proteins (IAPs) has been shown to be significantly up-regulated. The most potent among IAPs is probably the X-linked IAP (XIAP), which is characterized by three highly conserved tandem BIR domains (BIR1-3) able to block various members of the family of cysteine proteases (caspases) known as the main effectors of the apoptotic process. BIR3 domain selectively targets caspase 9, an initiator caspase, whereas the linker region between BIR1 and BIR2 binds with executioner caspases 3 and 7, thus inhibiting the activity of both initiator and effector of apoptosis. Smac-DIABLO (Second Mitochondria-derived Activator of Caspases - Direct IAp Binding protein with LOw pI) is a protein released from the mitochondria antagonizing the activity of XIAP. Therefore, small molecules mimicking Smac (Smac-mimetics) can induce apoptosis in tumor cells by displacing the interaction between XIAP and caspases. At the University of Milan, Center for biomolecular Interdisciplinary Studies and Industrial applications (CISI), new cell permeable Smac-mimetic compounds (monomers and dimers) binding to XIAP with high affinity were recently synthetized. Here we report the activity of a selection of these new agents in human lymphocytes from 32 CLL patients and from 6 healthy controls. The patient population included only subjects with active disease (11 females and 21 males; median age 72 years; Rai-Binet stage 0-A to II-B), who, at the time of samples collection, were either untreated or out of therapy since several months. Patients were characterized for the mutational IGVH status, cytogenetics (by FISH analysis), and the expression of both CD38 and ZAP 70. Following isolation from peripheral blood samples by density gradient centrifugation, mononuclear cells were treated for 16 hours at 37°C and 5% CO2 with low micromolar concentrations (1-10 microM) of a selection of our new Smac mimetic compounds, including 4 monomers (34, 55, 66 and 73), and 2 dimers (83, 85) which were have been previously shown to be active in leukemic cell lines overexpressing XIAP. Combination treatments with the proteasome inhibitor bortezomib (10nM) and with Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) (50 ng/ml) were also tested. Apoptosis rate was evaluated by flow cytometric analysis following annexin V and propidium iodide staining. Overall, the monomers were more cytotoxic than the dimeric compounds on leukemic cells. The most active agent was the monomer Smac66, which, after 16 hours of treatment at a 10 microM concentration promoted apoptosis in up to 72,5% (median value 40% ranging from 11% to 72,5%) of CLL lymphocytes. In comparison to normal lymphocytes from healthy controls, where the median rate of apoptosis was 10.1% (range1.2% to 18%), the cytotoxic effect of the Smac66 compound was significantly higher (p=0.0004) in CLL cells. No synergistic effect was observed in combined treatment with bortezomib nor with TRAIL. Rate of apoptosis did not correlate with clinical parameters, cytogenetics, mutational IGVH status, and expression of CD38 and ZAP70 was observed, but the limited size of our patient population should be taken into account. In conclusion, among our Smac mimetics selected molecules seem be very active in CLL lymphocytes, suggesting a promising therapeutic potential in this malignancy. Further molecular investigations as well as in vivo studies in animal models are currently ongoing respectively to better elucidate the mechanism of action of these compounds and to promote their preclinical development. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2756 Poster Board II-732 The Inhibitor of Apoptosis Proteins (IAP) are important regulators of programmed cell death. Among them, XIAP, which is characterized by 3 tandem BIR domains selectively blocking caspases 3, 7 and 9, is the most potent and is over-expressed in several hematological malignancies. Its activity is antagonized by Second Mitochondria-derived Activator of Caspases (Smac) and also by small molecules mimicking Smac able to induce apoptosis in tumor cells, alone or in combination with other drugs. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine, is capable of triggering programmed death in cancer cells, where it synergizes with chemotherapeutic agents and/or radiation by several malignant cell-type specific mechanisms, and it is currently under evaluation in clinical trials for its strong pro-apoptotic activity. Here we describe the pro-apoptotic effect of newly synthesized monovalent and bivalent Smac-mimetic compounds tested for cytotoxicity on a cohort of human leukemic cell lines over-expressing XIAP (HL60, K562 and Jurkat) as well as on normal CD34+ hematopoietic progenitor cells, alone or in combination with TRAIL. The Smac-mimetics, designed and produced by the University of Milan Center for biomolecular Interdisciplinary Studies and Industrial applications, were dissolved in dimethylsulfoxide (DMSO) to obtain a 10 mM solution and stored at −20°C. Drug stocks were diluted with phosphate buffer (PBS) prior to their use. The cells were treated with 0.1 nM – 50 μM doses for up to 72 hours. TRAIL was kindly provided by Prof. Henning Walczak (Imperial College, London, UK). In combined treatments, Smac-mimetics and TRAIL were used simultaneously. The cytotoxic effect was evaluated by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect on fresh human CD34+ hematopoietic normal progenitor cells selected from healthy donors' peripheral blood stem cells (PBSC) was assayed by myeloid colony (CFU-GM) formation. Apoptosis was determined by flow cytometric analysis of DNA fragmentation and by Western blot analysis of caspases activation and PARP cleavage. The combination of some of our Smac-mimetics (both monomers and dimers) with TRAIL highly enhanced the inhibition of proliferation of the chronic myelogenous leukemia cell line K562, which was shown to be resistant to TRAIL single agent. In particular, a synergistic effect was observed in combined treatment with Smac012 10 μM and TRAIL 50 ng/ml (R Kern index = 6.2). Our Smac-mimetics, particularly the dimeric ones, were capable of inducing apoptosis, as demonstrated by DNA fragmentation and accumulation of cleaved PARP, caspase 8 and caspase 3, especially when administrated in combination with TRAIL. No cytotoxic effect was observed on normal CD34+ progenitor cells by Smac mimetics at doses ranging from 40 μM to 70 μM, even normal controls when were treated simultaneously with TRAIL. As many hematological malignancies are resistant to TRAIL alone, thus limiting its therapeutic effectiveness, the observed strong synergistic effect with Smac mimetic compounds not affecting the normal hematopoietic progenitor cell compartment might be of great consequence for the development of innovative potent pro-apoptotic drug combinations in myeloid leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2–BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.