Development of Novel Amides as Noncovalent Inhibitors of Immunoproteasomes.

The development of immunoproteasome-selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with Ki values in the low micromolar or sub-micromolar ranges toward the ß5i and/or ß1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibitor with a Ki value of 21 nm against the single ß1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.

Drug combination studies of curcumin and genistein against rhodesain of Trypanosoma brucei rhodesiense.

Curcumin and genistein are two natural products obtained from Curcuma longa L. and soybeans, endowed with many biological properties. Within the last years they were shown to possess also a promising antitrypanosomal activity. In the present paper, we investigated the activity of both curcumin and genistein against rhodesain, the main cysteine protease of Trypanosoma brucei rhodesiense; drug combination studies, according to Chou and Talalay method, allowed us to demonstrate a potent synergistic effect for the combination curcumin-genistein. As a matter of fact, with our experiments we observed that the combination index of curcumin-genistein is < 1 for the reduction from 10 to 90% of rhodesain activity.

Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors.

This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the treatment of hematological malignancies. In particular, we focused our efforts on the design of noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of drawbacks and side-effects related to irreversible inhibition. Among all the synthesized compounds, we identified a panel of active inhibitors with Ki values towards one or two chymotrypsin-like activities of proteasome (ß5c) and immunoproteasome (ß5i and ß1i subunits) in the low micromolar range. Docking studies suggested a unique binding mode of the molecules in the catalytic site of immunoproteasome proteolytic subunits.

Immunoproteasome-selective and non-selective inhibitors: A promising approach for the treatment of multiple myeloma.

The ubiquitin-proteasome system (UPS) is the major non-lysosomal proteolytic system for the degradation of abnormal or damaged proteins no longer required. The proteasome is involved in degradation of numerous proteins which regulate the cell cycle, indicating a role in controlling cell proliferation and maintaining cell survival. Defects in the UPS can lead to anarchic cell proliferation and to tumor development. For these reasons UPS inhibition has become a significant new strategy for drug development in cancer treatment. In addition to the constitutive proteasome, which is expressed in all cells and tissues, higher organisms such as vertebrates possess two immune-type proteasomes, the thymoproteasome and the immunoproteasome. The thymoproteasome is specifically expressed by thymic cortical epithelial cells and has a role in positive selection of CD8+ T cells, whereas the immunoproteasome is predominantly expressed in monocytes and lymphocytes and is responsible for the generation of antigenic peptides for cell-mediated immunity. Recent studies demonstrated that the immunoproteasome has a preservative role during oxidative stress and is up-regulated in a number of pathological disorders including cancer, inflammatory and autoimmune diseases. As a consequence, immunoproteasome-selective inhibitors are currently the focus of anticancer drug design. At present, the commercially available proteasome inhibitors bortezomib and carfilzomib which have been validated in multiple myeloma and other model systems, appear to target both the constitutive and immunoproteasomes, indiscriminately. This lack of specificity may, in part, explain some of the side effects of these agents, such as peripheral neuropathy and gastrointestinal effects, which may be due to targeting of the constitutive proteasome in these tissues. In contrast, by selectively inhibiting the immunoproteasome, it may be possible to maintain the antimyeloma and antilymphoma efficacy while reducing these toxicities, thereby increasing the therapeutic index. This review article will be focused on the discussion of the most promising immunoproteasome specific inhibitors which have been developed in recent years. Particular attention will be devoted to the description of their mechanism of action, their structure-activity relationship, and their potential application in therapy.

Development of Novel Peptide-Based Michael Acceptors Targeting Rhodesain and Falcipain-2 for the Treatment of Neglected Tropical Diseases (NTDs).

This paper describes the development of a class of peptide-based inhibitors as novel antitrypanosomal and antimalarial agents. The inhibitors are based on a characteristic peptide sequence for the inhibition of the cysteine proteases rhodesain of Trypanosoma brucei rhodesiense and falcipain-2 of Plasmodium falciparum. We exploited the reactivity of novel unsaturated electrophilic functions such as vinyl-sulfones, -ketones, -esters, and -nitriles. The Michael acceptors inhibited both rhodesain and falcipain-2, at nanomolar and micromolar levels, respectively. In particular, the vinyl ketone 3b has emerged as a potent rhodesain inhibitor (k2nd = 67 × 106 M-1 min-1), endowed with a picomolar binding affinity (Ki = 38 pM), coupled with a single-digit micromolar activity against Trypanosoma brucei brucei (EC50 = 2.97 µM), thus being considered as a novel lead compound for the discovery of novel effective antitrypanosomal agents.

Malignant Mixed Müllerian Tumour of the Uterus: Analysis of 44 Cases.

OBJECTIVES: The aim of our study was to evaluate the clinicopathological features and prognostic factors of uterine carcinosarcoma. PATIENTS AND METHODS: In this retrospective study, the clinical characteristics of 44 patients with uterine MMMT were evaluated. Survival curves were estimated by the Kaplan-Meier method and compared by the log-rank test. RESULTS: Forty-four patients with uterine carcinosarcoma were referred to our unit between 1995 and 2015. Their median age was 66.5 years. All women underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. Twenty-five percent had omental resection. Pelvic lymphadenectomy was performed in 18.2% of the cases. Twenty-six of the patients presented with stage I/II disease, 17 with advanced stages. In 20.5% of the cases there were metastases at diagnosis. Forty women received adjuvant chemotherapy, with complete remission in 67.9% of the cases. Recurrences were observed in 27.3% of the women. Disease-free and overall survival was 27 and 103 months, respectively. The FIGO stage, histological type, tumour size, chemotherapy regimen, pelvic lymphadenectomy, and myometrial invasion did not affect survival. CONCLUSIONS: Uterine MMMT is an aggressive tumour, often diagnosed at an advanced stage and with a high rate of metastases or recurrences. Because of its rarity, its management is controversial and fixed prognostic factors cannot be defined.

Development of novel 1,4-benzodiazepine-based Michael acceptors as antitrypanosomal agents.

Novel 1,4-benzodiazepines, endowed with a Michael acceptor moiety, were designed taking advantage of a computational prediction of their pharmacokinetic parameters. Among all the synthesized derivatives, we identified a new lead compound (i.e., 4a), bearing a vinyl ketone warhead and endowed with a promising antitrypanosomal activity against Trypanosoma brucei brucei (IC50=5.29µM), coupled with a lack of cytotoxicity towards mammalian cells (TC50 >100µM).

Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.

Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 µM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the ß5/ß6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (Ki = 0.42 µM). The solution structure of 9 and 38 was solved by (1)H NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 µM) is slightly more potent than 9 (IC50 = 34.3 µM) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors.

The Inhibition of Cysteine Proteases Rhodesain and TbCatB: A Valuable Approach to Treat Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) is an endemic parasitic disease of sub-Saharan Africa, caused by two subspecies of protozoa belonging to Trypanosoma genus: T. brucei gambiense and T. brucei rhodesiense. In this context the inhibition of the papain-family cysteine proteases rhodesain and TbCatB has to be considered a promising strategy for HAT treatment. Rhodesain, the major cathepsin L-like cysteine protease of T. brucei rhodesiense, is a lysosomal protease essential for parasite survival. It is involved in parasite invasivity, allowing it to cross the blood-brain barrier (BBB) of the human host, causing the second lethal stage of the disease. Moreover, it plays an important role in immunoevasion, being involved in the turnover of variant surface glycoproteins of the T. brucei coat and in the degradation of immunoglobulins, avoiding a specific immune response by the host cells. On the other hand TbCatB, a cathepsin B-like cysteine protease, present in minor abundance in T. brucei, showed a key role in the degradation of host transferrin, which is necessary for iron acquisition by the parasite. In this review article we now discuss the most active peptide, peptidomimetic and non-peptide rhodesain and TbCatB inhibitors as valuable strategy to treat HAT, due also to the complementary role of the two T. brucei proteases.

Immunoproteasome-Selective Inhibitors: A Promising Strategy to Treat Hematologic Malignancies, Autoimmune and Inflammatory Diseases.

The immunoproteasome is predominantly expressed in monocytes and lymphocytes and is responsible for the generation of antigenic peptides for cell-mediated immunity. Upon the exposure of inflammatory cytokines IFN-γ and TNF-α, constitutive subunits can be replaced by the synthesis of the immuno-core particles ß1i, ß2i and ß5i. Recent studies demonstrated that the immunoproteasome function is not only limited to MHC class I presentation, but it is also implicated in a number of pathological disorders including hematological malignancies, inflammatory and autoimmune diseases. At present the commercially available proteasome inhibitors Bortezomib and Carfilzomib, which have been validated in multiple myeloma and other diseases, appear to target both the constitutive and immunoproteasomes indiscriminately. This lack of specificity may, in part, explain some of the side effects of these agents. In contrast, by selectively targeting the immunoproteasome, it may be possible to keep the antimyeloma and antilymphoma efficacy unchanged and, at the same time, to increase the therapeutic index. The aim of this review article is to discuss the most promising immunoproteasome core particle-selective inhibitors which have been developed in the recent years, with a particular attention to their structural features, mechanism of action and therapeutic application.

Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors.

Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L.

Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation.

Novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation were developed; some of them possess K(i) values in the micromolar range. We studied the structure-activity relationship of these derivatives and we performed docking studies, which allowed us to find out the key interactions established by the inhibitors with the target enzyme. Biological results indicate that the nature of the P2 and P3 substituents and their binding to the S2/S3 pockets is strictly interdependent.

Development of novel selective peptidomimetics containing a boronic acid moiety, targeting the 20S proteasome as anticancer agents.

This paper describes the design, synthesis, and biological evaluation of peptidomimetic boronates as inhibitors of the 20S proteasome, a validated target in the treatment of multiple myeloma. The synthesized compounds showed a good inhibitory profile against the ChT-L activity of 20S proteasome. Compounds bearing a ß-alanine residue at the P2 position were the most active, that is, 3-ethylphenylamino and 4-methoxyphenylamino (R)-1-{3-[4-(substituted)-2-oxopyridin-1(2H)-yl]propanamido}-3-methylbutylboronic acids (3 c and 3 d, respectively), and these derivatives showed inhibition constants (Ki ) of 17 and 20 nM, respectively. In addition, they co-inhibited post glutamyl peptide hydrolase activity (3 c, Ki=2.57 µM; 3 d, Ki=3.81 µM). No inhibition was recorded against the bovine pancreatic α-chymotrypsin, which thus confirms the selectivity towards the target enzyme. Docking studies of 3 c and related inhibitors into the yeast proteasome revealed the structural basis for specificity. The evaluation of growth inhibitory effects against 60 human tumor cell lines was performed at the US National Cancer Institute. Among the selected compounds, 3 c showed 50% growth inhibition (GI50) values at the sub-micromolar level on all cell lines.

Optimization of peptidomimetic boronates bearing a P3 bicyclic scaffold as proteasome inhibitors.

A new series of pseudopeptide boronate proteasome inhibitors (2-3) was developed, through optimization of our previously described analogs of bortezomib, bearing a bicyclic 1,6-naphthyridin-5(6H)-one scaffold as P3 fragment (1). The biological evaluation on human 20S proteasome displayed a promising inhibition profile, especially for compounds bearing a P2 ethylene fragment, which exhibited Ki values in the nanomolar range for the ChT-L activity (e.g. 2a, Ki = 0.057 µM) and considerable selectivity for proteasome over bovine pancreatic α-chymotrypsin. Docking experiments into the yeast 20S proteasome revealed that the ligands are accommodated predominantly into the ChT-L site and that they covalently bind to the active site threonine residue via boron atom. Within the cellular assays performed against a 60 cancer cell line panel, compounds 3e and 3f demonstrated also good antiproliferative activity and compound 3f emerged as promising lead compound for the development of anticancer agents targeting melanoma and non-small cell lung cancer.

Synthesis and biological evaluation of papain-family cathepsin†L-like cysteine protease inhibitors containing a 1,4-benzodiazepine scaffold as antiprotozoal agents.

Novel papain-family cathepsin L-like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure-activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3-bromoisoxazoline moiety. Several rhodesain and falcipain-2 inhibitors displaying single-digit micromolar or sub-micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.

Peptide-based proteasome inhibitors in anticancer drug design.

The identification of the key role of the eukaryotic 26S proteasome in regulated intracellular proteolysis and its importance as a target in many pathological conditions wherein the proteasomal activity is defective (e.g., malignancies, autoimmune diseases, neurodegenerative diseases, etc.) prompted several research groups to the development of specific inhibitors of this multicatalytic complex with the aim of obtaining valid drug candidates. In regard to the anticancer therapy, the peptide boronate bortezomib (Velcade®) represents the first molecule approved by FDA for the treatment of multiple myeloma in 2003 and mantle cell lymphoma in 2006. Since then, a plethora of molecules targeting the proteasome have been identified as potential anticancer agents and a few of them reached clinical trials or are already in the market (i.e., carfilzomib; Kyprolis®). In most cases, the design of new proteasome inhibitors (PIs) takes into account a proven peptide or pseudopeptide motif as a base structure and places other chemical entities throughout the peptide skeleton in such a way to create an efficacious network of interactions within the catalytic sites. The purpose of this review is to provide an in-depth look at the current state of the research in the field of peptide-based PIs, specifically those ones that might find an application as anticancer agents.

Identification of a new series of amides as non-covalent proteasome inhibitors.

Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the ß5, ß2, and ß1 subunits of each heptameric ß rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the ß5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure.

Development of rhodesain inhibitors with a 3-bromoisoxazoline warhead.

Novel rhodesain inhibitors were obtained by combining an enantiomerically pure 3-bromoisoxazoline warhead with a specific peptidomimetic recognition moiety. All derivatives behaved as inhibitors of rhodesain, with low micromolar Ki values. Their activity against the enzyme was found to be paralleled by an in vitro antitrypanosomal activity, with IC50 values in the mid-micromolar range. Notably, a preference for parasitic over human proteases, specifically cathepsins B and L, was observed.

Two cases of complete hydatidiform mole and coexistent live fetus.

The aim of this study was to report the clinical features, management, and outcome of complete hydatidiform mole with a coexisting viable fetus. Two cases are reported. In both cases ultrasound examination demonstrated a normally growing live fetus alongside a normal placenta and an additional intrauterine echogenic mass with features of hydatidiform mole. The hCG levels were significantly increased and fetal karyotypes were normal. A cesarean section performed at 28 weeks' gestation in the first case and at 26 weeks' gestation in the second one resulted in the delivery of live normal infant and two adjoining placentas in both cases. Microscopic examination of the abnormal placentas confirmed complete hydatidiform mole. The babies did well and serial maternal serum hCG levels showed a declining trend and were undetectable by a few months after delivery. Continuation of a twin pregnancy with complete hydatidiform mole (CHMF) is an acceptable option. There is, although, an increased risk of developing maternal and fetal complications. Close surveillance of an ongoing pregnancy is compulsory to detect potential early signs of complications.