Dipeptide Boronic Acid Research Articles

Design and discovery of novel dipeptide boronic acid ester proteasome inhibitors, an oral slowly-released prodrug for the treatment of multiple myeloma

Multiple myeloma (MM), the second most common hematological malignancy, is a disease characterized by a clonal expansion of malignant plasma cells that accumulate in the bone marrow. Ixazomib citrate was the first commercially available oral proteasome inhibitor for the treatment of MM. However, it immediately hydrolyzed into the active form on exposure to aqueous solution and so it was a pseudo prodrug. Herein, a series of dipeptide boronic acid esters as novel oral proteasome inhibitors were designed, synthesized and biologically investigated for the inhibition of the β5 subunit of 20S proteasome. Based on the enzymatic results, structure-activity relationships (SAR) were discussed in detail. Some potent compounds were further evaluated to inhibit the proliferation of MM cell line RPMI-8226. The results showed that some compounds were active against RPMI-8226 with IC50 values of less than 10 nM. The solution stability showed that ixazomib citrate was completely hydrolyzed to its active form ixazomib within 2 min in the simulated gastric juice. However, among the screened compounds, prodrug 18u was stable enough in simulated gastric juice and simulated intestinal juice, and its hydrolysis rate was 59.7% and 3.6% after 2 h, respectively. In addition, 18u exhibited good microsome stabilities and pharmacokinetic properties and displayed strong antiproliferative activity against the RPMI-8226 cell line (5.6 nM). Furthermore, compound 18u exhibited strong in vivo anticancer efficacy in human MM (RPMI-8226) xenograft mouse model.

GLOMERULAR MICROANGIOPATHY WITH MARKED SYSTEMIC THROMBOTIC MICROANGIOPATHY SHORTLY AFTER BORTEZOMIB IN A NEWLY DIAGNOSED POEMS SYNDROME PATIENT

AbstractThe dipeptide boronic acid analogue bortezomib as a potent and selective inhibitor of the proteasome is used for the treatment of plasma cell dyscrasias such as multiple myeloma (MM). Bortezomib may induce glomerular microangiopathy (GMA) with or without systemic thrombotic microangiopathy (TMA) in which vascular endothelial growth factor-nuclear factor (VEGF) -κ B pathway could be involved. MM itself can cause TMA but primarily at presentation. Case reportWe present a case with GMA associated with clinical features supporting systemic TMA shortly after bortezomib. Case: A 54-year-old woman has been diagnosed as having POEMS syndrome. She had symmetric mild degree of peripheral neuropathy, scleroatrophic skin lesions, Raynaud's phenomenon, and retinopathy. IgG kappa type paraproteinemia with a monotypic increase of plasma cells and increased pulmonary artery pressure contributed to the diagnosis. Bortezomib based treatment was started. MethodologyAt the 20th day she developed severe dyspnea. Bilateral pleural effusion and acute kidney failure with thrombocytopenia and microangiopathic hemolytic anemia were documented. Urgent steroid and plasmapheresis were started. ADAMTS13 level proved to be within normal and plasmapheresis did not contribute to improvement. She commenced on hemodialysis and kidney biopsy was decided. Light microscopy findings revealed glomerular capillary thrombus, basement membrane thickening and segmental Resultsduplication. Hyperplastic arteriolar changes were present. No immune deposits were detected by immunofluorescence microscopy. Biopsy findings were diagnostic for thrombotic microangiopathy. The clinical picture deteriorated as sleepiness and confusion. Cranial imaging and cerebrospinal fluid analysis showed no abnormality. Eculizumab with off-label approval contributed to stabilization but no improvement. ConclusionConclusion: Proteasome inhibitors associated with TMA may be life-threatening along with organ dysfunction due to microangiopathy-related ischemia. Membrane attack complex (C5b-9) deposition was found on endothelial cells in culture exposed to plasma from patients during the acute phase of the disease which may point to complement blockade benefit.

Glutathione Ameliorates Bortezomib (Velcade) -Induced Neuronal Toxicity in N2a and SHSY5Y Cells

Bortezomib (Velcade, Millennium Pharmaceuticals, Inc.), a potent proteasome inhibitor, is a boronic acid dipeptide that induce cancer cell death. It is an effective drug for both newly diagnosed or relapsed multiple myeloma (MM) patients and is widely used in the treatment of various kinds of lymphomas and other diseases. Peripheral nerve damage is one of the most significant nonhematologic toxicities of bortezomib. The reported incidence of bortezomib-induced peripheral neuropathy (BIPN) ranges from 25 to 75 % . When it occurs, the painful sensory neuropathy can interfere with quality of life (QOL) and with performance of activities of daily living, and it may also adversely affect clinical outcomes by forcing dose modification and/or premature treatment discontinuation. Therefore there are many efforts to improve multiple symptoms associated with BIPN. But there are no reports on amelioration of neurotoxicity which occurred in patients with treatment of bortezomib .Therefore control of this toxicity by the concomitant use of other safe candidates can make it a better option for the cancer therapy with bortezomib.Glutathione (GSH) is a substance produced naturally by the liver and it is also found in fruits, vegetables, and meats. GSH is an important antioxidant and capable of preventing damage to important cellular components caused by reactive oxygen species (ROS) such as free radicals, peroxides, lipid peroxides, and heavy metals. Several small randomized trials have addressed the protective effect of GSH against chemotherapy induced peripheral neuropathy (CIPN )with a platinum agent .In this study we observed that treatment of GSH either in the pre-treatment or in the post treatment inhibited the bortezomib -induced neuronal cell death in both mouse neuronal cells (N2a) as well as dopaminergic neuron of human origin (SHSY5Y). Bortezomib treatment at the concentration of 100 and 200 µg/ml significantly increased the cell death with increased lactate dehydrogenase activity (LDH) and increased apoptotic proteins expressions in the 24 h of treatment of both cells. GSH treatment (1 mg/ml) not only inhibited the bortezomib -induced cell death but also inhibited the LDH activity, suggesting that GSH ameliorates bortezomib -induced neuronal toxicity. A clear activation of the autophagy was also observed in the bortezomib treatment that was lowered by the GSH treatment. In addition to this inhibition of the apoptotic proteins specially Cleaved caspase 3 was observed in the neuronal cells and also the increased Bcl2 and NRF2 protein which are responsible for the cell survival was upregulated with GSH treatment suggesting that GSH increase the cell survival against bortezomib -induced neurotoxicity. Inhibition of the cleaved caspase-3, Bax and LC3 protein are prominent in SHSY5Y cells while activation of the BCL2 and NRF2 is more significant in N2a cells. To elucidate the role of GSH in the development and maintenance of bortezomib -induced neurotoxicity, we will additionally assess ROS and antioxidant enzyme activity levels in the cellular system.This research suggests that GSH ameliorates BIPN and thus can improve the QOL and increase the survival rate of patients treated with bortezomib. DisclosuresNo relevant conflicts of interest to declare.

Bortezomib-Containing Regimens for the Treatment of Newly Diagnosed AL Amyloidosis: Experience of Two Centers in Canada

Introduction Bortezomib is a novel, dipeptide boronic acid molecule that has shown anti-tumor activity against Multiple Myeloma (MM) and more recently has been demonstrated to be efficacious in the treatment of Light-chain Amyloidosis (AL). Bortezomib-based regimens have changed the landscape of the treatment of AL Amyloidosis due to high levels of response as well as the adequate tolerance. Based on the above mentioned, we aimed to assess the role of Bortezomib-containing regimens (BCR) for the treatment of newly diagnosed AL amyloidosis. Methods 80 consecutive patients have been treated with bortezomib-containing regimens at BCCA and TBCC in Calgary, AB and Vancouver, BC, Canada from 01/2010 to 01/2016. Among these cases, 51 (63.8%) have received CyBorD, 19 (23.7%) bortezomib (1.3 mg/m2) and dexamethasone and the rest Bortezomib in different combinations (Table 1). The primary objective of the study was to assess hematological and organ response. Results Clinical characteristics are shown in Table 1. Median age at diagnosis was 66 years and 51.1% of patients were male. At the time of analysis, 56 patients are still alive and 14 patients have already progressed. A HR was seen in 65/72 evaluable patients (ORR, 90%) after a median of 6 cycles (range, 1-24). Fourteen patients (17.5%) did not have measurable disease based on light chain levels. CR (34.7%) and VGPR (26.4%) were seen in 61% of cases. Organ response was achieved in 49% of cases. Cardiac response was observed in 40% of cases. Median OS and PFS have not been reached (Estimate, 67 and 41 months, respectively). (Fig 1a) However, OS was shorter in the group with severe heart involvement (NT-pro-BNP>5000 or BNP>1100) (p=0.03) (Fig 1b). In conclusion, BCR are efficacious for the treatment of newly diagnosed AL Amyloidosis, providing with a high degree of organ and hematological responses. The impact of BCR on advanced heart dysfunction cases remains unclear but it seems that these patients still require a different approach maintaining a dismal prognosis. [Display omitted] [Display omitted] DisclosuresSong:Janssen: Honoraria; Otsuka: Honoraria; Celgene: Honoraria, Research Funding. Neri:Celgene and Jannsen: Consultancy, Honoraria. Bahlis:Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel Expenses, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria; BMS: Honoraria. Jimenez-Zepeda:Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Celgene, Janssen, Amgen, Onyx: Honoraria.

Bortezomib sensitizes esophageal squamous cancer cells to radiotherapy by suppressing the expression of HIF-1α and apoptosis proteins.

Radiation therapy is a typical treatment for esophageal squamous cell carcinoma (ESCC), especially middle and upper segment esophagus, and inoperable patients. However, how to promote radiation sensitivity in radio-resistant cancer cells is a conundrum. Here, our study investigated the radiosensitizing effect of bortezomib, a specific and reversible dipeptide boronic acid analog, in ESCC cells. Human esophageal squamous carcinoma cell lines Eca109 and TE-13 were exposed to hypoxia and/or ionizing radiation (IR) with or without treatment of bortezomib. Cell proliferation assay was performed with CCK8. Cell apoptosis and cell cycle assay were performed with flow cytometry. The radiosensitization effect of was assessed by clonogenic survival and progression of tumor xenograft. The expression of HIF-1α, VEGF, and apoptosis proteins was evaluated by Western blot. Radiation-induced DNA double strand break and homologous recombination repair were assessed by immunofluorescence. Our results show that bortezomib efficiently radiosensitizes ESCC cells by decreasing the expression of HIF- 1α and VEGF, inducing apoptosis by activating caspase, and delaying DNA damage repair after radiation.

Heme oxygenase-1 nuclear translocation regulates bortezomibinduced cytotoxicity and mediates genomic instability in myeloma cells.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells in the bone marrow leading to bone destruction and bone marrow failure. Several molecular mechanisms underlie chemoresistance among which heme oxygenase-1 (HO-1) could play a major role. The aim of the present research was to evaluate the impact of HO-1 in MM following bortezomib (BTZ) treatment and how HO-1 is implicated in the mechanisms of chemoresistance. MM cells were treated for 24h with BTZ (15 nM), a boronic acid dipeptide inhibitor of the 26S proteasome used in the treatment of patients with MM as first-line therapy. We evaluated cell viability, reactive oxygen species (ROS) formation, endoplasmic reticulum (ER) stress, HO-1 expression and compartmentalization and cellular genetic instability. Results showed that BTZ significantly reduced cell viability in different MM cell lines and induced ER-stress and ROS formation. Concomitantly, we observed a significant overexpression of both HO-1 gene and protein levels. This effect was abolished by concomitant treatment with 4-phenybutirric acid, a molecular chaperone, which is known to reduce ER-stress. Surprisingly, inhibition of HO activity with SnMP (10μM) failed to increase BTZ sensitivity in MM cells whereas inhibition of HO-1 nuclear translocation by E64d, a cysteine protease inhibitor, increased sensitivity to BTZ and decreased genetic instability as measured by cytokinesis-block micronucleus assay. In conclusion, our data suggest that BTZ sensitivity depends on HO-1 nuclear compartmentalization and not on its enzymatic activity and this finding may represent an important tool to overcome BTZ chemoresistance in MM patients.

An Overview of Bortezomib-Induced Neurotoxicity.

The boronic acid dipeptide bortezomib, able to induce tumor cell death by degradation of key proteins, is the first proteasome inhibitor drug to enter clinical practice. It is employed as first-line treatment in relapsed or resistant multiple myeloma (MM) patients. However, bortezomib often induces a dose-limiting toxicity in the form of painful sensory neuropathy, which can mainly be reduced by subcutaneous administration or dose modification. In this review we focus on the current understanding of the pathophysiological mechanisms of bortezomib-induced neuropathy to allow further studies in animal models and humans, including analysis of clinical and pharmacogenetic aspects, to optimize the treatment regimens.

A multi-stimuli responsive, self-assembling, boronic acid dipeptide.

Modification of the dipeptide of phenylalanine, FF, with a boronic acid (BA) functionality imparts unique aqueous self-assembly behavior that responds to multiple stimuli. Changes in pH and ionic strength are used to trigger hydrogelation via the formation of nanoribbon networks. Furthermore, we show for the first time that the binding of polyols to the BA functionality can modulate a peptide between its assembled and disassembled states.

B and H-NMR Evaluation of Chemical Interactions between Two Polyphenols; Epigallocatechingallate and Quercetin with Bortezomib

Bortezomib (Velcade) is a boronic acid dipeptide, proteasome inhibitor drug that its antimyeloma activity in treatment of patients with multiple myeloma (MM) has been approved by FDA. Studies show that consumption of daily diet rich in polyphenols affect Bortezomib’s bioavailability and reduce its antitumor activity. In the present study we made an attempt to investigate the interactions between Bortezomib and some common polyphenols to examine whether these polyphenols interact with the drug. For this purpose, interactions between four dietary polyphenols including: Emodin, Rhein, Epigallocatechingallate (EGCG) and Quercetin with methyl boronic acid, Bortezomib’s substitute, were studied using 1H and 11B- NMR spectroscopy. The results suggest that the vicinal diols in the structure of the two polyphenols (EGCG and Quercetin) interact with the boronic acid moiety of Bortezomib and convert the active triangular boronic acid to an inactive tetrahedral boronate through direct chemical interaction, and this conversion abolishes the antimyeloma activity of Bortezomib. In conclusion, our study suggests that the consumption of dietary sources enriched in EGCG and Quecetin during treatment with Bortezomib in MM patients should be considered.

Bortezomib overcomes MGMT-related resistance of glioblastoma cell lines to temozolomide in a schedule-dependent manner

Development of drug resistance after standard chemotherapy for glioblastoma multiforme (GBM) with temozolomide (TMZ) is associated with poor prognosis of GBM patients and is at least partially mediated by a direct DNA repair pathway involving O6-methylguanine methyltransferase (MGMT). This enzyme is under post-translational control by a multisubunit proteolytic cellular machinery, the 26S proteasome. Inhibition of the proteasome by bortezomib (BZ), a boronic acid dipeptide already in clinical use for the treatment of myeloma, has been demonstrated to induce growth arrest and apoptosis in GBM cells. In this study we investigated the effect of sequential treatment with BZ and TMZ on cell proliferation-viability and apoptosis of the human T98G and U87 GBM cell lines. We also tested for an effect of treatment on MGMT expression and important upstream regulators of the latter, including nuclear factor kappa B (NFκB), p44/42 mitogen-activated protein kinase (MAPK), p53, signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). The sequence of drug administration for maximal cytotoxicity favored BZ prior to TMZ in T98G cells while the opposite was the case for U87 cells. Maximal efficacy was associated with downregulation of MGMT, reduced IκBα-mediated proteasome-dependent nuclear accumulation of NFκB, attenuation of p44/42 MAPK, AKT and STAT3 activation, and stabilization of p53 and inactive HIF-1α. Collectively, these results suggest that proteasome inhibition by BZ overcomes MGMT-mediated GBM chemoresistance, with scheduling of administration being critical for obtaining the maximal tumoricidal effect of combination with TMZ.

Bortezomib downregulates MGMT expression in T98G glioblastoma cells.

The efficacy of treatment for glioblastoma multiforme is currently limited by the development of resistance, particularly, but not exclusively, due to the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) in a significant proportion of astrocytic tumors. MGMT is post-translationally regulated by the 26S proteasome, a multi-subunit organelle responsible for degradation of misfolded cellular proteins. The boronic acid dipeptide bortezomib is the first and only proteasome inhibitor in clinical use so far, and has been reported as a strategy to restrict growth and promote apoptosis of glioblastoma cells. In this study we investigated the effect of bortezomib on MGMT expression in T98G cells, looking for an effect on the nuclear factor kappa B (NFκB) pathway, which is a major player in MGMT regulation and is also under tight control by the ubiquitin-proteasome system. Administration of bortezomib led to a significant reduction of T98G cell viability and induction of DNA fragmentation. These effects coincided with reduced expression of MGMT transcript levels, and a decrease in cellular amount and IκBα-mediated, proteasomal activity-dependent nuclear translocation of NFκB. In addition, bortezomib-induced phosphorylation of the translation initiation factor 2alpha (eIF2α) was in parallel with translational repression of MGMT. Taken together, these results suggest a novel role for bortezomib as a potent MGMT inhibitor and support its ongoing testing as a chemosensitizer in glioblastoma.

Selective growth inhibition of human malignant melanoma cells by syringic acid-derived proteasome inhibitors

BackgroundIt has been shown that proteasome inhibition leads to growth arrest in the G1 phase of the cell cycle and/or induction of apoptosis. However, it was found that some of these inhibitors do not induce apoptosis in several human normal cell lines. This selective activity makes proteasome inhibition a promising target for new generation of anticancer drugs. Clinical validation of the proteasome, as a therapeutic target in oncology, has been provided by the dipeptide boronic acid derivative; bortezomib. Bortezomib has proven to be effective as a single agent in multiple myeloma and some forms of non-Hodgkin’s lymphoma. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid, 1), a known phenolic acid, was isolated from the methanol extract of Tamarix aucheriana and was shown to possess proteasome inhibitory activity.MethodsUsing Surflex-Dock program interfaced with SYBYL, the docking affinities of syringic acid and its proposed derivatives to 20S proteasome were studied. Several derivatives were virtually proposed, however, five derivatives: benzyl 4-hydroxy-3,5-dimethoxybenzoate (2), benzyl 4-(benzyloxy)-3,5-dimethoxybenzoate (3), 3'-methoxybenzyl 3,5-dimethoxy-4-(3'-methoxybenzyloxy)benzoate (4), 3'-methoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (5) and 3',5'-dimethoxybenzyl 4-hydroxy-3,5-dimethoxybenzoate (6), were selected based on high docking scores, synthesized, and tested for their anti-mitogenic activity against human colorectal, breast and malignant melanoma cells as well as normal human fibroblast cells.ResultsDerivatives 2, 5, and 6 showed selective dose-dependent anti-mitogenic effect against human malignant melanoma cell lines HTB66 and HTB68 with minimal cytotoxicity on colorectal and breast cancer cells as well as normal human fibroblast cells. Derivatives 2, 5 and 6 significantly (p ≤ 0.0001) inhibited the various proteasomal chymotrypsin, PGPH, and trypsin like activities. They growth arrested the growth of HTB66 cells at G1 and G2-phases. They also arrested the growth of HTB68 cells at S- and G2-phase, respectively. Moreover, derivatives 2, 5, and 6 markedly induced apoptosis (≥ 90%) in both HTB66 and HTB68.ConclusionsComputer-derived syringic acid derivatives possess selective anti-mitogenic activity on human malignant melanoma cells that may be attributed to perturbation of cell cycle, induction of apoptosis and inhibition of various 26S proteasomal activities.

Bortezomib interferes with C-KIT processing and transforms the t(8;21)-generated fusion proteins into tumor-suppressing fragments in leukemia cells

The boronic acid dipeptide bortezomib inhibits the chymotrypsin-like activity of the 26S proteasome and shows significant therapeutic efficacy in multiple myeloma. However, recent studies suggest that bortezomib may have more complex mechanisms of action in treating cancer. We report here that the endocytosis and lysosomal degradation of the receptor tyrosine kinase C-KIT are required for bortezomib- but not tyrosine kinase inhibitor imatinib-caused apoptosis of t(8;21) leukemia and gastrointestinal stromal tumor cells, suggesting that C-KIT may recruit an apoptosis initiator. We show that C-KIT binds and phosphorylates heat shock protein 90β (Hsp90β), which sequestrates apoptotic protease activating factor 1 (Apaf-1). Bortezomib dephosphorylates pHsp90β and releases Apaf-1. Although the activated caspase-3 is not sufficient to cause marked apoptosis, it cleaves the t(8;21) generated acute myeloid leukemia 1-eight twenty one (AML1-ETO) and AML1-ETO9a fusion proteins, with production of cleavage fragments that perturb the functions of the parental oncoproteins and further contribute to apoptosis. Notably, bortezomib exerts potent therapeutic efficacy in mice bearing AML1-ETO9a-driven leukemia. These data show that C-KIT-pHsp90β-Apaf-1 cascade is critical for some malignant cells to evade apoptosis, and the clinical therapeutic potentials of bortezomib in C-KIT-driven neoplasms should be further explored.

Pharmacophore Modeling, Docking Studies, and Synthesis of Novel Dipeptide Proteasome Inhibitors Containing Boron Atoms

A 3D pharmacophore model had been generated for a series of dipeptide proteasome inhibitors containing boron atoms using Catalyst. A data set consisting of 24 inhibitors was selected on the basis of the information content of the structures and activity data as required by the Catalyst/HypoGen program. The built model was able to predict the activity of other known proteasome inhibitors not included in the model generation. Based on the analysis of the best hypotheses, some novel proteasome inhibitors were designed and predicted. Three dipeptide boronic acid inhibitors SMNT 1, SMNT 2, and SMNT 3, were synthesized and biologically assayed. It turned out that the three designed molecules were all more potent than the marketed MG341, and the experimental values were consistent with the predicted ones, indicating that the theoretical model was reliable enough to predict and design novel proteasome inhibitors. The covalent interaction mode between the boron atom of the inhibitor and O(gamma)-Thr1 residue of the 20S proteasome was studied for the first time by employing the most potent inhibitor SMNT 2 with the Insight II 2005/Affinity program. The docking results agreed well with the experimental ones.

Bortezomib Induces the Formation of Nuclear poly(A) RNA Granules Enriched in Sam68 and PABPN1 in Sensory Ganglia Neurons

The ubiquitin-dependent proteasome system (UPS) is the major pathway responsible for selective nuclear and cytoplasmic protein degradation. Bortezomib, a boronic acid dipeptide, is a reversible 20S proteasome inhibitor used as novel anticancer drug, particularly in the treatment of multiple myeloma and certain lymphomas. Bortezomib-induced peripheral neuropathy (BIPN) is a widely recognized dose-limiting neurotoxicity of this proteasome inhibitor, which causes a significant negative impact on the quality of life. The pathogenic mechanisms underlying bortezomib neurotoxicity are little known. In this study a rat was used as our animal model to investigate the bortezomib-induced nuclear changes in dorsal root ganglia (DRG) neurons. Our results indicate that this neuronal population is an important target of bortezomib neurotoxicity. Nuclear changes include accumulation of ubiquitin-protein conjugates, reduction of transcriptional activity, and nuclear retention of poly(A) RNAs in numerous spherical or ring-shaped dense granules. They also contained the RNA-binding proteins PABPN1 (poly(A) binding protein nuclear 1) and Sam68, but lacked the mRNA nuclear export factors REF and Y14. At the cytoplasmic level, most neurons exhibited chromatolysis, supporting the inhibition of mRNA translation. Our results indicate that bortezomib interferes with transcription, nuclear processing and transport, and cytoplasmic translation of mRNAs in DRG neurons. They also support that this neuronal dysfunction is an essential pathogenic mechanism in the BIPN, which is characterized by sensory impairment including sensory ataxia.

Dipeptide Boronic Acid Inhibitors of Dipeptidyl Peptidase IV: Determinants of Potency and in Vivo Efficacy and Safety

Dipeptidyl peptidase IV (DPP-IV; E.C. 3.4.14.5), a serine protease that degrades the incretin hormones GLP-1 and GIP, is now a validated target for the treatment of type 2 diabetes. Dipeptide boronic acids, among the first, and still among the most potent DPP-IV inhibitors known, suffer from a concern over their safety. Here we evaluate the potency, in vivo efficacy, and safety of a selected set of these inhibitors. The adverse effects induced by boronic acid-based DPP-IV inhibitors are essentially limited to what has been observed previously for non-boronic acid inhibitors and attributed to cross-reactivity with DPP8/9. While consistent with the DPP8/9 hypothesis, they are also consistent with cross-reactivity with some other intracellular target. The results further show that the potency of simple dipeptide boronic acid-based inhibitors can be combined with selectivity against DPP8/9 in vivo to produce agents with a relatively wide therapeutic index (>500) in rodents.

Proteasome inhibitor Bortezomib induces cell-cycle arrest and apoptosis in cell lines derived from Ewing’s sarcoma family of tumors and synergizes with TRAIL

Bortezomib (VELCADE®), formerly known as PS-341, is a novel dipeptide boronic acid proteasome inhibitor with in vitro and in vivo anti-tumor activity. Bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma. In this report, we examined the sensitivity of cell lines derived from Ewing’s sarcoma-family of tumors (ESFT) to Bortezomib. Five ESFT-derived cell lines, TC-71, TC-32, SK-N-MC, A4573, and GRIMES, were highly sensitive to Bortezomib (IC50 = 20 to 50 nM), and underwent cell-cycle arrest and apoptosis following drug treatment. Bortezomib-induced apoptosis was associated with activation of caspase 3, cleavage of PARP and induction of p27 and p21 expression. Moreover, Bortezomib exhibited synergistic activity against the TC-71 and TC-32 cell lines when combined with TRAIL. Our results suggest that Bortezomib might be a useful agent for treatment of ESFT, when used alone or in combination with TRAIL.

Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapeutic agents in gastric cancer cell lines

The proteasome plays a pivotal role in controlling cell proliferation, apoptosis, and differentiation in a variety of tumor cells. Bortezomib is a boronic acid dipeptide derivative, which is a selective and potent inhibitor of the proteasome and has prominent effects in vitro and in vivo against several solid tumors. We examined the anti-proliferative and apoptotic effects of bortezomib in three gastric cancer cell lines (SNU638, MUGC-3 and MKN-28), along with its antitumor combination effects with other chemotherapeutic agents. Tumor cell growth inhibition and apoptosis was measured by MTT assay and FACS analysis, respectively. Apoptosis- and cell cycle-associated protein expression levels were measured by Western blot assay. Bortezomib induced the suppression of tumor cell growth and apoptosis in a dose-dependent manner with an inhibitory dose (ID)50 of approximately 0.5 microg/ml in all gastric cancer cell lines tested. Further combination treatment with cisplatin and docetaxel, in particular with docetaxel displayed dramatically increased tumor cell growth suppression in all three gastric cancer cell lines, as compared to single drug treatment alone. This was concomitant with the induction patterns of apoptotic cells. Bortezomib treatment increased the Bax protein expression. Moreover, combination treatment of bortezomib plus docetaxel resulted in a dramatic increase in the Bax expression. In contrast, Bcl-2 expression was decreased by combination treatment with bortezomib plus docetaxel in SNU638 cells. Finally, bortezomib, docetaxel and to a greater degree bortezomib plus docetaxel increased the expression levels of p27 proteins even without influencing p53 expression levels. Bortezomib has profound effects on tumor cell growth inhibition and induction of apoptosis in human gastric cancer cells, suggesting that bortezomib may be an effective therapeutic drug for patients with gastric cancer. Further combination studies with other chemotherapeutic drugs, in particular docetaxel showing more tumor cell growth inhibition and apoptosis suggest that combining bortezomib with docetaxel might be more effective for displaying tumor cell growth inhibitory effects in gastric cancer cells through regulation of Bcl-2, Bax and p27 proteins in vitro.

Use of novel proteosome inhibitors as a therapeutic strategy in lymphomas current experience and emerging concepts

Precedent from preclinical experiments coupled with two pivotal phase 2 studies in myeloma has focused attention on a potential role for ubiquitin–proteasome pathway in modulating a number of events that occur commonly in the neoplastic process involving proteins in the regulation of cells cycling, growth and differentiation. This influence is vested in the proteasomes which are large complexes of proteolytic enzymes responsible for degradation of many of these intracellular messengers. Logically interest has centred on molecules having the capacity to influence, by degradation, such molecules and although a number of agents are in development bortezomib is the only one currently in clinical use. Velcade, formerly PS-341, is a novel dipeptide boronic acid capable of reversibly inhibiting the 26S proteasome through a range of activities. The latter are anti-proliferative and proapoptotic with the latter blocking nuclear transcription via NF- κ B in addition to down regulating adhesion and inhibiting angiogenesis. Additional changes are mediated in protein folding within the endoplasmic reticulum and contribute to cell death. These concepts are given focus by considering their introduction into treatment of lymphoreticular malignancy.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-κB in the cytoplasm and inhibit cell growth (IC 50 = 22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p < 0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-κB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.