Inhibitory effects of a maleimide compound on the virulence factors of Candida albicans.

Candidiasis caused by Candida albicans infection has long been a serious human health problem. The pathogenicity of C. albicans is mainly due to its virulence factors, which are novel targets of antifungal drugs for low risk of resistance development. In this study, we identified a maleimide compound [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] that exerts effective anti-virulence activity. It could inhibit the process of adhesion, filamentation, and biofilm formation in C. albicans. In addition, it exhibited low cytotoxicity, hemolytic activity, and drug resistance development. Moreover, in Galleria mellonella-C. albicans (in vivo) infection model, the survival time of infected larvae was significantly prolonged under the treatment of MPD. Further, mechanism research revealed that MPD increased farnesol secretion by upregulating the expression of Dpp3. The increased farnesol inhibited the activity of Cdc35, which then decreased the intracellular cAMP content resulting in the inhibition of virulence factors via the Ras1-cAMP-Efg1 pathway. In all, this study evaluated the inhibitory effect of MPD on various virulence factors of C. albicans and identified the underlying mechanisms. This suggests a potential application of MPD to overcome fungal infections in clinics.

Cell Penetrating Peptides Conjugated to Anti-Carcinoembryonic Antigen "Catch-and-Release" Monoclonal Antibodies Alter Plasma and Tissue Pharmacokinetics in Colorectal Cancer Xenograft Mice.

Cell penetrating peptides conjugated to delivery vehicles, such as nanoparticles or antibodies, can enhance the cytosolic delivery of macromolecules. The present study examines the effects of conjugation to cell penetrating and endosomal escape peptides (i.e., TAT, GALA, and H6CM18) on the pharmacokinetics and distribution of an anti-carcinoembryonic antigen "catch-and-release" monoclonal antibody, 10H6, in a murine model of colorectal cancer. GALA and TAT were conjugated to 10H6 using SoluLINK technology that allowed the evaluation of peptide-to-antibody ratio by ultraviolet spectroscopy. H6CM18 was conjugated to either NHS or maleimide-modified 10H6 using an azide-modified valine-citrulline linker and copper-free click chemistry. Unmodified and peptide-conjugated 10H6 preparations were administered intravenously at 6.67 nmol/kg to mice-bearing MC38CEA+ tumors. Unconjugated 10H6 demonstrated a clearance of 19.9 ± 1.36 mL/day/kg, with an apparent volume of distribution of 62.4 ± 7.78 mL/kg. All antibody-peptide conjugates exhibited significantly decreased plasma and tissue exposure, increased plasma clearance, and increased distribution volume. Examination of tissue-to-plasma exposure ratios showed an enhanced selectivity of 10H6-TAT for the GI tract (+25%), kidney (+24%), liver (+38%), muscle (+3%), and spleen (+33%). 10H6-GALA and 10H6-H6CM18 conjugates demonstrated decreased exposure in all tissues, relative to unmodified 10H6. All conjugates demonstrated decreased tumor exposure and selectivity; however, differences in tumor selectivity between 10H6 and 10H6-H6CM18 (maleimide) were not statistically significant. Relationships between the predicted peptide conjugate isoelectric point (pI) and pharmacokinetic parameters were bell-shaped, where pI values around 6.8-7 exhibit the slowest plasma clearance and smallest distribution volume. The data and analyses presented in this work may guide future efforts to develop immunoconjugates with cell penetrating and endosomal escape peptides.

Structure-Activity Relationships of Triple-Action Platinum(IV) Prodrugs with Albumin-Binding Properties and Immunomodulating Ligands.

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure-activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment.

A Protein-Binding Molecular Photothermal Agent for Tumor Ablation.

Photothermal therapy usually requires a high power density to activate photothermal agent for effective treatment, which inevitably leads to damage to normal tissues and inflammation in tumor tissues. Herein, we rationally design a protein-binding strategy to build a molecular photothermal agent for photothermal ablation of tumor. The synthesized photothermal agent can covalently bind to the thiol groups on the intracellular proteins. The heat generated by the photothermal agent directly destroyed the bioactive proteins in the cells, effectively reducing the heat loss and the molecular leakage. Under a low power density of 0.2 W cm-2 , the temperature produced by the photothermal agent was sufficient to induce apoptosis. In vitro and in vivo experiments showed that the therapeutic effect of photothermal therapy can be efficiently improved with the protein-binding strategy.

Ruboxistaurin maintains the bone mass of subchondral bone for blunting osteoarthritis progression by inhibition of osteoclastogenesis and bone resorption activity.

Osteoarthritis (OA) is a common degenerative disease with a series of changes occurring in aging cartilage, such as increased oxidative stress, decreased markers of healthy cartilage and alterations in the autophagy pathway. And increasing evidence indicates that osteoarthritis affects the whole joint, including both cartilage and subchondral bone. The agents that can effectively suppress chondrocyte degradation and subchondral bone deterioration are crucial for the prevention and treatment of OA. Ruboxistaurin (RU), an orally active protein kinase C inhibitor, can reduce macrophage adhesion to endothelial cells and relieve the local inflammation when applicating in diabetes and kinds of aging-related vasculopathy, which were realized by its effects on decreasing inflammatory cytokines' expression and increasing cell anti-oxidative stress ability. However, whether ruboxistaurin protects against OA remains unknown. In this study, we investigated the therapeutic effects of ruboxistaurin in an anterior cruciate ligament transection (ACLT)-induced OA model by preventing the bone mass loss of subchondral bone. We found that ruboxistaurin can effectively alleviate ACLT-induced osteoarthritis, as demonstrated by the phenomenon of correcting pathological bone loss caused by osteoclasts overactivated in the early stage of osteoarthritis and protecting against articular cartilage degeneration. Moreover, we found that ruboxistaurin inhibited osteoclast formation and resorption activity by suppressing the expressions of osteoclast-related genes and (PKCδ/MAPKs) signaling cascade. Taken together, these results show that ruboxistaurin may be a potential therapeutic agent for rescuing abnormal subchondral bone deterioration and cartilage degradation in OA and reverses the vicious cycle related to osteoarthritis.

Inhibitors of metalloprotease, γ-sectretase, protein kinase C and Rho kinase inhibit wild-type adenoviral replication.

Adenoviruses cause upper respiratory infections, conjunctivitis, keratitis, and gastrointestinal illness. These can be fatal in immunocompromised individuals. Adenoviruses have also been engineered into viral vectors to deliver therapeutic genes or induce immunity as vaccine carriers. The success of ocular gene therapy is driven partly by the immunologic and biochemical influences of the intraocular environment. We have shown that versican and hyaluronan modulate adenoviral vector transgene expression through CD44 signaling. Herein we explored the role of these pathways on virus replication and viral protein expression of wild type adenovirus. We report that the addition of vitreous humor (which contains both versican and hyaluronan) increases viral hexon protein levels. Vitreous humor also increased wild type adenovirus DNA replication in vitro. Metalloproteinase and γ-secretase inhibitors, which inhibit CD44 proteolytic activation, blocked adenoviral replication in vitro. Similarly, protein kinase C and RhoA kinase inhibitors, both proteins associated with CD44 mediated pathways, also inhibited wild type adenoviral replication in vitro. Application of metalloproteinase and γ-secretase inhibitors to human conjunctival explants sharply decreased adenoviral vector gene expression. Our results demonstrate that pharmacologic delivery of these inhibitors is easily achievable. The inhibition of these enzymes should be explored as potential therapies of wild type adenoviral infections.

Inhibiting Glycogen Synthase Kinase-3 Mitigates the Hematopoietic Acute Radiation Syndrome in a Sex- and Strain-dependent Manner in Mice.

The Radiation and Nuclear Countermeasures Program at the National Institute of Allergy and Infectious Diseases (NIAID) mandated that medical countermeasures for treating Acute Radiation Syndrome (ARS) must have efficacy when administered at least 24 h after radiation exposure. At this time point, many cells within key target tissues, such as the hematopoietic system and the gastrointestinal (GI) tract, will already be dead. Therefore, drugs that promote the regeneration of surviving cells may improve outcomes. The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) regulates stem and progenitor cell self-renewal and regeneration in the hematopoietic and GI compartments. We tested inhibition of GSK-3ß by SB216763 24 h after total body irradiation (TBI) and sub-total body irradiation (SBI). Here, we show that subcutaneous administration of SB216763 promotes the regeneration of surviving hematopoietic stem/progenitor cells (HSPCs), including myeloid progenitor cells, and improves survival of C57Bl/6 male mice when administered 24 h after TBI. However, these results were not recapitulated in female C57Bl/6 animals, suggesting a sex difference in GSK-3ß signaling in HSPCs. Subcutaneous administration of SB216763 in male mice stimulated activation of Sox2 transcription but failed to induce Sox2 transcription in female C57Bl/6 mice. Using TCF/lef-GFP reporter mice, we examined Wnt signaling in HSPCs of irradiated male and female mice treated with SB216763. GSK-3 inhibition elevated Wnt reporter activity in HSPCs isolated from male but not female mice. SB216763 did not mitigate hematopoietic ARS in males or females of a second strain of wild-type mice, C3H. In addition, administration of SB216763 did not mitigate hematopoietic ARS beyond the currently available standard approved therapy of ciprofloxacin and granulocyte-colony stimulating factor (G-CSF) in male C57Bl/6 mice. Further, SB216763 did not mitigate GI-ARS after SBI in C57Bl/6 male mice. The lack of efficacy in both sexes and multiple strains of mice indicate that SB216763 is not suitable for further drug development as a mitigator of ARS. Our studies demonstrate that activation of Wnt signaling in HSPCs promotes hematopoietic regeneration following radiation exposure, and targeting this pathway downstream of GSK-3ß may mitigate ARS in a sex- and strain-independent manner.

Gö6983 attenuates breast cancer-induced osteolysis by the apoptotic pathway.

Bone metastasis caused by breast cancer leads to significant complications in treatment, and the resulting osteolysis considerably affects patients' overall survival and quality of life. Gö6983 is a broad spectrum protein kinase C inhibitor. In this study, based on our finding that the Gö6983 inhibits osteolysis, we applied Gö6983 to the MDA-MB-231 breast cancer-induced mouse bone metastasis model. And we found that Gö6983 has a strong inhibitory effect on the tumorigenic model of breast cancer by promoting the mitochondrial apoptosis pathway. Our study, therefore, demonstrates that Gö6983 has a potential inhibitory effect on breast cancer-induced osteoclast activation and provides mechanistic insight that may prove useful for designing future treatments.

A Comparative Study on Albumin-Binding Molecules for Targeted Tumor Delivery through Covalent and Noncovalent Approach.

Various types of albumin-binding molecules have been conjugated to anticancer drugs, and these modified prodrugs could be effective in cancer treatments compared to free anticancer drugs. However, the tumor targeting of albumin-binding prodrugs has not been clearly investigated. Herein, we examined the in vitro and in vivo tumor-targeting efficiency of three different albumin-binding molecules including albumin-binding peptide (DICLPRWGCLW: PEP), fatty acid (palmitic acid: PA), and maleimide (MI), respectively. In order to characterize the different targeting efficiency of albumin-binding molecules, PEP, PA, or MI was chemically labeled with near-infrared fluorescence (NIRF) dye, Cy5.5, in resulting PEP-Cy5.5, PA-Cy5.5, and MI-Cy5.5. These NIRF dye-labeled albumin-binding molecules were physically or chemically bound to albumin via gentle incubation in aqueous conditions in vitro. Notably, PA-Cy5.5 with reversible and multivalent binding affinities formed stable albumin complexes, compared to PEP-Cy5.5 and MI-Cy5.5, confirmed via surface plasmon resonance measurement, gel electrophoresis assay, and albumin-bound column-binding test. In tumor-bearing mice model, the different albumin-binding affinities of PA-Cy5.5, PEP-Cy5.5, and MI-Cy5.5 greatly contributed to their tumor-targeting ability. Even though the binding affinity of PEP-Cy5.5 and MI-Cy5.5 to albumin is higher than that of PA-Cy5.5 in vitro, intravenous PA-Cy5.5 showed a higher tumor-targeting efficiency in tumor-bearing mice compared to that of PEP-Cy5.5 and MI-Cy5.5. The reversible and multivalent affinities of albumin-binding molecules to native serum albumin greatly increased the pharmacokinetics and tumor-targeting efficiency in vivo.

Differential regulation of GSK-3ß in spinal dorsal horn and in hippocampus mediated by interleukin-1beta contributes to pain hypersensitivity and memory deficits following peripheral nerve injury.

Accumulating evidence shows that inhibition of glycogen synthase kinase-3beta (GSK-3ß) ameliorates cognitive impairments caused by a diverse array of diseases. Our previous work showed that spared nerve injury (SNI) that induces neuropathic pain causes short-term memory deficits. Here, we reported that GSK-3ß activity was enhanced in hippocampus and reduced in spinal dorsal horn following SNI, and the changes persisted for at least 45 days. Repetitive applications of selective GSK-3ß inhibitors (SB216763, 5 mg/kg, intraperitoneally, three times or AR-A014418, 400 ng/kg, intrathecally, seven times) prevented short-term memory deficits but did not affect neuropathic pain induced by SNI. Surprisingly, we found that the repetitive SB216763 or AR-A014418 induced a persistent pain hypersensitivity in sham animals. Mechanistically, both ß-catenin and brain-derived neurotrophic factor (BDNF) were upregulated in spinal dorsal horn but downregulated in hippocampus following SNI. Injections of SB216763 prevented the BDNF downregulation in hippocampus but enhanced its upregulation in spinal dorsal horn in SNI rats. In sham rats, SB216763 upregulated both ß-catenin and BDNF in spinal dorsal horn but affect neither of them in hippocampus. Finally, intravenous injection of interleukin-1beta that induces pain hypersensitivity and memory deficits mimicked the SNI-induced the differential regulation of GSK-3ß/ß-catenin/BDNF in spinal dorsal horn and in hippocampus. Accordingly, the prolonged opposite changes of GSK-3ß activity in hippocampus and in spinal dorsal horn induced by SNI may contribute to memory deficits and neuropathic pain by differential regulation of BDNF in the two regions. GSK-3ß inhibitors that treat cognitive disorders may result in a long-lasting pain hypersensitivity.

Efficacy and safety of NAD+ ADP-ribosyltransferase 1 agonist versus Donepezil in elderly Chinese patients with Alzheimer disease: A novel target for effective therapy.

This pilot study designed to evaluate the efficacy and safety of NAD+ ADP-ribosyl transferase 1 (NART) agonist in comparison with Donepezil (DNP) in elderly Chinese patients with Alzheimer disease (AD). In the present clinical trial, Chinese elderly patients aged >65 years with a confirmed diagnosis of AD were enrolled. The patients received NART agonist (test, DAG-structured PKC blockers (GF109203X)) or DNP 10mg daily (reference) for 6 months. The efficacy and safety data were collected from 120 patients (60 patients in each group) every 3 weeks until 6 months. The primary endpoints were to assess the change in cognitive score from baseline in both the treatment group. The result of the present study showed that the patients treated with DNP and NART agonist have similar efficacy and safety profile. Considering the clinical benefit, improvement in sign and symptoms of was numerically greater in DNP-treated patients as compared to NART agonist. However, a statistical difference in terms of clinical benefit was similar between both the treatment groups. Overall, both the study drugs were found comparable in relieving the symptoms of AD. This indicates that NART is a potential target for the treatment of AD in China. The results of the present study may help to design a large clinical trial to evaluate the efficacy and safety of NART agonist in comparison with DNP in AD patients.

GSK-3 inhibitors enhance TRAIL-mediated apoptosis in human gastric adenocarcinoma cells.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Reducing this resistance might shed light on the treatment of human gastric adenocarcinoma. In this study, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors can restore TRAIL responsiveness in gastric adenocarcinoma cells. The effect of two GSK-3 inhibitors, SB-415286, and LiCl, on apoptosis signaling of TRAIL in human gastric adenocarcinoma cell lines and primary gastric epithelial cells was analyzed. Both inhibitors can sensitize gastric adenocarcinoma cells, but not primary gastric epithelial cells, to TRAIL-induced apoptosis by increasing caspase-8 activity and its downstream signal transmission. Adding p53 siRNA can downregulate GSK-3 inhibitor-related sensitization to TRAIL-induced apoptosis and caspase-3 activity. GSK-3 inhibitors strongly activate the phosphorylation of JNK. Inhibition of JNK leads to earlier and more intense apoptosis, showing that the activation of JNK may provide anti-apoptotic equilibrium of pro-apoptotic cells. Our observations indicate that GSK-3 inhibitors can sentize AGS gastric adenocarcinoma cells to TRAIL-induced apoptosis. Therefore, in certain types of gastric adenocarcinoma, GSK-3 inhibitor might enhance the antitumor activity of TRAIL and mightbe a promising candidate for the treatment of certain types of gastric adenocarcinoma.

Wip1 knockout inhibits neurogenesis by affecting the Wnt/ß-catenin signaling pathway in focal cerebral ischemia in mice.

Neurogenesis correlates closely with the recovery of neural function after brain ischemia but the critical proteins and signaling pathways involved remain unclear. The phosphatase WIP1 has been shown to regulate neurogenesis in models of aging. However, it is not known if WIP1 affects neurogenesis and functional recovery after brain ischemia. To explore these questions, we performed permanent middle cerebral artery occlusion (MCAO) in mice and performed BrdU labeling, neurobehavioral testing, western blotting, and immunofluorescence staining. We found that ischemia induced WIP1 expression in the area bordering the injury. Compared to wild-type mice, the knockout of the Wip1 gene inhibited neurological functional recovery, reduced the expression of doublecortin, and inactivated the Wnt/ß-Catenin signaling pathway in cerebral ischemia in mice. Pharmacological activation of the Wnt/ß-Catenin signaling pathway compensated for the Wip1 knockout-induced deficit in neuroblast formation in animals with MCAO. These findings indicate that WIP1 is essential for neurogenesis after brain injury by activating the Wnt/ß-Catenin signaling pathway.

IPEM topical report: the first UK survey of dose indices from radiotherapy treatment planning computed tomography scans for adult patients.

CT scans are an integral component of modern radiotherapy treatments, enabling the accurate localisation of the treatment target and organs-at-risk, and providing the tissue density information required for the calculation of dose in the treatment planning system. For these reasons, it is important to ensure exposures are optimised to give the required clinical image quality with doses that are as low as reasonably achievable. However, there is little guidance in the literature on dose levels in radiotherapy CT imaging either within the UK or internationally. This IPEM topical report presents the results of the first UK wide survey of dose indices in radiotherapy CT planning scans. Patient dose indices were collected for prostate, gynaecological, breast, lung 3D, lung 4D, brain and head and neck scans. Median values per scanner and examination type were calculated and national dose reference levels and 'achievable levels' of CT dose index (CTDIvol), dose-length-product (DLP) and scan length are proposed based on the third quartile and median values of these distributions, respectively. A total of 68 radiotherapy CT scanners were included in this audit. The proposed dose reference levels for CTDIvol and DLP are; prostate 16 mGy and 570 mGy · cm, gynaecological 16 mGy and 610 mGy · cm, breast 10 mGy and 390 mGy · cm, lung 3D 14 mGy and 550 mGy · cm, lung 4D 63 mGy and 1750 mGy · cm, brain 50 mGy and 1500 mGy · cm and head and neck 49 mGy and 2150 mGy · cm. Significant variations in dose indices were noted, with head and neck and lung 4D yielding a factor of eighteen difference between the lowest and highest dose scanners. There was also evidence of some clustering in the data by scanner manufacturer, which may be indicative of a lack of local optimisation of individual systems to the clinical task. It is anticipated that providing this data to the UK and wider radiotherapy community will aid the optimisation of treatment planning CT scan protocols.

Tuning the Diels-Alder Reaction for Bioconjugation to Maleimide Drug-Linkers.

The thiol-maleimide linkage is widely used for antibody-drug conjugate (ADC) production; however, conjugation of maleimide-drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels-Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol-maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.

Antiproliferative and apoptotic effect of LY2090314, a GSK-3 inhibitor, in neuroblastoma in vitro.

BACKGROUND: Neuroblastoma (NB) is a devastating disease. Despite recent advances in the treatment of NB, about 60% of high-risk NB will have relapse and therefore long-term event free survival is very minimal. We have reported that targeting glycogen synthase kinase-3 (GSK-3) may be a potential strategy to treat NB. Consequently, investigating LY2090314, a clinically relevant GSK-3 inhibitor, on NB cellular proliferation and may be beneficial for NB treatment. METHODS: The effect of LY2090314 was compared with a previously studied GSK-3 inhibitor, Tideglusib. Colorimetric, clonogenic, and live-cell image confluency assays were used to study the proliferative effect of LY2090314 on NB cell lines (NGP, SK-N-AS, and SH-SY-5Y). Western blotting and caspase glo assay were performed to determine the mechanistic function of LY2090314 in NB cell lines. RESULTS: LY2090314 treatment exhibited significant growth reduction starting at a 20 nM concentration in NGP, SK-N-AS, and SH-SY-5Y cells. Western blot analysis indicated that growth suppression was due to apoptosis as evidenced by an increase in pro-apoptotic markers cleaved PARP and cleaved caspase-3 and a reduction in the anti-apoptotic protein, survivin. Further, treatment significantly reduced the level of cyclin D1, a key regulatory protein of the cell cycle and apoptosis. Functionally, this was confirmed by an increase in caspase activity. LY2090314 treatment reduced the expression levels of phosphorylated GSK-3 proteins and increased the stability of ß-catenin in these cells. CONCLUSIONS: LY2090314 effectively reduces growth of both human MYCN amplified and non-amplified NB cell lines in vitro. To our knowledge, this is the first study to look at the effect of LY2090314 in NB cell lines. These results indicate that GSK-3 may be a therapeutic target for NB and provide rationale for further preclinical analysis using LY2090314.

2-fluoro-5-maleimidobenzoic acid-linked albumin drug (MAD) delivery for selective systemic targeting of metastatic prostate cancer.

BACKGROUND: The SH-group at Cys-34 of human serum albumin (HSA) is a unique and accessible functional group that can be exploited for efficient linkage of a maleimide containing cytotoxic drug derivative to albumin. The specific maleimide chemistry used for production of the maleimide-linked albumin drug (MAD) is critical, however, to minimize the plasma concentration of "free" cytotoxic drug spontaneously released from albumin carrier thus decreasing dose-limiting host toxicity while enhancing the plasma half-life from minutes to days (ie, pharmacokinetic effect) and tissue concentration of the MAD in the extracellular cellular fluid at sites of cancer (ie, EPR effect). METHODS: To accomplish this goal, a chemical synthesis was developed using 2-fluoro-5-maleimidobenzoic acid to stably link the potent cytotoxic chemically modified analogue of the naturally occurring sesquiterpene γ-lactone, thapsigargin, 8-O-(12-aminododecanoyl)-8-O-debutanoyl thapsigargin (12ADT), to Cys-34 of albumin to produce 12ADT-MAD. RESULTS: Using FITC-labeling, LC/MS analysis, and in vitro growth and clonogenic survival assays on a series of 6 human prostate cancer lines (LNCaP, LAPC-4, VCap, CWR22Rv 1, PC3, and Du145), we documented that 12ADT-MAD is endocytosed by prostate cancer cells where it is degraded into its amino acids liberating cysteinyl-maleimide-12ADT which is both chemically stable at the acidic pH of 5.5 present in the endosome while retaining its high killing ability (IC50 50 nM) via SERCA inhibition. CONCLUSIONS: Based upon these positive in vitro validation results, the in vivo efficacy versus host toxicity of this 12-ADT-MAD approach is presently being evaluated against a series of patient derived androgen responsive and castration resistant human xenografts in immune-deficient mice.

Bisindolylmaleimide alkaloid BMA-155Cl induces autophagy and apoptosis in human hepatocarcinoma HepG-2 cells through the NF-κB p65 pathway.

Bisindolylmaleimides, a series of derivatives of a PKC inhibitor staurosporine, exhibit potential as anti-cancer drugs and have received considerable attention in clinical trials. This study aims to investigate the effects of a bisindolylmaleimide alkaloid 155Cl (BMA-155Cl) with a novel structure on autophagy and apoptosis in human hepatocarcinoma HepG-2 cells in vitro and in vivo. The cell poliferation was assessed with a MTT assay. Autophagy was evaluated by MDC staining and TEM analysis. Apoptosis was investigated using Annexin V-FITC/PI and DAPI staining. The antitumor effects were further evaluated in nude mice bearing HepG-2 xenografts, which received BMA-155Cl (10, 20 mg/kg, ip) for 18 days. Autophagy- and apoptosis-associated proteins and their mRNA levels were examined with Western blotting, immunohistochemistry, and RT-PCR. BMA-155Cl (2.5-20 µmol/L) inhibited the growth of HepG-2 cells with IC50 values of 16.62±1.34, 12.21±0.83, and 8.44±1.82 µmol/L at 24, 48, and 72 h, respectively. Furthermore, BMA-155Cl (5-20 µmol/L) dose-dependently induced autophagy and apoptosis in HepG-2 cells. The formation of autophagic vacuoles was induced by BMA-155Cl (10 µmol/L) at approximately 6 h and peaked at approximately 15 h. Pretreatment with 3-MA potentiated BMA-155Cl-mediated apoptotic cell death. This compound dose-dependently increased the mRNA and protein levels of Beclin-1, NF-κB p65, p53, and Bax, but decreased the expression of IκB and Bcl-2. Pretreatment with BAY 11-7082, a specific inhibitor of NF-κB p65, blocked BMA-155Cl-induced expression of autophagy- and apoptosis-associated proteins. BMA-155Cl administration effectively suppressed the growth of HepG-2 xenografts in vivo, and increased the protein expression levels of LC3B, Beclin-1, NF-κB p65, and Bax in vivo. We conclude that the NF-κB p65 pathway is involved in BMA-155Cl-triggered autophagy, followed by apoptosis in HepG-2 cells in vitro and in vivo. Hence, BMA-155Cl could be a promising pro-apoptotic candidate for developing as a novel anti-cancer drug.

Identification of Bisindolylmaleimide IX as a potential agent to treat drug-resistant BCR-ABL positive leukemia.

Chronic myeloid leukemia (CML) treatment with BCR-ABL inhibitors is often hampered by development of drug resistance. In a screen for novel chemotherapeutic drug candidates with genotoxic activity, we identified a bisindolylmaleimide derivative, IX, as a small molecule compound with therapeutic potential against CML including drug-resistant CML. We show that Bisindolylmaleimide IX inhibits DNA topoisomerase, generates DNA breaks, activates the Atm-p53 and Atm-Chk2 pathways, and induces cell cycle arrest and cell death. Interestingly, Bisindolylmaleimide IX is highly effective in targeting cells positive for BCR-ABL. BCR-ABL positive cells display enhanced DNA damage and increased cell cycle arrest in response to Bisindolylmaleimide IX due to decreased expression of topoisomerases. Cells positive for BCR-ABL or drug-resistant T315I BCR-ABL also display increased cytotoxicity since Bisindolylmaleimide IX inhibits B-Raf and the downstream oncogene addiction pathway. Mouse cancer model experiments showed that Bisindolylmaleimide IX, at doses that show little side effect, was effective in treating leukemia-like disorders induced by BCR-ABL or T315I BCR-ABL, and prolonged the lifespan of these model mice. Thus, Bisindolylmaleimide IX presents a novel drug candidate to treat drug-resistant CML via activating BCR-ABL-dependent genotoxic stress response and inhibiting the oncogene addiction pathway activated by BCR-ABL.

Combination of long-acting HIV fusion inhibitor albuvirtide and LPV/r showed potent efficacy in HIV-1 patients.

BACKGROUND: Long acting antiretroviral drugs represent a promising approach for chronic treatment of HIV infection. Here, we study the efficacy and safety of albuvirtide (ABT), an HIV-1 fusion inhibitor with a half life of 11-12 days in human. METHODS: ABT was evaluated in a 7-week, open-label and randomized trial, combining with LPV/r. Twenty HIV-1-infected adults were assigned to two dose groups, receiving ABT (160 or 320 mg) given weekly and LPV/r given twice daily. RESULTS: At week 7, the decline of HIV-1 RNA from baseline was 1.9 (1.3-2.3) log10 and 2.2 (1.6-2.7) log10 copies/ml, and suppression of HIV-1 RNA to below 50 copies/ml was achieved in 11.1 % (1/9) and 55.6 % (5/9) patients, for the 160 and 320 mg dose group respectively. CONCLUSION: A clear dose-efficacy correlation of ABT was demonstrated. ABT combining with LPV/r is a promising two-drug regimen to be tested in larger patient population.