Role of Mitogen-activated protein kinase kinase kinase kinase 4 signaling in liver and metabolic diseases.

MAP4K4 is a serine/threonine protein kinase belonging to the germinal center kinase (GCK) sub-group of sterile 20 protein (Ste20p) family of kinases. MAP4K4 has been involved in regulating multiple biological processes and a plethora of pathologies, including systemic inflammation, cardiovascular diseases, cancers, metabolic and hepatic diseases. Recently, multiple reports have indicated the upregulation of MAP4K4 expression and signaling in hyperglycemia and liver diseases. This review summarizes our current understanding of MAP4K4 structure, expression, as well as its regulation and signaling, specifically in metabolic and hepatic diseases. Reviewing these promising studies will enrich our understanding of MAP4K4 signaling which will help us design innovative therapeutic interventions against metabolic and liver diseases using MAP4K4 as a target. Significance Statement Although most studies on the involvement of MAP4K4 in human pathologies are related to cancers, only recently its role in liver and other metabolic diseases is beginning to unravel. This mini review discusses recent advancements in MAP4K4 signaling and comprehensively characterizes MAP4K4 as a clinically relevant therapeutic target against liver diseases.

Advancements in metabolic-associated steatotic liver disease research: Diagnostics, small molecule developments, and future directions.

Metabolic (dysfunction)-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease, is a growing global health concern with no approved pharmacological treatments. At the same time, there are no standard methods to definitively screen for the presence of MASLD because of its progressive nature and symptomatic commonality with other disorders. Recent advances in molecular understanding of MASLD pathophysiology have intensified research on development of new drug molecules, repurposing of existing drugs approved for other indications, and an educated use of dietary supplements for its treatment and prophylaxis. This review focused on depicting the latest advancements in MASLD research related to small molecule development for prophylaxis or treatment and diagnosis, with emphasis on mechanistic basis at the molecular level.

Evaluation of anti-inflammatory diphenyldihaloketone EF24 in transient ischemic stroke model.

OBJECTIVES: Revascularization is necessary in patients with ischemic stroke, however it does not address inflammation that contribute to reperfusion injury and the early growth of ischemic core. We investigated EF24, an anti-inflammatory agent, in a stroke model. METHODS: Ischemic stroke was induced in mice by occluding middle cerebral artery for 1 h followed by reperfusion. EF24 was given either 10 min post-reperfusion (EF24Post) or 10 min before occlusion (prophylactic, EF24Pro). Survival, ipsilateral uptake of radioactive infarct marker 18F-fluoroglucaric acid (FGA), inflammatory cytokines, and tetrazolium chloride (TTC) staining were assessed. RESULTS: Survival was increased in both EF24-treated groups compared to the stroke+vehicle group. Ipsilateral 18F-FGA uptake increased 2.6-fold in stroke+vehicle group compared to sham group (p < 0.05); the uptake in EF24-treated groups and sham group was not significantly different. TTC-staining also showed reduction in infarct size by EF24 treatment. Plasma IL-6, TNF-α, and corticosterone did not show significant changes among groups. However, ipsilateral tissue in stroke+vehicle mice showed increased IL-6 (>90-fold) and TNF-α (3-fold); the tissue IL-6 and TNF-α were significantly reduced in stroke+EF24Pro and stroke+EF24Post groups. 18F-FGA uptake significantly correlated with tissue IL-6 levels. CONCLUSIONS: EF24 controls infarct growth and suppresses tissue inflammation in ischemic stroke, which can be monitored by 18F-FGA uptake.

Positron Emission Tomography (PET) with 18F-FGA for Diagnosis of Myocardial Infarction in a Coronary Artery Ligation Model.

Location and extent of necrosis are valuable information in the management of myocardial infarction (MI). Methods. We investigated 2-deoxy-2-18F-fluoro glucaric acid (FGA), a novel infarct-avid agent, for positron emission tomography (PET) of MI. We synthesized FGA from commercially available 18F-fluoro-2-deoxy-2-D-glucose (FDG). MI was induced in mice by permanently occluding the left anterior descending coronary artery. Biodistribution of FGA was assessed 1 h after FGA injection (11 MBq). PET/CT was conducted 1 h, 6 h, 1 d, 3 d, and 4 d after MI. Subcellular compartment of FGA accumulation in necrosis was studied by tracing the uptake of biotin-labeled glucaric acid with streptavidin-HRP in H2O2-treated H9c2 cardiomyoblasts. Streptavidin-reactive protein bands were identified by LC-MS/MS. Results. We obtained a quantitative yield of FGA from FDG within 7 min (radiochemical purity > 99%). Cardiac uptake of FGA was significantly higher in MI mice than that in control mice. Imaging after 1 h of FGA injection delineated MI for 3 days after MI induction, with negligible background signal from surrounding tissues. Myocardial injury was verified by tetrazolium staining and plasma troponin (47.63 pg/mL control versus 311.77 pg/mL MI). In necrotic H9c2 myoblasts, biotinylated glucaric acid accumulated in nuclear fraction. LC-MS/MS primarily identified fibronectin in necrotic cells as a putative high fidelity target of glucaric acid. Conclusion. FGA/PET detects infarct early after onset of MI and FGA accumulation in infarct persists for 3 days. Its retention in necrotic cells appears to be a result of interaction with fibronectin that is known to accumulate in injured cardiac tissue.

Design of 99mTc-labeled zinc-chelating imaging probe for SPECT imaging of the pancreas.

Early and sensitive diagnosis of pancreatic diseases is a contemporary clinical challenge. Zinc level in pancreatic tissue and its secretion in pancreatic juice has long been considered a surrogate marker of pancreatic function. The objective of this study was to design a Zn-chelating imaging probe (ZCIP) which could be labeled with 99mTc radionuclide for imaging of pancreas using single photon emission tomography (SPECT). We synthesized ZCIP as a bifunctional chelate consisting of diethylene triamine pentaacetic acid for 99mTc-chelation at one end and bispicolylethylamine for Zn-complexation at the other end. ZCIP was labeled with 99mTc by standard Sn2+-based reduction method. The 99mTc-labeled ZCIP was studied in normal mice (0.3 mCi) for SPECT imaging. We found that ZCIP consistently labeled with 99mTc radionuclide with over 95% efficiency. Addition of ZCIP altered the spectrum of standard dithizone-Zn complex, indicating its ability to chelate Zn. SPECT data demonstrated the ability of 99mTc-ZCIP to image pancreas with high sensitivity in a non-invasive manner; liver and spleen were the other major organs of 99mTc-ZCIP uptake. Based on these results, we conclude that 99mTc-ZCIP presents as a novel radiotracer for pancreas imaging for diagnosis of diseases such as pancreatitis.

Mechanism of Anti-Inflammatory Activity of TLR4-Interacting SPA4 Peptide.

The TLR4-interacting SPA4 peptide suppresses inflammation. We assessed the structural and physicochemical properties and binding of SPA4 peptide to TLR4-MD2. We also studied the changes at the whole transcriptome level, cell morphology, viability, secreted cytokines and chemokines, and cell influx in cell systems and mouse models challenged with LPS and treated with SPA4 peptide. Our results demonstrated that the SPA4 peptide did not alter the cell viability and size and only moderately affected the transcriptome of the cells. Computational docking and rendering suggested that the SPA4 peptide intercalates with LPS-induced TLR4-MD2 complex. Results with alanine mutations of D-2 amino acid and NYTXXXRG-12-19 motif of SPA4 peptide suggested their role in binding to TLR4 and in reducing the cytokine response against LPS stimulus. Furthermore, therapeutically administered SPA4 peptide significantly suppressed the secreted levels of cytokines and chemokines in cells and bronchoalveolar lavage fluids of LPS-challenged mice. The results suggest that the SPA4 peptide intercalates with LPS-induced TLR4 complex and signaling for the suppression of inflammation.

Biodegradable Polymeric Nanoparticles for Drug Delivery to Solid Tumors.

Advances in nanotechnology have favored the development of novel colloidal formulations able to modulate the pharmacological and biopharmaceutical properties of drugs. The peculiar physico-chemical and technological properties of nanomaterial-based therapeutics have allowed for several successful applications in the treatment of cancer. The size, shape, charge and patterning of nanoscale therapeutic molecules are parameters that need to be investigated and modulated in order to promote and optimize cell and tissue interaction. In this review, the use of polymeric nanoparticles as drug delivery systems of anticancer compounds, their physico-chemical properties and their ability to be efficiently localized in specific tumor tissues have been described. The nanoencapsulation of antitumor active compounds in polymeric systems is a promising approach to improve the efficacy of various tumor treatments.

Tumor suppressive activities of solvatochromic 3,3'-azadimethylene dinaphthospiropyran in colon cancer model.

Spiropyrans have been extensively investigated because of their thermo- and photochromic characteristics, but their biotherapeutic properties have not been explored much. We report anti-proliferative properties of a novel 3,3'-azadimethylene dinaphthospiropyran 11. Dibenzospiropyrans and dinaphthospiropyrans were synthesized by a simple and expedient method using acid-catalyzed aldol condensation of salicylaldehyde and 2-hydroxy-1-naphthaldehyde, respectively, with cyclic ketones. Together with structural elucidation by 2D NMR and X-ray crystallography studies, we provide a putative mechanism for their formation. Compound 11 showed solvatochromism and exhibited altered spectral characteristics depending on the pH. In acidic conditions, 11 remains in open form, whereas upon alkalinization it reverts back to closed form. Based on the in vitro anti-proliferative activity in H441, HCT-116, MiaPaCa-2, and Panc-1 cancer cell lines, 11 was submitted to further investigation. It reduced HCT116 colonosphere formation and demonstrated induction of caspase cascade, suggesting apoptosis. In vitro proliferation assays also suggested that HCl and trifluoroacetate salts of 11 are more effective. Treatment of mice carrying HCT-116 xenografts with 11 (5 µg/day, intraperitoneal for 3 weeks) suppressed tumor growth by 62%. Overall, the results reveal a new series of structurally complex, but relatively easy to synthesize molecules of which compound 11 represents a lead for anticancer development.

Tubulin inhibitory activity of a novel colchicine-binding compounds based on a dinaphthospiropyranran scaffold.

Spiropyrans have been investigated for their thermo- and photochromic characteristics, but their biotherapeutic properties have not been addressed. We report anti-proliferative properties of a novel dinaphthospiropyran analogue (1). The compound 1 was synthesized by a simple and expedient method using a one-pot acid-catalyzed aldol condensation of 2-hydroxy-1-naphthaldehyde with 4-piperidone followed by an acetalization reaction. Compound 1 was submitted to anticancer drug screen in the National Cancer Institute's panel of 60 human tumor cell lines. The average concentration of 1 to inhibit 50% cell growth was 5.4 ± 0.23 µM. All cell lines responded at almost the same concentration, suggesting that the action of 1 is not selective for cancer of origin. COMPARE analysis of dose-response data revealed interaction with tubulin as the possible mechanism of action of 1. At molecular level, 1 induced tubulin reorganization in colon cancer HCT-116 cells. Under cell-free conditions, the efficacy of 1 to inhibit tubulin polymerization was comparable to that of paclitaxel and vinblastine. Molecular docking showed that compound 1 binds to the colchicine-binding site of tubulin. We conclude that dinaphthospiropyrans present a novel scaffold for the development of tubulin inhibitors.

Nanoparticles for Targeting of Prostate Cancer.

Prostate cancer (PCa) is the leading cause of death by cancer in men. Because of the drastic decline in the survival rate of PCa patients with advanced/metastatic disease, early diagnosis of disease and therapy without toxic side effects is crucial. Chemotherapy is widely used to control the progression of PCa at the later stages; however, it is associated with off-target toxicities and severe adverse effects due to the lack of specificity. Delivery of therapeutic or diagnostic agents by using targeted nanoparticles is a promising strategy to enhance accuracy and sensitivity of diagnosis of PCa and to increase efficacy and specificity of therapeutic agents. Numerous efforts have been made in past decades to create nanoparticles with different architectural bases for specific delivery payloads to prostate tumors. Major PCa associated cell membrane protein markers identified as targets for such purposes include folate receptor, sigma receptors, transferrin receptor, gastrin-releasing peptide receptor, urokinase plasminogen activator receptor, and prostate specific membrane antigen. Among these markers, prostate specific membrane antigen has emerged as an extremely specific and sensitive targetable marker for designing targeted nanoparticle-based delivery systems for PCa. In this article, we review contemporary advances in design, specificity, and efficacy of nanoparticles functionalized against PCa. Whenever feasible, both diagnostic as well as therapeutic applications are discussed.

Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide.

BACKGROUND: A surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model. METHODS: The outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625-10 µg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 µg LPS/g body weight and treated with 50 µg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test. RESULTS: The results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation. CONCLUSIONS: These findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 µg SPA4 peptide, comparable to 2.5 µg/g body weight, alleviates LPS-induced lung inflammation.

PET Detection of Cerebral Necrosis Using an Infarct-Avid Agent 2-Deoxy-2-[18F]Fluoro-D-Glucaric Acid (FGA) in a Mouse Model of the Brain Stroke.

PURPOSE: Ischemic stroke is a leading cause of disability worldwide. The volume of necrotic core in affected tissue plays a major role in selecting stroke patients for thrombolytic therapy or endovascular thrombectomy. In this study, we investigated a recently reported positron emission tomography (PET) agent 2-deoxy-2-[18F]fluoro-D-glucaric acid (FGA) to determine necrotic core in a model of transient middle cerebral artery occlusion (t-MCAO) in mice. PROCEDURES: The radiopharmaceutical, FGA, was synthesized by controlled, rapid, and quantitative oxidation of clinical doses of 2-deoxy-2-[18F]fluoro-D-glucose (FDG) in a one-step reaction using a premade kit. Brain stroke was induced in the left cerebral hemisphere of CD-1 mice by occluding the middle cerebral artery for 1 h, and then allowing reperfusion by removing the occlusion. One day post-ictus, perfusion single-photon emission tomography (SPECT) was performed with 99mTc-lableled hexamethylpropyleneamine oxime (HMPAO), followed by PET acquisition with FGA. Plasma and brain tissue homogenates were assayed for markers of inflammation and neurotrophins. RESULTS: The kit-based synthesis was able to convert up to 2.2 GBq of FDG into FGA within 5 min. PET images showed 375 % more accumulation of FGA in the ipsilateral hemisphere than in the contralateral hemisphere. SPECT images showed that the ipsilateral HMPAO accumulation was reduced to 55 % of normal levels; there was a significant negative correlation between the ipsilateral accumulation of FGA and HMAPO (p < 0.05). FGA accumulation in stroke also correlated with IL-6 levels in the ipsilateral hemisphere. There was no change in IL-6 or TNFα in the plasma of stroke mice. CONCLUSIONS: Accumulation of FGA correlated well with the perfusion defect and inflammatory injury. As a PET agent, FGA has potential to image infarcted core in the brain stroke injury with high sensitivity, resolution, and specificity.

Drug-induced cardiomyopathy: Characterization of a rat model by [18F]FDG/PET and [99mTc]MIBI/SPECT.

BACKGROUND: Drug-induced cardiomyopathy is a significant medical problem. Clinical diagnosis of myocardial injury is based on initial electrocardiogram, levels of circulating biomarkers, and perfusion imaging with single photon emission computed tomography (SPECT). Positron emission tomography (PET) is an alternative imaging modality that provides better resolution and sensitivity than SPECT, improves diagnostic accuracy, and allows therapeutic monitoring. The objective of this study was to assess the detection of drug-induced cardiomyopathy by PET using 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and compare it with the conventional SPECT technique with [99mTc]-Sestamibi (MIBI). METHODS: Cardiomyopathy was induced in Sprague Dawley rats using high-dose isoproterenol. Nuclear [18F]FDG/PET and [99mTc]MIBI/SPECT were performed before and after isoproterenol administration. [18F]FDG (0.1 mCi, 200-400 µL) and [99mTc]MIBI (2 mCi, 200-600 µL) were administered via the tail vein and imaging was performed 1 hour postinjection. Isoproterenol-induced injury was confirmed by the plasma level of cardiac troponin and triphenyltetrazolium chloride (TTC) staining. RESULTS: Isoproterenol administration resulted in an increase in circulating cardiac troponin I and showed histologic damage in the myocardium. Visually, preisoproterenol and postisoproterenol images showed alterations in cardiac accumulation of [18F]FDG, but not of [99mTc]MIBI. Image analysis revealed that myocardial uptake of [18F]FDG reduced by 60% after isoproterenol treatment, whereas that of [99mTc]MIBI decreased by 45%. CONCLUSION: We conclude that [18F]FDG is a more sensitive radiotracer than [99mTc]MIBI for imaging of drug-induced cardiomyopathy. We theorize that isoproterenol-induced cardiomyopathy impacts cellular metabolism more than perfusion, which results in more substantial changes in [18F]FDG uptake than in [99mTc]MIBI accumulation in cardiac tissue.

Anti-inflammatory mediators ST2 and SIGIRR are induced by diphenyldifluoroketone EF24 in lipopolysaccharide-stimulated dendritic cells.

The objective of this study was to investigate the effect of EF24, an NF-κB-inhibitor, on the expression of negative regulators in IL-1R pathway, namely ST2 and SIGIRR. Murine JAWS II dendritic cells (DC) were challenged with lipopolysaccharide (LPS, 100 ng/ml) for 4 h, followed by treatment with 10 µM EF24 for 1 h. ST2 and SIGIRR expression was monitored by qRT-PCR and immunoblotting. ST2L and MyD88 interaction was studied by co-immunoprecipitation, and IL-33, a ST2L ligand, was assayed by ELISA. Activation of transcription factor SP1 was examined by confocal microscopy, immunoblotting, and EMSA. The effect of EF24 on accumulation of ubiquitinated proteins in DCs and proteolysis of fluorogenic peptides by purified proteasome was studied. We found that EF24 upregulated the expression of ST2 and SIGIRR and decreased the interaction of the membrane-bound ST2 (ST2L) with MyD88, and significantly reduced IL-33 levels in LPS-stimulated DCs. Simultaneously it increased the activation of transcription factor SP1and restored the basal level of ubiquitinated proteins in LPS-stimulated DCs. Moreover, EF24 inhibited trypsin- and chymotrypsin-like activity of proteasome by directly interacting with 26S proteasome. The results suggest that EF24 activates endogenous anti-inflammatory arm of IL-1R signaling, most likely by stabilizing SP1 against proteasomal degradation.

Anchoring Property of a Novel Hydrophilic Lipopolymer, HDAS-SHP, Post-Inserted in Preformed Liposomes.

Polyethylene glycol (PEG)-phospholipids in long-circulating liposomes cause non-specific immune reactions; mainly attributable to negatively-charged phosphoryl s at the interface of PEG and phospholipid. We investigated a novel lipopolymer, by which a superhydrophilic polymer (SHP) is conjugated to a non-phospholipid N1-(2-aminoethyl)-N4-hexadecyl-2-tetradecylsuccinamide (HDAS). The modification of preformed liposomes HDAS-SHP, HDAS-PEG2000, and DSPE-PEG2000 were performed by post-insertion techniques. The efficiency of post-insertion and desorption rates, from the liposome surface, were determined. HDAS-SHP micelles showed highly positive zeta potential (+28.4 mV); zeta potentials of DSPE-PEG2000 and HDAS-PEG2000 micelles were -34.4 mV, and -3.7 mV, respectively. Critical micelle concentration predicted amphiphilicity of HDAS-SHP (CMC 2.58 µM) as close to that of DSPE-PEG2000 (CMC 2.44 µM). Both HDAS-SHP and HDAS-PEG2000 post-inserted with comparable efficiency (79%, and 73%, respectively), but noticeably lower than DSPE-PEG2000 (90%). The desorption rate of HDAS-SHP was close to that of DSPE-PEG2000 (0.53%/h, and 0.45%/h, respectively); the desorption rate for HDAS-PEG2000 was slightly more at 0.67%/h. Compared to plain liposomes, both HDAS-SHP- and DSPE-PEG2000-liposomes showed significant leakage of encapsulated Na-fluorescein isothiocyanate (FITC) upon incubation with serum. At the same time, both modified liposomes were found to suppress serum levels of the complement proteins, Bb and C4d. We infer that HDAS-SHP is a viable alternative to commonly-used PEG-phospholipid derivatives for stealth purposes.

Production of [13N]ammonia from [13C]methanol on a 7.5 MeV cyclotron using 13C(p, n)13N reaction: Detection of myocardial infarction in a mouse model.

[13N]Ammonia is commonly produced using 16O(p, α)13N reaction but one of the limiting factor of this reaction is the relatively small nuclear cross-section at proton energies of <10 MeV. An alternative production method using 13C(p, n)13N reaction, which has a higher nuclear cross-section at low proton energies, is more suitable for a preclinical PET imaging facility equipped with a <10 MeV cyclotron. Here, we report a novel method to produce [13N]ammonia from [13C]methanol for preclinical use on a 7.5 MeV cyclotron. A tantalum solution target (80 µl) consisting of a havar window supplied by the cyclotron manufacturer for the production of [18F]fluoride was used without any modifications. The final bombardment parameters were optimized as follow: [13C]methanol concentration in target solution - 10%, bombardment time - 8 min, and beam current - 2.2 µA. These parameters provided doses of [13N]ammonia which were sufficient to conduct preclinical PET imaging studies in a mouse model of myocardial infarction. Under optimized conditions, the operational lifetime of the target was approximately 150 µAmin. Radionuclide identity of the product as 13N was confirmed by measuring the decay half-life and its radionuclide purity was confirmed by γ-ray spectroscopic analysis. Gas chromatography revealed that the final [13N]ammonia dose was not distinguishable from water, showing no traces of methanol. As expected, PET/CT imaging in healthy CD-1 mice indicated the accumulation of [13N]ammonia in myocardial tissue; mice with myocardial infarction created by left ascending coronary ligation showed clear perfusion deficit in affected tissue. This work demonstrates the proof-of-concept of using 13C(p, n)13N reaction to produce [13N]ammonia from [13C]methanol with a <10 MeV cyclotron, and its diagnostic application in imaging cardiac perfusion.

Surface Modification of Liposomes by a Lipopolymer Targeting Prostate Specific Membrane Antigen for Theranostic Delivery in Prostate Cancer.

Prostate specific membrane antigen (PSMA) is a marker for diagnosis and targeted delivery of therapeutics to advanced/metastasized prostate cancer. We report a liposome-based system for theranostic delivery to PSMA-expressing (PSMA⁺) LNCaP cells. A lipopolymer (P³) comprising of PSMA ligand (PSMAL), polyethylene glycol (PEG2000), and palmitate was synthesized and post-inserted into the surface of preformed liposomes. These P³-liposomes were loaded with doxorubicin and radiolabeled with 99mTc radionuclide to study their theranostic characteristics. Differential expression of PSMA on LNCaP and PC3 cells was confirmed by immunoblotting as well as by uptake of PSMAL labeled with 18F radionuclide. We found that the uptake of 99mTc-labeled P³-liposomes by LNCaP cells was >3-fold higher than 99mTc-labeled Plain-liposomes; the amount of doxorubicin delivered to LNCaP cells was also found to be >3-fold higher by P³-liposomes. Cell-based cytotoxicity assay results showed that doxorubicin-loaded P³-liposomes were significantly more toxic to LNCaP cells (p < 0.05), but not to PSMA-negative PC3 cells. Compared to doxorubicin-loaded Plain-liposomes, the IC50 value of doxorubicin-loaded P³-liposomes was reduced by ~5-fold in LNCaP cells. Together, these results suggest that surface functionalization of liposomes with small PSMA-binding motifs, such as PSMAL, can provide a viable platform for specific delivery of theranostics to PSMA⁺ prostate cancer.

Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome.

The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities.

Induction of gut proteasome activity in hemorrhagic shock and its recovery by treatment with diphenyldihaloketones CLEFMA and EF24.

Multiorgan failure in hemorrhagic shock is triggered by gut barrier dysfunction and consequent systemic infiltration of proinflammatory factors. Our previous study has shown that diphenyldihaloketone drugs 4-[3,5-bis[(2-chlorophenyl)methylene]-4-oxo-1-piperidinyl]-4-oxo-2-butenoic acid (CLEFMA) and 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF24) restore gut barrier dysfunction and reduce systemic inflammatory response in hemorrhagic shock. We investigated the effect of hemorrhagic shock on proteasome activity of intestinal epithelium and how CLEFMA and EF24 treatments modulate proteasome function in hemorrhagic shock. CLEFMA or EF24 (0.4 mg/kg) were given 1 h after withdrawing 50% of blood from Sprague-Dawley rats; no other resuscitation was provided. After another 5 h of compensation, small gut was collected to process tissue for proteasome activity, immunoblotting, and mRNA levels of genes responsible for unfolded-protein response (XBP1, ATF4, glucose-regulated protein of 78/95 kDa, and growth arrest and DNA damage inducible genes 153/34), polyubiquitin B and C, and immunoproteasome subunits ß type-8 and -10 and proteasome activator subunit 1. We found that hemorrhagic shock induced proteasome activity in gut tissue and reduced the amounts of ubiquitinated proteins displayed on antiubiquitin immunoblots. However, simultaneous induction of unfolded-protein response or immunoproteasome genes was not observed. CLEFMA and EF24 treatments abolished the hemorrhagic shock-induced increase in proteasome activity. Further investigations revealed that the induction of proteasome in hemorrhagic shock is associated with disassembly of 26S proteasome; CLEFMA and EF24 prevented this disassembly. Consistent with these data, CLEFMA and EF24 reduced hemorrhagic shock-induced degradation of 20S substrate ornithine decarboxylase in gut tissue. These results suggest that activated proteasome plays an important role in ischemic gut pathophysiology, and it can be a druggable target in shock-induced gut dysfunction. NEW & NOTEWORTHY Ischemic injury to the gut is a trigger for the systemic inflammatory response and multiple organ failure in trauma and hemorrhagic shock. We show for the first time that hemorrhagic shock induces the gut proteasome activity by engendering 26S proteasome disassembly. Diphenyldihaloketones 4-[3,5-bis[(2-chlorophenyl)methylene]-4-oxo-1-piperidinyl]-4-oxo-2-butenoic acid and 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone treatment prevented the 26S disassembly. Understanding the role of proteasome in shock-associated gut injury will assist in the development of therapeutic means to address it.