Cutting Edge Bionics in Highly Impaired Individuals: A Case of Challenges and Opportunities.

Highly impaired individuals stand to benefit greatly from cutting-edge bionic technology, however concurrent functional deficits may complicate the adaptation of such technology. Here, we present a case in which a visually impaired individual with bilateral burn injury amputation was provided with a novel transradial neuromusculoskeletal prosthesis comprising skeletal attachment via osseointegration and implanted electrodes in nerves and muscles for control and sensory feedback. Difficulties maintaining implant hygiene and donning and doffing the prosthesis arose due to his contralateral amputation, ipsilateral eye loss, and contralateral impaired vision necessitating continuous adaptations to the electromechanical interface. Despite these setbacks, the participant still demonstrated improvements in functional outcomes and the ability to control the prosthesis in various limb positions using the implanted electrodes. Our results demonstrate the importance of a multidisciplinary, iterative, and patient-centered approach to making cutting-edge technology accessible to patients with high levels of impairment.

A redox-responsive prodrug for tumor-targeted glutamine restriction.

Modulating the metabolism of cancer cells, immune cells, or both is a promising strategy to potentiate cancer immunotherapy in the nutrient-competitive tumor microenvironment. Glutamine has emerged as an ideal target as cancer cells highly rely on glutamine for replenishing the tricarboxylic acid cycle in the process of aerobic glycolysis. However, non-specific glutamine restriction may induce adverse effects in unconcerned tissues and therefore glutamine inhibitors have achieved limited success in the clinic so far. Here we report the synthesis and evaluation of a redox-responsive prodrug of 6-Diazo-5-oxo-L-norleucine (redox-DON) for tumor-targeted glutamine inhibition. When applied to treat mice bearing subcutaneous CT26 mouse colon carcinoma, redox-DON exhibited equivalent antitumor efficacy but a greatly improved safety profile, particularly, in spleen and gastrointestinal tract, as compared to the state-of-the-art DON prodrug, JHU083. Furthermore, redox-DON synergized with checkpoint blockade antibodies leading to durable cures in tumor-bearing mice. Our results suggest that redox-DON is a safe and effective therapeutic for tumor-targeted glutamine inhibition showing promise for enhanced metabolic modulatory immunotherapy. The approach of reversible chemical modification may be generalized to other metabolic modulatory drugs that suffer from overt toxicity.

Effectiveness of Virtual Reality Training in Teaching Personal Protective Equipment Skills: A Randomized Clinical Trial.

Importance: Training on the proper use of personal protective equipment (PPE) is critical for infection prevention among health care workers. Traditional methods, such as face-to-face and video-based training, can strain resources and present challenges. Objective: To determine the effectiveness of 360° virtual reality (VR) training for PPE donning and doffing compared with face-to-face and video training in enhancing the PPE use skills of prospective health care practitioners. Design, Setting, and Participants: A blinded, prospective, and randomized noninferiority clinical trial was conducted from August to December 2021 at Teikyo University School of Medicine in Tokyo, Japan, with a mixed population of medical students. Participants were second- to fourth-year medicine, medical technology, or pharmacy students aged 20 years or older with no prior PPE training. Participants were randomized into 1 of 3 training groups (VR, face-to-face, or video) based on their enrollment order. An intention-to-treat analysis was conducted. Intervention: A 30-minute lecture on PPE procedures was delivered to all participants before the training. After the lecture, the VR group trained with an immersive 360° VR tool, the face-to-face group trained with actual PPE, and the video group trained by watching video footage on a computer and a projector. After 3 days, a standardized practical skills test was administered. Main Outcomes and Measures: The primary outcome was the mean score on a 20-point practical skills test, and the secondary outcome was the percentage of correct execution. Results: A total of 91 participants were recruited and randomized into 3 groups: VR (n = 30), face-to-face (n = 30), and video (n = 31) training. After excluding 1 participant due to illness, 90 participants (mean [SD] age, 24.2 [3.15] years; 54 males [60.0%]) completed the assessment. The mean (SD) scores were 17.70 (2.10) points for the VR group, 17.57 (2.45) points for the face-to-face group, and 15.87 (2.90) points for the video group. The VR group demonstrated no significant difference in performance from the face-to-face group. However, the VR group had significantly higher effectiveness than the video group (17.70 vs 15.87 points; P = .02). Conclusions and Relevance: Results of this trial indicate that VR training was as effective as face-to-face training in enhancing PPE donning and doffing skills and was superior to video training. The findings suggest that VR training is a viable resource-conserving training option. Trial Registration: Japan Registry of Clinical Trials Identifier: jRCT103021029.

DON of Hope: Starving Pancreatic Cancer by Glutamine Antagonism.

A promising approach to treat solid tumors involves disrupting their reliance on glutamine, a key component for various metabolic processes. Traditional attempts using glutamine inhibitors like 6-diazo-5-oxo-L-norleucine (DON) and CB-839 were unsuccessful, but new hope arises with DRP-104, a prodrug of DON. This compound effectively targets tumor metabolism while minimizing side effects. In a recent study published in Nature Cancer, Encarnación-Rosado and colleagues demonstrated in preclinical models that pancreatic ductal adenocarcinoma (PDAC) responds well to DRP-104, although tumors adapt through the MEK/ERK signaling pathway, which can be countered by the MEK inhibitor trametinib. In a related study, Recouvreux and colleagues found that DON is effective against pancreatic tumors, revealing that PDAC tumors upregulate asparagine synthesis in response to DON, making them susceptible to asparaginase treatment. Both studies underscore the potential of inhibiting glutamine metabolism and adaptive pathways as a promising strategy against PDAC. These findings pave the way for upcoming clinical trials utilizing DRP-104 and similar glutamine antagonists in the battle against solid tumors.

Extremity Tourniquet Self-Application by Antarctica Zodiac Crew Members.

Search and rescue teams and Antarctic research groups use protective cold-water anti-exposure suits (AES) when cruising on Zodiacs. Extremity tourniquet (ET) self-application (SA) donned with AESs has not been previously studied. Our study therefore assessed the SA of 5 commercial ETs (CAT, OMNA, RATS, RMT, and SWAT-T) among 15 volunteers who donned these suits. Tourniquet's SA ability, ease of SA, tolerance, and tourniquet preference were measured. All ETs tested were self-applied to the upper extremity except for the SWAT, which was self-applied with the rest to the lower extremity. Ease- of- SA mean values were compared using the Friedman and Durbin-Conover post hoc tests (P < 0.001). Regarding the upper extremity, OMNA achieved the highest score of 8.5 out of 10, while RMT, and SWAT received lower scores than other options (P < 0.001). For lower extremities, SWAT was found to be inferior to other options (P < 0.01). Overall, OMNA was the best performer. The RATS showed significantly lower tolerance than the other groups in repeated- measures ANOVA with a Tukey post hoc test (P < 0.01). Additionally, out of the 5 ETs tested, 60% of subjects preferred OMNA. The study concluded that SA commercial ETs are feasible over cold-water anti-exposure suits in the Antarctic climate.

Discovery of tert-Butyl Ester Based 6-Diazo-5-oxo-l-norleucine Prodrugs for Enhanced Metabolic Stability and Tumor Delivery.

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) exhibits remarkable anticancer efficacy; however, its therapeutic potential is hindered by its toxicity to gastrointestinal (GI) tissues. We recently reported the discovery of DRP-104, a tumor-targeted DON prodrug with excellent efficacy and tolerability, which is currently in clinical trials. However, DRP-104 exhibits limited aqueous solubility, and the instability of its isopropyl ester promoiety leads to the formation of an inactive M1-metabolite, reducing overall systemic prodrug exposure. Herein, we aimed to synthesize DON prodrugs with various ester and amide promoieties with improved solubility, GI stability, and DON tumor delivery. Twenty-one prodrugs were synthesized and characterized in stability and pharmacokinetics studies. Of these, P11, tert-butyl-(S)-6-diazo-2-((S)-2-(2-(dimethylamino)acetamido)-3-phenylpropanamido)-5-oxo-hexanoate, showed excellent metabolic stability in plasma and intestinal homogenate, high aqueous solubility, and high tumor DON exposures and preserved the ideal tumor-targeting profile of DRP-104. In conclusion, we report a new generation of glutamine antagonist prodrugs with improved physicochemical and pharmacokinetic attributes.

Effectiveness of an artificial intelligence-based training and monitoring system in prevention of nosocomial infections: A pilot study of hospital-based data.

This work describes a novel artificial intelligence-based training and monitoring system (AITMS) that was used to control and prevent nosocomial infections (NIs) by improving the skills of donning/removing personal protective equipment (PPE). The AITMS has two working modes, namely an AI-based protective equipment surveillance mode and an AI-based training mode, that were used for routine surveillance and training, respectively. Data revealed that the accuracy rate of donning/removing PPE improved as a result of the AITMS. Interestingly, the frequency of NIs decreased with the use of the AITMS. This study suggested the key role of using PPE in controlling and preventing NIs. Data preliminarily proved that appropriate donning/removing PPE may help to reduce the risk of NIs. In addition, the newest computerized technologies, such as AI, have proven to be useful in controlling and preventing NIs. These findings should helpful to formulate a better strategy against NIs in the future.

Validation of a portable shoe tread scanner to predict slip risk.

PROBLEM: Worn shoes are an important contributor to occupational slip and fall injuries. Tools to assess worn tread are emerging; imaging tools offer the potential to assist. The aim of this study was to develop a shoe tread scanner and evaluate its effectiveness to predict slip risk. METHODS: This study analyzed data from two previous studies in which worn or new slip-resistant shoes were donned during an unexpected slip condition. The shoe tread for each shoe was scanned using a portable scanner that utilized frustrated total internal reflection (FTIR) technology. The shoe tread parameters of the worn region size (WRS) for worn shoes and total contact area for new shoes were measured. These parameters were then used to predict slip risk from the unexpected slip conditions. RESULTS: The WRS was able to accurately predict slip risk, but the contact area was not. DISCUSSION: These findings support that increased WRS on the shoe outsole is associated with worse slip outcomes. Furthermore, the tool was able to offer robust feedback across a wide range of tread designs, but the results of this study show that the tool may be more applicable for slip-resistant shoes that are worn compared to their new counterparts. SUMMARY: This study shows that FTIR technology utilized in this tool may be a useful and portable method for determining slip risk for worn shoes. PRACTICAL APPLICATIONS: This tool has the potential to be an efficient, objective, end-user tool that improves timely replacement of shoes and prevention of injuries.

E-learning as a strategy in dentistry in the context of COVID-19: a path to follow?

The current study aims to assess the effectiveness of e-learning in compliance with the new biosafety recommendations in dentistry in the context of COVID-19 applied to the clinical staff of a dental school in Brazil. A quasi-experimental epidemiological study was carried out by means of a structured, pre-tested online questionnaire, applied before and after an educational intervention, using an e-learning format. After data collection, statistical tests were performed. A total of 549 members of the clinical staff participated in the study in the two collection phases, with a return rate of 26.9%. After the e-learning stage, a reduction was found in the reported use of disposable gloves, protective goggles, and surgical masks. The course had no impact on the staff's knowledge concerning the proper sequence for donning PPE and showed 100% effectiveness regarding proper PPE doffing sequence. Knowledge about avoiding procedures that generate aerosols in the clinical setting was improved. Despite the low rate of return, it can be concluded that online intervention alone was ineffective in significantly improving learning about the new clinical biosafety guidelines. Therefore, the use of hybrid teaching and repetitive training is highly recommended.

Differential Effects of Glutamine Inhibition Strategies on Antitumor CD8 T Cells.

Activated T cells undergo metabolic reprogramming to meet anabolic, differentiation, and functional demands. Glutamine supports many processes in activated T cells, and inhibition of glutamine metabolism alters T cell function in autoimmune disease and cancer. Multiple glutamine-targeting molecules are under investigation, yet the precise mechanisms of glutamine-dependent CD8 T cell differentiation remain unclear. We show that distinct strategies of glutamine inhibition by glutaminase-specific inhibition with small molecule CB-839, pan-glutamine inhibition with 6-diazo-5-oxo-l-norleucine (DON), or by glutamine-depleted conditions (No Q) produce distinct metabolic differentiation trajectories in murine CD8 T cells. T cell activation with CB-839 treatment had a milder effect than did DON or No Q treatment. A key difference was that CB-839-treated cells compensated with increased glycolytic metabolism, whereas DON and No Q-treated cells increased oxidative metabolism. However, all glutamine treatment strategies elevated CD8 T cell dependence on glucose metabolism, and No Q treatment caused adaptation toward reduced glutamine dependence. DON treatment reduced histone modifications and numbers of persisting cells in adoptive transfer studies, but those T cells that remained could expand normally upon secondary Ag encounter. In contrast, No Q-treated cells persisted well yet demonstrated decreased secondary expansion. Consistent with reduced persistence, CD8 T cells activated in the presence of DON had reduced ability to control tumor growth and reduced tumor infiltration in adoptive cell therapy. Overall, each approach to inhibit glutamine metabolism confers distinct effects on CD8 T cells and highlights that targeting the same pathway in different ways can elicit opposing metabolic and functional outcomes.

Determining the nurses' perception regarding the effectiveness of COVID-19 protocols implemented in Eastern Province: Saudi Arabia.

Background: The global impact of Coronavirus Disease 2019 (COVID-19) has been profound, affecting public health, the global economy, and overall human life. Past experiences with global pandemics underscored the significance of understanding the perception of HCWs and hospital staff in developing and implementing preventive measures. The World Health Organization (WHO) provided protocols to manage the spread of COVID-19 and assist healthcare workers and health systems globally in maintaining high-quality health services. Objective: This study aims to assess nurses' perception, awareness, and compliance regarding the implementation of COVID-19 protocols and explore factors influencing their perception. Methodology: A quantitative cross-sectional survey-based study was conducted, distributing a constructed survey among nurses in the Eastern Province of Saudi Arabia. Results: Out of 141 participants, most adhered to protocols such as hand sanitization, social distancing, and proper personal protective equipment (PPE) usage. The predominant age group among respondents was 31 to 40 years (n = 71, 50%). A significant portion of participants reported holding a bachelor's degree (n = 86, 61%), with only 14% possessing advanced degrees (n = 19). Nearly a third of the nurses in the study had accumulated 6 to 10 years of professional experience (n = 49, 34.8%). A noteworthy percentage of nurses were engaged in daily shifts exceeding 8 h (n = 98, 70%). Gender differences were observed, with females exhibiting a higher tendency to avoid shaking hands and social gatherings. Saudi nationals were more inclined to shake hands and engage in gatherings. Non-Saudi nurses and those aged between <25 to 40 years demonstrated proper donning/doffing practices. Nurses with over 6 years of experience avoided social gatherings, while those working >8 h adhered better to PPE usage, proper donning/doffing, and disposal of PPE in designated bins. Conclusion: Understanding COVID-19 protocols is crucial for tailoring interventions and ensuring effective compliance with COVID-19 preventive measures among nurses. More efforts should be made toward preparing the healthcare nursing to deal with the outbreak. Preparing healthcare nursing with the right knowledge, attitude, and precautionary practices during the COVID-19 outbreak is very essential to patient and public safety.

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Targeting Glutaminase by Natural Compounds: Structure-Based Virtual Screening and Molecular Dynamics Simulation Approach to Suppress Cancer Progression.

Cancer cells change their glucose and glutamine (GLU) metabolism to obtain the energy required to continue growing. Glutaminase (GLS) plays a crucial role in promoting cell metabolism for cancer cell growth; targeting GLU metabolism by inhibiting GLS has attracted interest as a potential cancer management strategy. Herein, we employed a sequential screening of traditional Chinese medicine (TCM) database followed by drug-likeness and molecular dynamics simulations against the active site of GLS. We report 12 potent compounds after screening the TCM database against GLS, followed by a drug-likeness filter with Lipinski and Veber rule criteria. Among them, ZINC03978829 and ZINC32296657 were found to have higher binding energy (BE) values than the control compound 6-Diazo-5-Oxo-L-Norleucine, with BEs of -9.3 and -9.7 kcal/mol, respectively, compared to the BE of 6-Diazo-5-Oxo-L-Norleucine (-4.7 kcal/mol) with GLS. Molecular dynamics simulations were used to evaluate the results further, and a 100 ns MD simulation revealed that the hits form stable complexes with GLS and formed 2-5 hydrogen bond interactions. This study indicates that these hits might be employed as GLS inhibitors in the battle against cancer. However, more laboratory tests are a prerequisite to optimize them as GLS inhibitors.

Sirpiglenastat (DRP-104) Induces Antitumor Efficacy through Direct, Broad Antagonism of Glutamine Metabolism and Stimulation of the Innate and Adaptive Immune Systems.

Glutamine is a conditionally essential amino acid consumed by rapidly proliferating cancer cells, which deprives the same fuel from immune cells and contributes to tumor immune evasion. As such, the broad antagonism of glutamine in tumors and the tumor microenvironment may lead to direct antitumor activity and stimulation of antitumoral immune responses. DRP-104 (sirpiglenastat) was designed as a novel prodrug of the broad-acting glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). DRP-104 is an inactive form that is preferentially converted to DON within tumors. Metabolomic profiling of tumors treated with DRP-104 revealed widespread changes indicative of the disruption of tumor anabolism and canonical cancer metabolism pathways; including altered glutamine metabolism while several immunosuppressive metabolites were decreased. Gene expression profiling revealed broad immunological modulation, confirmed by flow cytometry indicating that DRP-104 treatment resulted in substantial and broad changes in various immune cell infiltrates, such as increased TIL, T, NK, and NK T cells. Functionally, T cells became more proliferative and less exhausted; tumor-associated macrophages were polarized to the M1 phenotype; MDSCs and protumorigenic proteins were decreased in TME. Finally, DRP-104 demonstrated significant antitumor activity as a monotherapy, which was further enhanced in combination with checkpoint blockade therapies, leading to improved survival and long-term durable cures. In summary, DRP-104 broadly remodels the tumor microenvironment by inducing extensive tumor metabolism effects and enhancing the infiltration and function of multiple immune cells distinct from those obtained by checkpoint inhibitor therapy. This unique mechanism of action supports the ongoing clinical development of DRP-104 alone and in combination with checkpoint inhibitors.

Hypoxia-triggered O-GlcNAcylation in the brain drives the glutamate-glutamine cycle and reduces sensitivity to sevoflurane in mice.

BACKGROUND: Hypersensitivity to general anaesthetics predicts adverse postoperative outcomes in patients. Hypoxia exerts extensive pathophysiological effects on the brain; however, whether hypoxia influences sevoflurane sensitivity and its underlying mechanisms remain poorly understood. METHODS: Mice were acclimated to hypoxia (oxygen 10% for 8 h day-1) for 28 days and anaesthetised with sevoflurane; the effective concentrations for 50% of the animals (EC50) showing loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR) were determined. Positron emission tomography-computed tomography, O-glycoproteomics, seahorse analysis, carbon-13 tracing, site-specific mutagenesis, and electrophysiological techniques were performed to explore the underlying mechanisms. RESULTS: Compared with the control group, the hypoxia-acclimated mice required higher concentrations of sevoflurane to present LORR and LTWR (EC50LORR: 1.61 [0.03]% vs 1.46 [0.04]%, P<0.01; EC50LTWR: 2.46 [0.14]% vs 2.22 [0.06]%, P<0.01). Hypoxia-induced reduction in sevoflurane sensitivity was correlated with elevation of protein O-linked N-acetylglucosamine (O-GlcNAc) modification in brain, especially in the thalamus, and could be abolished by 6-diazo-5-oxo-l-norleucine, a glutamine fructose-6-phosphate amidotransferase inhibitor, and mimicked by thiamet-G, a selective O-GlcNAcase inhibitor. Mechanistically, O-GlcNAcylation drives de novo synthesis of glutamine from glucose in astrocytes and promotes the glutamate-glutamine cycle, partially via glycolytic flux and activation of glutamine synthetase. CONCLUSIONS: Intermittent hypoxia exposure decreased mouse sensitivity to sevoflurane anaesthesia through enhanced O-GlcNAc-dependent modulation of the glutamate-glutamine cycle in the brain.

Pharmacologically Inferred Glycolysis and Glutaminolysis Requirement of B Cells in Lupus-Prone Mice.

Several studies have shown an enhanced metabolism in the CD4+ T cells of lupus patients and lupus-prone mice. Little is known about the metabolism of B cells in lupus. In this study, we compared the metabolism of B cells between lupus-prone B6.Sle1.Sle2.Sle3 triple-congenic mice and C57BL/6 controls at steady state relative to autoantibody production, as well as during T cell-dependent (TD) and T cell-independent (TI) immunizations. Starting before the onset of autoimmunity, B cells from triple-congenic mice showed an elevated glycolysis and mitochondrial respiration, which were normalized in vivo by inhibiting glycolysis with a 2-deoxy-d-glucose (2DG) treatment. 2DG greatly reduced the production of TI-Ag-specific Abs, but showed minimal effect with TD-Ags. In contrast, the inhibition of glutaminolysis with 6-diazo-5-oxo-l-norleucine had a greater effect on TD than TI-Ag-specific Abs in both strains. Analysis of the TI and TD responses in purified B cells in vitro suggests, however, that the glutaminolysis requirement is not B cell-intrinsic. Thus, B cells have a greater requirement for glycolysis in TI than TD responses, as inferred from pharmacological interventions. B cells from lupus-prone and control mice have different intrinsic metabolic requirements or different responses toward 2DG and 6-diazo-5-oxo-l-norleucine, which mirrors our previous results obtained with follicular Th cells. Overall, these results predict that targeting glucose metabolism may provide an effective therapeutic approach for systemic autoimmunity by eliminating both autoreactive follicular Th and B cells, although it may also impair TI responses.

The α/ß Hydrolase AzpM Catalyzes Dipeptide Synthesis in Alazopeptin Biosynthesis Using Two Molecules of Carrier Protein-Tethered Amino Acid.

During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6-diazo-5-oxo-L-norleucine (DON) and one of alanine, the α/ß hydrolase AzpM synthesizes the DON-DON dipeptide using DON tethered to the carrier protein AzpF (DON-AzpF). However, whether AzpM catalyzes the condensation of DON-AzpF with DON or DON-AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS-AzpF and DON-AzpF, but not between AZS-AzpF and DON. Possible reaction intermediates, DON-DON-AzpF and AZS-AZS-AzpF, were also detected during AzpM-catalyzed dipeptide formation from DON-AzpF and AZS-AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON-AzpF and subsequent hydrolysis to produce DON-DON. Thus, AzpM is an unprecedented α/ß hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein-tethered amino acid.

Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

Model studies towards prodrugs of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) containing a diazo precursor.

Two distinct diazo precursors, imidazotetrazine and nitrous amide, were explored as promoieties in designing prodrugs of 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist. As a model for an imidazotetrazine-based prodrug, we synthesized (S)-2-acetamido-6-(8-carbamoyl-4-oxoimidazo[5,1-d][1,2,3,5]tetrazin-3(4H)-yl)-5-oxohexanoic acid (4) containing the entire scaffold of temozolomide, a precursor of the DNA-methylating agent clinically approved for the treatment of glioblastoma multiforme. For a nitrous amide-based prodrug, we synthesized 2-acetamido-6-(((benzyloxy)carbonyl)(nitroso)amino)-5-oxohexanoic acid (5) containing a N-nitrosocarbamate group, which can be converted to a diazo moiety via a mechanism similar to that of streptozotocin, a clinically approved diazomethane-releasing drug containing an N-nitrosourea group. Preliminary characterization confirmed formation of N-acetyl DON (6), also known as duazomycin A, from compound 4 in a pH-dependent manner while compound 5 did not exhibit sufficient stability to allow further characterization. Taken together, our model studies suggest that further improvements are needed to translate this prodrug approach into glutamine antagonist-based therapy.

Inhibitory Effect of a Glutamine Antagonist on Proliferation and Migration of VSMCs via Simultaneous Attenuation of Glycolysis and Oxidative Phosphorylation.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the development of atherosclerosis and restenosis. Glycolysis and glutaminolysis are increased in rapidly proliferating VSMCs to support their increased energy requirements and biomass production. Thus, it is essential to develop new pharmacological tools that regulate metabolic reprogramming in VSMCs for treatment of atherosclerosis. The effects of 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist, have been broadly investigated in highly proliferative cells; however, it is unclear whether DON inhibits proliferation of VSMCs and neointima formation. Here, we investigated the effects of DON on neointima formation in vivo as well as proliferation and migration of VSMCs in vitro. DON simultaneously inhibited FBS- or PDGF-stimulated glycolysis and glutaminolysis as well as mammalian target of rapamycin complex I activity in growth factor-stimulated VSMCs, and thereby suppressed their proliferation and migration. Furthermore, a DON-derived prodrug, named JHU-083, significantly attenuated carotid artery ligation-induced neointima formation in mice. Our results suggest that treatment with a glutamine antagonist is a promising approach to prevent progression of atherosclerosis and restenosis.