HIV-associated neurocognitive impairment persists despite combination antiretroviral therapy, largely driven by chronic microglial activation that sustains neuroinflammation and neuronal injury. Activated microglia contribute to HIV-associated brain pathology by releasing proinflammatory mediators that disrupt synaptic integrity and impair cognition. N-acetylaspartylglutamate (NAAG), an abundant neuropeptide that maintains glutamatergic homeostasis, is hydrolyzed by glutamate carboxypeptidase II (GCPII) to glutamate. We previously demonstrated that reduced brain and cerebrospinal fluid NAAG levels in people living with HIV correlate with cognitive impairment, and that pharmacological GCPII inhibition with 2-(phosphonomethyl)-pentanedioic acid (2-PMPA) elevates brain NAAG and improves cognition in EcoHIV-infected mice. To enhance brain delivery and preferentially target activated microglia, we conjugated 2-PMPA to a generation 4 hydroxyl poly(amidoamine) (PAMAM) dendrimer (D-2-PMPA). Our findings demonstrate that D-2-PMPA achieves preferential microglial drug delivery, resulting in a >600% increase in cerebrospinal fluid NAAG levels. At doses 8.3-fold lower than free 2-PMPA, this formulation reversed EcoHIV-induced deficits in social interaction, novel object recognition, and fear-conditioned memory without altering locomotor activity or anxiety-like behavior. D-2-PMPA also restored prefrontal cortex synaptic density and preserved dendritic architecture. Together, these findings demonstrate that microglia-targeted GCPII inhibition represents a potent nanotherapeutic strategy to restore synaptic integrity and cognitive function in HIV-associated neurocognitive impairment.

BACKGROUND:Cerebral malaria (CM) is a common cause of febrile coma among African children. Despite treatment with highly efficacious intravenous artesunate, CM remains associated with high mortality and neurologic sequelae. In a mouse CM model, the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON) demonstrated robust efficacy as a potential adjuvant therapy. Before testing DON in children with CM, we investigated tolerability in African adults, including individuals with malaria. METHODS:We conducted an open-label, prospective, dose-escalation, phase 1 clinical trial of single-dose intravenous DON in Blantyre, Malawi. Participants included healthy adults and adults with uncomplicated malaria, enrolled in dose-cohorts of 10 and administered DON at 0.1, 1.0, 5.0, or 10 mg/kg. We assessed adverse events (AEs) and plasma pharmacokinetics. RESULTS:Forty healthy adults and 38 adults with uncomplicated malaria received DON. Transient, asymptomatic creatinine elevation occurred 12 hours postinfusion in 18% of all participants. Nausea and vomiting were uncommon at 0.1 and 1.0 mg/kg but occurred more frequently at 5.0 and 10 mg/kg DON. All DON-related AEs resolved rapidly, without sequelae, and did not significantly differ between healthy and uncomplicated malaria participants. In healthy adults, maximum plasma concentrations increased proportional to dose, but adults with malaria had greater than dose-proportional increases. Terminal half-life ranged from 1.7 to 4.1 hours. CONCLUSIONS:DON was well tolerated in healthy adults and adults with uncomplicated malaria. Higher doses were associated with transient gastrointestinal AEs. Pharmacokinetics were minimally influenced by malaria disease. These results provide critical data to guide dosing strategies for adjunctive DON in children with CM. Clinical Trials Registration. NCT05478720.

Despite viral suppression, many people with HIV (PWH) experience persistent cognitive difficulties. We previously demonstrated that cerebrospinal fluid (CSF) N-acetyl-aspartyl-glutamate (NAAG) was associated with spatial attention and working memory. Here, we report that CSF NAAG also correlates with an inflammatory signature (M-CSF, IL-15, MCP-1, sCD40L, IL-18, MMP-9) that relates to spatial attention and working memory. These results suggest that CSF NAAG may serve as an immunomodulatory biomarker relevant to cognition in PWH.

Background: Extracellular vesicles (EVs) can carry pathological cargo, contributing to disease progression. The enzyme neutral sphingomyelinase 2 (nSMase2) plays a critical role in EV biogenesis, making it a promising therapeutic target. Our lab previously identified a potent and selective inhibitor of nSMase2, named DPTIP (IC50 = 30 nM). Although promising, DPTIP exhibits poor pharmacokinetics (PKs) with a low oral bioavailability (%F < 5), and a short half-life (t1/2 ≤ 0.5 h). To address these limitations, we previously developed DPTIP prodrugs by masking its phenolic hydroxyl group, demonstrating improved plasma exposure in mice. Recognizing that species-specific metabolic differences can influence prodrug PK, we expanded our studies to evaluate selected prodrugs in both mice and dogs. Methods: The scaleup of selected prodrugs was completed and two additional valine- ester based prodrugs were synthesized. Mice were dosed prodrugs via peroral route (10 mg/kg equivalent). For dog studies DPTIP was dosed via intravenous (1 mg/kg) or peroral route (2 mg/kg) and prodrugs were given peroral at a dose 2 mg/kg DPTIP equivalent. Plasma samples were collected at predetermined points and analyzed using developed LC/MS-MS methods. Results: In mice, several of the tested prodrugs showed similar or improved plasma exposures compared to DPTIP. However, in dog studies, the double valine ester prodrug 9, showed significant improvement with an almost two-fold increase in DPTIP plasma exposure (AUC0–t = 1352 vs. 701 pmol·h/mL), enhancing oral bioavailability from 8.9% to 17.3%. Conclusions: These findings identify prodrug 9 as a promising candidate for further evaluation and underscore the critical role of species-specific differences in prodrug PKs.

Despite effective antiretroviral therapy, many people living with HIV (PLH) experience cognitive impairments, particularly in executive function and working memory. These deficits have been linked to dysregulation of brain circuits involving the neuropeptide N-acetyl-aspartyl glutamate (NAAG), which is catabolized by the enzyme glutamate carboxypeptidase II (GCPII). Inhibiting GCPII elevates brain NAAG levels and improves cognition in preclinical models. In prior magnetic resonance spectroscopy (MRS) studies, we demonstrated that higher brain NAAG levels in PLH correlate with better cognitive performance, highlighting NAAG as a potential biomarker and GCPII as a potential therapeutic target. In this study, we used EcoHIV-infected mice to model HIV-associated neurocognitive disorders and evaluated the therapeutic potential of the selective GCPII inhibitor 2-PMPA. We found that 2-PMPA treatment increased cerebrospinal fluid (CSF) NAAG levels by 800 ​% and reversed EcoHIV-induced deficits in social interaction, recognition memory, and fear conditioning, without affecting general locomotion or anxiety-like behavior. Furthermore, 2-PMPA restored synaptic density and preserved dendritic structure in EcoHIV-infected mice, indicating a neuroprotective effect. These findings provide strong evidence that GCPII inhibition represents a viable therapeutic strategy for HIV-associated cognitive dysfunction by elevating NAAG and protecting neural circuits critical for cognition.

OBJECTIVES:Despite effective antiretroviral therapy, many people with HIV (PWH) experience persistent deficits in attention and working memory. Identifying neurometabolic drivers of these impairments is critical for precision diagnostics and targeted interventions. N-acetylaspartylglutamate (NAAG), the most abundant brain dipeptide and endogenous agonist of metabotropic glutamate receptor 3 (mGluR3), regulates glutamatergic transmission central to these cognitive domains. While prior magnetic resonance spectroscopy (MRS) studies have associated higher NAAG with better cognition, NAAG has never been quantified in cerebrospinal fluid (CSF) of PWH or linked to cognition in this population. DESIGN:We tested whether CSF NAAG levels relate to domain-specific cognitive function in 28 PWH (plasma viral load <200 cp/ml). METHODS:NAAG was quantified by a sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS-MS) method. Cognition was measured using a Research Domain Criteria (RDoC)-based battery, with principal component analysis deriving domain scores. Pearson correlations and age/viral load adjusted regressions were used to assess NAAG-cognition associations. RESULTS:Higher CSF NAAG was significantly associated with better spatial attention and working memory ( r  = 0.479, P  = 0.01), independent of age and viral load. In contrast, NAAG levels showed no relationship with verbal attention and working memory or other domains such as verbal episodic memory and motor function. CONCLUSION:This is the first study to identify a CSF-based neurometabolic marker linked to specific cognitive domains in PWH, bridging MRS findings to a scalable fluid biomarker platform. NAAG CSF measurement opens new translational pathways for early detection, risk profiling, and glutamatergic-targeted interventions in neuroHIV. Longitudinal studies will determine its prognostic and therapeutic utility.

Multiple lines of evidence show that the microRNA system plays a prominent role in regulating behavioral responses to psychostimulants. Suppressing microRNA degradation is an effective strategy for elucidating the impact of these intracellular messengers on cellular function. The translin/trax complex is an RNase that appears to mediate degradation of a small number of microRNAs. In this study we investigated the effect of deleting the translin/trax microRNA-degrading enzyme on cocaine-induced behavioral responses in mice. Wild type and Translin (Tsn) KO mice were injected with cocaine and their open-field locomotor activity was monitored. We found that the locomotor activity in response to repeated (5, 10 and 20 mg/kg, i.p.), but not acute (20 mg/kg, i.p.), cocaine exposure was significantly impaired in Tsn KO mice. We identified several microRNAs (412–5p, 412–3p, 93–3p, 7b–3p, and 204–5p) that were significantly increased in the NAc of Tsn KO mice. As regulator of G-protein signaling 8 (RGS8) is a predicted target gene shared by three of these microRNAs, and expressed in the NAc, we confirmed its reduced expression in this region in Tsn KO mice. Moreover, shRNA-mediated knockdown of RGS8 in the NAc attenuated locomotor sensitization to repeated cocaine administration. Taken together, our results suggest that microRNAs targeted by the translin/trax RNase inhibit cocaine-induced locomotor sensitization, in part, by silencing expression of RGS8.

IntroductionThere is an unmet need for therapeutics with a novel mechanism to address Q9 symptoms associated with conditions where aberrant glutamatergic neurotransmission is presumed pathogenic. One enzyme of potential relevance is glutamate carboxypeptidase II (GCPII), a brain metallopeptidase with significantly upregulated activity in nervous tissues following neurodegeneration or injury. Current inhibitors are too polar and charged leading to minimal brain penetration necessitating high systemic doses or direct brain injection. Our efforts are focused on identifying new inhibitor scaffolds with favorable brain penetration.MethodsHerein, we used a newly developed dual-stream liquid chromatography mass spectrometry (LC/MS/MS) substrate cleavage assay to screen two small molecule libraries. The two top confirmed hits were cefsulodin (IC50 = 2 ± 0.1 μM) and amaranth (IC50 = 0.3 ± 0.01 μM). The interactions of Amaranth and cefsulodin with GCPII were characterized with mode of inhibition (MOI) studies, nano differential scanning fluorimetry (DSF) thermal shift assay, and binding site was modeled with in-silico docking. As cefsulodin is an antibiotic used clinically to treat bacterial meningitis, we tested the compound’s brain pharmacokinetics (PK) in mice using a sensitive LC/MS method we developed. Moreover, following confirmation and characterization of cefsulodin and amaranth as viable hits an SAR investigation was conducted with analogs of both compounds.ResultsA first derivative analysis of the DSF data revealed a shift in melting temperature of Δ 0.76 °C (±0.04) for amaranth at 25 μM and 80.41 °C (±0.05) for cefsulodin at 250 μM, suggesting both compounds are acting as stabilizers for the enzyme. Increasing concentrations of cefsulodin increased the Km of N-acetyl-aspartyl-glutamate (NAAG) as a substrate with no change in Vmax, suggesting active site competitive inhibition. In contrast, increasing concentrations of amaranth led to reductions in Vmax while the Km remained constant, suggesting a non-competitive MOI. Results from in-silico docking studies complemented this MOI data, suggesting cefsulodin likely binds in the active site while amaranth likely binds in an allosteric site. Our PK study demonstrated that administration of cefsulodin (100 mg/kg IP) led to a Cmax of 4 μM in the brain, exceeding its GCPII IC50 value.DiscussionOur new screening approaches identified novel inhibitors of GCPII that could serve as molecular templates for further structural optimization.

Itaconate, an endogenous immunomodulator from the tricarboxylic acid (TCA) cycle, shows therapeutic effects in various disease models, but is highly polar with poor cellular permeability. We previously reported a novel, topical itaconate derivative, SCD-153, for the treatment of alopecia areata. Here, we present the discovery of orally available itaconate derivatives for systemic and skin disorders. Four sets of prodrugs were synthesized using pivaloyloxymethyl (POM), isopropyloxycarbonyloxymethyl (POC), (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl (ODOL), and 3-(hexadecyloxy)propyl (HDP) pro-moieties pairing with itaconic acid (IA), 1-methyl itaconate (1-MI), and 4-methyl itaconate (4-MI). Among these, POC-based prodrugs (P2, P9, P13) showed favorable stability, permeability, and pharmacokinetics. Notably, P2 and P13 significantly inhibited Poly(I:C)/IFNγ-induced inflammatory cytokines in human epidermal keratinocytes. Oral studies demonstrated favorable pharmacokinetics releasing micromolar concentrations of IA or 4-MI from P2 and P13, respectively. These findings highlight the potential of prodrug strategies to enhance itaconate’s cellular permeability and oral bioavailability, paving the way for clinical translation.

Human Immunodeficiency Viruses (HIV) are lentiviruses that cause a chronic, progressive infection, which can ultimately lead to acquired immunodeficiency syndrome (AIDS). Despite the development of antiretroviral therapy (ART), which has improved and extended the lives of people living with HIV (PLH), these individuals often experience neurocognitive deficits, especially in working memory and executive function. These domains are linked to brain circuits regulated by N-acetyl-aspartyl glutamate (NAAG), the most prevalent brain dipeptide. NAAG is cleaved by the enzyme glutamate carboxypeptidase II (GCPII) into N-acetyl aspartate (NAA) and glutamate. Inhibition of GCPII has been shown to elevate brain NAAG levels and improve learning and memory in both rodents and primate models. Using magnetic resonance spectroscopy (MRS) data in PLH, we recently reported that higher brain NAAG levels were associated with better attention/working memory, cognitive function and verbal fluency. These data suggest that brain NAAG may serve as a biomarker of cognition in PLH, and modulation of brain NAAG could represent a novel therapeutic strategy. To explore this potential, we utilized EcoHIV-infected mice as a mouse model of HIV as these mice have been shown to have cognitive performance deficits. Mice were infected with EcoHIV and received daily treatment of either vehicle or the GCPII inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA; 100 mg/kg IP) beginning 3 weeks post-infection. After 2 weeks of treatment, mice underwent behavioral tests, including open field (OF), novel object recognition test (NORT), light-dark box (LDB), social interaction test (SIT) and fear conditioning (FC). We found no difference in locomotion activities measured by OF, recognitive function measured by NORT, or anxiety-like behaviors measured by LDB in EcoHIV-infected mice comparing to age-matched WT controls. However, EcoHIV-infected mice displayed social avoidance behavior in the SIT and significant impairments in cued fear memory in the FC, both of which were reversed by 2-PMPA treatment. After completing the behavioral tests, we measured NAAG levels in the cerebrospinal fluid (CSF) and found that treatment with 2-PMPA significantly increased NAAG levels in the CSF of EcoHIV-infected mice. In conclusion, inhibiting GCPII with 2-PMPA increases NAAG levels in the CSF and improves specific cognitive and social deficits in EcoHIV-infected mice. Further studies are needed to elucidate the precise molecular mechanisms of these effects. This work was supported by P30 CAHN Pilot 90108414 (MH), P30 CAHN Pilot 90101781 (XZ) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Glutamine metabolism in tumor microenvironments critically regulates antitumor immunity. Using the glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes. We show JHU083-mediated glutamine antagonism in tumor microenvironments induced by TNF, proinflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibited increased tumor cell phagocytosis and diminished proangiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken tricarboxylic acid (TCA) cycle, and purine metabolism disruption. Although the antitumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell–like phenotype in CD8+ T cells and decreased the abundance of regulatory T cells. Finally, JHU083 caused a global shutdown in glutamine-utilizing metabolic pathways in tumor cells, leading to reduced HIF-1α, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key antitumor features. Altogether, our findings demonstrate that targeting glutamine with JHU083 led to suppressed tumor growth as well as reprogramming of immunosuppressive TAMs within prostate and bladder tumors that promoted antitumor immune responses. JHU083 can offer an effective therapeutic benefit for tumor types that are enriched in immunosuppressive TAMs.

JHU083-induced glutamine antagonism affects tumor cell metabolism and induces cell death in urologic tumors. A, Log-fold changes of glutamine utilizing enzymes after JHU083 treatment vs. control tumors in CD45− sorted cells from B6CaP tumors, followed by scRNA-seq. B, Western blot showing qualitative changes in the levels of glutamine synthesizing/utilizing enzymes and transporters in the CD45− fraction of MB49 tumors. C, Percentage of GLUT1+ CD45− live cells determined by flow cytometry in B6CaP tumors (n = 7/group). D, Targeted metabolomic analysis of B6CaP tumors by LC-MS/MS (n = 3/group). E, Volcano plot showing key metabolite levels of JHU083-treated vs. nontreated control tumors based on the metabolomic analysis shown in D. F, Absolute quantification of metabolites by LC/MS-MS (n = 3 or 5/group). G and H, Western blot images showing qualitative changes in c-MYC, phospho-c-MYC, and HIF-1ɑ in MB49 tumors following JHU083 treatment, and (H and I) MTT assay in DON-treated MB49 cells and immunoblot of cleaved caspase 3 quantification in CD45− fraction MB49 tumors (J). Statistical analyses were performed using the unpaired t test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

AbstractCytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume‐regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl− influx‐dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA‐approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.

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.

Supplemental Figure 2: JHU395 DON delivery to P493B lymphoma cells and in vivo time-dependent pharmacokinetics of JHU395 in CD-1 mouse plasma and jejunum

Supplementary Materials and Figures

AbstractPurpose:Glioblastoma (GBM) is the most common brain malignancy with median survival <2 years. Standard-of-care temozolomide has marginal efficacy in approximately 70% of patients due to MGMT expression. LP-184 is an acylfulvene-derived prodrug activated by the oxidoreductase PTGR1 that alkylates at N3-adenine, not reported to be repaired by MGMT. This article examines LP-184 efficacy against preclinical GBM models and identifies molecular predictors of LP-184 efficacy in clinical GBM.Experimental Design:LP-184 effects on GBM cell viability and DNA damage were determined using cell lines, primary PDX-derived cells and patient-derived neurospheres. GBM cell sensitivities to LP-184 relative to temozolomide and MGMT expression were examined. Pharmacokinetics and CNS bioavailability were evaluated in mice with GBM xenografts. LP-184 effects on GBM xenograft growth and animal survival were determined. Machine learning, bioinformatic tools, and clinical databases identified molecular predictors of GBM cells and tumors to LP-184 responsiveness.Results:LP-184 inhibited viability of multiple GBM cell isolates including temozolomide-resistant and MGMT-expressing cells at IC50 = approximately 22–310 nmol/L. Pharmacokinetics showed favorable AUCbrain/plasma and AUCtumor/plasma ratios of 0.11 (brain Cmax = 839 nmol/L) and 0.2 (tumor Cmax = 2,530 nmol/L), respectively. LP-184 induced regression of GBM xenografts and prolonged survival of mice bearing orthotopic xenografts. Bioinformatic analyses identified PTGR1 elevation in clinical GBM subtypes and associated LP-184 sensitivity with EGFR signaling, low nucleotide excision repair (NER), and low ERCC3 expression. Spironolactone, which induces ERCC3 degradation, decreased LP-184 IC50 3 to 6 fold and enhanced GBM xenograft antitumor responses.Conclusions:These results establish LP-184 as a promising chemotherapeutic for GBM with enhanced efficacy in intrinsic or spironolactone-induced TC-NER–deficient tumors.