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Despite advances in HIV‐1 management with antiretroviral therapy, drug resistance and toxicities with multidrug regimens can result in treatment failure. Hence, there is a continuing demand for antiretroviral agents (ARVs) with novel mechanisms of action. Maturation inhibitors inhibit HIV‐1 replication via a unique mechanism of action and can be combined with other ARVs. Two phase I randomized clinical trials were conducted for a maturation inhibitor, GSK3640254, to determine safety, pharmacokinetics (NCT03231943), and relative bioavailability (NCT03575962) in healthy adults. The first trial was conducted in two parts. Part 1 was conducted in a two‐cohort, interlocking, eight‐period fashion in 20 participants with single ascending doses of GSK3640254 (1‐700 mg) or placebo. In Part 2, 58 participants were randomized to receive GSK3640254 (n = 44) or placebo (n = 14). Four participants reported adverse events (AEs) leading to study discontinuation, with one adverse drug reaction (maculopapular rash). There was no relationship between frequency or severity of AEs and dose. Pharmacokinetic assessments showed that GSK3640254 was slowly absorbed, with time to maximum concentration (tmax) occurring between 3.5 and 4 hours and half‐life of ~24 hours. In the relative bioavailability study of GSK3640254 mesylate salt vs bis‐hydrochloride salt capsules in 14 healthy adults, the mesylate salt performed slightly better than the bis‐hydrochloride formulation (12%‐16% increase in area under the concentration‐time curve and maximum concentration); tmax (5 hours) was similar between the formulations. Initial pharmacokinetic and safety data from these healthy‐participant studies informed further development of GSK3640254 for once‐daily dosing for the treatment of HIV‐1 infection. A comparison of GSK3640254 levels in plasma on Day 14 of multiple‐dose administration showed 1.9‐ to 2.6‐fold accumulation compared with Day 1. Statistical assessment of the attainment of steady state was performed on the trough concentrations from all participants. By Day 8, slope of trough concentration vs time data was close to zero with the 90% CI of the slope containing zero.

Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring 1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite 2 . Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines 2 , including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase 2 , 3 , yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of IκBζ protein induction. The regulation of IκBζ is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17–IκBζ-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI–IκBζ regulatory axis could be an important new strategy for the treatment of IL-17–IκBζ-mediated autoimmune diseases. The immunoregulatory metabolite itaconate and its dimethyl derivative induce electrophilic stress and react with glutathione to induce both Nrf2-dependent and Nrf2-independent responses, resulting in AF3-mediated inhibition of the inflammation-related protein IκBζ.

WebTreatment of lipopolysaccharide-activated macrophages with the cell-permeable itaconate derivative 4-octyl itaconate activates the anti-inflammatory transcription factor Nrf2 by alkylating key cysteine residues on the KEAP1 protein. Anti-inflammatory effects of itaconate Macrophages are white blood cells that recognize and destroy invading bacterial pathogens, and later tone down inflammation to enable tissue repair. The endogenous metabolite itaconate inhibits a number of inflammatory cytokines during macrophage activation. Luke O'Neill and colleagues investigate the mechanism underlying this process. Treatment of lipopolysaccharide (LPS)-activated macrophages with the cell-permeable itaconate derivative 4-octyl itaconate activates the anti-oxidant and anti-inflammatory transcription factor Nrf2. This activation occurs via alkylation of key cysteine residues on the KEAP1 protein, which blocks KEAP1-dependent proteolysis of Nrf2. Pre-treating mouse models of LPS with the itaconate derivative activates Nrf2 and prolongs the survival of the animals after a lethal dose of LPS. The authors suggest that itaconate derivatives may prove useful in the treatment of inflammatory diseases. The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood 1 , 2 , 3 . Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1 ) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Dimethyl fumarate (DMF) is an immunomodulatory compound used to treat multiple sclerosis and psoriasis whose mechanisms of action remain only partially understood. Kornberg et al. found that DMF and its metabolite, monomethyl fumarate, succinate the glycolytic enzyme GAPDH (see the Perspective by Matsushita and Pearce). After DMF treatment, GAPDH was inactivated, and aerobic glycolysis was down-regulated in both myeloid and lymphoid cells. This resulted in down-modulated immune responses because inflammatory immune-cell subsets require aerobic glycolysis. Thus, metabolism can serve as a viable therapeutic target in autoimmune disease. Science , this issue p. 449 ; see also p. 377 An immunomodulatory drug suppresses immune responses by modulating metabolism in activated immune cells. Activated immune cells undergo a metabolic switch to aerobic glycolysis akin to the Warburg effect, thereby presenting a potential therapeutic target in autoimmune disease. Dimethyl fumarate (DMF), a derivative of the Krebs cycle intermediate fumarate, is an immunomodulatory drug used to treat multiple sclerosis and psoriasis. Although its therapeutic mechanism remains uncertain, DMF covalently modifies cysteine residues in a process termed succination. We found that DMF succinates and inactivates the catalytic cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in mice and humans, both in vitro and in vivo. It thereby down-regulates aerobic glycolysis in activated myeloid and lymphoid cells, which mediates its anti-inflammatory effects. Our results provide mechanistic insight into immune modulation by DMF and represent a proof of concept that aerobic glycolysis is a therapeutic target in autoimmunity.

The development of metabolic tracers reveals that malic enzyme is the main contributor of NADPH production in differentiating adipocytes. During hypoxic conditions, the source of NADPH shifts to the oxidative pentose phosphate pathway. The critical cellular hydride donor NADPH is produced through various means, including the oxidative pentose phosphate pathway (oxPPP), folate metabolism and malic enzyme. In growing cells, it is efficient to produce NADPH via the oxPPP and folate metabolism, which also make nucleotide precursors. In nonproliferating adipocytes, a metabolic cycle involving malic enzyme holds the potential to make both NADPH and two-carbon units for fat synthesis. Recently developed deuterium ( 2 H) tracer methods have enabled direct measurement of NADPH production by the oxPPP and folate metabolism. Here we enable tracking of NADPH production by malic enzyme with [2,2,3,3- 2 H]dimethyl-succinate and [4- 2 H]glucose. Using these tracers, we show that most NADPH in differentiating 3T3-L1 mouse adipocytes is made by malic enzyme. The associated metabolic cycle is disrupted by hypoxia, which switches the main adipocyte NADPH source to the oxPPP. Thus, 2 H-labeled tracers enable dissection of NADPH production routes across cell types and environmental conditions.

HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, 3D microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-Å MicroED structure revealed that a single BVM molecule stabilizes the six-helix bundle via both electrostatic interactions with the dimethylsuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.

The bioproduction of high-value chemicals such as itaconic and fumaric acids (IA and FA, respectively) from renewable resources via solid-state fermentation (SSF) represents an alternative to the current bioprocesses of submerged fermentation using refined sugars. Both acids are excellent platform chemicals with a wide range of applications in different market, such as plastics, coating, or cosmetics. The use of lignocellulosic biomass instead of food resources (starch or grains) in the frame of a sustainable development for IA and FA bioproduction is of prime importance. Filamentous fungi, especially belonging to the Aspergillus genus, have shown a great capacity to produce these organic dicarboxylic acids. This study attempts to develop and optimize the SSF conditions with lignocellulosic biomasses using A. terreus and A. oryzae to produce IA and FA. First, a kinetic study of SSF was performed with non-food resources (wheat bran and corn cobs) and a panel of pH and moisture conditions was studied during fermentation. Next, a new process using an enzymatic cocktail simultaneously with SSF was investigated in order to facilitate the use of the biomass as microbial substrate. Finally, a large-scale fermentation process was developed for SSF using corn cobs with A. oryzae; this specific condition showed the best yield in acid production. The yields achieved were 0.05 mg of IA and 0.16 mg of FA per gram of biomass after 48 h. These values currently represent the highest reported productions for SSF from raw lignocellulosic biomass.

In the present work, polybutylene succinate (PBS)/stearate modified magnesium-aluminium layered double hydroxide (St-Mg-Al LDH) composites were prepared via melt processing and the effect of different loadings of St-Mg-Al LDH on the degradation behaviour of PBS under marine conditions was investigated. The morphological, mechanical and thermal characteristics of the composites were studied using different characterisation techniques. Optical imaging and scanning electron microscopy revealed that the incorporation of St-Mg-Al LDH accelerates the degradation of PBS along with the activity of microorganisms adhered to the composite films. PBS/St-Mg-Al LDH composites are found to have lower thermal degradation temperatures than those of pure PBS. The decrease in thermal stability is correlated with the degradation of PBS due to the catalytic action Mg and Al present in LDH. Tensile and DMA analysis revealed that the addition of St-Mg-Al LDH did not have a significant impact on the mechanical properties of PBS.

Militarine, a natural glucosyloxybenzyl 2-isobutylmalate, isolated from Bletilla striata, was reported with a prominent neuroprotective effect recently. The limited information on the metabolism of militarine impedes comprehension of its biological actions and pharmacology. This study aimed to investigate the metabolite profile and the distribution of militarine in vivo, which help to clarify the action mechanism further. A total of 71 metabolites (57 new metabolites) in rats were identified with a systematic method by ultra-high-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). The proposed metabolic pathways of militarine include hydrolyzation, oxidation, glycosylation, esterification, sulfation, glucuronidation and glycine conjugation. Militarine and its metabolites were distributed extensively in the treated rats. Notably, six metabolites of militarine were identified in cerebrospinal fluid (CSF), which were highly consistent with the metabolites after oral administration of gastrodin in rats. Among the metabolites in CSF, five of them were not reported before. It is the first systematic metabolic study of militarine in vivo, which is very helpful for better comprehension of the functions and the central nervous system (CNS) bioactivities of militarine. The findings will also provide an essential reference for the metabolism of other glucosylated benzyl esters of succinic, malic, tartaric and citric acids.

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.