A prototrophic suppressor of a Vibrio fischeri D-glutamate auxotroph reveals a member of the periplasmic broad-spectrum racemase family (BsrF).

Although bacterial peptidoglycan (PG) is highly conserved, some natural variations in PG biosynthesis and structure have evolved. Understanding the mechanisms and limits of such variation will inform our understanding of antibiotic resistance, innate immunity, and the evolution of bacteria. We have explored the constraints on PG evolution by blocking essential steps in PG biosynthesis in Vibrio fischeri and then selecting mutants with restored prototrophy. Here, we attempted to select prototrophic suppressors of a D-glutamate auxotrophic murI racD mutant. No suppressors were isolated on unsupplemented lysogeny broth salts (LBS), despite plating >1011 cells, nor were any suppressors generated through mutagenesis with ethyl methanesulfonate. A single suppressor was isolated on LBS supplemented with iso-D-gln, although the iso-D-gln subsequently appeared irrelevant. This suppressor has a genomic amplification formed by the creation of a novel junction that fuses proB to a gene encoding a putative broad-spectrum racemase of V. fischeri, bsrF. An engineered bsrF allele lacking the putative secretion signal (ΔSS-bsrF) also suppressed D-glu auxotrophy, resulting in PG that was indistinguishable from the wild type. The ΔSS-bsrF allele similarly suppressed the D-alanine auxotrophy of an alr mutant and restored prototrophy to a murI alr double mutant auxotrophic for both D-ala and D-glu. The ΔSS-bsrF allele increased resistance to D-cycloserine but had no effect on sensitivity to PG-targeting antibiotics penicillin, ampicillin, or vancomycin. Our work helps define constraints on PG evolution and reveals a periplasmic broad-spectrum racemase in V. fischeri that can be co-opted for PG biosynthesis, with concomitant D-cycloserine resistance. IMPORTANCE: D-Amino acids are used and produced by organisms across all domains of life, but often, their origins and roles are not well understood. In bacteria, D-ala and D-glu are structural components of the canonical peptidoglycan cell wall and are generated by dedicated racemases Alr and MurI, respectively. The more recent discovery of additional bacterial racemases is broadening our view and deepening our understanding of D-amino acid metabolism. Here, while exploring alternative PG biosynthetic pathways in Vibrio fischeri, we unexpectedly shed light on an unusual racemase, BsrF. Our results illustrate a novel mechanism for the evolution of antibiotic resistance and provide a new avenue for exploring the roles of non-canonical racemases and D-amino acids in bacteria.

Safety and Effectiveness of 3 Novel All-Oral Shortened Regimens for Rifampicin- or Multidrug-Resistant Tuberculosis in Kazakhstan.

BACKGROUND: In 2019, the World Health Organization called for operational research on all-oral shortened regimens for multidrug- and rifampicin-resistant tuberculosis (MDR/RR-TB). We report safety and effectiveness of three 9-month all-oral regimens containing bedaquiline (Bdq), linezolid (Lzd), and levofloxacin (Lfx) and reinforced with cycloserine (Cs) and clofazimine (Cfz), delamanid (Dlm) and pyrazinamide (Z), or Dlm and Cfz. METHODS: We conducted a prospective cohort study of patients initiating treatment for pulmonary MDR/RR-TB under operational research conditions at public health facilities in Kazakhstan. Participants were screened monthly for adverse events. Participants with baseline resistance were excluded from the study and treated with a longer regimen. We analyzed clinically relevant adverse events of special interest in all participants and sputum culture conversion and end-of-treatment outcomes among individuals who were not excluded. RESULTS: Of 510 participants, 41% were women, the median age was 37 years (25th-75th percentile: 28-49), 18% had a body mass index <18.5 kg/m2, and 51% had cavitary disease. A total of 399 (78%) initiated Bdq-Lzd-Lfx-Cs-Cfz, 83 (16%) started Bdq-Lzd-Lfx-Dlm-Z, and 28 (5%) initiated Bdq-Lzd-Lfx-Dlm-Cfz. Fifty-eight individuals (11%) were excluded from the study, most commonly due to identification of baseline drug resistance (n = 52; 90%). Among the remaining 452 participants, treatment success frequencies were 92% (95% CI: 89-95%), 89% (95% CI: 80-94%), and 100% (95% CI: 86-100%) for regimens with Cs/Cfz, Dlm/Z, and Dlm/Cfz, respectively. Clinically relevant adverse events of special interest were uncommon. CONCLUSIONS: All regimens demonstrated excellent safety and effectiveness, expanding the potential treatment options for patients, providers, and programs.

Drug Exposure and Treatment Outcomes in Patients With Multidrug-Resistant Tuberculosis and Diabetes Mellitus: A Multicenter Prospective Cohort Study From China.

BACKGROUND: The management of multidrug-resistant tuberculosis (MDR-TB) remains challenging. Treatment outcome is influenced by multiple factors; the specific roles of diabetes and glycemic control remain uncertain. This study aims to assess the impact of glycemic control on drug exposure, to investigate the association between drug exposure and treatment outcomes, and to identify clinically significant thresholds predictive of treatment outcome, among patients with diabetes. METHODS: This multicenter prospective cohort study involved patients with confirmed MDR-TB and diabetes. Drug exposure level was estimated by noncompartmental analysis. The minimum inhibitory concentrations (MICs) were determined for the individual Mycobacterium tuberculosis isolates. The influence of poor glycemic control (glycated hemoglobin ≥7%) on drug exposure and the associations between drug exposure and treatment outcome were evaluated by univariate and multivariate analysis. Classification and regression tree analysis was used to identify the drug exposure/susceptibility thresholds. RESULTS: Among the 131 diabetic participants, 43 (32.8%) exhibited poor glycemic control. Poor glycemic control was independently associated with decreased exposure to moxifloxacin, linezolid, bedaquiline, and cycloserine, but not clofazimine. Additionally, a higher ratio of drug exposure to susceptibility was found to be associated with a favorable MDR-TB treatment outcome. Thresholds predictive of 6-month culture conversion and favorable outcome were bedaquiline area under the concentration-time curve (AUC)/MIC ≥245 and moxifloxacin AUC/MIC ≥67, demonstrating predictive accuracy in patients, regardless of their glycemic control status. CONCLUSIONS: Glycemic control and optimal TB drug exposure are associated with improved treatment outcomes. This dual management strategy should be further validated in randomized controlled trials of patients with MDR-TB and diabetes.

Assessing potential drug-drug interactions between clofazimine and other frequently used agents to treat drug-resistant tuberculosis.

Clofazimine is included in drug regimens to treat rifampicin/drug-resistant tuberculosis (DR-TB), but there is little information about its interaction with other drugs in DR-TB regimens. We evaluated the pharmacokinetic interaction between clofazimine and isoniazid, linezolid, levofloxacin, and cycloserine, dosed as terizidone. Newly diagnosed adults with DR-TB at Klerksdorp/Tshepong Hospital, South Africa, were started on the then-standard treatment with clofazimine temporarily excluded for the initial 2 weeks. Pharmacokinetic sampling was done immediately before and 3 weeks after starting clofazimine, and drug concentrations were determined using validated liquid chromatography-tandem mass spectrometry assays. The data were interpreted with population pharmacokinetics in NONMEM v7.5.1 to explore the impact of clofazimine co-administration and other relevant covariates on the pharmacokinetics of isoniazid, linezolid, levofloxacin, and cycloserine. Clofazimine, isoniazid, linezolid, levofloxacin, and cycloserine data were available for 16, 27, 21, 21, and 6 participants, respectively. The median age and weight for the full cohort were 39 years and 52 kg, respectively. Clofazimine exposures were in the expected range, and its addition to the regimen did not significantly affect the pharmacokinetics of the other drugs except levofloxacin, for which it caused a 15% reduction in clearance. A posteriori power size calculations predicted that our sample sizes had 97%, 90%, and 87% power at P < 0.05 to detect a 30% change in clearance of isoniazid, linezolid, and cycloserine, respectively. Although clofazimine increased the area under the curve of levofloxacin by 19%, this is unlikely to be of great clinical significance, and the lack of interaction with other drugs tested is reassuring.

Structure and function of the pyridoxal 5'-phosphate-dependent (PLP) threonine deaminase IlvA1 from Pseudomonas aeruginosa PAO1.

IlvA1, a pyridoxal phosphate-dependent (PLP) enzyme, catalyzes the deamination of l-threonine and l-serine to yield 2-ketobutyric acid or pyruvate. To gain insights into the function of IlvA1, we determined its crystal structure from Pseudomonas aeruginosa to 2.3 Å. Density for a 2-ketobutyric acid product was identified in the active site and a putative allosteric site. Activity and substrate binding assays confirmed that IlvA1 utilizes l-threonine, l-serine, and L-allo-threonine as substrates. The enzymatic activity is regulated by the end products l-isoleucine and l-valine. Additionally, the efficiency of d-cycloserine and l-cycloserine inhibitors on IlvA1 enzymatic activity was examined. Notably, site-directed mutagenesis confirmed the active site residues and revealed that Gln165 enhances the enzyme activity, emphasizing its role in substrate access. This work provides crucial insights into the structure and mechanism of IlvA1 and serves as a starting point for further functional and mechanistic studies of the threonine deaminase in P. aeruginosa.

Nine months of bedaquiline, linezolid, levofloxacin, clofazimine, and cycloserine chemotherapy for rifampicin/multidrug-resistant tuberculosis: a multicenter, randomized, open-label non-inferiority trial in China.

BACKGROUND: We concurrently developed a prospective study to assess clinical outcomes among patients receiving 9-month bedaquiline (BDQ)-containing regimens, aiming to provide valuable data on the use of this short-course regimen in China. METHODS: This open-label, randomized, controlled, multicenter, non-inferiority trial was conducted at sixteen hospitals, and enrolled participants aged 18 years and older with pulmonary rifampicin/multidrug tuberculosis. Participants were randomly assigned, in a 1:1 ratio. Individuals within the standard-regimen group received 6 months of BDQ, linezolid, levofloxacin, clofazimine, and cycloserine plus 12 months of levofloxacin, and any three potentially effective drugs from clofazimine, cycloserine pyrazinamide, ethambutol and protionamide, whereas individuals within shorter-regimen group received 9 months of BDQ, linezolid, levofloxacin, clofazimine and cycloserine. The primary outcome was the percentage of participants with a composite unfavorable outcome (treatment failure, death, treatment discontinuation, or loss to follow-up) by the end of the treatment course after randomization in the modified intention-to-treat population. The noninferiority margin was 10%. This trial was registered with www.chictr.org.cn , ChiCTR2000029012. RESULTS: Between Jan 1, 2020, and Dec 31, 2023, 264 were screened and randomly assigned, 132 of 264 participants were assigned to the standard-regimen group and 132 were assigned to the shorter-regimen. Thirty-three (12.55%) of 264 participants were excluded from the modified intention-to-treat analysis. As a result, 231 participants were included in the modified intention-to-treat analysis (116 in the standard-regimen group and 115 in the shorter-regimen group).In the modified intention-to-treat population, unfavorable outcomes were reported in 19 (16.5%) of 115 participants for whom the outcome was assessable in the shorter-regimen group and 26 (22.4%) of 116 participants in the standard care group (risk difference 5.9 percentage points (97.5% CI - 5.8 to 17.5)). One death was reported in the standard-regimen group. The incidence of QTcF prolongation in the shorter-regimen group (22.6%, 26/115) was similar to the standard-regimen group (24.1%, 28/116). CONCLUSIONS: The 9-month, all-oral regimen is safe and efficacious for the treatment of pulmonary rifampicin/multidrug-resistant tuberculosis. The high incidence of QTc prolongation associated with the use of BDQ highlights the urgent need of routine electrocardiogram monitoring under treatment with BDQ-containing regimens in the Chinese population.

The mitochondrial pyruvate carrier (MPC) complex mediates one of three pyruvate-supplying pathways that sustain Arabidopsis respiratory metabolism.

Malate oxidation by plant mitochondria enables the generation of both oxaloacetate and pyruvate for tricarboxylic acid (TCA) cycle function, potentially eliminating the need for pyruvate transport into mitochondria in plants. Here, we show that the absence of the mitochondrial pyruvate carrier 1 (MPC1) causes the co-commitment loss of its putative orthologs, MPC3/MPC4, and eliminates pyruvate transport into Arabidopsis thaliana mitochondria, proving it is essential for MPC complex function. While the loss of either MPC or mitochondrial pyruvate-generating NAD-malic enzyme (NAD-ME) did not cause vegetative phenotypes, the lack of both reduced plant growth and caused an increase in cellular pyruvate levels, indicating a block in respiratory metabolism, and elevated the levels of branched-chain amino acids at night, a sign of alterative substrate provision for respiration. 13C-pyruvate feeding of leaves lacking MPC showed metabolic homeostasis was largely maintained except for alanine and glutamate, indicating that transamination contributes to the restoration of the metabolic network to an operating equilibrium by delivering pyruvate independently of MPC into the matrix. Inhibition of alanine aminotransferases when MPC1 is absent resulted in extremely retarded phenotypes in Arabidopsis, suggesting all pyruvate-supplying enzymes work synergistically to support the TCA cycle for sustained plant growth.

Sphingolipid biosynthetic inhibitor L-Cycloserine prevents oxidative-stress-mediated death in an in vitro model of photoreceptor-derived 661W cells.

Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.

Impaired Hippocampal Long-Term Potentiation and Memory Deficits upon Haploinsufficiency of MDGA1 Can Be Rescued by Acute Administration of D-Cycloserine.

The maintenance of proper brain function relies heavily on the balance of excitatory and inhibitory neural circuits, governed in part by synaptic adhesion molecules. Among these, MDGA1 (MAM domain-containing glycosylphosphatidylinositol anchor 1) acts as a suppressor of synapse formation by interfering with Neuroligin-mediated interactions, crucial for maintaining the excitatory-inhibitory (E/I) balance. Mdga1-/- mice exhibit selectively enhanced inhibitory synapse formation in their hippocampal pyramidal neurons, leading to impaired hippocampal long-term potentiation (LTP) and hippocampus-dependent learning and memory function; however, it has not been fully investigated yet if the reduction in MDGA1 protein levels would alter brain function. Here, we examined the behavioral and synaptic consequences of reduced MDGA1 protein levels in Mdga1+/- mice. As observed in Mdga1-/- mice, Mdga1+/- mice exhibited significant deficits in hippocampus-dependent learning and memory tasks, such as the Morris water maze and contextual fear-conditioning tests, along with a significant deficit in the long-term potentiation (LTP) in hippocampal Schaffer collateral CA1 synapses. The acute administration of D-cycloserine, a co-agonist of NMDAR (N-methyl-d-aspartate receptor), significantly ameliorated memory impairments and restored LTP deficits specifically in Mdga1+/- mice, while having no such effect on Mdga1-/- mice. These results highlight the critical role of MDGA1 in regulating inhibitory synapse formation and maintaining the E/I balance for proper cognitive function. These findings may also suggest potential therapeutic strategies targeting the E/I imbalance to alleviate cognitive deficits associated with neuropsychiatric disorders.

Population Pharmacokinetics and Dose Evaluation of Cycloserine among Patients with Multidrug-Resistant Tuberculosis under Standardized Treatment Regimens.

Although cycloserine is a recommended drug for the treatment of multidrug-resistant tuberculosis (MDR-TB) according to World Health Organization (WHO), few studies have reported on pharmacokinetics (PK) and/or pharmacodynamics (PD) data of cycloserine in patients with standardized MDR-TB treatment. This study aimed to estimate the population PK parameters for cycloserine and to identify clinically relevant PK/PD thresholds, as well as to evaluate the current recommended dosage. Data from a large cohort with full PK curves was used to develop a population PK model. This model was used to estimate drug exposure in patients with MDR-TB from a multicentre prospective study in China. The classification and regression tree was used to identify the clinically relevant PK/PD thresholds. Probability of target attainment was analyzed to evaluate the currently recommended dosing strategy. Cycloserine was best described by a two-compartment disposition model. A percentage of time concentration above MICs (T>MIC) of 30% and a ratio of area under drug concentration-time curve (AUC0-24h) over MIC of 36 were the valid predictors for 6-month sputum culture conversion and final treatment outcome. Simulations showed that with WHO-recommended doses (500 mg and 750 mg for patients weighing <45 kg and ≥45 kg), the probability of target attainment exceeded 90% at MIC ≤16 mg/L in MGIT for both T>MIC of 30% and AUC0-24h/MIC of 36. New clinically relevant PK/PD thresholds for cycloserine were identified in patients with standardized MDR-TB treatment. WHO-recommended doses were considered adequate for the MGIT MIC distribution in our cohort of Chinese patients with MDR-TB.

Optimizing dosing of the cycloserine pro-drug terizidone in children with rifampicin-resistant tuberculosis.

There are no pharmacokinetic data in children on terizidone, a pro-drug of cycloserine and a World Health Organization (WHO)-recommended group B drug for rifampicin-resistant tuberculosis (RR-TB) treatment. We collected pharmacokinetic data in children <15 years routinely receiving 15-20 mg/kg of daily terizidone for RR-TB treatment. We developed a population pharmacokinetic model of cycloserine assuming a 2-to-1 molecular ratio between terizidone and cycloserine. We included 107 children with median (interquartile range) age and weight of 3.33 (1.55, 5.07) years and 13.0 (10.1, 17.0) kg, respectively. The pharmacokinetics of cycloserine was described with a one-compartment model with first-order elimination and parallel transit compartment absorption. Allometric scaling using fat-free mass best accounted for the effect of body size, and clearance displayed maturation with age. The clearance in a typical 13 kg child was estimated at 0.474 L/h. The mean absorption transit time when capsules were opened and administered as powder was significantly faster compared to when capsules were swallowed whole (10.1 vs 72.6 min) but with no effect on bioavailability. Lower bioavailability (-16%) was observed in children with weight-for-age z-score below -2. Compared to adults given 500 mg daily terizidone, 2022 WHO-recommended pediatric doses result in lower exposures in weight bands 3-10 kg and 36-46 kg. We developed a population pharmacokinetic model in children for cycloserine dosed as terizidone and characterized the effects of body size, age, formulation manipulation, and underweight-for-age. With current terizidone dosing, pediatric cycloserine exposures are lower than adult values for several weight groups. New optimized dosing is suggested for prospective evaluation.

Brain activity measured by functional brain imaging predicts breathlessness improvement during pulmonary rehabilitation.

BACKGROUND: Chronic breathlessness in chronic obstructive pulmonary disease (COPD) is effectively treated with pulmonary rehabilitation. However, baseline patient characteristics predicting improvements in breathlessness are unknown. This knowledge may provide better understanding of the mechanisms engaged in treating breathlessness and help to individualise therapy. Increasing evidence supports the role of expectation (ie, placebo and nocebo effects) in breathlessness perception. In this study, we tested functional brain imaging markers of breathlessness expectation as predictors of therapeutic response to pulmonary rehabilitation, and asked whether D-cycloserine, a brain-active drug known to influence expectation mechanisms, modulated any predictive model. METHODS: Data from 71 participants with mild-to-moderate COPD recruited to a randomised double-blind controlled experimental medicine study of D-cycloserine given during pulmonary rehabilitation were analysed (ID: NCT01985750). Baseline variables, including brain-activity, self-report questionnaires responses, clinical measures of respiratory function and drug allocation were used to train machine-learning models to predict the outcome, a minimally clinically relevant change in the Dyspnoea-12 score. RESULTS: Only models that included brain imaging markers of breathlessness-expectation successfully predicted improvements in Dyspnoea-12 score (sensitivity 0.88, specificity 0.77). D-cycloserine was independently associated with breathlessness improvement. Models that included only questionnaires and clinical measures did not predict outcome (sensitivity 0.68, specificity 0.2). CONCLUSIONS: Brain activity to breathlessness related cues is a strong predictor of clinical improvement in breathlessness over pulmonary rehabilitation. This implies that expectation is key in breathlessness perception. Manipulation of the brain's expectation pathways (either pharmacological or non-pharmacological) therefore merits further testing in the treatment of chronic breathlessness.

Early correction of synaptic long-term depression improves abnormal anxiety-like behavior in adult GluN2B-C456Y-mutant mice.

Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.

NMDA receptor-related mechanisms of dopaminergic modulation of tDCS-induced neuroplasticity.

Dopamine is a key neuromodulator of neuroplasticity and an important neuronal substrate of learning, and memory formation, which critically involves glutamatergic N-methyl-D-aspartate (NMDA) receptors. Dopamine modulates NMDA receptor activity via dopamine D1 and D2 receptor subtypes. It is hypothesized that dopamine focuses on long-term potentiation (LTP)-like plasticity, i.e. reduces diffuse widespread but enhances locally restricted plasticity via a D2 receptor-dependent NMDA receptor activity reduction. Here, we explored NMDA receptor-dependent mechanisms underlying dopaminergic modulation of LTP-like plasticity induced by transcranial direct current stimulation (tDCS). Eleven healthy, right-handed volunteers received anodal tDCS (1 mA, 13 min) over the left motor cortex combined with dopaminergic agents (the D2 receptor agonist bromocriptine, levodopa for general dopamine enhancement, or placebo) and the partial NMDA receptor agonist D-cycloserine (dosages of 50, 100, and 200 mg, or placebo). Cortical excitability was monitored by transcranial magnetic stimulation-induced motor-evoked potentials. We found that LTP-like plasticity was abolished or converted into LTD-like plasticity via dopaminergic activation, but reestablished under medium-dose D-cycloserine. These results suggest that diffuse LTP-like plasticity is counteracted upon via D2 receptor-dependent reduction of NMDA receptor activity.

Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and ß-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

New Role of D-Cycloserine: Shorten Adaptation Process and Extend Maintenance Time of Motion Sickness.

INTRODUCTION: The aim of the study was to investigate the role of D-cycloserine (DCS) in the adaptation process and maintenance of motion sickness (MS). METHODS: In experiment 1, 120 SD rats were used to study the promoting effect of DCS on the adaptation process of MS in rats. They were randomly divided into four groups, DCS-rotation (DCS-Rot), DCS-static, saline-rotation (Sal-Rot), and saline-static, and further divided into three subgroups according to the adaptation time (4 days, 7 days, and 10 days) in each group. After being given DCS (0.5 mg/kg) or 0.9% saline, they were rotated or kept static according to the group. Their fecal granules, total distance, and total activity of spontaneous activity were recorded and analyzed. In experiment 2, other 120 rats were used. The experimental grouping and specific experimental method were the same as experiment 1. According to the grouping of the adaptive maintenance duration, the animals of 14 days, 17 days, and 21 days groups were measured on the corresponding date of the changes in the animals' exploratory behavior. RESULTS: In experiment 1, the fecal granules, total distance, and total activity of spontaneous activity of Sal-Rot returned to the control level on 9 days, and the DCS-Rot group returned to the control level on 6 days, indicating that DCS could shorten the adaptation time of MS rats from 9 days to 6 days. In experiment 2, the Sal-Rot could not maintain the adaptive state after 14 days' absence from the seasickness environment. The fecal granules of DCS-Rot increased significantly, and total distance and total activity of spontaneous activity of DCS-Rot decreased significantly from 17 days. These illustrate that DCS can prolong the adaptive maintenance time from within 14 days to 17 days in MS rats. CONCLUSION: 0.5 mg/kg DCS injected intraperitoneally can shorten the MS adaptation process and extend the maintenance time of adaptation of SD rats.

NRX-101, a Rapid-Acting Anti-Depressant, Does Not Cause Neurotoxicity Following Ketamine Administration in Preclinical Models.

Agents that act at the N-methyl-D-aspartate receptor (NMDAR), such as ketamine, have gained increasing attention as rapid-acting antidepressants; however, their use has been limited by potential neurotoxicity. Recent FDA guidance requires a demonstration of safety on histologic parameters prior to the initiation of human studies. D-cycloserine (DCS) is a partial NMDA agonist that, along with lurasidone, is being investigated as a treatment for depression. The current study was designed to investigate the neurologic safety profile of DCS. To this end, female Sprague Dawley rats (n = 106) were randomly divided into 8 study groups. Ketamine was administered via tail vein infusion. DCS and lurasidone were administered via oral gavage in escalating doses to a maximum of 2000 mg/kg DCS. To ascertain toxicity, dose escalation with 3 different doses of D-cycloserine/lurasidone was given in combination with ketamine. MK-801, a known neurotoxic NMDA antagonist, was administered as a positive control. Brain tissue was sectioned and stained with H&E, silver, and Fluoro-Jade B stains. No fatalities were observed in any group. No microscopic abnormalities were found in the brain of animal subjects given ketamine, ketamine followed by DCS/lurasidone, or DCS/lurasidone alone. Neuronal necrosis, as expected, was seen in the MK-801 (positive control) group. We conclude that NRX-101, a fixed-dose combination of DCS/lurasidone, when administered with or without prior infusion of IV ketamine was tolerated and did not induce neurotoxicity, even at supratherapeutic doses of DCS.

Crystal structure of O-ureidoserine racemase found in the d-cycloserine biosynthetic pathway.

The O-ureidoserine racemase (DcsC) is an enzyme found from the biosynthetic gene cluster of antitubercular agent d-cycloserine. Although DcsC is homologous to diaminopimelate epimerase (DapF) that catalyzes the interconversion between ll- and dl-diaminopimelic acid, it specifically catalyzes the interconversion between O-ureido-l-serine and its enantiomer. Here we determined the crystal structure of DcsC at a resolution of 2.12 Å, implicating that the catalytic mechanism of DcsC shares similarity with that of DapF. Comparing the structure of the active center of DcsC to that of DapF, Thr72, Thr198, and Tyr219 of DcsC are likely to be involved in the substrate specificity.

Cycloserine and Linezolid for Tuberculosis Meningitis: Pharmacokinetic Evidence of Potential Usefulness.

BACKGROUND: The ability of antituberculosis drugs to cross the blood-brain barrier and reach the central nervous system is critical to their effectiveness in treating tuberculosis meningitis (TBM). We sought to fill a critical knowledge gap by providing data on the ability of new and repurposed antituberculosis drugs to penetrate into the cerebrospinal fluid (CSF). METHODS: We conducted a clinical pharmacology study among patients treated for TBM in Tbilisi, Georgia, from January 2019 until January 2020. Serial serum and CSF samples were collected while patients were hospitalized. CSF was collected from routine lumbar punctures with the timing of the lumbar puncture alternating between 2 and 6 hours to capture early and late CSF penetration. RESULTS: A total of 17 patients treated for TBM (8 with confirmed disease) were included; all received linezolid, with a subset receiving cycloserine (5), clofazimine (5), delamanid (4), and bedaquiline (2). All CSF measurements of bedaquiline (12), clofazimine (24), and delamanid (19) were below the limit of detection. The median CSF concentrations of cycloserine at 2 and 6 hours were 15.90 and 15.10 µg/mL with adjusted CSF/serum ratios of 0.52 and 0.66. CSF concentrations of linezolid were 0.90 and 3.14 µg/mL at 2 and 6 hours, with adjusted CSF/serum ratios of 0.25 and 0.59, respectively. CSF serum linezolid concentrations were not affected by rifampin coadministration. CONCLUSIONS: Based on moderate to high CSF penetration, linezolid and cycloserine may be effective drugs for TBM treatment, whereas the utility of bedaquiline, delamanid, and clofazimine is uncertain given their low CSF penetration.

Bone Penetration of Cycloserine in Osteoarticular Tuberculosis Patients of China.

The cycloserine concentrations in plasma and bone that were collected during operations on 28 osteoarticular tuberculosis (TB) patients treated daily with a 500-mg cycloserine-containing regimen were determined. The median concentrations in plasma and bone were 16.29 µg/mL (interquartile range [IQR], 6.47 µg/mL) and 24.33 µg/g (IQR, 14.68 µg/g), respectively. The median bone/plasma penetration ratio was 0.76 (range, 0.33 to 1.98). Cycloserine could effectively penetrate bone and acquire concentrations comparable to those in plasma, which favors its usage in osteoarticular TB treatment.