Integrative structural and functional analysis of human malic enzyme 3: A potential therapeutic target for pancreatic cancer.

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to determine the structures of ME3 in various ligand-binding states at near-atomic resolutions. ME3 is captured in the open form existing as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 Domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

Efficacy of ready-to-use therapeutic food in malnourished children with cancer: Results of a randomized, open-label phase 3 trial.

BACKGROUND: The adverse influence of undernutrition in children with cancer may be remediated by early nutritional intervention. This study assessed the efficacy of ready-to-use therapeutic food (RUTF) in improving nutritional status and reducing treatment-related toxicities (TRTs) in such children. METHODS: In a randomized controlled phase-3 open-label trial, severely and moderately undernourished children with cancer were randomized 1:1 to receive standard nutritional therapy (SNT) or SNT+RUTF for 6 weeks. The primary outcome (weight gain >10%) and secondary outcomes (improved/maintained nutritional status, improved body composition) were assessed after 6 weeks. TRTs were assessed over 6 months. RESULTS: Between July 2015 and March 2018, 260 subjects were enrolled, 126 were analyzable in both arms at 6 weeks. More children on RUTF had weight gain (98 [77.8%] vs. 81 [64.2%], p = .025) with a greater increase in fat mass as a percentage of body mass (median 2% [IQR -0.12 to 4.9] vs. 0.5% [IQR -1.45 to 2.27, p = .005]) but a greater loss of lean mass (median -1.86% [IQR -4.4 to 0.50] vs. -0.4% [IQR -2.4 to 1.4, p = .007]) compared to the SNT arm. Fewer subjects on the RUTF arm had episodes of severe infection (10.6% vs. 31%, p < .0001), treatment delays (17.7% vs. 39%, p < .0001), and severe mucositis (11% vs. 23.8%, p = .006) compared to the SNT arm. The odds of developing TRTs on the RUTF arm were lower even after adjusting for improvement in nutritional status. CONCLUSIONS: RUTF is efficacious in improving weight gain and nutritional status in undernourished children with cancer and decreases TRTs. Incorporating RUTF into a healthy, balanced diet should be considered in undernourished children with cancer.

Structure of a ternary complex of lactoperoxidase with iodide and hydrogen peroxide at 1.77 Å resolution.

Lactoperoxidase (LPO) is a mammalian heme peroxidase which catalyzes the conversion of thiocyanate (SCN¯) and iodide (I-) by hydrogen peroxide (H2O2) into antimicrobial hypothiocyanite (OSCN¯) and hypoiodite (IO-). The prosthetic heme group is covalently attached to LPO through two ester linkages involving conserved glutamate and aspartate residues. On the proximal side, His351 is coordinated to heme iron while His 109 is located in the substrate binding site on the distal heme side. We report here the first structure of the ternary complex of LPO with iodide (I-) and H2O2 at 1.77 Å resolution. LPO was crystallized with ammonium iodide and the crystals were soaked in the reservoir solution containing H2O2. Structure determination showed the presence of an iodide ion and a H2O2 molecule in the substrate binding site. The iodide ion occupied the position which is stabilized by the interactions with heme moiety, His109, Arg255 and Glu258 while H2O2 was held between the heme iron and His109. The presence of I- in the distal heme cavity seems to screen the positive charge of Arg255 thus suppressing the proton transfer from H2O2 to His109. This prevents compound I formation and allows trapping of a stable enzyme-substrate (LPO-I--H2O2) ternary complex. This stable geometrical arrangement of H2O2 in the distal heme cavity of LPO is similar to that of H2O2 in the structure of the transient intermediate of the palm tree heme peroxidase. The biochemical studies showed that the catalytic activity of LPO decreased when the samples of LPO were preincubated with ammonium iodide.

A comparative study of transdermal nitroglycerine patch and oral nifedipine in preterm labor.

Background: Currently, the main goal for the use of tocolytic therapy is to delay the birth so as to allow the use of corticosteroids for accelerating fetal lung maturity and maternal transfer to a tertiary care center and thereby reducing neonatal morbidity and mortality. Aims and Objectives: The aims amd objectives were to compare the safety and efficacy of transdermal nitroglycerine patch with oral nifedipine as a tocolytic agent to arrest preterm labor and prevent preterm birth. Materials and Methods: Based on the selection criteria, 50 patients were selected randomly in Group A and Group B. Group A women were given transdermal nitroglycerin patch, which delivered 10 mg Nitroglycerin (NTG) over 24 h and it was applied to the woman's abdomen followed by another patch of 10 mg after 1 h if contractions persisted. After 24 h, it was replaced by a fresh patch. Group B women were given an oral loading dose of nifedipine 20 mg followed by a similar dose if contractions persisted after 1 h. A maintenance dose of 10 mg thrice daily was given if contractions were suppressed. Patients were monitored from the time of admission to the time of discharge. Results: The mean duration of prolongation of pregnancy in Group B (3.68 ± 1.91 days) was significantly more than Group A (2.78 ± 1.39 days). Headache was seen significantly more in Group A (42%) than group B (6%). Tachycardia, hypotension, and palpitation showed no statistically significant difference between them. There was no statistically significant difference in the birth weight of the babies in both the groups. Conclusion: Nifedipine is a safe and effective drug in prolonging preterm labor and has minimal maternal and neonatal side effects.

RésuméContexte: Actuellement, le principal objectif de l'utilisation de la thérapie tocolytique est de retarder la naissance afin de permettre l'utilisation de corticostéroïdes pour accélérer la maturité pulmonaire fœtale et le transfert maternel vers un centre de soins tertiaires et ainsi réduire la morbidité et la mortalité néonatales. Buts et objectifs: Les buts et objectifs étaient de comparer l'innocuité et l'efficacité du timbre transdermique de nitroglycérine avec la nifédipine par voie orale comme agent tocolytique pour arrêter le travail prématuré et prévenir l'accouchement prématuré. Matériel et méthodes: Sur la base des critères de sélection, 50 patientes ont été sélectionnées au hasard dans les groupes A et B.Les femmes du groupe A ont reçu un patch transdermique de nitroglycérine, qui a administré 10 mg de NTG en 24 h et appliqué sur l'abdomen de la femme suivi d'un autre patch de 10 mg après 1 h si les contractions ont persisté. Après 24 h, il a été remplacé par un nouveau patch. Les femmes du groupe B ont reçu une dose de charge orale de 20 mg de nifédipine suivie d'une dose similaire si les contractions persistaient après 1 h. Une dose d'entretien de 10 mg trois fois par jour était administrée si les contractions étaient supprimées. Les patients ont été suivis du moment de l'admission au moment de la sortie. Résultats: La durée moyenne de prolongation de la grossesse dans le groupe B (3,68 ± 1,91 jours) était significativement plus élevée que dans le groupe A (2,78 ± 1,39 jours). Les céphalées étaient significativement plus observées dans le groupe A (42%) que dans le groupe B (6%). La tachycardie, l'hypotension et les palpitations n'ont montré aucune différence statistiquement significative entre elles. Il n'y avait pas de différence statistiquement significative du poids à la naissance des bébés dans les deux groupes. Conclusion: La nifédipine est un médicament sûr et efficace pour prolonger le travail prématuré et a des effets secondaires maternels et néonatals minimes.

Vitamin C Deficiency and Oxidant Levels in Children With Transfusion-Dependent b-Thalassemia.

OBJECTIVES: To study vitamin C levels in children with transfusion-dependent b-thalassemia and correlate with age, transfusions received and iron overload; and to study the effect of administering vitamin C on its levels and Malondialdehyde (MDA) in deficient patients. METHODS: This case-control study enrolled 100 children with transfusion-dependent b-thalassemia and 30 healthy controls. MDA levels before and after administration of vitamin C were performed randomly in 36 children with low vitamin C levels. RESULTS: 81/95 (85.3%) study subjects vs none in control group, had low plasma vitamin C levels (P<0.001). Vitamin C levels were low in 64 of 71 (74.7%) subjects with dietary deficiency, while none of the 19 (63.3%) controls with dietary deficiency had low levels (P=0.04). Increasing serum ferritin values correlated with vitamin C deficiency (P=0.02). The mean level of MDA reduced (P<0.001) with vitamin C supplementation. CONCLUSIONS: Low levels of vitamin C are common in children with thalassemia. Dietary counseling along with supplementation with vitamin C, in those with low levels may prevent oxidative stress.

Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Structure of Yak Lactoperoxidase at 1.55 Å Resolution.

Lactoperoxidase (LPO) is a heme containing oxido-reductase enzyme. It is secreted from mammary, salivary, lachrymal and mucosal glands. It catalyses the conversion of thiocyanate into hypothiocyanate and halides into hypohalides. LPO belongs to the superfamily of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The heme prosthetic group is covalently linked in LPO through two ester bonds involving conserved residues Glu258 and Asp108. It was isolated from colostrum of yak (Bos grunniens), purified to homogeneity and crystallized using ammonium iodide as a precipitating agent. The crystals belonged to monoclinic space group P21 with cell dimensions of a = 53.91 Å, b = 78.98 Å, c = 67.82 Å and ß = 92.96°. The structure was determined at 1.55 Å resolution. This is the first structure of LPO from yak. Also, this is the highest resolution structure of LPO determined so far from any source. The structure determination revealed that three segments (Ser1-Cys15), (Thr117-Asn138) and (Cys167-Leu175) were disordered and formed one surface of LPO structure. In the substrate binding site, the iodide ions were observed in three subsites which are formed by (1) heme moiety and residues, Gln105, Asp108, His109, Phe113, Arg255, Glu258, Phe380 and Phe381, (2) residues, Asn230, Lys232, Pro236, Cys248, Phe254, Phe381 and Pro424 and (3) residues, Ser198, Leu199 and Arg202. The structure determination also revealed that the side chain of Phe254 was disordered. It was observed to adopt two conformations in the structures of LPO.

Identification of bioactive molecule from Withania somnifera (Ashwagandha) as SARS-CoV-2 main protease inhibitor.

SARS-CoV-2 is the causative agent of COVID-19 and has been declared as pandemic disease by World Health Organization. Lack of targeted therapeutics and vaccines for COVID-2019 have triggered the scientific community to develop new vaccines or drugs against this novel virus. Many synthetic compounds and antimalarial drugs are undergoing clinical trials. The traditional medical practitioners widely use Indian medicinal plant Withania somnifera (Ashwagandha) natural constituents, called withanolides for curing various diseases. The main protease (Mpro) of SARS-CoV-2 plays a vital role in disease propagation by processing the polyproteins which are required for its replication. Hence, it denotes a significant target for drug discovery. In the present study, we evaluate the potential of 40 natural chemical constituents of Ashwagandha to explore a possible inhibitor against main protease of SARS-CoV-2 by adopting the computational approach. The docking study revealed that four constituents of Ashwagandha; Withanoside II (-11.30 Kcal/mol), Withanoside IV (-11.02 Kcal/mol), Withanoside V (-8.96 Kcal/mol) and Sitoindoside IX (-8.37 Kcal/mol) exhibited the highest docking energy among the selected natural constituents. Further, MD simulation study of 100 ns predicts Withanoside V possess strong binding affinity and hydrogen-bonding interactions with the protein active site and indicates its stability in the active site. The binding free energy score also correlates with the highest score of -87.01 ± 5.01 Kcal/mol as compared to other selected compounds. In conclusion, our study suggests that Withanoside V in Ashwagandha may be serve as a potential inhibitor against Mpro of SARS-CoV-2 to combat COVID-19 and may have an antiviral effect on nCoV.Communicated by Ramaswamy H. Sarma.

From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9.

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.

Biological Properties and Characterization of ASL50 Protein from Aged Allium sativum Bulbs.

Allium sativum is well known for its medicinal properties. The A. sativum lectin 50 (ASL50, 50 kDa) was isolated from aged A. sativum bulbs and purified by gel filtration chromatography on Sephacryl S-200 column. Agar well diffusion assay were used to evaluate the antimicrobial activity of ASL50 against Candida species and bacteria then minimal inhibitory concentration (MIC) was determined. The lipid A binding to ASL50 was determined by surface plasmon resonance (SPR) technology with varying concentrations. Electron microscopic studies were done to see the mode of action of ASL50 on microbes. It exerted antimicrobial activity against clinical Candida isolates with a MIC of 10-40 µg/ml and clinical Pseudomonas aeruginosa isolates with a MIC of 10-80 µg/ml. The electron microscopic study illustrates that it disrupts the cell membrane of the bacteria and cell wall of fungi. It exhibited antiproliferative activity on oral carcinoma KB cells with an IC50 of 36 µg/ml after treatment for 48 h and induces the apoptosis of cancer cells by inducing 2.5-fold higher caspase enzyme activity than untreated cells. However, it has no cytotoxic effects towards HEK 293 cells as well as human erythrocytes even at higher concentration of ASL50. Biological properties of ASL50 may have its therapeutic significance in aiding infection and cancer treatments.

Evaluation of single epidural bolus dose of magnesium as an adjuvant to epidural fentanyl for postoperative analgesia: A prospective, randomized, double-blind study.

OBJECTIVE: Magnesium has been used as an adjuvant by various routes, including intravenous, intrathecal, and epidural in different dosage regimens. The effect of single bolus dose of magnesium as an adjuvant to fentanyl for postoperative analgesia has not been studied. This prospective randomized controlled trial was done to evaluate the efficacy of single bolus administration of magnesium epidurally as an adjuvant to epidural fentanyl for postoperative analgesia in patients undergoing total hip replacement under combined spinal epidural anesthesia. METHODS: Sixty patients received combined spinal-epidural anesthesia with 2 mL of 0.5% hyperbaric bupivacaine intrathecally. After the surgery, patients were randomized into Group F [epidural fentanyl (1 µg/kg) in 10 mL saline] and Group FM [epidural magnesium (75 mg) along with fentanyl (1 µg/kg) in 10 mL saline]. Supplementary analgesia was provided by 50 mg intravenous tramadol if Verbal Rating Score (VRS) >4. Patient's first analgesic requirement and duration of analgesia were recorded. RESULTS: The duration of analgesia was significantly longer for Group FM, 340±28.8 min, compared with Group F, 164±17.1 min (P=0.001). The frequency of rescue analgesics required in 24-h postoperative period in Group FM (2.3±0.5) was significantly less than that in Group F (4.3±0.5) (P=0.001). VRS was significantly lower in Group FM up to 4 h in the postoperative period (P=0.001). Bromage scale was statistically insignificant at all points of time. CONCLUSIONS: The administration of magnesium (75 mg) as an adjuvant to epidural fentanyl (1 µg/ kg) for postoperative analgesia results in significantly lower VRS with prolonged duration of analgesia as compared with epidural fentanyl (1 µg/kg) alone. Concomitant administration of magnesium also reduces the requirement of breakthrough analgesics with no increased incidence of side effects.

Isolation and characterization of novel protein with anti-fungal and anti-inflammatory properties from Aloe vera leaf gel.

The Aloe protein of 14 kDa from the Aloe vera leaf gel was isolated by an ion exchange chromatography using DEAE-cellulose and CM-cellulose column. The purified Aloe protein exhibited a potent anti-fungal activity against Candida paraprilosis, Candida krusei and Candida albicans. In addition, the purified Aloe protein also showed an anti-inflammatory property against pure lipoxygenase and cyclooxygenase-2 with 84% and 73% inhibition, respectively, and was verified by binding with these proteins by real time method by the phenomenon of surface plasmon resonance. This Aloe protein is a novel protein possessing antifungal and anti-inflammatory properties and thus sets a platform to be used as a medicinal plant product.

The structural basis for the prevention of nonsteroidal antiinflammatory drug-induced gastrointestinal tract damage by the C-lobe of bovine colostrum lactoferrin.

Nonsteroidal antiinflammatory drugs (NSAIDs), due to their good efficacy in the treatment of pain, inflammation, and fever, are among the most prescribed class of medicines in the world. The main drawback of NSAIDs is that they induce gastric complications such as peptic ulceration and injury to the intestine. Four NSAIDs, indomethacin, diclofenac, aspirin, and ibuprofen were selected to induce gastropathy in mouse models. It was found that the addition of C-terminal half of bovine lactoferrin (C-lobe) reversed the NSAID-induced injuries to the extent of 47-70% whereas the coadministration of C-lobe prevented it significantly. The C-lobe was prepared proteolytically using serine proteases. The binding studies of C-lobe with NSAIDs showed that these compounds bind to C-lobe with affinities ranging from 2.6 to 4.8 x 10(-4) M. The complexes of C-lobe were prepared with the above four NSAIDs. All four complexes were crystallized and their detailed three-dimensional structures were determined using x-ray crystallographic method. The structures showed that all the four NSAID molecules bound to C-lobe at the newly identified ligand binding site in C-lobe that is formed involving two alpha-helices, alpha10 and alpha11. The ligand binding site is separated from the well known iron binding site by the longest and the most stable beta-strand, betaj, in the structure. Similar results were also obtained with the full length lactoferrin molecule. This novel, to our knowledge, binding site in C-lobe of lactoferrin shows a good complementarity for the acidic and lipophilic compounds such as NSAIDs. We believe this indicates that C-lobe of lactoferrin can be exploited for the prevention of NSAID-induced gastropathy.

Saponins as novel TNF-alpha inhibitors: isolation of saponins and a nor-pseudoguaianolide from Parthenium hysterophorus.

Two novel saponins and a 13-nor-pseudoguaianolide designated as hysterolactone were isolated from Parthenium hysterophorus. The two saponins were found to be potent inhibitors of TNF-alpha. Their mode of inhibition was studied through molecular modeling. The wet lab results were in concordance with the data obtained from docking experiments.

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site.

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.

Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution.

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.