Identification of small-molecule protein-protein interaction inhibitors for NKG2D.

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ+, and CD8+ T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein-protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure-activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

Convergence of immune escape strategies highlights plasticity of SARS-CoV-2 spike.

The global spread of the SARS-CoV-2 virus has resulted in emergence of lineages which impact the effectiveness of immunotherapies and vaccines that are based on the early Wuhan isolate. All currently approved vaccines employ the spike protein S, as it is the target for neutralizing antibodies. Here we describe two SARS-CoV-2 isolates with unusually large deletions in the N-terminal domain (NTD) of the spike. Cryo-EM structural analysis shows that the deletions result in complete reshaping of the NTD supersite, an antigenically important region of the NTD. For both spike variants the remodeling of the NTD negatively affects binding of all tested NTD-specific antibodies in and outside of the NTD supersite. For one of the variants, we observed a P9L mediated shift of the signal peptide cleavage site resulting in the loss of a disulfide-bridge; a unique escape mechanism with high antigenic impact. Although the observed deletions and disulfide mutations are rare, similar modifications have become independently established in several other lineages, indicating a possibility to become more dominant in the future. The observed plasticity of the NTD foreshadows its broad potential for immune escape with the continued spread of SARS-CoV-2.

Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59 Å resolution.

Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4'-phosphopantetheine (PNS) yielding 3'-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1 M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82 Å and c = 200.37 Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a ß/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10-4 M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.

Septum site placement in Mycobacteria - identification and characterisation of mycobacterial homologues of Escherichia coli MinD.

A major virulence trait of Mycobacterium tuberculosis (M. tb) is its ability to enter a dormant state within its human host. Since cell division is intimately linked to metabolic shut down, understanding the mechanism of septum formation and its integration with other events in the division pathway is likely to offer clues to the molecular basis of dormancy. The M. tb genome lacks obvious homologues of several conserved cell division proteins, and this study was aimed at identifying and functionally characterising mycobacterial homologues of the E. coli septum site specification protein MinD (Ec MinD). Sequence homology based analyses suggested that the genomes of both M. tb and the saprophyte Mycobacterium smegmatis (M. smegmatis) encode two putative Ec MinD homologues - Rv1708/MSMEG_3743 and Rv3660c/ MSMEG_6171. Of these, Rv1708/MSMEG_3743 were found to be the true homologues, through complementation of the E. coli ∆minDE mutant HL1, overexpression studies, and structural comparisons. Rv1708 and MSMEG_3743 fully complemented the mini-cell phenotype of HL1, and over-expression of MSMEG_3743 in M. smegmatis led to cell elongation and a drastic decrease in c.f.u. counts, indicating its essentiality in cell-division. MSMEG_3743 displayed ATPase activity, consistent with its containing a conserved Walker A motif. Interaction of Rv1708 with the chromosome associated proteins ScpA and ParB, implied a link between its septum formation role, and chromosome segregation. Comparative structural analyses showed Rv1708 to be closer in similarity to Ec MinD than Rv3660c. In summary we identify Rv1708 and MSMEG_3743 to be homologues of Ec MinD, adding a critical missing piece to the mycobacterial cell division puzzle.

High dietary iodine intake may contribute to the low death rate from COVID-19 infection in Japan with activation by the lactoperoxidase system.

We draw the attention of readers and governments to the death rate from coronavirus disease 2019 in Japan, continuing as a fraction of that experienced by many other developed nations. We think this is due to the activity of the powerful, protective lactoperoxidase system (LPO) which prevents serious airborne infections. The LPO system requires iodine, which is liberally provided by the typical Japanese diet but lacking in many others. One might consider the Japanese experience an incredibly large, open-label study exhibiting the preventative power of a high-iodine diet. We predict this favourable trend will continue for Japan because deadly variants of the severe, acute respiratory syndrome coronavirus 2 will be with us, forever.

Discovery of arylsulfonamides as a novel class of allosteric integrase inhibitors with antiviral activity.

Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.

Development of small molecule inhibitors of natural killer group 2D receptor (NKG2D).

Natural killer group 2D (NKG2D) is a homodimeric activating immunoreceptor whose function is to detect and eliminate compromised cells upon binding to the NKG2D ligands (NKG2DL) major histocompatibility complex (MHC) molecules class I-related chain A (MICA) and B (MICB) and UL16 binding proteins (ULBP1-6). While typically present at low levels in healthy cells and tissue, NKG2DL expression can be induced by viral infection, cellular stress or transformation. Aberrant activity along the NKG2D/NKG2DL axis has been associated with autoimmune diseases due to the increased expression of NKG2D ligands in human disease tissue, making NKG2D inhibitors an attractive target for immunomodulation. Herein we describe the discovery and optimization of small molecule PPI (protein-protein interaction) inhibitors of NKG2D/NKG2DL. Rapid SAR was guided by structure-based drug design and accomplished by iterative singleton and parallel medicinal chemistry synthesis. These efforts resulted in the identification of several potent analogs (14, 21, 30, 45) with functional activity and improved LLE.

Structural basis for selectivity and diversity in angiotensin II receptors.

The angiotensin II receptors AT1R and AT2R serve as key components of the renin-angiotensin-aldosterone system. AT1R has a central role in the regulation of blood pressure, but the function of AT2R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT2R bound to an AT2R-selective ligand and to an AT1R/AT2R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or ß-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.

What are the reasons for abandonment of low vision devices prescribed in a large tertiary eye care centre?

PURPOSE: To investigate abandonment rates of near-vision low vision devices (LVDs) and factors that influence abandonment among patients attending a tertiary low vision rehabilitation centre in South India. METHODS: Two hundred and eighty-six adults with low vision completed the modified device abandonment survey 1 year following device prescription. The survey included six questions: possession of device, timing of last use, reasons for abandonment, tasks for which the device was used, payment type and change in quality of life (QoL) from device use. The primary outcome measure was abandonment. Multivariate logistic regression analysis was used to investigate factors for abandonment. RESULTS: Three hundred and twelve near-vision devices were prescribed (mean, 1.09 device per patient.) Stand magnifiers (35%) followed by hand-held magnifiers (24%) were most frequently prescribed. Mean logMAR visual acuity (Snellen) in the better-seeing eye was 0.80 (6/38). Of the prescribed near-vision devices, 22% (95% CI, 17 to 27) were abandoned. Patients who abandoned the device were significantly older than those who did not (49.3 ± 17.2 vs. 43.5 ± 18.1 years; p = 0.03). In multivariable analysis, patients reporting no change in their QoL from device use had higher odds of abandoning the device (OR: 63.97; 95% CI, 23.77 to 172.12). Device-related (31%) and psychological (30%) factors were the most frequent reasons for abandonment. Among device-related issues, the most frequent reason was that patients felt the device was too complex to use (50%) followed by being too cumbersome to use (25%). CONCLUSION: The abandonment rate for near-vision LVDs in South India was comparable with that reported in high-income countries. Patients reporting no change in their QoL had a higher likelihood of abandoning the device compared with those who reported some change. Device-related and psychological factors were the most frequent reasons for abandonment. These results can be used to develop strategies to improve compliance with use of devices.

Early detection of glomerular dysfunction and renal tubulopathy in children with sickle cell disease in India.

BACKGROUND: Sickle cell disease causes microvascular occlusion in different vascular beds. In kidneys, it leads to occult glomerular dysfunction causing asymptomatic microalbuminuria, proximal tubulopathy causing hyposthenuria and increased free water loss and distal tubulopathy causing poor urine acidification. We studied the prevalence of various types of renal dysfunction, the ability of different tests to detect it at an early stage and the correlation of these parameters in children receiving hydroxyurea (HU). PROCEDURE: Fifty-six children (sample size calculated using SAS9.2 package) attending paediatric clinical services in a tertiary care hospital between 2 and 12 years of age diagnosed by high-performance liquid chromatography (HPLC) were enrolled. Their demographic and laboratory data including renal and urine parameters were collected. Parameters like fractional excretion of sodium (FeNa), trans tubular potassium gradient (TtKg) and free water clearance (TcH2O) were derived by calculations. Data were analysed using IBM SPSS Version 21.0 and Microsoft Office Excel 2007. RESULTS: We found a significant number of children to have microalbuminuria (17.8%), hyposthenuria (30.4%) and impaired renal tubular potassium excretion (TtKg) (81.3%). A significant correlation was found between the dose of HU with urine osmolality (p < 0.0005) and free water clearance (p = 0.002), while all parameters showed a significant correlation with compliance with HU. Derangement in urine microalbumin and TcH2O correlated significantly with low mean haemoglobin levels (<9 g/dl). CONCLUSION: Renal dysfunction is common in children with SCD and can be detected early using simple urine parameters and can be prevented with an early and appropriate dosage of HU with good compliance.

Comments on the Discussion Forum: Oromucosal immunomodulation as clinical spectrum mitigating factor in SARS-CoV-2 infection.

The mammalian lactoperoxidase system, consisting of lactoperoxidase and the H2 O2 -producing enzyme duox, is our first line of defence against airborne microbes. This system catalyses the production of hypoiodite and hypoiodous acid in the presence of sufficient iodine. These products are highly efficient at destroying the H1N1 virus and the respiratory syncytial virus (RSV). Japan has not been affected as much as other nations during the COVID-19 pandemic (death rate about 10% of the United States), and we think this is due to a diet high in iodine. With this in mind, we suggest four actions to prevent SARS-CoV-2 infections. First, health professionals should study the preventative effect of increasing iodine in the diets of the aged, institutionalized, diabetics andsmokers. Second, the recommended daily intake (RDI) for iodine should be significantly increased, to at least double, the current RDI. Governments should encourage the use and distribution of cheap iodized salts, kelp and seaweed. Third, more research should be done around the physiology and the protective effects of the lactoperoxidase system. Finally, the degradation products of the SARS-CoV-2 viral particle by hypoiodite and hypoiodous acid should be characterized; portions of the damaged particle are likely to elicit stronger immunity and better vaccines.

Discovery of the Lead Molecules Targeting the First Step of the Histidine Biosynthesis Pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant, opportunistic, nosocomial pathogen for which a new line of treatments is desperately needed. We have targeted the enzyme of the first step of the histidine biosynthesis pathway, viz., ATP-phosphoribosyltransferase (ATP-PRT). The three-dimensional structure of ATP-PRT was predicted on the template of the known three-dimensional structure of ATP-PRT from Psychrobacter arcticus (PaATPPRT) using a homology modeling approach. High-throughput virtual screening (HTVS) of the antibacterial library of Life Chemicals Inc., Ontario, Canada was carried out followed by molecular dynamics simulations of the top hit compounds. In silico results were then biochemically validated using surface plasmon resonance spectroscopy. We found that two compounds, namely, F0843-0019 and F0608-0626, were binding with micromolar affinities to the ATP-phosphoribosyltransferase from Acinetobacter baumannii (AbATPPRT). Both of these compounds were binding in the same way as AMP in PaATPPRT, and the important residues of the active site, viz., Val4, Ser72, Thr76, Tyr77, Glu95, Lys134, Val136, and Tyr156, were also interacting via hydrogen bonds. The calculated binding energies of these compounds were -10.5 kcal/mol and -11.1 kcal/mol, respectively. These two compounds can be used as the potential lead molecules for designing antibacterial compounds in the future, and this information will help in drug discovery programs against Acinetobacter worldwide.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Factors affecting quality of life in keratoconus.

PURPOSE: To identify the factors affecting the quality of life (QoL) in adults with keratoconus, using the disease-specific Keratoconus Outcomes Research Questionnaire (KORQ). METHODS: In this cross-sectional study, 574 patients with keratoconus completed the 29-item KORQ (18-item activity limitations and 11-item symptoms subscales) and demographic information. Based on mean corneal curvature to grade keratoconus severity, participants were categorised into four groups: mild <48 dioptres [D]; moderate: 48 to 53 D; advanced: 54 to 55 D and severe: >55D. Rasch analysis was used to assess the psychometric properties and to score the KORQ. Multivariable regression was performed to determine the independent impact of age, sex, laterality, severity and duration of keratoconus, education, employment status and mode of treatment for keratoconus on activity limitations and symptoms. RESULTS: Mean (SD) age was 24.5 (5.1) years. 304 (53%), 160 (28%), 26 (4%) and 84 (15%) belonged to keratoconus groups 1, 2, 3 and 4, respectively. Rasch analysis demonstrated the 'activity limitations' and 'symptoms' KORQ subscales to possess good psychometric properties. Statistically significant associations were found between activity limitation score and the following measures: visual acuity (VA) in the better-seeing eye (r = -0.10, p = 0.01); VA in the worse-seeing eye (r = -0.09, p = 0.04) and ocular aberrations (r = -0.13, p = 0.008) in the worse-seeing eye. Statistically significant associations were also found between symptoms score and mean corneal curvature in the better-seeing eye (r = -0.10, p = 0.02). In regression models, female sex was associated with 21% worse symptom score than male (ß = -0.33, 95% CI, -0.09, -0.59, p = 0.01) and working people experienced clinically and statistically significantly greater trouble from symptoms compared to those not working (ß =0.44, 95% CI, 0.17, 0.70, p = 0.001). CONCLUSIONS: People with keratoconus have poorer QoL in terms of activity limitations if they have poorer VA and greater wavefront aberrations, and in terms of symptoms if they are female and employed.

Nanoformulation approach for improved antimicrobial activity of bovine lactoperoxidase.

Lactoperoxidase (LPO) is a glycosylated antimicrobial protein present in milk with a molecular mass of 78 kDa. LPO is included in many biological processes and is well-known to have biocidal actions, acting as an active antibiotic and antiviral agent. The wide spectrum biocidal activity of LPO is mediated via a definite inhibitory system named lactoperoxidase system which plays a potent role in the innate immune response. With the current advancement in nanotechnology, nanoformulations can be developed for stabilizing and potentiating the activity of LPO for several applications. In the research described in this Research Communication, fresh LPO purified from bovine mammary gland secretions was used for nanoparticle synthesis using a simple thermal process at different pH and temperatures. The round-shaped nanoparticles (average size 229 nm) were successfully synthesized at pH 7.0 and a temperature of 75°C. These nanoparticles were tested against four different bacterial species namely S. flexineri, P. aeruginosa, S. aureus, and E. coli. The prepared nanoparticles exhibited strong inhibition of the growth against all four bacterial species as stated by their MIC and ZOI values. These results may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO in drug discovery and industrial processes.

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.

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.

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with uracil and uridine.

Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.

Insights into activity and inhibition from the crystal structure of human O-GlcNAcase.

O-GlcNAc hydrolase (OGA) catalyzes removal of ßα-linked N-acetyl-D-glucosamine from serine and threonine residues. We report crystal structures of Homo sapiens OGA catalytic domain in apo and inhibited states, revealing a flexible dimer that displays three unique conformations and is characterized by subdomain α-helix swapping. These results identify new structural features of the substrate-binding groove adjacent to the catalytic site and open new opportunities for structural, mechanistic and drug discovery activities.

Search of multiple hot spots on the surface of peptidyl-tRNA hydrolase: structural, binding and antibacterial studies.

Peptidyl-tRNA hydrolase (Pth) catalyzes the breakdown of peptidyl-tRNA into peptide and tRNA components. Pth from Acinetobacter baumannii (AbPth) was cloned, expressed, purified and crystallized in a native unbound (AbPth-N) state and in a bound state with the phosphate ion and cytosine arabinoside (cytarabine) (AbPth-C). Structures of AbPth-N and AbPth-C were determined at 1.36 and 1.10 Šresolutions, respectively. The structure of AbPth-N showed that the active site is filled with water molecules. In the structure of AbPth-C, a phosphate ion is present in the active site, while cytarabine is bound in a cleft which is located away from the catalytic site. The cytarabine-binding site is formed with residues: Gln19, Trp27, Glu30, Gln31, Lys152, Gln158 and Asp162. In the structure of AbPth-N, the side chains of two active-site residues, Asn70 and Asn116, were observed in two conformations. Upon binding of the phosphate ion in the active site, the side chains of both residues were ordered to single conformations. Since Trp27 is present at the cytarabine-binding site, the fluorescence studies were carried out which gave a dissociation constant (KD) of 3.3 ± 0.8 × 10-7 M for cytarabine. The binding studies using surface plasmon resonance gave a KD value of 3.7 ± 0.7 × 10-7 M. The bacterial inhibition studies using the agar diffusion method and the biofilm inhibition assay established the strong antimicrobial potential of cytarabine. It also indicated that cytarabine inhibited Gram-negative bacteria more profoundly when compared with Gram-positive bacteria in a dose-dependent manner. Cytarabine was also effective against the drug-resistant bacteria both alone as well as in combination with other antibiotics.