Clinical Long-Term Outcomes of Patient-Reported Outcomes in the Prospective Real-World Tofacitinib Response in Ulcerative Colitis Registry.

INTRODUCTION: We previously reported the results of tofacitinib induction therapy in the prospective multisite US real-world Tofacitinib Response in Ulcerative Colitis registry. We now assessed patient-reported outcomes (PROs) and predictors of success during tofacitinib maintenance therapy. METHODS: Tofacitinib Response in Ulcerative Colitis included 103 patients with refractory ulcerative colitis (UC); 67% had failed ≥ 2 biologics. Patients reported the Simple Clinical Colitis Activity Index (SCCAI), Patient-Reported Outcome Measurement Information System measures for anxiety, depression, social satisfaction, and adverse events between weeks 8 and 52 using a web-based system. Paired t test and P for trend were used to compare changes in PRO measures over time. Bivariate analyses and logistic regression models were used to determine factors associated with response (SCCAI <5) or remission (SCCAI <2) at week 52. RESULTS: Of 103 patients, 82.5% entered the maintenance phase and 43.7% remained on tofacitinib at week 52. Tofacitinib de-escalation to 5 mg BID occurred in 15% of patients. At week 52, 42.7% and 31.1% of all patients reported an SCCAI <5 and SCCAI ≤2, respectively. Normalization of bowel frequency, rectal bleeding, and urgency occurred in 79%, 61%, and 48% of patients remaining on maintenance therapy. Social satisfaction improved significantly ( P < 0.001), while anxiety and depression scores only numerically improved. No consistent predictors for tofacitinib long-term treatment efficacy were identified, and safety findings were consistent with the known safety profile of tofacitinib. DISCUSSION: Tofacitinib is an effective maintenance therapy in patients with refractory UC. Dose reductions infrequently occurred during maintenance. Unmet needs in UC maintenance include improvement of urgency and psychosocial factors (NCT03772145).

Fortuitous In Vitro Compound Degradation Produces a Tractable Hit against Mycobacterium tuberculosis Dethiobiotin Synthetase: A Cautionary Tale of What Goes In Does Not Always Come Out.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here, the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by an in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD = 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here, we show that the lactone of 1 hydrolyzes to a carboxylic acid (2) under basic conditions, including those of the crystallography soak, with a subsequent ring opening of the component cyclobutane ring forming a cyclopentylacetic acid (3). Crystals soaked directly with authentic 3 produced an electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled the subsequent compound development of nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.

Structural Study of Potent Triazole-Based Inhibitors of Staphylococcus aureus Biotin Protein Ligase.

The rise of multidrug-resistant bacteria, such as Staphylococcus aureus, has highlighted global urgency for new classes of antibiotics. Biotin protein ligase (BPL), a critical metabolic regulatory enzyme, is an important target that shows significant promise in this context. Here we report the in silico docking, synthesis, and biological assay of a new series of N1-diphenylmethyl-1,2,3-triazole-based S. aureus BPL (SaBPL) inhibitors (8-19) designed to probe the adenine binding site and define whole-cell activity for this important class of inhibitor. Triazoles 13 and 14 with N1-propylamine and -butanamide substituents, respectively, were particularly potent with K i values of 10 ± 2 and 30 ± 6 nM, respectively, against SaBPL. A strong correlation was apparent between the K i values for 8-19 and the in silico docking, with hydrogen bonding to amino acid residues S128 and N212 of SaBPL likely contributing to potent inhibition.

Design and synthesis of benzochromene derivatives as AcrB inhibitors for the reversal of bacterial multidrug resistance.

A series of novel benzo[h]chromene compounds were designed, synthesized and evaluated for their biological activity as AcrB inhibitors. The compounds were assessed for their ability to potentiate the effect of antibiotics. Compounds with antibiotic-potentiating effects were then evaluated for inhibition of Nile Red efflux, and for off-target effects including activity on the outer and inner bacterial membranes and toxicity. Six compounds were identified to reduce the MIC values of at least one of the tested antibiotics by at least 4-fold, and further reduced the MICs in the presence of a membrane permeabilizer. The identified compounds were also able to inhibit Nile Red efflux at concentrations between 50 µM and 200 µM. The compounds did not disrupt the bacterial outer membrane nor display toxicity in a nematode model (Caenorhabditis elegans). The 4-methoxyphenoxy)propoxy derivative compound G6 possessed the most potent antibacterial potentiation with erythromycin by 8-fold even without the presence of a membrane permeabilizer. Furthermore, H6, G6, G10 and G11 completely abolished the Nile Red efflux at a concentration of 50 µM. The 3,4-dihydro-2H-benzo[h]chromen-5-yl)(morpholino)methanone core appears to be a promising chemical skeleton to be further studied in the discovery of more putative AcrB inhibitors.

Tofacitinib Response in Ulcerative Colitis (TOUR): Early Response After Initiation of Tofacitinib Therapy in a Real-world Setting.

BACKGROUND: Tofacitinib is an oral, small-molecule JAK inhibitor for the treatment of ulcerative colitis (UC). Using a novel electronic reporting tool, we aimed to prospectively describe the onset of tofacitinib efficacy during induction therapy in a real-world study. METHODS: Patient-reported outcome data (PROs) including the simple clinical colitis activity index (SCCAI), PRO Measurement Identification Systems (PROMIS) measures, and adverse events were collected daily for the first 14 days and at day 28 and 56. Paired t tests and P for trend were utilized to compare changes in SCCAI over time. Bivariate analyses and logistic regression models were performed to describe response (SCCAI <5) and remission (SCCAI ≤2) by clinical factors. RESULTS: Of all included patients (n = 96), 67% had failed ≥2 biologics, and 61.5% were on concomitant steroids. Starting at day 3, PROs showed significant and persistent decline of the mean SCCAI (-1.1, P < 000.1) including significantly lower SCCAI subscores for stool frequency (-0.3; P < .003), bleeding (-0.3; P < .0002) and urgency (-0.2; P < .001). Steroid-free remission at day 14, 28, and 56 was achieved in 25%, 30.2%, and 29.2% of patients, respectively. Neither prior biologics nor endoscopic severity were independently predictive of response or remission in multivariate models. Numeric improvements in all PROMIS measures (anxiety, depression, social satisfaction) were seen through day 56. Rates of discontinuation due to adverse events were low. CONCLUSIONS: In this prospective real-world study, tofacitinib resulted in a rapid and persistent improvement in UC disease activity PROs. The safety findings were consistent with the established safety profile of tofacitinib.

A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase.

Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7-13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 µg/mL, respectively.

Cinnamaldehyde derivatives act as antimicrobial agents against Acinetobacter baumannii through the inhibition of cell division.

Acinetobacter baumannii is a pathogen with high intrinsic antimicrobial resistance while multidrug resistant (MDR) and extensively drug resistant (XDR) strains of this pathogen are emerging. Treatment options for infections by these strains are very limited, hence new therapies are urgently needed. The bacterial cell division protein, FtsZ, is a promising drug target for the development of novel antimicrobial agents. We have previously reported limited activity of cinnamaldehyde analogs against Escherichia coli. In this study, we have determined the antimicrobial activity of six cinnamaldehyde analogs for antimicrobial activity against A. baumannii. Microscopic analysis was performed to determine if the compounds inhibit cell division. The on-target effect of the compounds was assessed by analyzing their effect on polymerization and on the GTPase activity of purified FtsZ from A. baumannii. In silico docking was used to assess the binding of cinnamaldehyde analogs. Finally, in vivo and in vitro safety assays were performed. All six compounds displayed antibacterial activity against the critical priority pathogen A. baumannii, with 4-bromophenyl-substituted 4 displaying the most potent antimicrobial activity (MIC 32 µg/mL). Bioactivity was significantly increased in the presence of an efflux pump inhibitor for A. baumannii ATCC 19606 (up to 32-fold) and significantly, for extensively drug resistant UW 5075 (greater than 4-fold), suggesting that efflux contributes to the intrinsic resistance of A. baumannii against these agents. The compounds inhibited cell division in A. baumannii as observed by the elongated phenotype and targeted the FtsZ protein as seen from the inhibition of polymerization and GTPase activity. In silico docking predicted that the compounds bind in the interdomain cleft adjacent to the H7 core helix. Di-chlorinated 6 was devoid of hemolytic activity and cytotoxicity against mammalian cells in vitro, as well as adverse activity in a Caenorhabditis elegans nematode model in vivo. Together, these findings present halogenated analogs 4 and 6 as promising candidates for further development as antimicrobial agents aimed at combating A. baumannii. This is also the first report of FtsZ-targeting compounds with activity against an XDR A. baumannii strain.

Role of intracellular energy metabolism in Mn(â ¡) removal by the novel bacterium Stenotrophomonas sp. MNB17.

Microorganism-mediated Mn(Ⅱ) removal has gained increasing attention as a valuble bioremediation approach. In this study, a novel strain Stenotrophomonas sp. MNB17 - obtained from marine sediments - was found to show Mn(Ⅱ) removal efficiencies of 98.51-99.38% within 7 days and 92.24% within 20 days at Mn(Ⅱ) concentrations of 10-40 mM and 50 mM, respectively. On day 7, 80.44% of 50 mM Mn(Ⅱ) was oxidized to Mn(Ⅲ/Ⅳ), whereas only 2.11-2.86% of 10-40 mM Mn(Ⅱ) was oxidized. This difference in the proportion of Mn-oxides suggested that the strain MNB17 could remove soluble Mn(Ⅱ) via distinct mechanisms under different Mn(Ⅱ) concentrations. At 10 mM Mn(Ⅱ), indirect mechanisms were employed by strain MNB17 to remove Mn(Ⅱ). The sufficient energy generated by increased cellular respiration led to enhanced ammonification, and MnCO3 was the main component of the Mn-precipitates (97.27%). Meanwhile, intracellular fatty acids were degraded and served as an important carbon source for respiration. At 50 mM Mn(Ⅱ), most of the soluble Mn(Ⅱ) was oxidized, and Mn-oxides dominated the Mn-precipitates (80.44%). Mn(Ⅱ) oxidation likely contributed to electrons for energy production, as the down-regulation of respiratory pathways resulted in a deficit of electron supply, which warrants futher study. The exogenous addition of tricarboxylic acid cycle substrates (malate, α-ketoglutarate, oxaloacetate, succinate, and fumarate) was found to accelerate Mn(Ⅱ) removal as MnCO3 at a concentration of 50 mM. Overall, this study reports a novel strain MNB17 with the biotechnological potential of Mn(Ⅱ) removal and elucidates the function of cellular energy metabolism during the Mn(Ⅱ) removal process. In addition, it demonstrates the potential of aerobic respiration-related substrates in accelerating the removal of high concentrations of Mn(Ⅱ) for the first time.

Exploring Cost Savings with Specialty Biologic Drugs Administered to Adult Inpatients with Inflammatory Bowel Disease.

Background: Specialty infusion and self-injectable biologic drugs for the treatment of inflammatory bowel disease (IBD) are high-cost medications. When administered to hospital-admitted patients, these medications are not reimbursed on an individual basis but rolled into a per diem payment by most payers in the United States (US). Therefore, choosing to administer these medications in the inpatient setting may reveal negative financial implications for some health care institutions. Selecting an alternative site of care to administer these medications during the clinical management process may lead to cost savings. Objective: Review the clinical necessity of inpatient specialty biologic administrations for the treatment of IBD to identify and quantify potential cost saving opportunities. Methods: Using patient medical records at a US academic medical center, we retrospectively identified inpatient administrations of specialty infusion and self-injectable biologic medications for IBD treatment from June 1, 2016 to May 31, 2017. Guided by a standardized form, an evaluation team consisting of 3 of the investigators determined the clinical necessity of each specialty biologic medication administration within the inpatient setting. Costs and reimbursement rates for administration in both the inpatient and outpatient settings were procured and tabulated. Results: Seventeen inpatient specialty biologic administrations for IBD during the 12 month study period were identified. Of these, 11 administrations were given for the treatment of Crohn's disease (CD) and 6 for ulcerative colitis (UC). The evaluation team determined that 65% of these administrations were clinically necessary as inpatient administrations, and that 35% were not. The sum of the wholesale acquisition costs (WAC) for clinically necessary inpatient biologic administrations totaled $54 737, and the WAC for those administrations deemed not clinically necessary totaled $43 702. Further analysis of administration events revealed that the institution could have realized an estimated $13 817 in additional revenue above the cost of the drug if eligible inpatient biologic administrations had been received in the institution's outpatient clinic setting instead. Conclusion: Administering specialty biologic drugs for the treatment of IBD in the care setting best aligned with existing reimbursement structures may lead to institutional cost savings.

Differential growth inhibition, cell cycle arrest and apoptosis of MCF-7 and MDA-MB-231 cells to holocarboxylase synthetase suppression.

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin onto the biotin-dependent carboxylases. Recent studies have shown that HLCS is over-expressed in breast cancer patients. Here we investigated the functional roles of free biotin and HLCS in supporting growth and migration of breast cancer cell lines. Depletion of biotin from culture medium markedly reduced biotinylation of the two most abundant biotin-carboxylases, acetyl-CoA carboxylase and pyruvate carboxylase. This was accompanied by a marked decrease in cell growth. Suppression of HLCS expression in the low invasive breast cancer cell line MCF-7 resulted in an 80% reduction of biotinylated ACC, but not PC. HLCS knockdown MCF-7 cell lines showed 40-50% reduction of proliferation and 35% reduction of migration, accompanied by G1 cell cycle-arrest-induced apoptosis. In contrast, knockdown of HLCS expression in the highly invasive cell line MDA-MB-231 resulted in only marginal reduction of biotinylation of both ACC and PC, accompanied by 30% reduction of proliferation and 30% reduction of migration. Our studies provide new insights to use HLCS as a novel anti-cancer drug target.

Direct endoscopic placement of percutaneous endoscopic gastrostomies with jejunal extension tubes (PEG-J) using ultra-thin endoscopes - long-term outcomes from a U.S. tertiary referral center.

INTRODUCTION: Several techniques for PEG-J tube placement have been described, commonly requiring fluoroscopic guidance and/or fixation of the jejunostomy tube (J-tube) into the small intestine. We describe a modified technique for placing jejunostomy tubes under direct visualization through a PEG with the use of ultra-thin endoscopes and steel guidewire. METHODS: A retrospective study at a single tertiary academic center evaluating patients who underwent PEG-J placement between 2010 and 2020. All PEG tubes were placed with a pull-through technique. The Olympus GIF-N180 endoscope was advanced through the PEG to the jejunum and a Savary-Gilliard guidewire was used for placement of the J-tube extension. RESULTS: Fifty-eight patients underwent PEG-J placement (median age 61 years; women 52%). Surgically altered gastric anatomy was observed in 11 patients (19%). Median procedure time was 44 min for new PEG-J tube placement (range 26-103) and 20 min for placement of a J-tube extension through an existing PEG tube (range 9-86) or gastrostomy tract. Technical success rate was in 100%. Sixty-two repeat procedures were performed for J-tube exchange in 27 patients (46%, range 1-9 per patient), of which 51 procedures (82%) were done using the same technique. The most common indication for tube replacement was tube dysfunction (63%, n = 39). The median procedure time for tube exchange was 20 min (range 2-62). No major adverse events were encountered. CONCLUSION: PEG-J tubes can be placed effectively, rapidly, and safely using an ultra-thin caliber endoscope and a stiff steel wire through the PEG tube or mature gastrostomy site, precluding the need for fluoroscopy or oral access. J-tubes can be easily replaced utilizing the same technique.

Inhibition of Mycobacterium tuberculosis Dethiobiotin Synthase (MtDTBS): Toward Next-Generation Antituberculosis Agents.

Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis. Here, we report a binder of MtDTBS, cyclopentylacetic acid 2 (KD = 3.4 ± 0.4 mM), identified via in silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of MtDTBS. Appending an acidic group to the para-position of the aromatic ring of the scaffold revealed compounds 4c and 4d as more potent binders, with KD = 19 ± 5 and 17 ± 1 µM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder (KD = 57 ± 5 nM) and inhibitor (Ki = 5 ± 1 µM) of MtDTBS. Our findings highlight the first reported inhibitors of MtDTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.

New Isocoumarin Analogues from the Marine-Derived Fungus Paraphoma sp. CUGBMF180003.

Nine new secondary metabolites, including six isocoumarin analogues, 7-hydroxyoospolactone (1), 7-methoxyoospolactone (2), 7-methoxy-9-hydroxyoospolactone (3), 10-acetoxy-9-hydroxyoospolactone (4), 6-dehydroxysescandelin (5), parapholactone (6), and three compounds with a rare skeleton of isocoumarin coupled with phenylethylamine, namely paraphamide A (12), paraphamide B (13), and paraphamide C (14), together with five known compounds, oospolactone (7), 8-O-methyloospolactone (8), 10-hydroxyoospolactone (9), 9,10-dihydroxyoospolactone (10), and oospoglycol (11), were isolated and identified from the marine-derived fungus Paraphoma sp. CUGBMF180003. Their chemical structures were determined using spectroscopic data, including HRESIMS and 1D and 2D NMR techniques. Furthermore, the stereogenic carbons in 5 and 14 were determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra. The carbon skeleton of 12-14 was identified as the first example of isocoumarin coupled with phenylethylamine derivatives. All of these compounds were examined for antimicrobial activities against Candida albicans and Staphylococcus aureus. Both 1 and 6 showed antibacterial activity against S. aureus with MIC values of 12.5 µg/mL.

Design and structural optimization of novel 2H-benzo[h]chromene derivatives that target AcrB and reverse bacterial multidrug resistance.

Drug efflux pumps have emerged as a new drug targets for the treatment of bacterial infections in view of its critical role in promoting multidrug resistance. Herein, novel chromanone and 2H-benzo[h]chromene derivatives were designed by means of integrated molecular design and structure-based pharmacophore modeling in an attempt to identify improved efflux pump inhibitors that target Escherichia coli AcrB. The compounds were tested for their efflux inhibitory activity, ability to inhibit efflux, and the effect on bacterial outer and inner membranes. Twenty-three novel structures were identified that synergized with antibacterials tested, inhibited Nile Red efflux, and acted specifically on the AcrB. Among them, WK2, WL7 and WL10 exhibiting broad-spectrum and high-efficiency efflux inhibitory activity were identified as potential ideal AcrB inhibitors. Molecular modeling further revealed that the strong π-π stacking interactions and hydrogen bond networks were the major contributors to tight binding of AcrB.

Antimicrobial Action and Reversal of Resistance in MRSA by Difluorobenzamide Derivatives Targeted at FtsZ.

The bacterial cell division protein, FtsZ, has been identified as a target for antimicrobial development. Derivatives of 3-methoxybenzamide have shown promising activities as FtsZ inhibitors in Gram-positive bacteria. We sought to characterise the activity of five difluorobenzamide derivatives with non-heterocyclic substituents attached through the 3-oxygen. These compounds exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), with an isopentyloxy-substituted compound showing modest activity against vancomycin resistant Enterococcus faecium (VRE). The compounds were able to reverse resistance to oxacillin in highly resistant clinical MRSA strains at concentrations far below their MICs. Three of the compounds inhibited an Escherichia coli strain lacking the AcrAB components of a drug efflux pump, which suggests the lack of Gram-negative activity can partly be attributed to efflux. The compounds inhibited cell division by targeting S. aureus FtsZ, producing a dose-dependent increase in GTPase rate which increased the rate of FtsZ polymerization and stabilized the FtsZ polymers. These compounds did not affect the polymerization of mammalian tubulin and did not display haemolytic activity or cytotoxicity. These derivatives are therefore promising compounds for further development as antimicrobial agents or as resistance breakers to re-sensitive MRSA to beta-lactam antibiotics.

Design and synthesis of novel 4-substituted quinazoline-2-carboxamide derivatives targeting AcrB to reverse the bacterial multidrug resistance.

Novel 4-substituted quinazoline-2-carboxamide derivatives targeting AcrB were designed, synthesized and evaluated for their biological activity as AcrB inhibitors. In particular, the ability of the compounds to potentiate the activity of antibiotics, to inhibit Nile Red efflux and to target AcrB was investigated. In this study, 19 compounds were identified to reduce the MIC values of at least one tested antibacterial by 2- to 16-fold at a lower concentration. Identified modulating compounds also possessed considerable inhibition on Nile red efflux at concentrations as low as 50 µM and did not display off-target effects on the outer membrane. Among the above compounds with characteristics of ideal AcrB inhibitors, the most outstanding ones are A15 and B5-B7. In particular, A15 and B7 exhibited not only the most prominent performance in the synergistic effect, but also completely abolished Nile Red efflux at concentrations of 50 and 100 µM, respectively. In docking simulations, A15 was observed to have the most favorable docking score and was predicted to bind in the hydrophobic trap as has been noted with other inhibitors such as MBX2319. It is worth noting that the 4-morpholinoquinazoline-2-carboxamide core appears to be a promising chemical skeleton to be further optimized for the discovery of more potent AcrB inhibitors.

Pyrexia and acidosis act independently of neutrophil elastase reactive center loop cleavage to effect cortisol release from corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) transports cortisol and other steroids. High-affinity CBG (haCBG) undergoes proteolysis of the reactive center loop (RCL) by neutrophil elastase (NE) altering conformation to low-affinity CBG (laCBG). Elevated temperature reduces CBG:cortisol binding affinity. Surface plasmon resonance was used to determine binding profiles of 19 steroids to haCBG and laCBG at 25, 37, and 39°C mimicking pyrexia and pH 7.4 and 7.0 mimicking acidosis, pathophysiological conditions relevant to sepsis. An expected 4-8-fold reduction in affinity for cortisol, cortisone, corticosterone, 11-deoxycortisol, progesterone, 17-hydroxyprogesterone, and prednisolone occurred with NE-mediated haCBG-to-laCBG conversion. CBG:cortisol binding affinity was further reduced 3.5-fold at 39°C relative to 37°C, binding affinity was also reduced by acidosis for both haCBG and laCBG. Using a conformational antibody generated against the RCL, we confirmed RCL antibody binding was eliminated by NE cleavage, but preserved in pyrexia and acidosis. Molecular modeling studies performed at 40°C confirmed a critical role for Trp371, positioned within the steroid-binding pocket, in ligand binding. These studies demonstrated CBG binding affinity to range of steroids is ligand specific and is reduced with NE-mediated haCBG-to-laCBG transition. Reduced CBG:cortisol binding occurs with increased temperature and in acidosis. Increased flexibility of the Trp371 side chain is proposed in the thermo-coupling mechanism of cortisol release. The synergy of NE cleavage, pyrexia, and acidosis on CBG:cortisol binding may serve to enhance cortisol delivery to the interstitial space in inflammation.

Overexpression of Holocarboxylase Synthetase Predicts Lymph Node Metastasis and Unfavorable Prognosis in Breast Cancer.

BACKGROUND/AIM: Holocarboxylase synthetase (HLCS) catalyzes the specific attachment of biotin onto biotin-dependent carboxylases (BDCs) which play important roles in intermediary metabolism. Previous studies show that BDCs are overexpressed in many cancer types. However, expression of HLCS in cancerous tissues has not been reported. MATERIALS AND METHODS: Immunohistochemistry was used to investigate HLCS expression in breast tissue obtained from 65 Thai patients, and the correlation between its expression and key clinical-pathological parameters was assessed. The role of HLCS in supporting invasion was investigated in HLCS-knockdown MCF-7 cells. RESULTS: Overexpression of HLCS was significantly associated with metastasis of breast cancer cells to other lymph nodes but not the sentinel and axillary lymph nodes - a finding supported in cellular invasion assays using HLCS knockdown cells. Furthermore, overexpression of HLCS reduced survival time of patients with breast cancer. CONCLUSION: HLCS appears to be a prognostic marker for patients with breast cancer.

Biochemical characterisation of class III biotin protein ligases from Botrytis cinerea and Zymoseptoria tritici.

Biotin protein ligase (BPL) is an essential enzyme in all kingdoms of life, making it a potential target for novel anti-infective agents. Whilst bacteria and archaea have simple BPL structures (class I and II), the homologues from certain eukaryotes such as mammals, insects and yeast (class III) have evolved a more complex structure with a large extension on the N-terminus of the protein in addition to the conserved catalytic domain. The absence of atomic resolution structures of any class III BPL hinders structural and functional analysis of these enzymes. Here, two new class III BPLs from agriculturally important moulds Botrytis cinerea and Zymoseptoria tritici were characterised alongside the homologue from the prototypical yeast Saccharomyces cerevisiae. Circular dichroism and ion mobility-mass spectrometry analysis revealed conservation of the overall tertiary and secondary structures of all three BPLs, corresponding with the high sequence similarity. Subtle structural differences were implied by the different thermal stabilities of the enzymes and their varied Michaelis constants for their interactions with ligands biotin, MgATP, and biotin-accepting substrates from different species. The three BPLs displayed different preferences for fungal versus bacterial protein substrates, providing further evidence that class III BPLs have a 'substrate validation' activity for selecting only appropriate proteins for biotinylation. Selective, potent inhibition of these three BPLs was demonstrated despite sequence and structural homology. This highlights the potential for targeting BPL for novel, selective antifungal therapies against B. cinerea, Z. tritici and other fungal species.