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.

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.

Mechanisms Governing Precise Protein Biotinylation.

Protein biotinylation is a key post-translational modification found throughout the living world. The covalent attachment of a biotin cofactor onto specific metabolic enzymes is essential for their activity. This modification is distinctive, in that it is carried out by a single enzyme: biotin protein ligase (BPL), an enzyme that is able to biotinylate multiple target substrates without aberrant-off target biotinylation. BPL achieves this target selectivity by recognizing a sequence motif in the context of a highly conserved tertiary structure. One structural class of BPLs has developed an additional 'substrate verification' mechanism to further enable appropriate protein selection. This is crucial for the precise and selective biotinylation required for efficient biotin management, especially in organisms that are auxotrophic for biotin.

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.

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.

A hypoxia-activated antibacterial prodrug.

A prodrug based on a known antibacterial compound is reported to target Staphylococcus aureus and Escherichia coli under reductive conditions. The prodrug was prepared by masking the N-terminus and side chain amines of a component lysine residue as 4-nitrobenzyl carbamates. Activation to liberate the antibacterial was demonstrated on treatment with a model reductant, tin(II) chloride. The bioactivity of 1 was confirmed in antibacterial susceptibility assays whereas prodrug 2 was inactive.

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.

Methotrexate Is Not Superior to Placebo in Maintaining Steroid-Free Response or Remission in Ulcerative Colitis.

BACKGROUND & AIMS: Parenteral methotrexate induces clinical remission but not endoscopic improvement of mucosal inflammation in patients with ulcerative colitis (UC). We performed a randomized, placebo-controlled trial to assess the efficacy of parenteral methotrexate in maintaining steroid-free response or remission in patients with UC after induction therapy with methotrexate and steroids. METHODS: We performed a 48-week trial, from February 2012 through May 2016, of 179 patients with active UC (Mayo score of 6-12 with endoscopy subscore ≥ 2) despite previous conventional or biological therapy. The study comprised a 16-week open label methotrexate induction period followed by a 32-week double-blind, placebo-controlled maintenance period. Patients were given subcutaneous methotrexate (25 mg/wk) and a 12-week steroid taper. At week 16, steroid-free responders were randomly assigned to groups that either continued methotrexate (25 mg/wk, n = 44) or were given placebo (n = 40) until week 48. We compared the efficacy of treatment by analyzing the proportion of patients who remained relapse free and were in remission at week 48 without use of steroids or other medications to control disease activity. RESULTS: Ninety-one patients (51%) achieved response at week 16, and 84 patients were included in the maintenance period study. During this period, 60% of patients in the placebo group (24/40) and 66% in the methotrexate group (29/44) had a relapse of UC (P = .75). At week 48, 30% of patients in the placebo group (12/40) and 27% of patients in the methotrexate group (12/44) were in steroid-free clinical remission without need for additional therapies (P = .86). No new safety signals for methotrexate were detected. CONCLUSIONS: Parenteral methotrexate (25 mg/wk) was not superior to placebo in preventing relapses of UC in patients who achieved steroid-free response during induction therapy. ClinicalTrials.gov, Number: NCT01393405.

Design, synthesis and evaluation of a series of 5-methoxy-2,3-naphthalimide derivatives as AcrB inhibitors for the reversal of bacterial resistance.

A series of novel 5-methoxy-2,3-naphthalimide derivatives were designed, synthesized and evaluated for their biological activities. In particular, the ability of the compounds to synergize with antimicrobials, to inhibit Nile Red efflux, and to target AcrB was assayed. The results showed that the most of the tested compounds more sensitized the Escherichia coli BW25113 to the antibiotics than the parent compounds 7c and 15, which were able to inhibit Nile Red efflux. Significantly, compound A5 possessed the most potent antibacterial synergizing activity in combination with levofloxacin by 4 times and 16 times at the concentration of 8 and 16 µg/mL, respectively, whilst A5 could effectively abolish Nile Red efflux at 100 µM. Additionally, target effect of A5 was confirmed in the outer- or inner membrane permeabilization assays. Therefore, A5 is an excellent lead compound for further structural optimization.

Two New Piperazine-Triones from a Marine-Derived Streptomycetes sp. Strain SMS636.

Two new piperazine-triones lansai E and F (1, 2), together with four known secondary metabolites lansai D (3), 1-N-methyl-(E,Z)-albonoursin (4), imidazo[4,5-e]-1,2,4-triazine (5), and streptonigrin (6) were isolated from a deep-sea-derived Streptomycetes sp. strain SMS636. The structures of the isolated compounds were confirmed by comprehensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR. Compound 4 exhibited moderate antibacterial activities against Staphylococcus aureus and methicillin resistant S. aureus (MRSA) with Minimum Inhibitory Concentration (MIC) values of 12.5 and 25 µg/mL, respectively. Compound 6 displayed significant antibacterial activities against S. aureus, MRSA and Bacillus Calmette-Guérin (BCG) with MIC values of 0.78, 0.78 and 1.25 µg/mL, respectively.

Two New Spiro-Heterocyclic γ-Lactams from A Marine-Derived Aspergillus fumigatus Strain CUGBMF170049.

Two new spiro-heterocyclic γ-lactam derivatives, cephalimysins M (1) and N (2), were isolated from the fermentation cultures of the marine-derived fungus Aspergillus fumigatus CUGBMF17018. Two known analogues, pseurotin A (3) and FD-838 (4), as well as four previously reported helvolic acid derivatives, 16-O-propionyl-16-O-deacetylhelvolic acid (5), 6-O-propionyl-6-O-deacetylhelvolic acid (6), helvolic acid (7), and 1,2-dihydrohelvolic acid (8) were also identified. One-dimensional (1D), two-dimensional (2D) NMR, HRMS, and circular dichroism spectral analysis characterized the structures of the isolated compounds.

Photopharmacological Control of Cyclic Antimicrobial Peptides.

Gramicidin S is a naturally occurring antimicrobial cyclic peptide. Herein, we present a series of cyclic peptides based on gramicidin S that contain an azobenzene photoswitch to reversibly control secondary structure and, hence, antimicrobial activity. 1 H NMR spectroscopy and density functional theory calculations revealed a ß-sheet/ß-turn secondary structure for the cis configuration of each peptide, and an ill-defined conformation for all associated trans structures. The cis-enriched and trans-enriched photostationary states (PSSs) for peptides 1-3 were assayed against Staphylococcus aureus to reveal a clear relationship between well-defined secondary structure, amphiphilicity and optimal antimicrobial activity. Most notably, peptides 2 a and 2 b exhibited a fourfold difference in antimicrobial activity in the cis-enriched PSS over the trans-enriched equivalent. This photopharmacological approach allows antimicrobial activity to be regulated through photochemical control of the azobenzene photoswitch, thereby opening new avenues in the design and synthesis of future antibiotics.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus.

Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

Heterocyclic acyl-phosphate bioisostere-based inhibitors of Staphylococcus aureus biotin protein ligase.

Inhibitors of Staphylococcus aureus biotin protein ligase (SaBPL) are generated by replacing the acyl phosphate group of biotinyl-5'-AMP with either a 1,2,3-triazole (see 5/10a/10b) or a 1,2,4-oxadiazole (see 7) bioisostere. Importantly, the inhibitors are inactive against the human BPL. The nature of the 5-substituent in the component benzoxazolone of the optimum 1,2,3-triazole series is critical to activity, where this group binds in the ATP binding pocket of the enzyme.

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).

Selective inhibition of biotin protein ligase from Staphylococcus aureus.

There is a well documented need to replenish the antibiotic pipeline with new agents to combat the rise of drug resistant bacteria. One strategy to combat resistance is to discover new chemical classes immune to current resistance mechanisms that inhibit essential metabolic enzymes. Many of the obvious drug targets that have no homologous isozyme in the human host have now been investigated. Bacterial drug targets that have a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have very high selectivity for the bacterial enzyme over the human homolog. We have demonstrated that the essential enzyme biotin protein ligase (BPL) from the clinically important pathogen Staphylococcus aureus could be selectively inhibited. Linking biotin to adenosine via a 1,2,3 triazole yielded the first BPL inhibitor selective for S. aureus BPL over the human equivalent. The synthesis of new biotin 1,2,3-triazole analogues using click chemistry yielded our most potent structure (K(i) 90 nM) with a >1100-fold selectivity for the S. aureus BPL over the human homologue. X-ray crystallography confirmed the mechanism of inhibitor binding. Importantly, the inhibitor showed cytotoxicity against S. aureus but not cultured mammalian cells. The biotin 1,2,3-triazole provides a novel pharmacophore for future medicinal chemistry programs to develop this new antibiotic class.

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.