Mucin binding reduces colistin antimicrobial activity

Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance

Design, synthesis and biological evaluation of 2-nitroimidazopyrazin-one/-es with antitubercular and antiparasitic activity

Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that anti-tubercular bicyclic nitroimidazoles such as pretomanid and delamanid have potential as repurposed therapeutics for the treatment of visceral leishmaniasis. Here we show that pretomanid also possesses potent activity against Giardia lamblia and Entamoeba histolytica, thus expanding the therapeutic potential of nitroimidazo-oxazines. Synthetic analogs with the novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed, synthesized and structure activity relationships generated. Selected derivatives had potent antiparasitic and antitubercular activity whilst maintaining drug-like properties such as low cytotoxicity against mammalian cell lines (CC50 >100 μM), good metabolic stability in human and mouse liver microsomes and high apparent permeability in a Caco-2 model of intestinal absorption. The kinetic solubility of the new bicyclic derivatives varied, and was found to be a key parameter for future optimization. Taken together, these results suggest promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development

Fluorescent trimethoprim conjugate probes to assess drug accumulation in wild type and mutant Escherichia coli

Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (Delta tolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment

Structure-function studies of polymyxin B lipononapeptides

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of -acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the -terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the -terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins

Glycoproteomic measurement of site-specific polysialylation

Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings

Identification, synthesis, and biological evaluation of the major human metabolite of NLRP3 inflammasome inhibitor MCC950

MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoy1)-4-(2-hydroxypropan-2-y0furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1 beta from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety

Structural characterization of α-amino acid complexes of molybdates: A spectroscopic and DFT study

The reactions of L-enantiopure alpha-amino acids (aaH) with Na2MoO4 led to the high-yield isolation of Mo(VI) complexes of general formula Mo2O4(OH)(4)(aaH). A similar outcome was achieved by using (NH4)(6)Mo7O24 in the place of Na2MoO4. Solid-state IR and NMR spectroscopy indicated the presence of cis-MoO2 units linked by a zwitterionic amino acid ligand, via a bidentate bridging coordination through the carboxylate group. Thus possible dinuclear and polynuclear structures are proposed on the basis of DFT calculations