INTRODUCTION:Polymyxins are considered last-resort antibiotics for treating Gram-negative bacterial infections. The alarming global rise in polymyxin-resistant bacteria coupled with the slowness of conventional antimicrobial susceptibility testing methods leads to an unmet need for rapid susceptibility testing to facilitate timely and targeted therapeutic interventions. OBJECTIVES:In this study, we aimed to develop novel polymyxin-derived fluorescent probes and improve the identification of polymyxin resistance by using these pathway-specific tools. METHODS:We designed, synthesized, and profiled a polymyxin-fluorophore derivative (PMX-NBD) with the small fluorophore NBD attached the central cyclic core of polymyxin via a triazole linker. We then applied PMX-NBD to develop RAPIDFC, a flow cytometry method that can rapidly and accurately profile polymyxin susceptibility of various Gram-negative pathogens. RESULTS:In contrast to many previously reported polymyxin probes, the fluorescent PMX-NBD retains the antimicrobial activity and profile of the parent polymyxin. Furthermore, we tested probe labeling in 49 strains encompassing E. coli, K. pneumoniae, P. aeruginosa, A. baumannii and S. aureus with a range of polymyxin sensitivity, leading to a susceptibility testing method RAPIDFC. Susceptibility to polymyxin B was defined by the difference between the Geometric Mean of PMX-NBD fluorescence intensity at the two concentrations, with sensitive strains showing a greater variation compared to resistant strains. The RAPIDFC method is easy to implement and only requires 45 min compared to the broth microdilution method, which requires at least 24 h. However, the standard protocol failed to provide clear discrimination forA. baumannii, a key WHO-priority pathogen, necessitating a modified, species-specific approach for this organism. CONCLUSIONS:The RAPIDFC method, based on the polymyxin-derived fluorescent probe, is fast and applicable to multiple species, making it a promising antimicrobial susceptibility method to guide appropriate therapy in the clinic. The case ofA. baumanniidemonstrates that species-specific resistance mechanisms may require tailored optimization of detection protocols.

Parkinson's disease is one of the fastest-growing neurodegenerative disorders, with no effective treatments to modify its progression. Microglial-driven neuroinflammation, mediated by NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activation, plays a key role in disease onset and progression. The NLRP3 inflammasome is upregulated in microglia from Parkinson's disease patients and activated by oxidative stress and α-synuclein aggregates, triggering the release of pro-inflammatory mediators that contribute to neuroinflammation and neuronal death. MCC950, the first described specific NLRP3 inhibitor, has shown promise in Parkinson's disease models but is limited by suboptimal pharmacokinetics and safety, hindering its clinical development. Here, we developed a novel NLRP3 inflammasome inhibitor, MCC7840 (also known as Inzomelid or Emlenoflast), and utilized clinically relevant PET-MRI imaging biomarkers to assess its therapeutic efficacy in preclinical models of Parkinson's disease. MCC7840 inhibited NLRP3 in human and mouse microglia with nanomolar potency, while demonstrating improved systemic exposure, half-life, brain permeability and bioavailability compared with MCC950. In a murine NLRP3 gain-of-function model of Muckle-Wells syndrome, MCC7840 effectively inhibited mortality and demonstrated superior potency compared with MCC950. Chronic oral administration of MCC7840 protected against neuroinflammation, motor deficits and dopamine loss in both 6-hydroxydopamine and preformed α-synuclein fibril mouse models of Parkinson's disease. Radiotracer imaging of multiple PET markers in the same mouse revealed that MCC7840 attenuated neuroinflammation (translocator protein ligand; 18F-DPA-714), preserved dopamine uptake (fluorodopa; 18F-FDOPA), mitigated dopamine transporter (DAT) loss (DAT ligand; 18F-FBCTT) and reduced blood-brain barrier leakage (gadolinium contrast MRI). Notably, MCC7840 was effective in a slowly progressing 12-month α-synuclein model, even when administered after symptom onset, 4 months post-α-synuclein injection. These findings highlight the utility of PET/MRI as a non-invasive tool to evaluate drug efficacy and support MCC7840, and other brain-penetrant NLRP3 inhibitors, as promising disease-modifying therapies for Parkinson's disease, warranting future clinical investigation.

A commercial vaccine to address the high global burden of Group A Streptococcus (GAS) disease is an urgent and unmet medical need. Messenger RNA (mRNA) lipid-nanoparticle (LNP) vaccines represent a largely untapped platform for targeting bacterial pathogens. Here, we evaluate the immunogenicity and preclinical efficacy of a multicomponent mRNA-LNP vaccine formulation based on the GAS vaccine, Combo#5. Combo#5 mRNA-LNP antigens confer protection from infection in mouse intraperitoneal and subcutaneous challenge models. Combo#5 mRNA-LNP vaccination generates significantly increased frequencies and numbers of effector type CD4+ and CD8 + T cells in the spleen, enhances T follicular helper cells, germinal center B cells and memory B cells in the spleen and draining lymph nodes, and boosts the production of antigen-specific antibodies. These findings demonstrate the potential of the mRNA-LNP platform for the development of vaccines against bacterial pathogens.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria ( Staphylococcus aureus and Streptococcus pneumoniae ) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC 90 ≤ 0.06 μg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae , as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

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.

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 N-acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N-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.

The public health threat posed by a looming ‘post-antibiotic’ era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature’s control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N -terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the ‘vancapticins’, possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to ‘revitalise’ antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Tuberculosis and parasitic diseases, such as giardiasis, amebiasis, leishmaniasis, and trypanosomiasis, all urgently require improved treatment options. Recently, it has been shown that antitubercular 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 nitroimidazooxazines. Synthetic analogues with a novel nitroimidazopyrazin-one/-e bicyclic nitroimidazole chemotype were designed and synthesized, and structure-activity relationships were generated. Selected derivatives had potent antiparasitic and antitubercular activity while maintaining drug-like properties such as low cytotoxicity, good metabolic stability in liver microsomes and high apparent permeability across Caco-2 cells. 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 that promising subclasses of bicyclic nitroimidazoles containing different core architectures have potential for further development.

Insulin-secretory sulfonylureas are widely used, cost-effective treatments for type 2 diabetes (T2D). However, pancreatic beta-cells are continually depleted as T2D progresses, thereby rendering the sulfonylurea drug class ineffective in controlling glycaemia. Dysregulation of the innate immune system via activation of the NLRP3 inflammasome, and the consequent production of interleukin-1 beta, has been linked to pancreatic beta-cell death and multiple inflammatory complications of T2D disease. One proposed strategy for treating T2D is the use of sulfonylurea insulin secretagogues that are also NLRP3 inhibitors. We report the synthesis and biological evaluation of nine sulfonylureas that inhibit NLRP3 activation in murine bone-marrowderived macrophages in a potent, dose-dependent manner. Six of these compounds inhibited NLRP3 at nanomolar concentrations and can also stimulate insulin secretion from a murine pancreatic cell line (MIN6). These novel compounds possess unprecedented dual modes of action, paving the way for a new generation of sulfonylureas that may be useful as therapeutic candidates and/ or tool compounds in T2D and its associated inflammatory complications.

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)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-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-1β 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.

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 (ΔtolC). 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.

The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately, their utility is compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have conducted a systematic activity-toxicity investigation by varying eight of the nine polymyxin amino acid free side chains, preparing over 30 analogues using a novel solid-phase synthetic route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened for in vitro cell toxicity. Promising compounds underwent additional testing against primary kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of the new compounds possessed equal or better antimicrobial potency compared to polymyxin B, and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and human primary kidney cells. These initial structure-activity and structure-toxicity studies set the stage for further improvements to the polymyxin class of antibiotics.

Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica constitute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and amebiasis, with metronidazole 1 being the most commonly used drug worldwide. However, treatment failures in giardiasis occur in up to 20% of cases and development of resistance to metronidazole is of concern. We have re-examined 'old' nitroimidazoles as a foundation for the systematic development of next-generation derivatives. Using this approach, derivatisation of the nitroimidazole carboxamide scaffold provided improved antiparasitic agents. Thirty-three novel nitroimidazole carboxamides were synthesised and evaluated for activity against G. lamblia and E. histolytica. Several of the new compounds exhibited potent activity against G. lamblia strains, including metronidazole-resistant strains of G. lamblia (EC50 = 0.1-2.5 μM cf. metronidazole EC50 = 6.1-18 μM). Other compounds showed improved activity against E. histolytica (EC50 = 1.7-5.1 μM cf. metronidazole EC50 = 5.0 μM), potent activity against Trichomonas vaginalis (EC50 = 0.6-1.4 μM cf. metronidazole EC50 = 0.8 μM) and moderate activity against the intestinal bacterial pathogen Clostridium difficile (0.5-2 μg/mL, cf. metronidazole = 0.5 μg/mL). The new compounds had low toxicity against mammalian kidney and liver cells (CC50 > 100 μM), and selected antiparasitic hits were assessed for human plasma protein binding and metabolic stability in liver microsomes to demonstrate their therapeutic potential.

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.