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The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens 1 , 2 . These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds 3 , 4 . As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s 2 . We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics. Bacterial symbionts of animals may contain antibiotics that are particularly suitable for development into therapeutics; one such compound, darobactin, is active against important Gram-negative pathogens both in vitro and in animal models of infection.

Gadolinium is a paramagnetic relaxation enhancement (PRE) agent that accelerates the relaxation of metabolite nuclei. In this study, we noted the ability of gadolinium to improve the sensitivity of two-dimensional, non-uniform sampled NMR spectral data collected from metabolomics samples. In time-equivalent experiments, the addition of gadolinium increased the mean signal intensity measurement and the signal-to-noise ratio for metabolite resonances in both standard and plasma samples. Gadolinium led to highly linear intensity measurements that correlated with metabolite concentrations. In the presence of gadolinium, we were able to detect a broad array of metabolites with a lower limit of detection and quantification in the low micromolar range. We also observed an increase in the repeatability of intensity measurements upon the addition of gadolinium. The results of this study suggest that the addition of a gadolinium-based PRE agent to metabolite samples can improve NMR-based metabolomics.

Parkinson’s disease (PD) is a progressive neurodegenerative disease, causing loss of motor and nonmotor function. Diagnosis is based on clinical symptoms that do not develop until late in the disease progression, at which point the majority of the patients’ dopaminergic neurons are already destroyed. While many PD cases are idiopathic, hereditable genetic risks have been identified, including mutations in the gene for LRRK2, a multidomain kinase with roles in autophagy, mitochondrial function, transcription, molecular structural integrity, the endo-lysosomal system, and the immune response. A definitive PD diagnosis can only be made post-mortem, and no noninvasive or blood-based disease biomarkers are currently available. Alterations in metabolites have been identified in PD patients, suggesting that metabolomics may hold promise for PD diagnostic tools. In this study, we sought to identify metabolic markers of PD in plasma. Using a 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) NMR spectroscopy metabolomics platform coupled with machine learning (ML), we measured plasma metabolites from approximately age/sex-matched PD patients with G2019S LRRK2 mutations and non-PD controls. Based on the differential level of known and unknown metabolites, we were able to build a ML model and develop a Biomarker of Response (BoR) score, which classified male LRRK2 PD patients with 79.7% accuracy, 81.3% sensitivity, and 78.6% specificity. The high accuracy of the BoR score suggests that the metabolomics/ML workflow described here could be further utilized in the development of a confirmatory diagnostic for PD in larger patient cohorts. A diagnostic assay for PD will aid clinicians and their patients to quickly move toward a definitive diagnosis, and ultimately empower future clinical trials and treatment options.

The antimicrobial resistance crisis requires the introduction of novel antibiotics. The use of conventional broad-spectrum compounds selects for resistance in off-target pathogens and harms the microbiome. This is especially true for Mycobacterium tuberculosis, where treatment requires a 6-month course of antibiotics. Here we show that a novel antimicrobial from Photorhabdus noenieputensis, which we named evybactin, is a potent and selective antibiotic acting against M. tuberculosis. Evybactin targets DNA gyrase and binds to a site overlapping with synthetic thiophene poisons. Given the conserved nature of DNA gyrase, the observed selectivity against M. tuberculosis is puzzling. We found that evybactin is smuggled into the cell by a promiscuous transporter of hydrophilic compounds, BacA. Evybactin is the first, but likely not the only, antimicrobial compound found to employ this unusual mechanism of selectivity.Evybactin is an antimicrobial natural product that targets DNA gyrase, where it binds to a site overlapping with synthetic thiophene poisons and exerts selectivity for Mycobacterium tuberculosis via its transport mechanism into the cell.

Lead optimization of drug candidates predominantly focus on improving the potency and selectivity of a drug, while other important aspects such as metabolic stability and bioavailability are optimized during the latter stages of the drug discovery process. This has often led to the early elimination of potential drug candidates due to various pharmacokinetic reasons, including poor absorption, high clearance, short biological half-life (t1/2), and extensive first-pass metabolism. Cytochrome P450 (CYP) mediated metabolism of a drug candidate by the host system plays a key role in determining a compound’s pharmacokinetic properties and in vivo efficacy/toxicity. Thus, factors that modify the rate and extent of the metabolism are likely to alter the overall disposition of the xenobiotic. In most cases, metabolism leads to either, 1) inactivation of xenobiotics, abolishing the pharmacological response, 2) bioactivation, through bioactive metabolites, prolonging the intensity and duration of the pharmacological response, or 3) drug-drug interactions, with potential toxicological implications of drugs. Together, these issues underscore the critical need to improve our understanding of the xenobiotic metabolism of a drug candidate early in the drug discovery process such that compounds with optimal drug metabolism and pharmacokinetic (DMPK) properties can be selected for further development. Moreover, knowledge of a compound’s metabolic fate early in drug discovery will provide key information for further metabolic optimization, which will facilitate the synthesis of metabolically stable, effective, and safe drug candidates. Obviously, identification and characterization of drug metabolites is the most important center piece in this drug discovery process. However, the unavailability of metabolite quantities from biological systems in early drug discovery poses considerable challenges in their Liquid chromatography mass spectrometry (LC-MS)/Nuclear magnetic resonance (NMR) based structural characterizations. This encumbers future metabolism studies and the discovery of druggable candidates. The overarching objective of this thesis is to address this problem by: a) using LC-MS in conjunction with microcoil-NMR to characterize very small quantities of metabolites isolated from liver microsomal preparations; and b) conducting critical drug metabolism and in vitro biochemical studies to systematically investigate the: i) pharmacological properties of metabolites and their formation kinetics, ii) involvement of key metabolizing enzymes, and iii) potential liabilities for drug-drug interactions.Notable discoveries in the last three decades in the cannabinoid field have provided valuable information about the physiological functions of the endocannabinoid system in the brain and periphery, and the contribution of this system in the etiology and therapy of many human diseases. Thus far, two cannabinoid receptors have been identified – the cannabinoid 1 receptor (CB1) and cannabinoid 2 receptor (CB2), which are expressed predominantly in the human brain and other tissues as well as immune system, respectively. Both receptors have received considerable attention as attractive pharmacotherapeutic targets for various indications, including neuropathic pain, neurodegenerative disorders, cardio-metabolic and inflammatory disorders. The research conducted in this thesis focused on drugs targeting the CB2 receptor, that have recently shown great potential for treating multiple sclerosis, inflammatory bowel disease, ischemia, renal fibrosis, liver cirrhosis, and neurodegenerative diseases. In particular, two CB2 selective agonists, AM9338 and AM1710 that are being developed for treating pain and inflammation were studied. First, micro-coil NMR technology in conjunction with mass spectrometry was employed for structural characterization of novel metabolites of AM9338 and AM1710 that were isolated in microgram quantities from liver microsomal preparations. Next, the metabolic disposition of these two CB2 compounds were investigated by conducting a series of key drug metabolism studies that included: a) CYP phenotyping, to identify whether CYP’s are involved in metabolism of CB2 selective agonists; b) CYP inhibition, to examine potential drug-drug interactions of these CB2 compounds; c) enzyme kinetics and CYP docking, to obtain insights into the interactions of these CB2 compounds with CYP enzymes that may be involved in their metabolism. Subsequently, chemistry based approaches were used to synthesize key metabolites and novel analogues in an attempt to block the primary metabolically labile site(s). Lastly, through stability and pharmacological assessment of metabolites and analogues, the structure-metabolism-activity relationship for these scaffolds was studied to generate critical information for developing future metabolic optimization strategies.The key findings of this thesis are: 1) discovery of novel metabolite of adamantyl moiety revealing secondary carbons as the site(s) of metabolic oxidation in contrast to widely reported tertiary adamantyl carbon as an oxidative site in other ligands. These data facilitated the synthesis of novel structural analogues of AM9338 in an attempt to block primary metabolic sites located on its adamantyl moiety; 2) identification of the structural requirements needed for metabolic stabilization of adamantyl moiety in AM9338. In addition, SAR points that are essential for preserving CB2 binding affinity, while optimizing metabolic stability of AM9338, were uncovered; 3) For the first time, identification and characterization of metabolites of AM1710 using LC-MS/microcoil NMR; 4) Using radioligand competition binding studies, identified key metabolites of AM1710 retaining CB2 binding affinities. This information can be potentially used as templates for generating novel bioactive and metabolically stable analogues of AM1710; and 5) Identified CYP1A2 and CYP2C9 as the major CYP enzymes involved in metabolism of AM1710. Taken together, the results presented in this thesis demonstrate that availability of structural and functional information on metabolites and their metabolic disposition will help identify structure-metabolic stability-activity relationships that will guide future metabolic optimization efforts in design and development of drug candidates.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.