N-methylation of a bactericidal compound as a resistance mechanism in Mycobacterium tuberculosis.

The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease Using RapidFire Mass Spectrometry.

LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson's disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.

Whole Cell Target Engagement Identifies Novel Inhibitors of Mycobacterium tuberculosis Decaprenylphosphoryl-ß-d-ribose Oxidase.

We have targeted the Mycobacterium tuberculosis decaprenylphosphoryl-ß-d-ribose oxidase (Mt-DprE1) for potential chemotherapeutic intervention of tuberculosis. A multicopy suppression strategy that overexpressed Mt-DprE1 in M. bovis BCG was used to profile the publically available GlaxoSmithKline antimycobacterial compound set, and one compound (GSK710) was identified that showed an 8-fold higher minimum inhibitory concentration relative to the control strain. Analogues of GSK710 show a clear relationship between whole cell potency and in vitro activity using an enzymatic assay employing recombinant Mt-DprE1, with binding affinity measured by fluorescence quenching of the flavin cofactor of the enzyme. M. bovis BCG spontaneous resistant mutants to GSK710 and a closely related analogue were isolated and sequencing of ten such mutants revealed a single point mutation at two sites, E221Q or G248S within DprE1, providing further evidence that DprE1 is the main target of these compounds. Finally, time-lapse microscopy experiments showed that exposure of M. tuberculosis to a compound of this series arrests bacterial growth rapidly followed by a slower cytolysis phase.

Demonstrating enhanced throughput of RapidFire mass spectrometry through multiplexing using the JmjD2d demethylase as a model system.

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

Monitoring clinical performance: the role of software architecture.

Methods of assessing and monitoring the performance of clinicians have received a lot of publicity in recent years. We review the main methodologies concentrating on the distinction between monitoring individual performance and monitoring aggregated performance. We also highlight the importance and difficulties associated with incorporating and assessing risk factors into the process. We discuss how software architecture can be developed to implement these methodologies. We illustrate this development by a case study involving the creation of a software tool to produce funnel plots to analyse surgeon performance. We discuss how such tools are currently evaluated and propose that in future assessments of usability would benefit from an experimental study.

A software system for clinical monitoring.

Three graphical methods for monitoring clinician performance - the Funnel Plot, the CUSUM and the Variable Life Adjusted Display - are described. In addition the problems associated with incorporating and assessing risk factors into the process are discussed. The software system produced to implement these methodologies is presented; the creation of the user friendly interfaces, the underling algorithms and the structure and connectivity of the database are described. The methodology for piloting and evaluating the system is described. Drawing on developments in experimental usability we propose an experimental procedure aimed at evaluating possible changes in design so as to maximise the use of the system in terms of its accuracy of input and clarity of output.

Optimisation of bile production during normothermic preservation of porcine livers.

Machine perfusion of livers may provide a mechanism for extended preservation of marginal donor organs before transplantation, as well as a method for viability assessment. It has proved possible in a series of experimental porcine liver perfusions to maintain liver viability for up to 72 h. However, a reduction in bile production with associated histological evidence of cholestasis was seen after 10 h of perfusion, damaging the biliary canaliculi during the preservation period and leaving these organs in an unacceptable condition for transplantation. It was proposed that reduction in bile production was the result of a relentless depletion of available bile salts, gut recirculation not being possible and de-novo synthesis being unable to keep up with loss. This was proved by measuring porcine native bile acids within serial perfusate and bile samples using gas chromatography mass spectrophotometry. It was shown that all three native pig bile acids were decreased to 30% of their original value by 20 h of unsupplemented perfusion. An infusion of taurocholate managed to maintain bile production at physiological levels throughout the 20-h period (8 mL/h +/- 0.75). It was successfully incorporated by the porcine livers into bile. We propose to use this circuit as a novel means of preserving donor livers for transplantation in which the organ is maintained at normal body temperature and perfused with blood. This will reduce ischaemia reperfusion injury and may enable prolonged preservation. The modification described ensures optimal bile production over the entire perfusion period, preventing inspissation and subsequent damage to the canaliculus.