High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a bacterial pathogen that claims roughly 1.4 million lives every year. Current drug regimens are inefficient at clearing infection, requiring at least 6 months of chemotherapy, and resistance to existing agents is rising. There is an urgent need for new drugs that are more effective and faster acting. The folate pathway has been successfully targeted in other pathogens and diseases, but has not yielded a lead drug against tuberculosis. We developed a high-throughput screening assay against Mtb dihydrofolate reductase (DHFR), a critical enzyme in the folate pathway, and screened a library consisting of 32,000 synthetic and natural product-derived compounds. One potent inhibitor containing a quinazoline ring was identified. This compound was active against the wild-type laboratory strain H37Rv (MIC99 = 207 µM). In addition, an Mtb strain with artificially lowered DHFR levels showed increased sensitivity to this compound (MIC99 = 70.7 µM), supporting that the inhibition was target-specific. Our results demonstrate the potential to identify Mtb DHFR inhibitors with activity against whole cells, and indicate the power of using a recombinant strain of Mtb expressing lower levels of DHFR to facilitate the discovery of antimycobacterial agents. With these new tools, we highlight the folate pathway as a potential target for new drugs to combat the tuberculosis epidemic

The Enduring Hypoxic Response of Mycobacterium tuberculosis

deletion mutant. mutant. for long-term bacteriostasis in the face of oxygen deprivation

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Characterization of NC00094221.

<p>A. Dose-response curves in low-throughput <i>Mtb</i> DHFR enzyme assay. NC00094221 (chemical structure shown inset into the graph) and MTX were tested at varied concentrations in the kinetic assay performed with 10 µM DHF and 125 ng/mL recombinant DHFR. Data were fitted to a non-linear least-squares curve and IC₅₀ values were calculated using GraphPad Prism™.<b>B</b>. Quanitification of DHFR transcript levels in a recombinant <i>Mtb</i> strain. The levels of DHFR transcript were assayed by qRT-PCR in wild-type <i>Mtb</i> strain H37Rv (wt- pale grey bar) and the engineered <i>Mtb</i> strain (H37Rv:<i>dfrA</i>-TetON, referred to as DHFR <i>kd</i>- dark grey bars) in the presence of varied levels of tetracycline (ATc). DHFR transcript levels from each sample were first normalized to SigA transcript levels and then shown as the fold change when compared to wild-type (wt) <i>Mtb</i>. C. Live <i>Mtb</i> growth inhibition assay. Varied concentrations of methotrexate (MTX, white symbols) or NC00094221 (black symbols) were incubated with wild-type <i>Mtb</i> (triangles) or the DHFR <i>kd</i> (circles) and grown for 6 days prior to assessment of cell growth using the commercially available Bactiter-Glo assay kit. For each strain, controls including wells with no drug (DMSO carrier was added instead), 1.22 µM rifampicin (10× MIC₉₉) and a 1/100 dilution of the starting culture were used to calculate 0%, 100% and 99% inhibition, respectively. Data were fitted to a non-linear least-squares curve and MIC₉₉ calculations were performed using GraphPad Prism™. D. Target-specific drug sensitivity in DHFR <i>kd</i>. The sensitivity of the wild-type <i>Mtb</i> and DHFR <i>kd</i> was tested against various inhibitors and displayed as the ratio of a compound’s MIC₉₉ when measured in wild-type <i>Mtb</i> over the MIC₉₉ measured in DHFR <i>kd</i> in the absence of ATc.</p

Development of a single-time point assay with a diaphorase-coupled fluorescence readout.

<p>A. Reaction schematic. Reaction 1: DHFR, in a NADPH-dependent reaction, converts dihydrofolate (DHF) to tetrahydrofolate (THF), causing a depletion of reduced NADPH. Reaction 2: Diaphorase utilizes NADPH that was unused from the DHFR reaction to generate fluorescence. Enzymes are italicized and bold. B. Kinetic NADPH-depletion assay. The rates of NADPH depletion in reactions performed with 200 µM NADPH, 0.2 mM DHF and varied levels of DHFR were monitored by measuring absorbance at 340 nm. C. Fluorescence coupling. The fluorescence generated after 4 minutes of incubating diaphorase and resazurin with various concentrations of NADPH were measured. D. End-point fluorescence assay. The NADPH-depletion assay run as in (B) was coupled to diaphorase and resazurin after 30 minutes, and the resulting fluorescence measured at excitation and emission wavelengths of 560 and 590 nM, respectively.</p

Confirming the robustness of the assay for HTS.

<p>A. Standard curve of methotrexate (MTX)<b>.</b> The concentration-dependent inhibitory activity of MTX was measured in the coupled assay. B. Statistical Factors. Sixteen replicates of each control reaction were performed in the optimized assay conditions. High-throughput screening (HTS) parameters, including Z’-factor (Z’) and signal to background ratio (S/B) were studied. Raw fluorescence units (right Y-axis) from each of the controls and % inhibition (left Y-axis) are shown. The (-) DHFR controls were used as a measure of complete inhibition, in order to calculate % inhibition.</p

List of primers used in this publication.

<p>List of primers used in this publication.</p