Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis.

Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645. Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.

A d-Phenylalanine-Benzoxazole Derivative Reveals the Role of the Essential Enzyme Rv3603c in the Pantothenate Biosynthetic Pathway of Mycobacterium tuberculosis.

New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.

Metabolomics of Mycobacterium tuberculosis.

Enzymes fuel the biochemical activities of all cells. Their substrates and products thus represent a potential window into the physiologic state of a cell. Metabolomics focuses on the global, or systems-level, study of small molecules in a given biological system and has thus provided an experimental tool with which to study cellular physiology, including the biochemistry within pathogenic microorganisms. While metabolomic studies of Mycobacterium tuberculosis are still in their infancy, recent studies have begun to deliver unique insights into the composition, organization, activity, and regulation of the bacterium's physiologic network not accessible by other approaches. Here, we outline practical methods for the culture, collection, and analysis of metabolomic samples from M. tuberculosis that emphasize minimally perturbing sample handling, broad and native metabolite recovery, and sensitive, biologically agnostic metabolite detection.

A novel method to determine residual detergent in biological samples post endotoxin reduction treatment and evaluation of strategies for subsequent detergent removal.

Endotoxin removal using detergent washes and extractions are well-established, efficient, and cost-effective methods; however, removing residual detergent post treatment has been shown to be a challenge. In this communication, we show a simple and fast method for determining the detergent concentration in a protein solution post treatment and highlight strategies for detergent removal to achieve levels below the critical micelle concentration (CMC), the minimum concentration at which detergent micelles form.