Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in () is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the de novo arginine biosynthesis pathway mutants Δ and Δ without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. Δ and Δ were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for and a promising drug target space for combating tuberculosis.

Translational frameshift errors are often deleterious to the synthesis of functional proteins and could therefore be promoted therapeutically to kill bacteria. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and represents an attractive potential target for the development of new antibiotics. Here, we describe the application of a structure-guided fragment-based drug discovery approach to the design of a new class of inhibitors against TrmD in Mycobacterium abscessus. Fragment library screening, followed by structure-guided chemical elaboration of hits, led to the rapid development of drug-like molecules with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and M. tuberculosis as well as against intracellular M. abscessus and M. leprae, indicating their potential as the basis for a novel class of broad-spectrum mycobacterial drugs.

Structure-guided drug discovery emerged in the 1970s and 1980s, stimulated by the three-dimensional structures of protein targets that became available, mainly through X-ray crystal structure analysis, assisted by the development of synchrotron radiation sources. Structures of known drugs or inhibitors were used to guide the development of leads. The growth of high-throughput screening during the late 1980s and the early 1990s in the pharmaceutical industry of chemical libraries of hundreds of thousands of compounds of molecular weight of approximately 500 Da was impressive but still explored only a tiny fraction of the chemical space of the predicted 10 40 drug-like compounds. The use of fragments with molecular weights less than 300 Da in drug discovery not only decreased the chemical space needing exploration but also increased promiscuity in binding targets. Here we discuss advances in X-ray fragment screening and the challenge of identifying sites where fragments not only bind but can be chemically elaborated while retaining their positions and binding modes. We first describe the analysis of fragment binding using conventional X-ray difference Fourier techniques, with Mycobacterium abscessus SAICAR synthetase (PurC) as an example. We observe that all fragments occupy positions predicted by computational hotspot mapping. We compare this with fragment screening at Diamond Synchrotron Light Source XChem facility using PanDDA software, which identifies many more fragment hits, only some of which bind to the predicted hotspots. Many low occupancy sites identified may not support elaboration to give adequate ligand affinity, although they will likely be useful in drug discovery as ‘warm spots’ for guiding elaboration of fragments bound at hotspots. We discuss implications of these observations for fragment screening at the synchrotron sources. This article is part of the theme issue ‘Fifty years of synchrotron science: achievements and opportunities’.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly growing infectious disease, widely spread with high mortality rates. Since the release of the SARS-CoV-2 genome sequence in March 2020, there has been an international focus on developing target-based drug discovery, which also requires knowledge of the 3D structure of the proteome. Where there are no experimentally solved structures, our group has created 3D models with coverage of 97.5% and characterized them using state-of-the-art computational approaches. Models of protomers and oligomers, together with predictions of substrate and allosteric binding sites, protein-ligand docking, SARS-CoV-2 protein interactions with human proteins, impacts of mutations, and mapped solved experimental structures are freely available for download. These are implemented in SARS CoV-2 3D, a comprehensive and user-friendly database, available at https://sars3d.com/. This provides essential information for drug discovery, both to evaluate targets and design new potential therapeutics.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.