74 research outputs found
SARS-CoV-2 3D database: Understanding the Coronavirus Proteome and Evaluating Possible Drug Targets.
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 characterised 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 imple- mented 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.This work is supported and funded by King Abdullah scholarship (Saudi Arabia research coun- cil), and American Leprosy Missions grants (G88726), SET is funded by the Cystic Fibrosis Trust (RG 70975) and Fondation Botnar (RG91317). A.R.J is funded by the Biotechnology and Biological Sciences Research Council (BBSRC) DTP studentship (BB/M011194/1). B.B. is funded by the Cystic Fibrosis Trust and L.C. on a studentship from Ipsen. T.L.B. is funded by a the Wellcome Trust Investigator Award, PHZJ/489 RG83114 (2016-2021
Structure-guided fragment-based drug discovery at the synchrotron: screening binding sites and correlations with hotspot mapping.
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 1040 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 Botnar Foundation (grant number: 6063), the Cystic Fibrosis Trust (Strategic Research Centre Awards 002, 010 & 201) and the Bill and Melinda Gates Foundation, Shorten-TB Award
Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs.
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
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Development of Inhibitors against Mycobacterium abscessus tRNA (m1G37) Methyltransferase (TrmD) Using Fragment-Based Approaches.
Mycobacterium abscessus (Mab) is a rapidly growing species of multidrug-resistant nontuberculous mycobacteria that has emerged as a growing threat to individuals with cystic fibrosis and other pre-existing chronic lung diseases. Mab pulmonary infections are difficult, or sometimes impossible, to treat and result in accelerated lung function decline and premature death. There is therefore an urgent need to develop novel antibiotics with improved efficacy. tRNA (m1G37) methyltransferase (TrmD) is a promising target for novel antibiotics. It is essential in Mab and other mycobacteria, improving reading frame maintenance on the ribosome to prevent frameshift errors. In this work, a fragment-based approach was employed with the merging of two fragments bound to the active site, followed by structure-guided elaboration to design potent nanomolar inhibitors against Mab TrmD. Several of these compounds exhibit promising activity against mycobacterial species, including Mycobacterium tuberculosis and Mycobacterium leprae in addition to Mab, supporting the use of TrmD as a target for the development of antimycobacterial compounds
A fragment-based approach to assess the ligandability of ArgB, ArgC, ArgD and ArgF in the L-arginine biosynthetic pathway of Mycobacterium tuberculosis.
The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes
Mycobacterial genomics and structural bioinformatics: opportunities and challenges in drug discovery.
Of the more than 190 distinct species of Mycobacterium genus, many are economically and clinically important pathogens of humans or animals. Among those mycobacteria that infect humans, three species namely Mycobacterium tuberculosis (causative agent of tuberculosis), Mycobacterium leprae (causative agent of leprosy) and Mycobacterium abscessus (causative agent of chronic pulmonary infections) pose concern to global public health. Although antibiotics have been successfully developed to combat each of these, the emergence of drug-resistant strains is an increasing challenge for treatment and drug discovery. Here we describe the impact of the rapid expansion of genome sequencing and genome/pathway annotations that have greatly improved the progress of structure-guided drug discovery. We focus on the applications of comparative genomics, metabolomics, evolutionary bioinformatics and structural proteomics to identify potential drug targets. The opportunities and challenges for the design of drugs for M. tuberculosis, M. leprae and M. abscessus to combat resistance are discussed
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Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification.
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
Investigating the effects of chronic low-dose radiation exposure in the liver of a hypothermic zebrafish model
Mankind's quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism
Cardiovascular safety of tocilizumab versus etanercept in rheumatoid arthritis: a randomized controlled trial
Objective:
To compare the risk for major adverse cardiovascular events (MACE) in RA patients treated with tocilizumab versus the tumor necrosis factor inhibitor etanercept.
Methods:
This randomized, open‐label, parallel‐group trial enrolled patients with active seropositive RA (N=3080), inadequate responses to conventional synthetic disease‐modifying antirheumatic drugs, and at least one cardiovascular risk factor. Patients were randomly assigned 1:1 to open‐label tocilizumab 8 mg/kg/month or etanercept 50 mg/week and followed up for an average of 3.2 years. The primary end point was comparison of time‐to‐first MACE. The trial was powered to exclude a 1.8 or higher relative hazard of MACE for tocilizumab versus etanercept.
Results:
By week 4, serum low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, and triglyceride levels were 11.1%, 5.7%, and 13.6% higher, respectively, for patients allocated to tocilizumab compared with etanercept (all P<.001). During follow‐up, 83 MACE occurred in the tocilizumab group compared with 78 in the etanercept group. The estimated hazard of MACE for tocilizumab relative to etanercept was 1.05 (95% confidence interval=0.77, 1.43). Result were similar in sensitivity analyses and the on‐treatment analysis. Adverse events that occurred more frequently in the tocilizumab group included serious infection and gastrointestinal perforation.
Conclusion:
The trial, which provides insights into the cardiovascular safety of tocilizumab versus etanercept, excluded a relative risk for MACE of 1.43 or higher. This result should be interpreted in the context of the clinical efficacy and the non‐cardiovascular safety of tocilizumab
Antibody decay, T cell immunity and breakthrough infections following two SARS-CoV-2 vaccine doses in inflammatory bowel disease patients treated with infliximab and vedolizumab
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