RESUMO
Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.
Assuntos
Azetidinas , Tuberculose Extensivamente Resistente a Medicamentos , Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Azetidinas/farmacologia , Azetidinas/uso terapêutico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Testes de Sensibilidade MicrobianaRESUMO
Early de-risking of drug targets and chemistry is essential to provide drug projects with the best chance of success. Target safety assessments (TSAs) use target biology, gene and protein expression data, genetic information from humans and animals, and competitor compound intelligence to understand the potential safety risks associated with modulating a drug target. However, there is a vast amount of information, updated daily that must be considered for each TSA. We have developed a data science-based approach that allows acquisition of relevant evidence for an optimal TSA. This is built on expert-led conventional and artificial intelligence-based mining of literature and other bioinformatics databases. Potential safety risks are identified according to an evidence framework, adjusted to the degree of target novelty. Expert knowledge is necessary to interpret the evidence and to take account of the nuances of drug safety, the modality, and the intended patient population for each TSA within each project. Overall, TSAs take full advantage of the most recent developments in data science and can be used within drug projects to identify and mitigate risks, helping with informed decision-making and resource management. These approaches should be used in the earliest stages of a drug project to guide decisions such as target selection, discovery chemistry options, in vitro assay choice, and end points for investigative in vivo studies.
Assuntos
Inteligência Artificial , Ciência de Dados , Animais , Humanos , Descoberta de Drogas , Biologia ComputacionalRESUMO
The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.
Assuntos
Regiões 5' não Traduzidas , Antivirais/farmacologia , Substâncias Macromoleculares/farmacologia , RNA/metabolismo , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/metabolismo , Chlorocebus aethiops , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Genoma Viral/efeitos dos fármacos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Metais Pesados/química , Simulação de Dinâmica Molecular , RNA/genética , SARS-CoV-2/química , Células VeroRESUMO
HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.
Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Alternativas aos Testes com Animais , Antracenos/toxicidade , Benzo(a)pireno/toxicidade , Ensaio Cometa , Hepatócitos/efeitos dos fármacos , Imidazóis/toxicidade , Fígado/efeitos dos fármacos , 4-Nitroquinolina-1-Óxido/metabolismo , Ativação Metabólica , Antracenos/metabolismo , Benzo(a)pireno/metabolismo , Dano ao DNA , Regulação Enzimológica da Expressão Gênica , Células Hep G2 , Hepatócitos/enzimologia , Hepatócitos/patologia , Histonas/metabolismo , Humanos , Imidazóis/metabolismo , Fígado/enzimologia , Fígado/patologia , Fosforilação , Esferoides Celulares , Fatores de TempoRESUMO
The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.
RESUMO
Reactive oxygen species (ROS) within the cell are rapidly detoxified by antioxidants such as glutathione. Depletion of glutathione will therefore increase levels of intracellular ROS, which can lead to oxidative DNA damage and the induction of apoptosis. The working hypothesis was that Ogg1 null mouse embryonic fibroblasts (mOgg1-/- MEFs) would be more sensitive in response to GSH depletion due to their deficiency in the removal of the oxidative DNA modification, 8-oxo-7,8-dihydroguanine (8-oxoG). Following GSH depletion, an increase in intracellular ROS and a subsequent induction of apoptosis was measured in mOgg1-/- MEFs; as expected. Unexpectedly, an elevated basal level of ROS was identified in mOgg1-/- MEFs compared to wild type MEFs; which we suggest is partly due to the differential expression of key anti-oxidant genes. The elevated basal ROS levels in mOgg1-/- MEFs were not accompanied by a deficiency in ATP production or a large increase in 8-oxoG levels. Although 8-oxoG levels did increase following GSH depletion in mOgg1-/- MEFs; this increase was significantly lower than observed following treatment with a non-toxic dose of hydrogen peroxide. Reconstitution of Ogg1 into mOgg1-/- MEFs resulted in an increased viability following glutathione depletion, however this rescue did not differ between a repair-proficient and a repair-impaired variant of Ogg1. The data indicates that induction of apoptosis in response to oxidative stress in mOgg1-/- MEFs is independent of DNA damage and OGG1-initiated DNA repair.