RESUMO
Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Isoxazóis/farmacologia , Testes de Sensibilidade Microbiana , Proteínas de BactériasRESUMO
The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.
Assuntos
Benzotiazóis/farmacologia , Tratamento Farmacológico da COVID-19 , Oligopeptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/genética , Animais , Benzamidinas/química , Benzotiazóis/farmacocinética , COVID-19/genética , COVID-19/virologia , Linhagem Celular , Desenho de Fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Ésteres/química , Guanidinas/química , Humanos , Pulmão/efeitos dos fármacos , Pulmão/virologia , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Oligopeptídeos/farmacocinética , SARS-CoV-2/patogenicidade , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/ultraestrutura , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 ( 4 ) has an IC 50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC 50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV SARS-CoV-2 chimeric virus, and an EC 50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East Respiratory Syndrome Coronavirus (MERS-CoV) cell entry with an EC 50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice with a half-life of 8.6 hours in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.
RESUMO
Annie Mayer Bridwell works in the field of tuberculosis pathogenesis from the host perspective. She is fascinated by comorbidities, and in this paper, she reflects on three publications that shaped her model of neutrophil-centric pathology in tuberculosis and type 2 diabetes comorbidity. She explains that "Systems immunology of diabetes-tuberculosis comorbidity reveals signatures of disease complications" (C. A. Prada-Medina, K. F. Fukutani, N. Pavan Kumar, L. Gil-Santana, et al., Sci Rep 7:1999, 2017, https://doi.org/10.1038/s41598-017-01767-4) led her to consider neutrophils as a central immunological player in comorbid patients. "Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis" (L. Moreira-Teixeira, P. J. Stimpson, E. Stavropoulos, S. Hadebe, et al., Nat Commun 11:5566, 2020, https://doi.org/10.1038/s41467-020-19412-6) and "Diabetes primes neutrophils to undergo NETosis, which impairs wound healing" (S. L. Wong, M. Demers, K. Martinod, M. Gallant, et al., Nat Med 21:815-819, 2015, https://doi.org/10.1038/nm.3887) then shed light on neutrophil extracellular trap (NET) formation as a common pathological feature of dysregulated neutrophils in tuberculosis and diabetes, respectively. Together, these works laid the foundation for Dr. Mayer Bridwell's interest in metabolic regulation of NETosis during TB infection and diabetes comorbidity.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/imunologia , Neutrófilos/imunologia , Tuberculose/epidemiologia , Tuberculose/imunologia , Comorbidade , Suscetibilidade a DoençasRESUMO
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a leading cause of death due to infection in humans. To more effectively combat this pandemic, many aspects of TB control must be developed, including better point of care diagnostics, shorter and safer drug regimens, and a protective vaccine. To address all these areas of need, better understanding of the pathogen, host responses, and clinical manifestations of the disease is required. Recently, the application of cutting-edge technologies to the study of Mtb pathogenesis has resulted in significant advances in basic biology, vaccine development, and antibiotic discovery. This leaves us in an exciting era of Mtb research in which our understanding of this deadly infection is improving at a faster rate than ever, and renews hope in our fight to end TB. In this review, we reflect on what is known regarding Mtb pathogenesis, highlighting recent breakthroughs that will provide leverage for the next leaps forward in the field.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antituberculosos/uso terapêutico , Humanos , Mycobacterium tuberculosis/patogenicidadeRESUMO
Antibiotic resistance is a global crisis that threatens our ability to treat bacterial infections, such as tuberculosis, caused by Mycobacterium tuberculosis Of the 10 million cases of tuberculosis in 2017, approximately 19% of new cases and 43% of previously treated cases were caused by strains of M. tuberculosis resistant to at least one frontline antibiotic. There is a clear need for new therapies that target these genetically resistant strains. Here, we report the discovery of a new series of antimycobacterial compounds, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit the growth of M. tuberculosis To elucidate the mechanism by which these compounds inhibit M. tuberculosis, we selected for mutants resistant to a representative 4-amino-thieno[2,3-d]pyrimidine and sequenced these strains to identify the mutations that confer resistance. We isolated a total of 12 resistant mutants, each of which harbored a nonsynonymous mutation in the gene qcrB, which encodes a subunit of the electron transport chain (ETC) enzyme cytochrome bc1 oxidoreductase, leading us to hypothesize that 4-amino-thieno[2,3-d]pyrimidines target this enzyme complex. We found that addition of 4-amino-thieno[2,3-d]pyrimidines to M. tuberculosis cultures resulted in a decrease in ATP levels, supporting our model that these compounds inhibit the M. tuberculosis ETC. Furthermore, 4-amino-thieno[2,3-d]pyrimidines had enhanced activity against a mutant of M. tuberculosis deficient in cytochrome bd oxidase, which is a hallmark of cytochrome bc1 inhibitors. Therefore, 4-amino-thieno[2,3-d]pyrimidines represent a novel series of QcrB inhibitors that build on the growing number of chemical scaffolds that are able to inhibit the mycobacterial cytochrome bc1 complex.IMPORTANCE The global tuberculosis (TB) epidemic has been exacerbated by the rise in drug-resistant TB cases worldwide. To tackle this crisis, it is necessary to identify new vulnerable drug targets in Mycobacterium tuberculosis, the causative agent of TB, and develop compounds that can inhibit the bacterium through novel mechanisms of action. The QcrB subunit of the electron transport chain enzyme cytochrome bc1 has recently been validated to be a potential drug target. In the current work, we report the discovery of a new class of QcrB inhibitors, 4-amino-thieno[2,3-d]pyrimidines, that potently inhibit M. tuberculosis growth in vitro These compounds are chemically distinct from previously reported QcrB inhibitors, and therefore, 4-amino-thieno[2,3-d]pyrimidines represent a new scaffold that can be exploited to inhibit this drug target.
Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Pirimidinas/farmacologia , Antibióticos Antituberculose/química , Proteínas de Bactérias/genética , Descoberta de Drogas , Complexo III da Cadeia de Transporte de Elétrons/genética , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Pirimidinas/químicaRESUMO
Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.