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Both the presence of an abnormal complement of chromosomes (aneuploidy) and an increased frequency of chromosome missegregation (chromosomal instability) are hallmarks of cancer. Analyses of cancer genome data have identified certain aneuploidy patterns in tumors; however, the bases behind their selection are largely unexplored. By establishing time-resolved long-term adaptation protocols, we found that human cells adapt to persistent spindle assembly checkpoint (SAC) inhibition by acquiring specific chromosome arm gains and losses. Independently adapted populations converge on complex karyotypes, which over time are refined to contain ever smaller chromosomal changes. Of note, the frequencies of chromosome arm gains in adapted cells correlate with those detected in cancers, suggesting that our cellular adaptation approach recapitulates selective traits that dictate the selection of aneuploidies frequently observed across many cancer types. We further engineered specific aneuploidies to determine the genetic basis behind the observed karyotype patterns. These experiments demonstrated that the adapted and engineered aneuploid cell lines limit CIN by extending mitotic duration. Heterozygous deletions of key SAC and APC/C genes recapitulated the rescue phenotypes of the monosomic chromosomes. We conclude that aneuploidy-induced gene dosage imbalances of individual mitotic regulators are sufficient for altering mitotic timing to reduce CIN.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Neoplasias , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/genética , Aneuploidia , Neoplasias/genética , Instabilidade Cromossômica/genética , Cariótipo , Fuso Acromático/genética , MitoseRESUMO
Over recent decades, the global burden of fungal disease has expanded dramatically. It is estimated that fungal disease kills approximately 1.5 million individuals annually; however, the true worldwide burden of fungal infection is thought to be higher due to existing gaps in diagnostics and clinical understanding of mycotic disease. The development of resistance to antifungals across diverse pathogenic fungal genera is an increasingly common and devastating phenomenon due to the dearth of available antifungal classes. These factors necessitate a coordinated response by researchers, clinicians, public health agencies, and the pharmaceutical industry to develop new antifungal strategies, as the burden of fungal disease continues to grow. This review provides a comprehensive overview of the new antifungal therapeutics currently in clinical trials, highlighting their spectra of activity and progress toward clinical implementation. We also profile up-and-coming intracellular proteins and pathways primed for the development of novel antifungals targeting their activity. Ultimately, we aim to emphasize the importance of increased investment into antifungal therapeutics in the current continually evolving landscape of infectious disease.
Assuntos
Antifúngicos , Micoses , Humanos , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Micoses/tratamento farmacológico , Micoses/microbiologia , Farmacorresistência FúngicaRESUMO
Preventing and controlling influenza virus infection remains a global public health challenge, as it causes seasonal epidemics to unexpected pandemics. These infections are responsible for high morbidity, mortality, and substantial economic impact. Vaccines are the prophylaxis mainstay in the fight against influenza. However, vaccination fails to confer complete protection due to inadequate vaccination coverages, vaccine shortages, and mismatches with circulating strains. Antivirals represent an important prophylactic and therapeutic measure to reduce influenza-associated morbidity and mortality, particularly in high-risk populations. Here, we review current FDA-approved influenza antivirals with their mechanisms of action, and different viral- and host-directed influenza antiviral approaches, including immunomodulatory interventions in clinical development. Furthermore, we also illustrate the potential utility of machine learning in developing next-generation antivirals against influenza.
Assuntos
Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Orthomyxoviridae , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Orthomyxoviridae/tratamento farmacológico , Vacinas contra Influenza/uso terapêuticoRESUMO
We used whole-genome sequencing to analyze a collection of 35 fluconazole-resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two persistent clinical lineages were identified. We identified copy number variation (CNV) of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show that the annotated telomeric gene CDR1B is actually an artifactual in silico fusion of two highly similar neighboring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased the expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.
RESUMO
In view of the urgent need for new antibiotics to treat human infections caused by multidrug-resistant pathogens, drug repurposing is gaining strength due to the relatively low research costs and shorter clinical trials. Such is the case of artemisinin, an antimalarial drug that has recently been shown to display activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To gain insight into how Mtb is affected by artemisinin, we used RNAseq to assess the impact of artemisinin on gene expression profiles, revealing the induction of several efflux pumps and the KstR2 regulon. To anticipate the artemisinin resistance-conferring mutations that could arise in clinical Mtb strains, we performed an in vitro evolution experiment in the presence of lethal concentrations of artemisinin. We obtained artemisinin-resistant isolates displaying different growth kinetics and drug phenotypes, suggesting that resistance evolved through different pathways. Whole-genome sequencing of nine isolates revealed alterations in the glpK and glpQ1 genes, both involved in glycerol metabolism, in seven and one strains, respectively. We then constructed a glpK mutant and found that loss of glpK increases artemisinin resistance only when glycerol is present as a major carbon source. Our results suggest that mutations in glycerol catabolism genes could be selected during the evolution of resistance to artemisinin when glycerol is available as a carbon source. These results add to recent findings of mutations and phase variants that reduce drug efficacy in carbon-source-dependent ways.
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The laboratory diagnosis of antiviral resistance is a quickly changing field due to new drug availability, the sunsetting of older drugs, the development of novel technologies, rapid viral evolution, and the financial/logistic pressures of the clinical laboratory. This mini-review summarizes the current state of clinically available antiviral resistance testing in the United States in 2024, covering the most commonly used test methods, mechanisms, and clinical indications for herpes simplex virus, cytomegalovirus, human immunodeficiency virus, influenza, hepatitis B virus, and hepatitis C virus drug resistance testing. Common themes include the move away from phenotypic to genotypic methods for first-line clinical testing, as well as uncertainty surrounding the clinical meaningfulness of minority variant detection as next-generation sequencing methods have become more commonplace.
Assuntos
Antivirais , Farmacorresistência Viral , Humanos , Farmacorresistência Viral/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Testes de Sensibilidade Microbiana , Viroses/diagnóstico , Viroses/virologia , Viroses/tratamento farmacológico , Estados Unidos , Vírus/efeitos dos fármacos , Vírus/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Several recent studies indicate that mutations in the human immunodeficiency virus type 1 (HIV-1) 3'polypurine tract (3'PPT) motif can reduce sensitivity to the integrase inhibitor dolutegravir (DTG). Using an in vivo systematic evolution of ligands by exponential enrichment (SELEX) approach, we discovered that multiple different mutations in this viral RNA element can confer DTG resistance, suggesting that the inactivation of this critical reverse transcription element causes resistance. An analysis of the viral DNA products formed upon infection by these 3'PPT mutants revealed that they replicate without integration into the host cell genome, concomitant with an increased production of 1-LTR circles. As the replication of these virus variants is activated by the human T-lymphotropic virus 1 (HTLV-1) Tax protein, a factor that reverses epigenetic silencing of episomal HIV DNA, these data indicate that the 3'PPT-mutated viruses escape from the integrase inhibitor DTG by switching to an integration-independent replication mechanism. IMPORTANCE The integrase inhibitor DTG is a potent inhibitor of HIV replication and is currently recommended in drug regimens for people living with HIV. Whereas HIV normally escapes from antiviral drugs by the acquisition of specific mutations in the gene that encodes the targeted enzyme, mutational inactivation of the viral 3'PPT sequence, an RNA element that has a crucial role in the viral reverse transcription process, was found to allow HIV replication in the presence of DTG in cell culture experiments. While the integration of the viral DNA into the cellular genome is considered one of the hallmarks of retroviruses, including HIV, 3'PPT inactivation caused integration-independent replication, which can explain the reduced DTG sensitivity. Whether this exotic escape route can also contribute to viral escape in HIV-infected persons remains to be determined, but our results indicate that screening for 3'PPT mutations in patients that fail on DTG therapy should be considered.
Assuntos
Infecções por HIV , Inibidores de Integrase de HIV , HIV-1 , Humanos , HIV-1/fisiologia , Replicação Viral/genética , DNA Viral , Mutação , Inibidores de Integrase de HIV/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Piridonas/farmacologia , Infecções por HIV/tratamento farmacológico , Farmacorresistência Viral/genéticaRESUMO
Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris.
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Antifúngicos , Candidíase , Humanos , Antifúngicos/farmacologia , Candidíase/microbiologia , Candida auris , Candida , Anfotericina B/farmacologia , Farmacorresistência Fúngica/genética , Testes de Sensibilidade MicrobianaRESUMO
Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.
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Interferon gama , Neoplasias , Humanos , Animais , Camundongos , Interferon gama/farmacologia , Interferon gama/metabolismo , Antígeno B7-H1 , Linhagem Celular Tumoral , Imunoterapia , Microambiente Tumoral , Neoplasias/genéticaRESUMO
Drug-resistant Mycobacterium tuberculosis is a worldwide health-care problem rendering current tuberculosis (TB) drugs ineffective. Drug efflux is an important mechanism in bacterial drug resistance. The MmpL4 and MmpL5 transporters form functionally redundant complexes with their associated MmpS4 and MmpS5 proteins and constitute the inner membrane components of an essential siderophore secretion system of M. tuberculosis. Inactivating siderophore secretion is toxic for M. tuberculosis due to self-poisoning at low-iron conditions and leads to a strong virulence defect in mice. In this study, we show that M. tuberculosis mutants lacking components of the MmpS4-MmpL4 and MmpS5-MmpL5 systems are more susceptible to bedaquiline, clofazimine, and rifabutin, important drugs for treatment of drug-resistant TB. While genetic deletion experiments revealed similar functions of the MmpL4 and MmpL5 transporters in siderophore and drug secretion, complementation experiments indicated that the MmpS4-MmpL4 proteins alone are not sufficient to restore drug efflux in an M. tuberculosis mutant lacking both operons, in contrast to MmpS5-MmpL5. Importantly, an M. tuberculosis mutant lacking the recently discovered periplasmic Rv0455c protein, which is also essential for siderophore secretion, is more susceptible to the same drugs. These results reveal a promising target for the development of dual-function TB drugs, which might poison M. tuberculosis by blocking siderophore secretion and synergize with other drugs by impairing drug efflux.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Animais , Camundongos , Sideróforos/metabolismo , Tuberculose/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Antituberculosos/uso terapêuticoRESUMO
The emergence and spread of antibiotic resistance in bacterial pathogens are serious and ongoing threats to public health. Since chromosome replication is essential to cell growth and pathogenesis, the essential DNA polymerases in bacteria have long been targets of antimicrobial development, although none have yet advanced to the market. Here, we use transient-state kinetic methods to characterize the inhibition of the PolC replicative DNA polymerase from Staphylococcus aureus by 2-methoxyethyl-6-(3'-ethyl-4'-methylanilino)uracil (ME-EMAU), a member of the 6-anilinouracil compounds that specifically target PolC enzymes, which are found in low-GC content Gram-positive bacteria. We find that ME-EMAU binds to S. aureus PolC with a dissociation constant of 14 nM, more than 200-fold tighter than the previously reported inhibition constant, which was determined using steady-state kinetic methods. This tight binding is driven by a very slow off rate of 0.006 s-1. We also characterized the kinetics of nucleotide incorporation by PolC containing a mutation of phenylalanine 1261 to leucine (F1261L). The F1261L mutation decreases ME-EMAU binding affinity by at least 3,500-fold but also decreases the maximal rate of nucleotide incorporation by 11.5-fold. This suggests that bacteria acquiring this mutation would be likely to replicate slowly and be unable to out-compete wild-type strains in the absence of inhibitors, reducing the likelihood of the resistant bacteria propagating and spreading resistance.
Assuntos
Antibacterianos , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Cinética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , NucleotídeosRESUMO
Helicobacter pylori (H. pylori) is a bacterial pathogen in the stomach, causing gastritis, gastric ulcer, duodenal ulcer and even gastric cancer. The triple therapy containing one bismuth-containing compound or a proton-pump inhibitor with two antibiotics was the cornerstone of the treatment of H. pylori infections. However the drug resistance of Helicobacter pylori is more and more common, which leads to the continued decline in the radical cure rate. The purpose of this study was to investigate the mechanism of metronidazole resistance of H. pylori through transcriptomics and biochemical characterizations. In this study, a 128-time-higher metronidazole-resistant H. pylori strain compared to the sensitive strain was domesticated, and 374 significantly differential genes were identified by transcriptomic sequencing as compared to the metronidazole-sensitive strain. Through GO and KEGG enrichment analysis, antibiotic-resistance pathways were found to be mainly involved in redox, biofilm formation and ABC transportation, and the results were verified by qRT-PCR. The subsequent biochemical analysis found that the urease activity of the drug-resistant strain decreased, and whereas the capabilities of bacterial energy production, membrane production and diffusion ability increased. The work here will drop hints for the mechanisms of antibiotic-resistance of H. pylori and provide promising biomarkers for the further development of new-kind drugs to treat metronidazole-resistant H. pylori.
Assuntos
Helicobacter pylori , Transcriptoma , Helicobacter pylori/genética , Metronidazol/farmacologia , Perfilação da Expressão Gênica , Antibacterianos/farmacologiaRESUMO
Breast cancer is one of the most prevalent cancers in women and a leading cause of mortality. As per the GLOBCAN report of 2021, breast cancer has surpassed lung cancer which until recently was the most commonly diagnosed cancer. Despite significant efforts to improve early detection and therapeutic efficacy of breast cancer, the frequent emergence of drug resistance remains the predominant basis for the poor prognosis of cancer patients harboring various malignancies. Long non-coding RNA (lncRNAs) are known to affect a variety of components of genome function, including epigenetics, gene transcription, splicing, translation, as well as many central biological processes like cell cycle progression, cell differentiation, development, and pluripotency. LncRNAs are dysregulated in various malignancies and interact with a multitude of RNAs and proteins to impact drug resistance. LncRNAs regulate chemoresistance in cancer by employing an assortment of molecular mechanisms including multidrug efflux, suppression of apoptosis, DNA damage response, epigenetic alterations, as well as functioning as competitive endogenous RNA. When combined with other regulatory mechanisms, these pathways form a complex orchestration of signaling that ultimately lead to chemoresistance. The current review delves into the role of lncRNAs in inducing drug resistance to conventional therapeutic anti-cancer drugs used for the treatment of breast cancer. We propose that lncRNAs that provoke drug resistance could be used to develop new targeted and tailored therapeutics providing a novel approach to introduce promising personalized treatment modalities to overcome chemoresistance in breast cancer patients. Hence, lncRNAs that drive anticancer drug resistance can be potentially explored as biomarkers of disease prognosis and may provide unique opportunities to circumvent chemoresistance in breast cancer patients.
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Antineoplásicos , Neoplasias da Mama , Neoplasias Pulmonares , RNA Longo não Codificante , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) catalyzes an essential step in purine salvage for parasite growth. 4'-Deaza-1'-Aza-2'-Deoxy-1'-(9-Methylene)-Immucillin-G (DADMe-ImmG) is a transition state analog inhibitor of this enzyme, and P. falciparum infections in an Aotus primate malaria model can be cleared by oral administration of DADMe-ImmG. P. falciparum cultured under increasing DADMe-ImmG drug pressure exhibited PfPNP gene amplification, increased protein expression, and point mutations involved in DADMe-ImmG binding. However, the weak catalytic properties of the M183L resistance mutation (â¼17,000-fold decrease in catalytic efficiency) are inconsistent with the essential function of PfPNP. We hypothesized that M183L subunits may form mixed oligomers of native and mutant PfPNP monomers to give hybrid hexameric enzymes with properties conferring DADMe-ImmG resistance. To test this hypothesis, we designed PfPNP constructs that covalently linked native and the catalytically weak M183L mutant subunits. Engineered hybrid PfPNP yielded trimer-of-dimer hexameric protein with alternating native and catalytically weak M183L subunits. This hybrid PfPNP gave near-native Km values for substrate, but the affinity for DADMe-ImmG and catalytic efficiency were both reduced approximately ninefold relative to a similar construct of native subunits. Contact between the relatively inactive M183L and native subunits is responsible for altered properties of the hybrid protein. Thus, gene amplification of PfPNP provides adequate catalytic activity while resistance to DADMe-ImmG occurs in the hybrid oligomer to promote parasite survival. Coupled with the slow development of drug resistance, this resistance mechanism highlights the potential for DADMe-ImmG use in antimalarial combination therapies.
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Adenosina/análogos & derivados , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/genética , Pirrolidinas/farmacologia , Adenosina/farmacologia , Resistência a Medicamentos , Humanos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/fisiologia , Mutação Puntual/efeitos dos fármacosRESUMO
The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.
Assuntos
Haemophilus influenzae , Quinolonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Mutação , Filogenia , Sinais Direcionadores de Proteínas/genética , Quinolonas/farmacologiaRESUMO
We generated highly chloroquine (CQ)-resistant (ResCQ) Plasmodium yoelii parasites by stepwise exposure to increasing concentrations of CQ and CQ-sensitive parasites (SenCQ) by parallel mock treatments. No mutations in genes that are associated with drug resistance were detected in ResCQ clones. Autophagy-related genes were highly upregulated in SenCQ compared to ResCQ parasites during CQ treatment. This indicates that CQ resistance can be developed in the malaria parasite by the inhibition of autophagy as an alternative drug resistance mechanism.
Assuntos
Antimaláricos , Cloroquina , Resistência a Medicamentos , Plasmodium yoelii , Proteínas de Protozoários , Humanos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Malária/tratamento farmacológico , Malária/parasitologia , Proteínas de Protozoários/genética , Plasmodium yoelii/efeitos dos fármacos , Plasmodium yoelii/genéticaRESUMO
Tuberculosis (TB) is the leading cause of death from any bacterial infection, causing 1.5 million deaths worldwide each year. Due to the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) there have been significant efforts aimed at developing novel drugs to treat TB. One promising drug target in Mtb is the arabinogalactan biosynthetic enzyme DprE1, and there have been over a dozen unique chemical scaffolds identified which inhibit the activity of this protein. Among the most promising lead compounds are the benzothiazinones BTZ043 and PBTZ169, both of which are currently in or have completed phase IIa clinical trials. Due to the potential clinical utility of these drugs, we sought to identify potential synergistic interactions and new mechanisms of resistance using a genome-scale CRISPRi chemical-genetic screen with PBTZ169. We found that knockdown of rv0678, the negative regulator of the mmpS5/L5 drug efflux pump, confers resistance to PBTZ169. Mutations in rv0678 are the most common form of resistance to bedaquiline and there is already abundant evidence of these mutations emerging in bedaquiline-treated patients. We confirmed that rv0678 mutations from clinical isolates confer low level cross-resistance to BTZ043 and PBTZ169. While it is yet unclear whether rv0678 mutations would render benzothiazinones ineffective in treating TB, these results highlight the importance of monitoring for clinically prevalent rv0678 mutations during ongoing BTZ043 and PBTZ169 clinical trials.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Antituberculosos/uso terapêutico , Diarilquinolinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/genética , Piperazinas , Compostos de Espiro , Tiazinas , Tuberculose/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Treatment failures with artemisinin combination therapies (ACTs) threaten global efforts to eradicate malaria. They highlight the importance of identifying drug targets and new inhibitors and of studying how existing antimalarial classes work. Here, we report the successful development of a heterologous expression-based compound-screening tool. The validated drug target Plasmodium falciparum ATPase 6 (PfATP6) and a mammalian orthologue (sarco/endoplasmic reticulum calcium ATPase 1a [SERCA1a]) were functionally expressed in Saccharomyces cerevisiae, providing a robust, sensitive, and specific screening tool. Whole-cell and in vitro assays consistently demonstrated inhibition and labeling of PfATP6 by artemisinins. Mutations in PfATP6 resulted in fitness costs that were ameliorated in the presence of artemisinin derivatives when studied in the yeast model. As previously hypothesized, PfATP6 is a target of artemisinins. Mammalian SERCA1a can be mutated to become more susceptible to artemisinins. The inexpensive, low-technology yeast screening platform has identified unrelated classes of druggable PfATP6 inhibitors. Resistance to artemisinins may depend on mechanisms that can concomitantly address multitargeting by artemisinins and fitness costs of mutations that reduce artemisinin susceptibility.
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Antimaláricos , Artemisininas , Malária Falciparum , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Artemisininas/farmacologia , Artemisininas/uso terapêutico , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/uso terapêutico , Resistência a Medicamentos , Malária Falciparum/tratamento farmacológico , Mamíferos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Contezolid (MRX-I), a safer antibiotic of the oxazolidinone class, is a promising new antibiotic with potent activity against Mycobacterium tuberculosis (MTB) both in vitro and in vivo. To identify resistance mechanisms of contezolid in MTB, we isolated several in vitro spontaneous contezolid-resistant MTB mutants, which exhibited 16-fold increases in the MIC of contezolid compared with the parent strain but were still unexpectedly susceptible to linezolid. Whole-genome sequencing revealed that most of the contezolid-resistant mutants bore mutations in the mce3R gene, which encodes a transcriptional repressor. The mutations in mce3R led to markedly increased expression of a monooxygenase encoding gene Rv1936. We then characterized Rv1936 as a putative flavin-dependent monooxygenase that catalyzes the degradation of contezolid into its inactive 2,3-dihydropyridin-4-one (DHPO) ring-opened metabolites, thereby conferring drug resistance. While contezolid is an attractive drug candidate with potent antimycobacterial activity and low toxicity, the occurrence of mutations in Mce3R should be considered when designing combination therapy using contezolid for treating tuberculosis.
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Mycobacterium tuberculosis , Oxazolidinonas , Linezolida , Antibacterianos , Mutação , Oxigenases de Função Mista/metabolismo , Flavinas/genética , Flavinas/metabolismo , Antituberculosos/farmacologia , Antituberculosos/metabolismo , Testes de Sensibilidade MicrobianaRESUMO
Cefiderocol is a promising novel siderophore cephalosporin for the treatment of multidrug-resistant Gram-negative bacilli and with stability against degradation by metallo-ß-lactamases. Nonetheless, the emergence of cefiderocol in metallo-ß-lactamase-producing Enterobacterales during therapy has been reported on more than one occasion. To understand the underlying mechanisms and factors facilitating the resistance development, we conducted an in vitro evolution experiment using clinical E. cloacae isolates via serial passaging under cefiderocol pressure. In this study, we showed that the presence of the New Delhi metallo-ß-lactamase (NDM) facilitates the emergence of resistance via nonsynonymous mutations of the CirA catecholate siderophore receptor. Inhibition of metallo-ß-lactamase activity using dipicolinic acid prevented the emergence of cefiderocol-resistant mutants successfully. This finding implies that caution should be taken when using cefiderocol for the treatment of infections caused by metallo-ß-lactamase-producing bacteria.