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1.
Science ; 385(6704): 91-99, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38963839

RESUMO

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.


Assuntos
Anemia Falciforme , Antidrepanocíticos , Hemoglobina Fetal , Fatores de Transcrição Kruppel-Like , Proteínas do Tecido Nervoso , Animais , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , Antidrepanocíticos/química , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Cristalografia por Raios X , Descoberta de Drogas , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Macaca fascicularis , Proteínas do Tecido Nervoso/metabolismo , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética
2.
APL Bioeng ; 8(2): 026129, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38938688

RESUMO

Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas.

3.
PLoS One ; 18(6): e0287809, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37384771

RESUMO

Cigarette smoking (CS) is the leading cause of COPD, and identifying the pathways that are driving pathogenesis in the airway due to CS exposure can aid in the discovery of novel therapies for COPD. An additional barrier to the identification of key pathways that are involved in the CS-induced pathogenesis is the difficulty in building relevant and high throughput models that can recapitulate the phenotypic and transcriptomic changes associated with CS exposure. To identify these drivers, we have developed a cigarette smoke extract (CSE)-treated bronchosphere assay in 384-well plate format that exhibits CSE-induced decreases in size and increase in luminal secretion of MUC5AC. Transcriptomic changes in CSE-treated bronchospheres resemble changes that occur in human smokers both with and without COPD compared to healthy groups, indicating that this model can capture human smoking signature. To identify new targets, we ran a small molecule compound deck screening with diversity in target mechanisms of action and identified hit compounds that attenuated CSE induced changes, either decreasing spheroid size or increasing secreted mucus. This work provides insight into the utility of this bronchopshere model to examine human respiratory disease impacted by CSE exposure and the ability to screen for therapeutics to reverse the pathogenic changes caused by CSE.


Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Humanos , Fumar Cigarros/efeitos adversos , Bioensaio , Transporte Biológico , Placas Ósseas , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico
4.
APL Bioeng ; 7(2): 026104, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37206658

RESUMO

Asthma is often characterized by tissue-level mechanical phenotypes that include remodeling of the airway and an increase in airway tightening, driven by the underlying smooth muscle. Existing therapies only provide symptom relief and do not improve the baseline narrowing of the airway or halt progression of the disease. To investigate such targeted therapeutics, there is a need for models that can recapitulate the 3D environment present in this tissue, provide phenotypic readouts of contractility, and be easily integrated into existing assay plate designs and laboratory automation used in drug discovery campaigns. To address this, we have developed DEFLCT, a high-throughput plate insert that can be paired with standard labware to easily generate high quantities of microscale tissues in vitro for screening applications. Using this platform, we exposed primary human airway smooth muscle cell-derived microtissues to a panel of six inflammatory cytokines present in the asthmatic niche, identifying TGF-ß1 and IL-13 as inducers of a hypercontractile phenotype. RNAseq analysis further demonstrated enrichment of contractile and remodeling-relevant pathways in TGF-ß1 and IL-13 treated tissues as well as pathways generally associated with asthma. Screening of 78 kinase inhibitors on TGF-ß1 treated tissues suggests that inhibition of protein kinase C and mTOR/Akt signaling can prevent this hypercontractile phenotype from emerging, while direct inhibition of myosin light chain kinase does not. Taken together, these data establish a disease-relevant 3D tissue model for the asthmatic airway, which combines niche specific inflammatory cues and complex mechanical readouts that can be utilized in drug discovery efforts.

5.
PLoS One ; 17(11): e0277937, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36409750

RESUMO

The importance of human cell-based in vitro tools to drug development that are robust, accurate, and predictive cannot be understated. There has been significant effort in recent years to develop such platforms, with increased interest in 3D models that can recapitulate key aspects of biology that 2D models might not be able to deliver. We describe the development of a 3D human cell-based in vitro assay for the investigation of nephrotoxicity, using RPTEC-TERT1 cells. These RPTEC-TERT1 proximal tubule organoids 'tubuloids' demonstrate marked differences in physiologically relevant morphology compared to 2D monolayer cells, increased sensitivity to nephrotoxins observable via secreted protein, and with a higher degree of similarity to native human kidney tissue. Finally, tubuloids incubated with nephrotoxins demonstrate altered Na+/K+-ATPase signal intensity, a potential avenue for a high-throughput, translatable nephrotoxicity assay.


Assuntos
Túbulos Renais Proximais , Organoides , Humanos , Linhagem Celular , Túbulos Renais Proximais/metabolismo , Túbulos Renais , Rim
6.
Virology ; 540: 195-206, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31929001

RESUMO

Respiratory syncytial virus (RSV) infection can cause mucus overproduction and bronchiolitis in infants leading to severe disease and hospitalization. As a therapeutic strategy, immune modulatory agents may help prevent RSV-driven immune responses that cause severe airway disease. We developed a high throughput screen to identify compounds that reduced RSV-driven mucin 5AC (Muc5AC) expression and identified dexamethasone. Despite leading to a pronounced reduction in RSV-driven Muc5AC, dexamethasone increased RSV infection in vitro and delayed viral clearance in mice. This correlated with reduced expression of a subset of immune response genes and reduced lymphocyte infiltration in vivo. Interestingly, dexamethasone increased RSV infection levels without altering antiviral interferon signaling. In summary, the immunosuppressive activities of dexamethasone had favorable inhibitory effects on RSV-driven mucus production yet prevented immune defense activities that limit RSV infection in vitro and in vivo. These findings offer an explanation for the lack of efficacy of glucocorticoids in RSV-infected patients.


Assuntos
Dexametasona/farmacologia , Interferons/metabolismo , Muco/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Redes Reguladoras de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Camundongos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-31658970

RESUMO

Upregulated expression of efflux pumps, lpxC target mutations, LpxC protein overexpression, and mutations in fabG were previously shown to mediate single-step resistance to the LpxC inhibitor CHIR-090 in P. aeruginosa Single-step selection experiments using three recently described LpxC inhibitors (compounds 2, 3, and 4) and mutant characterization showed that these mechanisms affect susceptibility to additional novel LpxC inhibitors. Serial passaging of P. aeruginosa wild-type and efflux pump-defective strains using the LpxC inhibitor CHIR-090 or compound 1 generated substantial shifts in susceptibility and underscored the interplay of efflux and nonefflux mechanisms. Whole-genome sequencing of CHIR-090 passage mutants identified efflux pump overexpression, fabG mutations, and novel mutations in fabF1 and in PA4465 as determinants of reduced susceptibility. Two new lpxC mutations, encoding A214V and G208S, that reduce susceptibility to certain LpxC inhibitors were identified in these studies, and we show that these and other target mutations differentially affect different LpxC inhibitor scaffolds. Lastly, the combination of target alteration (LpxCA214V) and upregulated expression of LpxC was shown to be tolerated in P. aeruginosa and could mediate significant decreases in susceptibility.


Assuntos
Pseudomonas aeruginosa/efeitos dos fármacos , Amidoidrolases/genética , Amidoidrolases/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana , Mutação/genética , Pseudomonas aeruginosa/genética , Sequenciamento Completo do Genoma
8.
Artigo em Inglês | MEDLINE | ID: mdl-31036686

RESUMO

Antibiotic hypersensitive bacterial mutants (e.g., Escherichia coliimp) are used to investigate intrinsic resistance and are exploited in antibacterial discovery to track weak antibacterial activity of novel inhibitor compounds. Pseudomonas aeruginosa Z61 is one such drug-hypersusceptible strain generated by chemical mutagenesis, although the genetic basis for hypersusceptibility is not fully understood. Genome sequencing of Z61 revealed nonsynonymous single-nucleotide polymorphisms in 153 genes relative to its parent strain, and three candidate mutations (in oprM, ampC, and lptE) predicted to mediate hypersusceptibility were characterized. The contribution of these mutations was confirmed by genomic restoration of the wild-type sequences, individually or in combination, in the Z61 background. Introduction of the lptE mutation or genetic inactivation of oprM and ampC genes alone or together in the parent strain recapitulated drug sensitivities. This showed that disruption of oprM (which encodes a major outer membrane efflux pump channel) increased susceptibility to pump substrate antibiotics, that inactivation of the inducible ß-lactamase gene ampC contributed to ß-lactam susceptibility, and that mutation of the lipopolysaccharide transporter gene lptE strongly altered the outer membrane permeability barrier, causing susceptibility to large antibiotics such as rifampin and also to ß-lactams.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Lipopolissacarídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico/genética , Permeabilidade da Membrana Celular/genética , Testes de Sensibilidade Microbiana/métodos , Mutação/genética , beta-Lactamas/farmacologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-30420483

RESUMO

Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several ß-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.


Assuntos
Piperidinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , beta-Lactamas/farmacologia , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Monobactamas/farmacologia , Pseudomonas aeruginosa/genética , Tazobactam/farmacologia , Tienamicinas/farmacologia , Transcriptoma/genética
10.
Nat Methods ; 15(11): 941-946, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30297964

RESUMO

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Genoma Humano , Células-Tronco Hematopoéticas/metabolismo , RNA Guia de Cinetoplastídeos/genética , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos
11.
Nature ; 537(7619): 229-233, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27501246

RESUMO

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.


Assuntos
Doença de Chagas/tratamento farmacológico , Kinetoplastida/efeitos dos fármacos , Kinetoplastida/enzimologia , Leishmaniose/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Pirimidinas/farmacologia , Triazóis/farmacologia , Tripanossomíase Africana/tratamento farmacológico , Animais , Doença de Chagas/parasitologia , Quimotripsina/antagonistas & inibidores , Quimotripsina/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Concentração Inibidora 50 , Leishmaniose/parasitologia , Camundongos , Estrutura Molecular , Terapia de Alvo Molecular , Inibidores de Proteassoma/efeitos adversos , Inibidores de Proteassoma/classificação , Pirimidinas/efeitos adversos , Pirimidinas/química , Pirimidinas/uso terapêutico , Especificidade da Espécie , Triazóis/efeitos adversos , Triazóis/química , Triazóis/uso terapêutico , Tripanossomíase Africana/parasitologia
12.
PLoS Pathog ; 11(7): e1005058, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26186534

RESUMO

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.


Assuntos
Antifúngicos/farmacologia , Doença de Chagas/tratamento farmacológico , Doença de Chagas/microbiologia , Citocromos b/metabolismo , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antimicina A/metabolismo , Doença de Chagas/genética , Citocromos b/genética , Transporte de Elétrons/efeitos dos fármacos , Transporte de Elétrons/imunologia , Genômica , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Consumo de Oxigênio/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/metabolismo
13.
Antimicrob Agents Chemother ; 58(9): 5060-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24913172

RESUMO

Renewed global efforts toward malaria eradication have highlighted the need for novel antimalarial agents with activity against multiple stages of the parasite life cycle. We have previously reported the discovery of a novel class of antimalarial compounds in the imidazolopiperazine series that have activity in the prevention and treatment of blood stage infection in a mouse model of malaria. Consistent with the previously reported activity profile of this series, the clinical candidate KAF156 shows blood schizonticidal activity with 50% inhibitory concentrations of 6 to 17.4 nM against P. falciparum drug-sensitive and drug-resistant strains, as well as potent therapeutic activity in a mouse models of malaria with 50, 90, and 99% effective doses of 0.6, 0.9, and 1.4 mg/kg, respectively. When administered prophylactically in a sporozoite challenge mouse model, KAF156 is completely protective as a single oral dose of 10 mg/kg. Finally, KAF156 displays potent Plasmodium transmission blocking activities both in vitro and in vivo. Collectively, our data suggest that KAF156, currently under evaluation in clinical trials, has the potential to treat, prevent, and block the transmission of malaria.


Assuntos
Antimaláricos/farmacologia , Imidazóis/farmacologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/transmissão , Piperazinas/farmacologia , Animais , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos ICR , Plasmodium falciparum/efeitos dos fármacos , Esporozoítos/efeitos dos fármacos
14.
Mol Microbiol ; 91(6): 1106-19, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24417450

RESUMO

Rifampicin resistance, a defining attribute of multidrug-resistant tuberculosis, is conferred by mutations in the ß subunit of RNA polymerase. Sequencing of rifampicin-resistant (RIF-R) clinical isolates of Mycobacterium tuberculosis revealed, in addition to RIF-R mutations, enrichment of potential compensatory mutations around the double-psi ß-barrel domain of the ß' subunit comprising the catalytic site and the exit tunnel for newly synthesized RNA. Sequential introduction of the resistance allele followed by the compensatory allele in isogenic Mycobacterium smegmatis showed that these mutations respectively caused and compensated a starvation enhanced growth defect by altering RNA polymerase activity. While specific combinations of resistance and compensatory alleles converged in divergent lineages, other combinations recurred among related isolates suggesting transmission of compensated RIF-R strains. These findings suggest nutrient poor growth conditions impose larger selective pressure on RIF-R organisms that results in the selection of compensatory mutations in a domain involved in catalysis and starvation control of RNA polymerase transcription.


Assuntos
Antituberculosos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Mutação de Sentido Incorreto , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Rifampina/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento
15.
J Antimicrob Chemother ; 69(1): 41-4, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23887866

RESUMO

OBJECTIVES: Current treatments for Clostridium difficile infection include vancomycin, metronidazole and fidaxomicin. LFF571 is an experimental agent undergoing evaluation in humans for the treatment of moderate C. difficile infection. Reduced susceptibility of C. difficile to fidaxomicin or LFF571 in vitro can be mediated by single point mutations in genes encoding the targets, whereas the mechanism(s) mediating reduced susceptibility to vancomycin in vitro remains elusive. To further characterize mechanisms reducing susceptibility of C. difficile to vancomycin, fidaxomicin or LFF571 in vitro, selections via serial passage at low cell density were performed, followed by whole-genome sequencing. METHODS: C. difficile strain ATCC 43255 and three clinical isolates were subjected to 10 passages on medium containing a range of concentrations of fidaxomicin, LFF571 or vancomycin. Genomic DNA from isolates with reduced susceptibility was sequenced using Illumina Whole Genome Sequencing. RESULTS: Clones exhibiting decreased susceptibility to fidaxomicin harboured mutations in rpoB and CD22120 (marR homologue). Clones exhibiting decreased susceptibility to vancomycin harboured mutations in rpoC and also in CD2725, CD3659 and sdaB, which encode a putative N-acetylglucosamine transferase, exonuclease and l-serine deaminase, respectively. All mutations resulted in non-synonymous substitutions. No clones with reduced susceptibility to LFF571 were selected in this study. CONCLUSIONS: Reduced susceptibility to fidaxomicin and vancomycin was associated with mutations mediating target modifications (RNA polymerase and cell wall, respectively), as well as with mutations that may contribute to reduced susceptibility via other mechanisms. The MIC of LFF571 was unaffected for those mutants with reduced susceptibility to fidaxomicin or vancomycin.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/crescimento & desenvolvimento , Farmacorresistência Bacteriana , Mutação , Vancomicina/farmacologia , Parede Celular/genética , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , RNA Polimerases Dirigidas por DNA/genética , Fidaxomicina , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Seleção Genética , Análise de Sequência de DNA , Inoculações Seriadas , Tiazóis/farmacologia
16.
Elife ; 2: e01020, 2013 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-24336796

RESUMO

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.


Assuntos
Autoanticorpos/imunologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Receptores de Hialuronatos/imunologia , Mutação , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/fisiologia , Animais , Motivos EF Hand , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos
17.
Malar J ; 12: 316, 2013 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24025732

RESUMO

BACKGROUND: One of the main causes of mortality from severe malaria in Plasmodium falciparum infections is cerebral malaria (CM). An important host genetic component determines the susceptibility of an individual to develop CM or to clear the infection and become semi-immune. As such, the identification of genetic loci associated with susceptibility or resistance may serve to modulate disease severity. METHODOLOGY: The Plasmodium berghei mouse model for experimental cerebral malaria (ECM) reproduces several disease symptoms seen in human CM, and two different phenotypes, a susceptible (FVB/NJ) and a resistant mouse strain (DBA/2J), were examined. RESULTS: FVB/NJ mice died from infection within ten days, whereas DBA/2J mice showed a gender bias: males survived on average nineteen days and females either died early with signs of ECM or survived for up to three weeks. A comparison of brain pathology between FVB/NJ and DBA/2J showed no major differences with regard to brain haemorrhages or the number of parasites and CD3+ cells in the microvasculature. However, significant differences were found in the peripheral blood of infected mice: For example resistant DBA/2J mice had significantly higher numbers of circulating basophils than did FVB/NJ mice on day seven. Analysis of the F2 offspring from a cross of DBA/2J and FVB/NJ mice mapped the genetic locus of the underlying survival trait to chromosome 9 with a Lod score of 4.9. This locus overlaps with two previously identified resistance loci (char1 and pymr) from a blood stage malaria model. CONCLUSIONS: Survival best distinguishes malaria infections between FVB/NJ and DBA/2J mice. The importance of char1 and pymr on chromosome 9 in malaria resistance to P. berghei was confirmed. In addition there was an association of basophil numbers with survival.


Assuntos
Cromossomos Humanos Par 9 , Resistência à Doença , Loci Gênicos , Malária Cerebral/genética , Malária Cerebral/imunologia , Plasmodium berghei/imunologia , Animais , Basófilos/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Análise de Sobrevida
18.
Proc Natl Acad Sci U S A ; 110(27): E2510-7, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23776209

RESUMO

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-ß-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.


Assuntos
Antituberculosos/farmacologia , Benzotiazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Tiofenos/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Oxirredutases do Álcool , Sequência de Aminoácidos , Animais , Antituberculosos/administração & dosagem , Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzotiazóis/administração & dosagem , Benzotiazóis/química , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carboidratos Epimerases/antagonistas & inibidores , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Farmacorresistência Bacteriana , Feminino , Genes Bacterianos , Ensaios de Triagem em Larga Escala , Isoniazida/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/genética , Rifampina/administração & dosagem , Tiofenos/administração & dosagem , Tiofenos/química , Tuberculose Pulmonar/microbiologia
19.
Nat Chem Biol ; 9(8): 499-506, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23770708

RESUMO

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Ácidos Micólicos/metabolismo , Policetídeo Sintases/antagonistas & inibidores , Policetídeo Sintases/metabolismo , Tiofenos/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Proteínas de Bactérias/genética , Biocatálise , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/citologia , Mycobacterium tuberculosis/metabolismo , Policetídeo Sintases/genética , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/química
20.
PLoS One ; 7(9): e42657, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22970117

RESUMO

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.


Assuntos
Oligopeptídeos/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Burkholderia/efeitos dos fármacos , Linhagem Celular Tumoral , Sequência Conservada , Cristalografia por Raios X , Humanos , Mamíferos , Testes de Sensibilidade Microbiana , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fator G para Elongação de Peptídeos/antagonistas & inibidores , Fator G para Elongação de Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
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