RESUMO
A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid ß-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.
Assuntos
Proteínas de Ligação a DNA , Shigella flexneri , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Shigella flexneri/genética , Transcrição Gênica , Proteínas de Bactérias/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/farmacologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fator de Transcrição AraC/genética , DNA/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Infection with Shigella, the organism responsible for the diarrheal disease shigellosis, leads to approximately 200,000 deaths globally annually. Virulence of this pathogen is primarily controlled by the DNA-binding transcriptional activator VirF. This AraC family protein activates transcription of two major virulence genes, virB and icsA, which lead to the pathogen's ability to invade and spread within colonic epithelial cells. While several AraC proteins have been studied, few studies of VirF's binding to its DNA promoters have been reported, and VirF's three-dimensional structure remains unsolved. Here, we used structures of two E. coli VirF homologs, GadX and MarA-marRAB, to generate homology models of the VirF DNA-binding domain in free and DNA-bound conformations. We conducted alanine scanning mutagenesis on seven residues within MarA that make base-specific interactions with its promoter, marRAB, and the corresponding residues within VirF (identified by sequence and structural homologies). In vitro DNA-binding assays studying both wild-type and mutant MarA and VirF proteins identified residues important for binding to the marRAB and virB promoters, respectively. Comparison of the effects of these DNA-binding domain mutants validated our MarA-based homology model, allowing us to identify crucial interactions between VirF and the virB promoter. Proteins with mutations to helix 3 within both MarA(W42A, R46A) and MalE-VirF(R192A, K193A) exhibited significant reductions in DNA binding, while the effects of mutations in helix 6 varied. This suggests the shared importance of helix 3 in the binding to these promoters, while helix 6 is transcription factor specific. These results can inform further development of virulence-targeting inhibitors as an alternative to traditional antimicrobial drug design. IMPORTANCE Globally, infection with Shigella flexneri is a leading cause of bacterial dysentery, particularly affecting children under the age of 5 years. The virulence of this pathogen makes it highly infectious, allowing it to spread easily within areas lacking proper sanitation or access to clean drinking water. VirF is a DNA-binding transcription factor that activates S. flexneri virulence once the bacteria infect the human colon. Development of drugs that target VirF's DNA-binding activity can be an effective treatment to combat shigellosis as an alternative or addition to traditional antibiotics. Due to the lack of structural data, analysis of VirF's DNA-binding activity is critical to the development of potent VirF inhibitors.
Assuntos
Água Potável , Disenteria Bacilar , Alanina/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Criança , Pré-Escolar , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Água Potável/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Shigella flexneri/genética , Shigella flexneri/metabolismo , Transcrição Gênica , Proteínas Virais , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, â¼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.
Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Sítios de Ligação/genética , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Humanos , Mutação/genética , Mycobacterium tuberculosis/genética , Conformação Proteica , RNA Bacteriano , Tuberculose Pulmonar/microbiologiaRESUMO
Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPr mutants with amino acid substitutions in the ß subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi ß-barrel (DPBB) domain of the ß' subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo, several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3'-RNA hydrolysis with the wild-type and RMPrM. tuberculosis RNAPs. We found that the less prevalent RMPr mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMPr mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the ßS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the ß' subunit.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Aptidão Genética/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Elongação Traducional da Cadeia Peptídica/genética , Rifampina/farmacologia , Rifamicinas/farmacologia , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificação , Terminação Traducional da Cadeia Peptídica/genética , Domínios Proteicos/genética , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
The enzyme tRNA-guanine transglycosylase (TGT) is involved in the queuosine modification of tRNAs in eukarya and eubacteria and in the archaeosine modification of tRNAs in archaea. However, the different classes of TGTs utilize different heterocyclic substrates (and tRNA in the case of archaea). Based on the X-ray structural analyses, an earlier study [Stengl et al. (2005) Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism. Chembiochem, 6, 1926-1939] has made a compelling case for the divergent evolution of the eubacterial and archaeal TGTs. The X-ray structure of the eukaryal class of TGTs is not known. We performed sequence homology and phylogenetic analyses, and carried out enzyme kinetics studies with the wild-type and mutant TGTs from Escherichia coli and human using various heterocyclic substrates that we synthesized. Observations with the Cys145Val (E. coli) and the corresponding Val161Cys (human) TGTs are consistent with the idea that the Cys145 evolved in eubacterial TGTs to recognize preQ(1) but not queuine, whereas the eukaryal equivalent, Val161, evolved for increased recognition of queuine and a concomitantly decreased recognition of preQ(1). Both the phylogenetic and kinetic analyses support the conclusion that all TGTs have divergently evolved to specifically recognize their cognate heterocyclic substrates.
Assuntos
Escherichia coli/enzimologia , Evolução Molecular , Pentosiltransferases/química , Sequência de Aminoácidos , Guanina/análogos & derivados , Guanina/síntese química , Guanina/química , Guanina/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Filogenia , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirróis/síntese química , Pirróis/química , Pirróis/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.
Assuntos
Disenteria Bacilar , Shigella , Humanos , Shigella flexneri , Virulência/genética , Disenteria Bacilar/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , MacrófagosRESUMO
There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60-70%) that is transcribed into non-coding RNA (Mattick [10]), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [(3)H] preQ(1) (a queuine precursor in eubacteria) clearly demonstrate preQ(1) incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ(1) incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.
Assuntos
Escherichia coli/metabolismo , Guanina/análogos & derivados , Pirimidinonas/química , Pirróis/química , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , Resinas Acrílicas/química , Eletroforese em Gel de Ágar , Guanina/análise , Marcação por Isótopo , RNA de Transferência/química , Trítio/químicaRESUMO
The eukaryotic tRNA-guanine transglycosylase (TGT) has been reported to exist as a heterodimer, in contrast to the homodimeric eubacterial TGT. While ubiquitin-specific protease 14 (USP14) has been proposed to act as a regulatory subunit of the eukaryotic TGT, the mouse TGT has recently been shown to be a queuine tRNA-ribosyltransferase 1 (QTRT1, eubacterial TGT homolog).queuine tRNA-ribosyltransferase domain-containing 1 (QTRTD1) heterodimer. We find that human QTRTD1 (hQTRTD1) co-purifies with polyhistidine-tagged human QTRT1 (ht-hQTRT1) via Ni(2+) affinity chromatography. Cross-linking experiments, mass spectrometry, and size exclusion chromatography results are consistent with the two proteins existing as a heterodimer. We have not been able to observe co-purification and/or association between hQTRT1 and USP14 when co-expressed in Escherichia coli. More importantly, under our experimental conditions, the transglycosylase activity of hQTRT1 is only observed when hQTRT1 and hQTRTD1 have been co-expressed and co-purified. Kinetic characterization of the human TGT (hQTRT1.hQTRTD1) using human tRNA(Tyr) and guanine shows catalytic efficiency (k(cat)/K(M)) similar to that of the E. coli TGT. Furthermore, site-directed mutagenesis confirms that the hQTRT1 subunit is responsible for the transglycosylase activity. Taken together, these results indicate that the human TGT is composed of a catalytic subunit, hQTRT1, and hQTRTD1, not USP14. hQTRTD1 has been implicated as the salvage enzyme that generates free queuine from QMP. Work is ongoing in our laboratory to confirm this activity.
Assuntos
Pentosiltransferases/química , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas , Primers do DNA/genética , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Técnicas In Vitro , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Multimerização Proteica , Subunidades Proteicas , RNA de Transferência de Tirosina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine's toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted nondisruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in Escherichia coli, and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs but also valuable for establishing a nondisruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.
Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Peptídeos Penetradores de Células/química , Hidrolases de Éster Carboxílico/genética , Cromatografia de Afinidade , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Protaminas/química , Recombinação Genética/genéticaRESUMO
Challenges to discovery and preclinical development of long-acting release systems for protein therapeutics include protein instability, use of organic solvents during encapsulation, specialized equipment and personnel, and high costs of proteins. We sought to overcome these issues by combining remote-loading self-healing encapsulation with binding HisTag protein to transition metal ions. Porous, drug-free self-healing microspheres of copolymers of lactic and glycolic acids with high molecular weight dextran sulfate and immobilized divalent transition metal (M2+) ions were placed in the presence of proteins with or without HisTags to bind the protein in the pores of the polymer before healing the surface pores with modest temperature. Using human serum albumin, insulin-like growth factor 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), encapsulated efficiencies of immunoreactive protein relative to nonencapsulation protein solutions increased from ~41%, ~23%, and ~9%, respectively, without Zn2+ and HisTags to ~100%, ~83%, and ~75% with Zn2+ and HisTags. These three proteins were continuously released in immunoreactive form over seven to ten weeks to 73%-100% complete release, and GM-CSF showed bioactivity >95% relative to immunoreactive protein throughout the release interval. Increased encapsulation efficiencies were also found with other divalent transition metals ions (Co2+, Cu2+, Ni2+, and Zn2+), but not with Ca2+. Ethylenediaminetetraacetic acid was found to interfere with this process, reverting encapsulation efficiency back to Zn2+-free levels. These results indicate that M2+-immobilized self-healing microspheres can be prepared for simple and efficient encapsulation by simple mixing in aqueous solutions. These formulations provide slow and continuous release of immunoreactive proteins of diverse types by using a amount of protein (e.g., <10 µg), which may be highly useful in the discovery and early preclinical development phase of new protein active pharmaceutical ingredients, allowing for improved translation to further development of potent proteins for local delivery.
RESUMO
Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (ß-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.
Assuntos
Infecções por HIV , Rifamicinas , Tuberculose , Adulto , Infecções por HIV/tratamento farmacológico , Humanos , Receptor de Pregnano X , Rifampina/farmacologia , Rifampina/uso terapêutico , Rifamicinas/farmacologia , Rifamicinas/uso terapêutico , Tuberculose/tratamento farmacológicoRESUMO
Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (â¼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is â¼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of â¼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.
Assuntos
Mycobacterium tuberculosis , Rifamicinas , Tuberculose , Animais , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana , Camundongos , Rifampina/farmacologia , Rifamicinas/farmacologia , Tuberculose/tratamento farmacológicoRESUMO
tRNA-guanine transglycosylases (TGTs) are responsible for incorporating 7-deazaguanine-modified bases into certain tRNAs in eubacteria (preQ(1)), eukarya (queuine) and archaea (preQ(0)). In each kingdom, the specific modified base is different. We have found that the eubacterial and eukaryal TGTs have evolved to be quite specific for their cognate heterocyclic base and that Cys145 (Escherichia coli) is important in recognizing the amino methyl side chain of preQ(1) (Chen et al., Nuc. Acids Res. 39 (2011) 2834 [15]). A series of mutants of the E. coli TGT have been constructed to probe the role of three other active site amino acids in the differential recognition of heterocyclic substrates. These mutants have also been used to probe the differential inhibition of E. coli versus human TGTs by pteridines. The results indicate that mutation of these active site amino acids can "open up" the active site, allowing for the binding of competitive pteridine inhibitors. However, even the "best" of these mutants still does not recognize queuine at concentrations up to 50µM, suggesting that other changes are necessary to adapt the eubacterial TGT to incorporate queuine into RNA. The pteridine inhibition results are consistent with an earlier hypothesis that pteridines may regulate eukaryal TGT activity (Jacobson et al., Nuc. Acids Res. 9 (1981) 2351 [8]).
Assuntos
Biopterinas/química , Escherichia coli/enzimologia , Pentosiltransferases/antagonistas & inibidores , Pentosiltransferases/química , Pteridinas/química , Biopterinas/farmacologia , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Cisteína/química , Cisteína/genética , Guanina/análogos & derivados , Guanina/química , Humanos , Mutação , Pentosiltransferases/genética , Conformação Proteica , Pteridinas/farmacologia , Especificidade por SubstratoRESUMO
A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyrazole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a sulfonate ester, followed by displacement with selected nitrogen and sulfur nucleophiles. The analogues were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions, and for inhibition of wild-type Mycobacterium tuberculosis (MTB) RNAP and rifamycin-resistant MTB RNAP (S450L) via an in vitro rolling circle transcription assay. Additionally, the MIC(90) values were determined for these analogues against Escherichia coli strains. Although none of the analogues displayed superior enzymatic or microbiological activity to their parent scaffolds, the results are consistent with the Rif C-8 hydroxyl acting as a hydrogen bond acceptor with S450 and that Rif resistance in the S450L mutant is due to loss of this hydrogen bond. Representative analogues were also evaluated in the human pregnane X receptor (PXR) activation assay.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/química , Rifampina/farmacologia , Rifamicinas/química , Rifamicinas/farmacologia , Antituberculosos/síntese química , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Pirazóis/síntese química , Pirazóis/química , Pirazóis/farmacologia , Rifampina/síntese química , Rifamicinas/síntese química , Tuberculose/tratamento farmacológicoRESUMO
Bioinformatics searches of eubacterial genomes have yielded many riboswitch candidates where the identity of the ligand is not immediately obvious on examination of associated genes. One of these motifs is found exclusively in the family Streptococcaceae within the 5' untranslated regions (UTRs) of genes encoding the hypothetical membrane protein classified as COG4708 or DUF988. While the function of this protein class is unproven, a riboswitch binding the queuosine biosynthetic intermediate pre-queuosine(1) (preQ(1)) has been identified in the 5' UTR of homologous genes in many Firmicute species of bacteria outside of Streptococcaceae. Here we show that a representative of the COG4708 RNA motif from Streptococcus pneumoniae R6 also binds preQ(1). Furthermore, representatives of this RNA have structural and molecular recognition characteristics that are distinct from those of the previously described preQ(1) riboswitch class. PreQ(1) is the second metabolite for which two or more distinct classes of natural aptamers exist, indicating that natural aptamers utilizing different structures to bind the same metabolite may be more common than is currently known. Additionally, the association of preQ(1) binding RNAs with most genes encoding proteins classified as COG4708 strongly suggests that these proteins function as transporters for preQ(1) or another queuosine biosynthetic intermediate.
Assuntos
Aptâmeros de Nucleotídeos/classificação , Aptâmeros de Nucleotídeos/metabolismo , Pirimidinonas/metabolismo , Pirróis/metabolismo , RNA Bacteriano/metabolismo , Streptococcaceae/metabolismo , Regiões 5' não Traduzidas , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Sítios de Ligação/genética , Biologia Computacional , Genes Bacterianos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , Homologia de Sequência do Ácido Nucleico , Streptococcaceae/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Polarização de Fluorescência , Mycobacterium tuberculosis/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaios de Triagem em Larga EscalaRESUMO
The modified RNA base queuine [7-(4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanine] is present in tRNA because of a unique base-exchange process catalyzed by tRNA-guanine transglycosylase (TGT). Previous studies have suggested the intermediacy of a covalent TGT-RNA complex. To exist on the reaction pathway, this covalent complex must be both chemically and kinetically competent. Chemical competence has been demonstrated by the crystal structure studies of Xie et al. [(2003) Nat. Struct. Biol. 10, 781-788]; however, evidence of kinetic competence had not yet been established. The studies reported here unequivocally demonstrate that the TGT-RNA covalent complex is kinetically capable of occurring on the TGT reaction pathway. These studies further suggest that dissociation of product RNA from the enzyme is overall rate-limiting in the steady state. Interestingly, studies comparing RNA with a 2'-deoxyriboside at the site of modification suggest a role for the 2'-hydroxyl group in stabilizing the growing negative charge on the nucleophilic aspartate (264) as the glycosidic bond to the aspartate is broken during the breakdown of the covalent complex.
Assuntos
Escherichia coli/enzimologia , Pentosiltransferases/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desoxirribose/química , Desoxirribose/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Cinética , Modelos Químicos , Pentosiltransferases/química , RNA/química , RNA/metabolismo , Especificidade por SubstratoRESUMO
Shigella flexneri is an enteropathogen responsible for severe dysentery in humans. VirF is a key transcriptional regulator that activates the expression of the downstream virulence factors required for cellular invasion and cell-to-cell spread of this pathogen. There are several environmental factors that induce the translation of VirF including temperature, pH, osmolarity and post-transcriptional RNA modification. Durand and colleagues (vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase of Escherichia coli K-12. J. Bacteriol., 176, 4627-4634) have demonstrated a correlation between VirF and tRNA-guanine transglycosylase (TGT), which catalyzes the exchange of the hypermodified base queuine for the guanine in the wobble position of certain tRNAs. They characterized tgt- mutant S. flexneri strains in which the translation of VirF is markedly reduced and the bacteria are unable to invade host cells. Although the function of TGT is to modify tRNA, we report that the virF mRNA is recognized by the Escherichia coli TGT (99% identity to the S. flexneri TGT) in vitro. Further, we show that this recognition results in the site-specific modification of a single base in the virF mRNA. In the context of previous reports that small molecule binding motifs ('riboswitches') in mRNAs modulate mRNA conformation and translation, our observations suggest that TGT may modulate the translation of VirF by base modification of the VirF encoding mRNA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , Pentosiltransferases/metabolismo , RNA Mensageiro/metabolismo , Shigella flexneri/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Anticódon/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Plasmídeos/genética , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirróis/química , Pirróis/metabolismo , RNA Mensageiro/química , RNA de Transferência de Tirosina/metabolismo , Shigella flexneri/enzimologia , Shigella flexneri/patogenicidade , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Within the large and diverse group of RNA-modifying enzymes, a number of enzymes seem to form stable covalent linkages to their respective RNA substrates. A complete understanding of the chemical and kinetic mechanisms of these enzymes, some of which have identified pathological roles, is lacking. As part of our ongoing work studying the posttranscriptional modification of tRNA with queuine, we wish to understand fully the chemical and kinetic mechanisms involved in this key transglycosylation reaction. In our previous investigations, we have used a gel mobility-shift assay to characterize an apparent covalent enzyme-RNA intermediate believed to be operative in the catalytic pathway. However, the simple observation of a covalent complex is not sufficient to prove intermediacy. To be a true intermediate, the complex must be both chemically and kinetically competent. As a case study for the proof of intermediacy, we report the use of this gel-shift assay under mildly denaturing conditions to probe the kinetic competency of the covalent association between RNA and the tRNA modifying enzyme tRNA-guanine transglycosylase (TGT).
Assuntos
Pentosiltransferases/metabolismo , RNA/metabolismo , Animais , Humanos , Cinética , Pentosiltransferases/química , RNA/químicaRESUMO
Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (ßS531L) containing a mutation (ß'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.