RESUMEN
Cell growth in mycobacteria involves cell wall expansion that is restricted to the cell poles. The DivIVA homolog Wag31 is required for this process, but the molecular mechanism and protein partners of Wag31 have not been described. In this study of Mycobacterium smegmatis, we identify a connection between wag31 and trehalose monomycolate (TMM) transporter mmpl3 in a suppressor screen, and show that Wag31 and polar regulator PlrA are required for MmpL3's polar localization. In addition, the localization of PlrA and MmpL3 are responsive to nutrient and energy deprivation and inhibition of peptidoglycan metabolism. We show that inhibition of MmpL3 causes delocalized cell wall metabolism, but does not delocalize MmpL3 itself. We found that cells with an MmpL3 C-terminal truncation, which is defective for localization, have only minor defects in polar growth, but are impaired in their ability to downregulate cell wall metabolism under stress. Our work suggests that, in addition to its established function in TMM transport, MmpL3 has a second function in regulating global cell wall metabolism in response to stress. Our data are consistent with a model in which the presence of TMMs in the periplasm stimulates polar elongation, and in which the connection between Wag31, PlrA and the C-terminus of MmpL3 is involved in detecting and responding to stress in order to coordinate synthesis of the different layers of the mycobacterial cell wall in changing conditions.
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Urine is assayed alongside blood in medicine, yet current clinical diagnostic tests utilize only a small fraction of its total biomolecular repertoire, potentially foregoing high-resolution insights into human health and disease. In this work, we characterized the joint landscapes of transcriptomic and metabolomic signals in human urine. We also compared the urine transcriptome to plasma cell-free RNA, identifying a distinct cell type repertoire and enrichment for metabolic signal. Untargeted metabolomic measurements identified a complementary set of pathways to the transcriptomic analysis. Our findings suggest that urine is a promising biofluid yielding prognostic and detailed insights for hard-to-biopsy tissues with low representation in the blood, offering promise for a new generation of liquid biopsies.
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Increasingly difficult-to-treat infections by antibiotic-resistant bacteria have become a major public health challenge. Rapid detection of common resistance mechanisms before empiric antibiotic usage is essential for optimizing therapeutic outcomes and containing further spread of resistance to antibiotics among other bacteria. Herein, we present a bioluminogenic probe, D-Bluco, for rapid detection of ß-lactamase activity in viable pathogenic bacteria. D-Bluco is a pro-luciferin caged by a ß-lactamase-responsive cephalosporin structure and further conjugated with a dabcyl quencher. The caging and quenching significantly decreased the initial background emission and increased the signal-to-background ratio by more than 1200-fold. D-Bluco was shown to detect a broad range of ß-lactamases at the femtomolar level. An ultrasensitive RAPID bioluminescence assay using D-Bluco can detect 102 to 103 colony forming unit per milliliter (cfu/mL) of ß-lactamase-producing Enterobacterales in urine samples within 30 min. The high sensitivity and rapid detection make the assay attractive for the use of point-of-care diagnostics for lactam-resistant pathogens.
Asunto(s)
Antibacterianos , Bacterias , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , beta-Lactamasas , CefalosporinasRESUMEN
PLP-dependent enzymes represent an important class of highly "druggable" enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. Hence, we developed a probe that contains an N-hydroxyalanine warhead that acts as a covalent inhibitor of PLP-dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP-dependent enzymes inâ vitro as well as look at whole-cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP-dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.
Asunto(s)
Fosfato de Piridoxal , Fosfato de Piridoxal/química , Modelos Moleculares , Espectrometría de MasasRESUMEN
Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10 % of new TB cases and present an urgent need for novel therapeutics. ß-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of ß-lactamases that destroy the electrophilic ß-lactam warhead. We have developed novel ß-lactam conjugates, which exploit this inherent ß-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the ß-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.
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Mycobacterium tuberculosis , Tuberculosis , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Pirazinamida/análogos & derivados , Pirazinamida/farmacología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , beta-Lactamas/farmacologíaRESUMEN
In this viewpoint, the concepts that chemistry transcends the laboratory into the clinic and beyond is explored from the perspective of a single individual who began strictly within synthetic chemistry. They learned through their training that in reality, chemists are capable of anything, requiring mentorship, open discussion, and some frontend work to learn something new.
Asunto(s)
Química Farmacéutica/educación , Conducta Cooperativa , Descubrimiento de Drogas , Aprendizaje , Mentores , HumanosRESUMEN
Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype; treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.
Asunto(s)
Membrana Celular/microbiología , Células Epiteliales/microbiología , Lípidos , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/patogenicidad , Factores de Virulencia/metabolismo , Células A549 , Animales , Animales Modificados Genéticamente , Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lípidos/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/microbiología , Estructura Molecular , Infecciones por Mycobacterium no Tuberculosas/metabolismo , Infecciones por Mycobacterium no Tuberculosas/prevención & control , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/genética , Mycobacterium marinum/metabolismo , Relación Estructura-Actividad , Células THP-1 , Virulencia , Factores de Virulencia/química , Pez CebraRESUMEN
A simple, one-pot, and high-yielding synthesis of 1,3-diphenyldisiloxane is presented. The preparation of similar symmetrical disiloxane materials is also accomplished with this same protocol. This mechano-chemical procedure is efficient and highly scalable, furnishing a convenient route to hydrido-disiloxanes from widely accessible commercially available silanes.
RESUMEN
Reduction of phosphine oxides to the corresponding phosphines represents the most straightforward method to prepare these valuable reagents. However, existing methods to reduce phosphine oxides suffer from inadequate chemoselectivity due to the strength of the P=O bond and/or poor atom economy. Herein, we report the discovery of the most powerful chemoselective reductant for this transformation to date, 1,3-diphenyl-disiloxane (DPDS). Additive-free DPDS selectively reduces both secondary and tertiary phosphine oxides with retention of configuration even in the presence of aldehyde, nitro, ester, α,ß-unsaturated carbonyls, azocarboxylates, and cyano functional groups. Arrhenius analysis indicates that the activation barrier for reduction by DPDS is significantly lower than any previously calculated silane reduction system. Inclusion of a catalytic Brønsted acid further reduced the activation barrier and led to the first silane-mediated reduction of acyclic phosphine oxides at room temperature.
RESUMEN
The ability to revitalize and re-purpose existing drugs offers a powerful approach for novel treatment options against Mycobacterium tuberculosis and other infectious agents. Antifolates are an underutilized drug class in tuberculosis (TB) therapy, capable of disrupting the biosynthesis of tetrahydrofolate, an essential cellular cofactor. Based on the observation that exogenously supplied p-aminobenzoic acid (PABA) can antagonize the action of antifolates that interact with dihydropteroate synthase (DHPS), such as sulfonamides and p-aminosalicylic acid (PAS), we hypothesized that bacterial PABA biosynthesis contributes to intrinsic antifolate resistance. Herein, we demonstrate that disruption of PABA biosynthesis potentiates the anti-tubercular action of DHPS inhibitors and PAS by up to 1000 fold. Disruption of PABA biosynthesis is also demonstrated to lead to loss of viability over time. Further, we demonstrate that this strategy restores the wild type level of PAS susceptibility in a previously characterized PAS resistant strain of M. tuberculosis. Finally, we demonstrate selective inhibition of PABA biosynthesis in M. tuberculosis using the small molecule MAC173979. This study reveals that the M. tuberculosis PABA biosynthetic pathway is responsible for intrinsic resistance to various antifolates and this pathway is a chemically vulnerable target whose disruption could potentiate the tuberculocidal activity of an underutilized class of antimicrobial agents.
Asunto(s)
Vías Biosintéticas/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Mycobacterium tuberculosis/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Ácido 4-Aminobenzoico/metabolismo , Proteínas Bacterianas/genética , Vías Biosintéticas/genética , Clonación Molecular , Reposicionamiento de Medicamentos , Sinergismo Farmacológico , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/síntesis químicaRESUMEN
The Mitsunobu reaction is renowned for its mild reaction conditions and broad substrate tolerance, but has limited utility in process chemistry and industrial applications due to poor atom economy and the generation of stoichiometric phosphine oxide and hydrazine by-products that complicate purification. A catalytic Mitsunobu reaction using innocuous reagents to recycle these by-products would overcome both of these shortcomings. Herein we report a protocol that is catalytic in phosphine (1-phenylphospholane) employing phenylsilane to recycle the catalyst. Integration of this phosphine catalytic cycle with Taniguchi's azocarboxylate catalytic system provided the first fully catalytic Mitsunobu reaction.
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Alcoholes/química , Ésteres/síntesis química , Fosfinas/química , Catálisis , Ésteres/química , Estructura MolecularRESUMEN
The synthesis of 2-alkylated pyridines by the nickel-catalyzed cross-coupling of two different electrophiles, 2-chloropyridines with alkyl bromides, is described. Compared to our previously published conditions for aryl halides, this method uses the different, more rigid bathophenanthroline ligand and is conducted at high concentration in DMF solvent. The method displays promising functional group compatibility and the conditions are orthogonal to the Stille coupling.
RESUMEN
A simple, ligand-free synthesis of the important bipyridyl ligand 4,4'-di-tert-butyl-2,2'-bipyridine is presented. 5,5'-bis(trifluoromethyl)-2,2'-bipyridine is also synthesized by the same protocol. The syntheses efficiently couple the parent 2-chlorpyridies by a nickel-catalyzed dimerization with manganese powder as the terminal reductant.