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BACKGROUND: Understanding the etiology of recurrent tuberculosis (rTB) is important for effective TB control. Prior to the advent of whole genome sequencing (WGS), attributing rTB to relapse or reinfection using genetic information was complicated by the limited resolution of conventional genotyping methods. METHODS: We applied a systematic method of evaluating whole genome single nucleotide polymorphism (wgSNP) distances and results of phylogenetic analyses to characterize the etiology of rTB in American Indian and Alaska Native (AIAN) persons in Alaska during 2008-2020. We contextualized our findings through descriptive analyses of surveillance data and results of a literature search for investigations that characterized rTB etiology using WGS. RESULTS: The percentage of TB cases in AIAN persons in Alaska classified as recurrent episodes (11.8%) was three times the national percentage (3.9%). Of 38 recurrent episodes included in genetic analyses, we attributed 25 (65.8%) to reinfection based on wgSNP distances and phylogenetic analyses; this proportion was the highest among 16 published point estimates identified through the literature search. By comparison, we attributed 11 of 38 (28.9%) and 6 of 38 (15.8%) recurrent episodes to reinfection based on wgSNP distances alone and on conventional genotyping methods, respectively. CONCLUSIONS: WGS and attribution criteria involving genetic distances and patterns of relatedness can provide an effective means of elucidating rTB etiology. Our findings indicate that rTB occurs at high proportions among AIAN persons in Alaska and is frequently attributable to reinfection, reinforcing the importance of active surveillance and control measures to limit the spread of TB disease in Alaskan AIAN communities.
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Background: Pyrazinamide is one of four first-line antibiotics used to treat tuberculosis; however, antibiotic susceptibility testing for pyrazinamide is challenging. Resistance to pyrazinamide is primarily driven by genetic variation in pncA, encoding an enzyme that converts pyrazinamide into its active form. Methods: We curated a dataset of 664 non-redundant, missense amino acid mutations in PncA with associated high-confidence phenotypes from published studies and then trained three different machine-learning models to predict pyrazinamide resistance. All models had access to a range of protein structural-, chemical- and sequence-based features. Results: The best model, a gradient-boosted decision tree, achieved a sensitivity of 80.2% and a specificity of 76.9% on the hold-out test dataset. The clinical performance of the models was then estimated by predicting the binary pyrazinamide resistance phenotype of 4027 samples harbouring 367 unique missense mutations in pncA derived from 24â231 clinical isolates. Conclusions: This work demonstrates how machine learning can enhance the sensitivity/specificity of pyrazinamide resistance prediction in genetics-based clinical microbiology workflows, highlights novel mutations for future biochemical investigation, and is a proof of concept for using this approach in other drugs.
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OBJECTIVE: This study describes characteristics of large tuberculosis (TB) outbreaks in the United States detected using novel molecular surveillance methods during 2014-2016 and followed for 2 years through 2018. METHODS: We developed 4 genotype-based detection algorithms to identify large TB outbreaks of ≥10 cases related by recent transmission during a 3-year period. We used whole-genome sequencing and epidemiologic data to assess evidence of recent transmission among cases. RESULTS: There were 24 large outbreaks involving 518 cases; patients were primarily U.S.-born (85.1%) racial/ethnic minorities (84.1%). Compared with all other TB patients, patients associated with large outbreaks were more likely to report substance use, homelessness, and having been diagnosed while incarcerated. Most large outbreaks primarily occurred within residences among families and nonfamilial social contacts. A source case with a prolonged infectious period and difficulties in eliciting contacts were commonly reported contributors to transmission. CONCLUSION: Large outbreak surveillance can inform targeted interventions to decrease outbreak-associated TB morbidity.
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Personas con Mala Vivienda , Mycobacterium tuberculosis , Tuberculosis , Brotes de Enfermedades , Genotipo , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Tuberculosis/epidemiología , Estados Unidos/epidemiologíaRESUMEN
A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 â¼ 1 µM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.
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Acetiltransferasas , Mycobacterium tuberculosis , Acetiltransferasas/química , Aminoglicósidos/farmacología , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Antituberculosos/química , Proteínas Bacterianas , Bencilaminas/farmacología , Kanamicina/química , Kanamicina/farmacología , Mamíferos/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.
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Fluoroquinolones (FQ) are crucial components of multidrug-resistant tuberculosis (MDR TB) treatment. Differing levels of resistance are associated with specific mutations within the quinolone-resistance-determining region (QRDR) of gyrA We sequenced the QRDR from serial isolates of MDR TB patients in the Preserving Effective TB Treatment Study (PETTS) with baseline FQ resistance (FQR) or acquired FQ resistance (FQACQR) using an Ion Torrent Personal Genome Machine (PGM) to a depth of 10,000× and reported single nucleotide polymorphisms in ≥1% of reads. FQR isolates harbored 15 distinct alleles with 1.3 (maximum = 6) on average per isolate. Eighteen alleles were identified in FQACQR isolates with an average of 1.6 (maximum = 9) per isolate. Isolates from 78% of FQACQR individuals had mutant alleles identified within 6 months of treatment initiation. Asp94Gly was the predominant allele in the initial FQ-resistant isolates followed by Ala90Val. Seventy-seven percent (36/47) of FQACQR group patients had isolates with FQ resistance alleles prior to changes to the FQ component of their treatment. Unlike the individuals treated initially with other FQs, none of the 21 individuals treated initially with levofloxacin developed genotypic or phenotypic FQ resistance, although country of residence was likely a contributing factor since 69% of these individuals were from a single country. Initial detection of phenotypic resistance and genotypic resistance occurred simultaneously for most; however, phenotypic resistance occurred earlier in isolates harboring mixtures of alleles of very low abundance (<1% of reads), whereas genotypic resistance often occurred earlier for alleles associated with low-level resistance. Understanding factors influencing acquisition and evolution of FQ resistance could reveal strategies for improved treatment success.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Antituberculosos/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Fluoroquinolonas/farmacología , Humanos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.
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Acetiltransferasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/enzimología , Diseño de Fármacos , Resistencia a la Kanamicina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
BACKGROUND: Mutations in the genes inhA, katG, and rpoB confer resistance to anti-tuberculosis (TB) drugs isoniazid and rifampin. We questioned whether specific mutations in these genes were associated with different clinical and microbiological characteristics. METHODS: In a multicountry prospective cohort study of multidrug-resistant TB, we identified inhA, katG, and rpoB mutations in sputum isolates using the Hain MTBDRplus line probe assay. For specific mutations, we performed bivariate analysis to determine relative risk of baseline or acquired resistance to other TB drugs. We compared time to sputum culture conversion (TSCC) using Kaplan-Meier curves and stratified Cox regression. RESULTS: In total, 447 participants enrolled from January 2005 to December 2008 from 7 countries were included. Relative to rpoB S531L, isolates with rpoB D516V had less cross-resistance to rifabutin, increased baseline resistance to other drugs, and increased acquired fluoroquinolone resistance. Relative to mutation of katG only, mutation of inhA promoter and katG was associated with baseline extensively drug resistant (XDR) TB, increased acquired fluoroquinolone resistance, and slower TSCC (125.5 vs 89.0 days). CONCLUSIONS: Specific mutations in inhA and katG are associated with differences in resistance to other drugs and TSCC. Molecular testing may make it possible to tailor treatment and assess additional drug resistance risk according to specific mutation profile.
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Antituberculosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Genes Bacterianos/genética , Mycobacterium tuberculosis/genética , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Antituberculosos/uso terapéutico , Proteínas Bacterianas/genética , Catalasa/genética , Análisis Mutacional de ADN , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/genética , Humanos , Isoniazida/farmacología , Isoniazida/uso terapéutico , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Oxidorreductasas/genética , Regiones Promotoras Genéticas/genética , Estudios Prospectivos , Rifampin/farmacología , Rifampin/uso terapéutico , Esputo/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Rapid advances in DNA sequencing technology ("next-generation sequencing") have inspired optimism about the potential of human genomics for "precision medicine." Meanwhile, pathogen genomics is already delivering "precision public health" through more effective investigations of outbreaks of foodborne illnesses, better-targeted tuberculosis control, and more timely and granular influenza surveillance to inform the selection of vaccine strains. In this article, we describe how public health agencies have been adopting pathogen genomics to improve their effectiveness in almost all domains of infectious disease. This momentum is likely to continue, given the ongoing development in sequencing and sequencing-related technologies.
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Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/epidemiología , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Gripe Humana/epidemiología , Salud Pública , Tuberculosis/epidemiología , Animales , Bacterias/genética , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/parasitología , Humanos , Gripe Humana/diagnóstico , Gripe Humana/microbiología , Metagenómica , Parásitos/genética , Tuberculosis/diagnóstico , Virus/genéticaRESUMEN
Tuberculosis (TB) is the leading killer among all infectious diseases worldwide despite extensive use of the Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine. A safer and more effective vaccine than BCG is urgently required. More than a dozen TB vaccine candidates are under active evaluation in clinical trials aimed to prevent infection, disease, and recurrence. After decades of extensive research, renewed promise of an effective vaccine against this ancient airborne disease has recently emerged. In two innovative phase 2b vaccine clinical trials, one for the prevention of Mycobacterium tuberculosis infection in healthy adolescents and another for the prevention of TB disease in M. tuberculosis-infected adults, efficacy signals were observed. These breakthroughs, based on the greatly expanded knowledge of the M. tuberculosis infection spectrum, immunology of TB, and vaccine platforms, have reinvigorated the TB vaccine field. Here, we review our current understanding of natural immunity to TB, limitations in BCG immunity that are guiding vaccinologists to design novel TB vaccine candidates and concepts, and the desired attributes of a modern TB vaccine. We provide an overview of the progress of TB vaccine candidates in clinical evaluation, perspectives on the challenges faced by current vaccine concepts, and potential avenues to build on recent successes and accelerate the TB vaccine research-and-development trajectory.
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Mycobacterium tuberculosis/inmunología , Investigación , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Mycobacterium bovis/inmunología , Evaluación de Resultado en la Atención de Salud , Vacunas contra la Tuberculosis/administración & dosificaciónRESUMEN
Resistance to the first-line antituberculosis (TB) drug isoniazid (INH) is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole-genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a MIC of ≥0.25 µg/ml and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.
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Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación/genética , Tuberculosis/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/genéticaRESUMEN
A common cause of resistance to kanamycin (KAN) in tuberculosis is overexpression of the enhanced intracellular survival (Eis) protein. Eis is an acetyltransferase that multiacetylates KAN and other aminoglycosides, rendering them unable to bind the bacterial ribosome. By high-throughput screening, a series of substituted 1,2,4-triazino[5,6 b]indole-3-thioether molecules were identified as effective Eis inhibitors. Herein, we purchased 17 and synthesized 22 new compounds, evaluated their potency, and characterized their steady-state kinetics. Four inhibitors were found not only to inhibit Eis in vitro, but also to act as adjuvants of KAN and partially restore KAN sensitivity in a Mycobacterium tuberculosis KAN-resistant strain in which Eis is upregulated. A crystal structure of Eis in complex with a potent inhibitor and CoA shows that the inhibitors bind in the aminoglycoside binding site snugly inserted into a hydrophobic cavity. These inhibitors will undergo preclinical development as novel KAN adjuvant therapies to treat KAN-resistant tuberculosis.
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Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Indoles/química , Indoles/farmacología , Resistencia a la Kanamicina/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Células A549 , Acetiltransferasas/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Indoles/síntesis química , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Unión Proteica , Estructura Secundaria de Proteína , Análisis de Regresión , Sulfuros/química , Triazinas/químicaRESUMEN
We previously reported use of genotype surveillance data to predict outbreaks among incident tuberculosis clusters. We propose a method to detect possible outbreaks among endemic tuberculosis clusters. We detected 15 possible outbreaks, of which 10 had epidemiologic data or whole-genome sequencing results. Eight outbreaks were corroborated.
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Brotes de Enfermedades , Modelos Estadísticos , Mycobacterium tuberculosis , Tuberculosis/epidemiología , Análisis por Conglomerados , Genoma Bacteriano , Genómica/métodos , Genotipo , Humanos , Incidencia , Epidemiología Molecular , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple , Prevalencia , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Estados UnidosRESUMEN
Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.
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Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Mycobacterium tuberculosis/metabolismo , Acetilación , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Clonación Molecular , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/fisiología , Histona Desacetilasas/genética , Histonas/genética , Cinética , Lisina/química , Modelos Moleculares , Mycobacterium tuberculosis/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espectrometría de Masas en TándemRESUMEN
Pyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosis and is included in treatment regimens for drug-resistant M. tuberculosis whenever possible. Therefore, it is imperative that susceptibility to PZA be assessed reliably prior to the initiation of therapy. Currently available growth-based PZA susceptibility tests are time-consuming, and results can be inconsistent. Molecular tests have been developed for most first-line antituberculosis drugs; however, a commercial molecular test is not yet available for rapid detection of PZA resistance. Recently, a line probe assay, the Nipro Genoscholar PZA-TB II assay, was developed for the detection of mutations within the pncA gene, including the promoter region, that are likely to lead to PZA resistance. The sensitivity and specificity of this assay were evaluated by two independent laboratories, using a combined total of 249 strains with mutations in pncA or its promoter and 21 strains with wild-type pncA Overall, the assay showed good sensitivity (93.2% [95% confidence interval, 89.3 to 95.8%]) and moderate specificity (91.2% [95% confidence interval, 77.0 to 97.0%]) for the identification of M. tuberculosis strains predicted to be resistant to PZA on the basis of the presence of mutations (excluding known PZA-susceptible mutations) in the pncA coding region or promoter. The assay shows promise for the molecular prediction of PZA resistance.
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Proteínas Bacterianas/genética , Bioensayo/métodos , Mutación/genética , Mycobacterium tuberculosis/genética , Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Pirazinamida/farmacología , Tuberculosis Resistente a Múltiples MedicamentosRESUMEN
The primary platform used for pyrazinamide (PZA) susceptibility testing of Mycobacterium tuberculosis is the MGIT culture system (Becton Dickinson). Since false-resistant results have been associated with the use of this system, we conducted a multicenter evaluation to determine the effect of using a reduced cell density inoculum on the rate of false resistance. Two reduced inoculum densities were compared with that prescribed by the manufacturer (designated as "BD" method). The reduced inoculum methods (designated as "A" and "C") were identical to the manufacturer's protocol in all aspects with the exception of the cell density of the inoculum. Twenty genetically and phenotypically characterized M. tuberculosis isolates were tested in duplicate by ten independent laboratories using the three inoculum methods. False-resistant results declined from 21.1% using the standard "BD" method to 5.7% using the intermediate ("A") inoculum and further declined to 2.8% using the most dilute ("C") inoculum method. The percentages of the resistant results that were false-resistant declined from 55.2% for the "BD" test to 28.8% and 16.0% for the "A" and "C" tests, respectively. These results represent compelling evidence that the occurrence of false-resistant MGIT PZA susceptibility test results can be mitigated through the use of reduced inoculum densities.
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Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure-activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.
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Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Resistencia a la Kanamicina/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/química , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Sitios de Unión , Inhibidores Enzimáticos/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Unión Proteica , Pirazinas/química , Pirazinas/farmacología , Relación Estructura-ActividadRESUMEN
Drug-resistant tuberculosis (TB) is a global threat and innovative approaches such as using adjuvants of anti-TB therapeutics are required to combat it. High-throughput screening yielded two lead scaffolds of inhibitors of Mycobacterium tuberculosis (Mtb) acetyltransferase Eis, whose upregulation causes resistance to the anti-TB drug kanamycin (KAN). Chemical optimization on these scaffolds resulted in potent Eis inhibitors. One compound restored the activity of KAN in a KAN-resistant Mtb strain. Model structures of Eis-inhibitor complexes explain the structure-activity relationship.
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A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.
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Acetiltransferasas/antagonistas & inhibidores , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Sulfonamidas/farmacología , Acetiltransferasas/metabolismo , Antibacterianos/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Heterologous prime-boosting has emerged as a powerful vaccination approach against tuberculosis. However, optimal timing to boost BCG-immunity using subunit vaccines remains unclear in clinical trials. Here, we followed the adhesin Apa-specific T-cell responses in BCG-primed mice and investigated its BCG-booster potential. The Apa-specific T-cell response peaked 32-52 weeks after parenteral or mucosal BCG-priming but waned significantly by 78 weeks. A subunit-Apa-boost during the contraction-phase of BCG-response had a greater effect on the magnitude and functional quality of specific cellular and humoral responses compared to a boost at the peak of BCG-response. The cellular response increased following mucosal BCG-prime-Apa-subunit-boost strategy compared to Apa-subunit-prime-BCG-boost approach. However, parenteral BCG-prime-Apa-subunit-boost by a homologous route was the most effective strategy in-terms of enhancing specific T-cell responses during waning in the lung and spleen. Two Apa-boosters markedly improved waning BCG-immunity and significantly reduced Mycobacterium tuberculosis burdens post-challenge. Our results highlight the challenges of optimization of prime-boost regimens in mice where BCG drives persistent immune-activation and suggest that boosting with a heterologous vaccine may be ideal once the specific persisting effector responses are contracted. Our results have important implications for design of prime-boost regimens against tuberculosis in humans.