En High-throughput Kompatibel Assay at evaluere Drug Effekt mod makrofag passeres<em> Mycobacterium tuberculosis</em
Summary
New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.
Abstract
The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.
Introduction
Tuberkulose (TB) er fortsat en alvorlig global sundhedstrussel trods af tilgængeligheden af anti-TB kemoterapi i over 40 år 1. Dette skyldes til dels, at kravet om lange behandlingsperioder på over seks måneder ved hjælp af flere lægemiddelkombinationer, hvilket fører til patient manglende overholdelse 2. Fremkomsten af multiresistent tuberkulose i de seneste år har yderligere forværret problemer på et område, hvor vellykket udvikling af klinisk godkendte lægemidler er nærmest ikke-eksisterende 3. Trods omfattende anti-TB lægemiddeludvikling, har kun et enkelt lægemiddel været FDA godkendt til klinisk brug i de sidste 40 år 4. Således er der et akut behov for nye generationer af anti-TB-medicin for at løse dette problem.
Et centralt problem i TB lægemiddelforskning er manglen på vellykket overførsel fra forbindelser med in vitro-aktivitet til effekten i kliniske omgivelser= "xref"> 5, 6, 7. I første omgang blev mål tilgange der anvendes til at screene for anti Mtb lægemidler 5, som har undladt at oversætte til hele bakterieceller. Selv når der anvendes Mtb-celler, er det ofte udført under anvendelse bouillon dyrket kulturer, som ikke nøjagtigt forudsige lægemiddel effektivitet in vivo 8, 9. Disse problemer er blevet anerkendt og lægemiddelscreeningsassays mod makrofager indeholder Mtb eller latent Mtb succes er blevet etableret 8, 10, 11, 12. Men selv disse mere avancerede analyser ikke give tilstrækkelig hensyn til de Indtrængningsbarriererne at narkotika møder i de ikke-vaskulariserede lungelæsioner, og i den nekrotiske foci på stedet for infektion. Ja, Selv for den første linje TB stof rifampicin, har suboptimal dosering sat spørgsmålstegn på grund af utilstrækkelig in vivo væv og cerebral spinalvæske (CSF) penetration 13, 14, 15 samt nedsat effektivitet mod intracellulær Mtb 8, 9. Som sådan, nye modeller og analyser, der ville tage hensyn til disse parametre i den tidlige bly udviklingsprocessen vil utvivlsomt forbedre TB drug discovery indsats.
For at imødekomme dette behov, vi for nylig etableret en billig, hurtig og BSL-2 kompatibel alternativ infektion model for Mtb narkotika effektivitetsforsoegene 16. Denne infektion model produceret tæt pakket Mtb inden for store makrofag aggregerede strukturer, som er gengivet fysiologisk relevante cellulære Indtrængningsbarriererne og genererede makrofag-passage <em> Mtb med en ændret fysiologiske status ligner latent Mtb. Mtb afledt af denne infektion model blev kombineret med resazurin mikrotiteranalyse (REMA) at evaluere lægemiddeleffektivitet, som producerede resultater konsistente med andre intracellulære infektionsmodeller og korrelerede godt med den rapporterede evne af fælles TB-medicin til at opnå høj CSF koncentrationer i forhold til serumkoncentrationer 16.
Her beskriver vi i detaljer generation af Mtb / makrofag samlede strukturer til at producere makrofag-passeret Mtb egnet til resistensbestemmelse hjælp REMA. Især viser vi, hvordan denne infektion system kan tilpasses til en 96-brønds format for kompatibilitet med throughput screening af kandidat anti-TB-medicin.
Protocol
Representative Results
Discussion
Her har vi beskrevet detaljeret et alternativ Mtb infektion model velegnet til lægemiddeleffektivitet testning. Denne model tager hensyn til to vigtige faktorer, der bør gives mere hensyn i den tidlige TB lægemiddeludvikling proces: tilstedeværelsen af fysiologisk relevante barrierer for indtrængen narkotika og metaboliske ændringer af Mtb under infektion. Mens vi tidligere har vist fordelene ved vores infektion model og foreslået muligheden for opskalering af infektionen for gennemløb kompatib…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.
Materials
| 7H9 | BD Difco | 271310 | Follow manufacturer's recommendations |
| Middlebrook OADC | BD Biosciences | 212351 | |
| Tyloxapol | Sigma | T8761 | Prepare 20% stock solution in H2O; filter sterilize |
| D-Pantothenic acid hemicalcium salt | Sigma | P5710 | Prepare 24 mg/ml stock solution in H2O; filter sterilize |
| L-leucine | MP Biomedicals | 194694 | Prepare 50 mg/ml stock solution in H2O; filter sterilize |
| Hygromycin B | EMD Millipore | 400051 | Prepare 200 mg/ml stock solution in H2O |
| Nalgene Square PETG media bottle | Thermo Fisher | 2019-0030 | |
| RPMI 1640 media | Hyclone | SH30027.01 | |
| Fetal Bovine Serum | Atlanta Biologicals | S12450H | |
| L-glutamine | Corning | MT25005CI | |
| HEPES | Hyclone | SH30237.01 | |
| Cytation 3 plate reader | Biotek | Interchangable with any fluorescent plate reader and microscope | |
| Gen5 Software | Biotek | Recording and analysis of rezasurin coversion | |
| Rifampicin | Fisher Scientific | BP2679250 | Prepare 10 mg/ml stock solution in H2O |
| Moxifloxacin Hydrochloride | Acros Organics | 457960010 | Prepare 10 mg/ml stock solution in H2O |
| Resazurin Sodium Salt | Sigma | R7017 | Prepare 800 μg/mL stock solution in H2O; filter sterilize |
| Tween-80 | Fisher Scientific | T164500 | Prepare 20% stock solution in H2O; filter sterilize |
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