Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

Brown, Alistair S.; Owen, Jeremy G.; Jung, James; Baker, Edward N.; Ackerley, David F.

doi:10.3390/pharmaceutics13071066

Cited by 6 publications

(3 citation statements)

References 41 publications

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“…Of various biochemical assays to measure PpTase activity ( 9 , 17 , 22 , 23 ), we opted for fluorescence polarization (FP)-based assay introduced for the surfactin synthetase activating protein Sfp, a PpTase from Bacillus subtilis ( 24 ). We used the N-terminal ACP domain of Mtb polyketide synthase 13 (N-ACP PKS13) as a cosubstrate (fig.…”

Section: Resultsmentioning

confidence: 99%

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase

Singh,

Ottavi,

Krieger

et al. 2024

Sci. Adv.

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There is a compelling need to find drugs active against Mycobacterium tuberculosis ( Mtb ). 4′-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed’s ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds’ intrabacterial accumulation and transformation.

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Section: Resultsmentioning

confidence: 99%

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase

Singh,

Ottavi,

Krieger

et al. 2024

Sci. Adv.

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“…Numerous in vitro molecular target-based HTS assays using the SML are reported to date that successfully identified inhibitors of various protein targets such as penicillin-binding protein (PBP) [ 178 ], mitogen-activated protein kinase (MKK) [ 179 ], the type III secretion system (T3SS) [ 180 ], the anthrax lethal factor (LF) [ 181 ], phosphopantetheinyl transferase (PPTase) [ 182 ], peptidoglycan O-acetyltransferase [ 183 ], telomere resolvase (ResT) [ 184 ], etc. Spicer et al screened ~292,000 compounds against M18 Aspartyl Aminopeptidase (PfM18AAP) and identified two compounds that were also active in whole cell-based assays [ 185 ].…”

Section: Synthetic Molecule Library (Sml) Screening For Antibacterial...mentioning

confidence: 99%

High-Throughput Screening of Natural Product and Synthetic Molecule Libraries for Antibacterial Drug Discovery

Ayon

2023

Metabolites

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Due to the continued emergence of resistance and a lack of new and promising antibiotics, bacterial infection has become a major public threat. High-throughput screening (HTS) allows rapid screening of a large collection of molecules for bioactivity testing and holds promise in antibacterial drug discovery. More than 50% of the antibiotics that are currently available on the market are derived from natural products. However, with the easily discoverable antibiotics being found, finding new antibiotics from natural sources has seen limited success. Finding new natural sources for antibacterial activity testing has also proven to be challenging. In addition to exploring new sources of natural products and synthetic biology, omics technology helped to study the biosynthetic machinery of existing natural sources enabling the construction of unnatural synthesizers of bioactive molecules and the identification of molecular targets of antibacterial agents. On the other hand, newer and smarter strategies have been continuously pursued to screen synthetic molecule libraries for new antibiotics and new druggable targets. Biomimetic conditions are explored to mimic the real infection model to better study the ligand–target interaction to enable the designing of more effective antibacterial drugs. This narrative review describes various traditional and contemporaneous approaches of high-throughput screening of natural products and synthetic molecule libraries for antibacterial drug discovery. It further discusses critical factors for HTS assay design, makes a general recommendation, and discusses possible alternatives to traditional HTS of natural products and synthetic molecule libraries for antibacterial drug discovery.

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“…1 b) that kills M. tuberculosis by inhibiting PptT in vitro and in a mouse model, with no toxicity to human cells, contributed to redefine PPTases as major contributors to the development of antimycobacterial agents 11 . This motivated the recent development of a simple high-throughput colorimetric assay to directly screen for inhibitors of PptT 12 .…”

Section: Introductionmentioning

confidence: 99%

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds

Carivenc

Maveyraud

Blanger

et al. 2021

Sci Rep

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Owing to their role in activating enzymes essential for bacterial viability and pathogenicity, phosphopantetheinyl transferases represent novel and attractive drug targets. In this work, we examined the inhibitory effect of the aminido-urea 8918 compound against the phosphopantetheinyl transferases PptAb from Mycobacterium abscessus and PcpS from Pseudomonas aeruginosa, two pathogenic bacteria associated with cystic fibrosis and bronchiectasis, respectively. Compound 8918 exhibits inhibitory activity against PptAb but displays no activity against PcpS in vitro, while no antimicrobial activity against Mycobacterium abscessus or Pseudomonas aeruginosa could be detected. X-ray crystallographic analysis of 8918 bound to PptAb-CoA alone and in complex with an acyl carrier protein domain in addition to the crystal structure of PcpS in complex with CoA revealed the structural basis for the inhibition mechanism of PptAb by 8918 and its ineffectiveness against PcpS. Finally, in crystallo screening of potent inhibitors from the National Cancer Institute library identified a hydroxypyrimidinethione derivative that binds PptAb. Both compounds could serve as scaffolds for the future development of phosphopantetheinyl transferases inhibitors.

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Inhibition of Indigoidine Synthesis as a High-Throughput Colourimetric Screen for Antibiotics Targeting the Essential Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT

Cited by 6 publications

References 41 publications

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase

High-Throughput Screening of Natural Product and Synthetic Molecule Libraries for Antibacterial Drug Discovery

Phosphopantetheinyl transferase binding and inhibition by amidino-urea and hydroxypyrimidinethione compounds

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