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The Application of Fragment-based Approaches to the Discovery of Drugs for Neglected Tropical Diseases
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
Christina Spry, Anthony G. Coyne
To validate an ultrafiltration-based fragment screening approach, Shibata et al. (2011) screened a library of 134 fragments against T. brucei riboflavin kinase (TbRFK). From 134 fragments screened in 23 cocktails, each containing five to nine fragments, three fragment hits were identified (Table 1). Subsequent competitive binding assays revealed that flavin mononucleotide (FMN) decreased binding of each of the fragments, while ADP-Mg increased binding. These observations are consistent with the fragments binding in the FMN binding site, which is reported to be stabilized upon ADP-Mg binding (Bauer et al. 2003, Karthikeyan et al. 2003).
Acids produced by lactobacilli inhibit the growth of commensal Lachnospiraceae and S24-7 bacteria
Published in Gut Microbes, 2022
Emma J. E. Brownlie, Danica Chaharlangi, Erin Oi-Yan Wong, Deanna Kim, William Wiley Navarre
For quantitative PCR (qPCR), individual reactions were set up with 10 μL SsoFast EvaGreen Supermix (Bio-Rad, 1725203), 1 μL each of 10 μM forward and reverse primers, and 8 μL template DNA diluted 10X in nuclease-free water, to a total reaction volume of 20 μL. Primers for L. reuteri NM11 (forward: 5’-GGACTACCAGGGTATCTAA-3’; reverse: 5’-TCTCAACACCCGCCTTAATC-3’), L. murinus NM26 (forward: 5’-CCACATGCTAGTGAGCGTATC-3’; reverse: 5’-GTCCAGTTTCTTCTCGCTTCT-3’), and NM74_B14 (forward: 5’-GTGGAAACGAGAAGACTGTAGAA-3’; reverse: 5’-TTTCGTCTCTCAATCGGGAATAG-3’) were designed for this study. These primer sets targeted genes unique to each species (hypothetical proteins for L. reuteri and L. murinus, and a FMN adenylyltransferase/riboflavin kinase for NM74_B14) to ensure specificity, which was checked using PrimerBlast and confirmed experimentally. qPCR was carried out on an Eppendorf Mastercycler ep realplex in a 96-well format. Cycling conditions were 30 seconds at 95°C, 40 cycles of 5 seconds at 95°C and 10 seconds at 60°C, 15 seconds at 95°C followed by 15 seconds at 60°C, and a 20-minute ramp up to 95°C for 15 seconds. Five-point standard curves of 10X dilutions (20 ng to 0.002 ng or 10 ng to 0.001 ng) were set up in duplicate, while each sample was run in triplicate. Analysis of qPCR efficiency and accuracy was carried out using Eppendorf realplex software.
The RFK catalytic cycle of the pathogen Streptococcus pneumoniae shows species-specific features in prokaryotic FMN synthesis
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
María Sebastián, Adrián Velázquez-Campoy, Milagros Medina
Streptococcus pneumoniae is the causative agent of human pneumonia disease1, meningitis and bacteremia in children and adults. It is estimated that 1.6 millions of people, including 1 million of children under age five die every year of pneumonia disease2,3. The irruption during the last decades of multi-drug resistant pneumococci has revealed the need of finding new drugs, as well as novel drug targets. The bifunctional flavin-adenine dinucleotide (FAD) synthetase (FADS) from S. pneumoniae (SpnFADS) arises as a potential drug target4,5, since it synthesises the essential cofactors flavin mononucleotide (FMN) and FAD6, involved in a plethora of vital processes as part of flavoproteins and flavoenzymes7–9. As other bacterial FADSs, SpnFADS produces FMN and FAD through two sequential activities. First, a riboflavin kinase activity (RFK) at its C-terminus module phosphorylates riboflavin (RF) to FMN, and then the adenosine 5′-triphosphate (ATP):FMN:adenylyltransferase (FMNAT) activity of the enzyme N-terminus module transforms FMN into FAD10–12. Two characteristics stand out among the suitable properties of FADSs as drug targets. First, bacterial FADSs differ from the human proteins that synthesise FMN and particularly FAD. Thus, the eukaryotic FMNAT activity is catalysed by an enzyme with a completely different protein folding and chemistry relative to the N-terminus of bacterial FADS13–16. Regarding FMN production, the monofunctional Homo sapiens RFK has been hardly characterised so far. Overall, it is structurally homologous to the RFK module of bacterial enzymes, but only structures containing bound ligands are available17,18. Nonetheless, structural data predict differences in conformational changes to achieve the catalytic complex19,20, and the scarce biochemical information suggests differences in redox environmental requirements for maximal activity11,12. Second, the members of the prokaryotic FADSs family studied up to now differ catalytically among them, which might facilitate the design of new species-specific medicines. Structurally, when comparing SpnFADS with the member of the family so far best characterised—which is that from the organism Corynebacterium ammoniagenes (CaFADS)—it presents a very similar structure, with little differences in the position of some key loops. However, these two proteins only share the 26% of sequence homology6. Despite the overall structural similitude among prokaryotic FADSs6,12,18, SpnFADS shows three main distinctive functional behaviors; (i) it mainly stabilises monomers—which are the functional form6—or traces of dimers, during catalysis; (ii) its FMNAT activity requires reduced FMN as a substrate; and (iii) its RFK activity is not regulated by the RF substrate6.