Application of Fragment Screening and Merging to the Discovery of Inhibitors of the <i>Mycobacterium tuberculosis</i> Cytochrome P450 CYP121

Hudson, Sean A.; McLean, Kirsty J.; Surade, Sachin; Yang, Yong Qing; Leys, D.; Ciulli, Alessio; Munro, Andrew W.; Abell, Chris

doi:10.1002/anie.201202544

Cited by 73 publications

(93 citation statements)

References 23 publications

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“…The cyp121 gene is essential for mycobacterial growth, so inhibiting CYP121 is a promising approach to treating tuberculosis (11,31). Our findings contribute to the development of inhibitors with potential clinical applications.…”

Section: Discussionmentioning

confidence: 85%

“…The organization of its active site and features of the reaction it catalyzes suggest novel mechanisms (12,16), and it is also essential in vivo such that it is a potential target for the development of novel antimycobacterial agents (11,28,31). Here, we report an investigation of the substrate and reaction specificities of CYP121 involving synthesizing substrate analogues and studying their binding to and transformation by CYP121.…”

Section: Discussionmentioning

confidence: 99%

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Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121

Fonvielle

Lequin

et al. 2013

Journal of Biological Chemistry

107

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Background:Little is known about the substrate specificity of the essential P450 CYP121 from Mycobacterium tuberculosis. Results: CYP121 substrate analogues either display impaired binding to CYP121 or are poorly transformed by CYP121. Conclusion: CYP121 is a highly specific P450. Significance: This work contributes to the understanding of the function and the catalytic mechanism of CYP121 and provides novel data for the design of inhibitors.

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Section: Discussionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121

Fonvielle

Lequin

et al. 2013

Journal of Biological Chemistry

107

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“…TxtE has two B′ helices, which is an uncommon conformation. Besides TxtE, there are five known CYP structures which have two B′ helices, but only AurH[30], CYP121[31] and CalO 2 [32] form two B′ helices bundle similar to TxtE. This B′ helices bundle creates a hydrophobic “wall” that shields the substrate binding pocket from solvent, and seals the substrate binding pocket.…”

Section: Discussionmentioning

confidence: 99%

Structural Insights into the Mechanism for Recognizing Substrate of the Cytochrome P450 Enzyme TxtE

et al. 2013

PLoS ONE

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Thaxtomins, a family of phytotoxins produced by Streptomyces spp., can cause dramatic plant cell hypertrophy and seedling stunting. Thaxtomin A is the dominant form from Streptomyces scabies and has demonstrated herbicidal action. TxtE, a cytochrome P450 enzyme from Streptomyces scabies 87.22, catalyzes direct nitration of the indolyl moiety of L-tryptophan to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH. The crystal structure of TxtE was determined at 2.1 Å resolution and described in this work. A clearly defined substrate access channel is observed and can be classified as channel 2a, which is common in bacteria cytochrome P450 enzymes. A continuous hydrogen bond chain from the active site to the external solvent is observed. Compared with other cytochrome P450 enzymes, TxtE shows a unique proton transfer pathway which crosses the helix I distortion. Polar contacts of Arg59, Tyr89, Asn293, Thr296, and Glu394 with L-tryptophan are seen using molecular docking analysis, which are potentially important for substrate recognition and binding. After mutating Arg59, Asn293, Thr296 or Glu394 to leucine, the substrate binding ability of TxtE was lost or decreased significantly. Based on the docking and mutation results, a possible mechanism for substrate recognition and binding is proposed.

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“…TSA has been successfully employed as the primary screening tool in three recent academic FBDD campaigns against myoinositol-3-phosphate synthase from Trypanosoma brucei (TbINO1) (The University of St Andrews, 2011), [15] a cytochrome P450 protein (CYP121) from Mycobacterium tuberculosis (University of Cambridge, 2012) , [16] and a human p53 mutant Y220C (Eberhard-Karls-University, and MRC LMB, Cambridge, 2012). [17] TSA screening of a 600 fragment rule-of-three compliant library at a concentration of 1 mM, identified 38 fragments (,6 % hit rate) that induced a DTm .1.58C in TbINO1.…”

Section: Tsa In Fragment-based Drug Discoverymentioning

confidence: 99%

“…[16] Four independent ligand-crystal structures were solved revealing that each of the fragments bound in an overlapping manner within the relatively large (1350 Å 3 ) active site cavity. This allowed the authors to immediately merge the overlapping structural moieties to generate a moderate affinity (28 mM) lead compound in a textbook application of TSA screening, hit validation by orthogonal methods, and successful SAR development.…”

Section: Tsa In Fragment-based Drug Discoverymentioning

confidence: 99%

Interrogating Fragments Using a Protein Thermal Shift Assay

McMahon¹,

Scanlon²,

Martin³

2013

Aust. J. Chem.

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Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand–protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein–drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein–protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD campaign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree.

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Application of Fragment Screening and Merging to the Discovery of Inhibitors of the Mycobacterium tuberculosis Cytochrome P450 CYP121

Cited by 73 publications

References 23 publications

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121

Structural Insights into the Mechanism for Recognizing Substrate of the Cytochrome P450 Enzyme TxtE

Interrogating Fragments Using a Protein Thermal Shift Assay

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