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Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

Kavanagh ME, Coyne AG, McLean KJ, James GG, Levy CW, Marino LB, de Carvalho LP, Chan DS, Hudson SA, Surade S, Leys D, Munro AW, Abell C - J. Med. Chem. (2016)

Bottom Line: Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM).Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands.Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, U.K.

ABSTRACT
The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

No MeSH data available.


Related in: MedlinePlus

Optical titration difference spectra (top panel) and concentration-dependentchanges in CYP121 absorbance (bottom panel) for representative hemebinding ligands (a) 24a, (b) 25b, and (c) 19a. Absorption spectra of CYP121 (5 μM) were collectedbetween 250 and 800 nm after sequential additions of 0.2 μLaliquots of DMSO-d6 stock solutions (0.15–83mM) of ligands. Difference spectra were obtained from the subtractionof the ligand-free CYP121 spectra from those obtained over the courseof the titration. The maximum change in absorbance from each differencespectrum was plotted against ligand concentration and data were fittedusing a modified version of the Morrison equation for tight bindingligands 24a and 25b(17) or a hyperbolic function for 19a.
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fig5: Optical titration difference spectra (top panel) and concentration-dependentchanges in CYP121 absorbance (bottom panel) for representative hemebinding ligands (a) 24a, (b) 25b, and (c) 19a. Absorption spectra of CYP121 (5 μM) were collectedbetween 250 and 800 nm after sequential additions of 0.2 μLaliquots of DMSO-d6 stock solutions (0.15–83mM) of ligands. Difference spectra were obtained from the subtractionof the ligand-free CYP121 spectra from those obtained over the courseof the titration. The maximum change in absorbance from each differencespectrum was plotted against ligand concentration and data were fittedusing a modified version of the Morrison equation for tight bindingligands 24a and 25b(17) or a hyperbolic function for 19a.

Mentions: All subsequent Ar2 analogues containing a3-aminophenyl substituent(19b,c, 24a, 25a,b) caused type II shifts in the CYP121 optical spectrum,allowing their binding affinity to be determined by optical titrationin addition to ITC (Table 2 and Figure 5). The magnitude of the type II shift decreasedwith increasing steric bulk ortho to the 3-aminophenylsubstituent. These results supported the hypothesis that torsionalstrain in the biphenyl system, or the steric bulk of the benzyl ormethyl hydroxy protecting groups, in analogues 19b and 19c, respectively, prevented the ligands approaching the cofactorfor heme coordination.


Fragment-Based Approaches to the Development of Mycobacterium tuberculosis CYP121 Inhibitors.

Kavanagh ME, Coyne AG, McLean KJ, James GG, Levy CW, Marino LB, de Carvalho LP, Chan DS, Hudson SA, Surade S, Leys D, Munro AW, Abell C - J. Med. Chem. (2016)

Optical titration difference spectra (top panel) and concentration-dependentchanges in CYP121 absorbance (bottom panel) for representative hemebinding ligands (a) 24a, (b) 25b, and (c) 19a. Absorption spectra of CYP121 (5 μM) were collectedbetween 250 and 800 nm after sequential additions of 0.2 μLaliquots of DMSO-d6 stock solutions (0.15–83mM) of ligands. Difference spectra were obtained from the subtractionof the ligand-free CYP121 spectra from those obtained over the courseof the titration. The maximum change in absorbance from each differencespectrum was plotted against ligand concentration and data were fittedusing a modified version of the Morrison equation for tight bindingligands 24a and 25b(17) or a hyperbolic function for 19a.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835159&req=5

fig5: Optical titration difference spectra (top panel) and concentration-dependentchanges in CYP121 absorbance (bottom panel) for representative hemebinding ligands (a) 24a, (b) 25b, and (c) 19a. Absorption spectra of CYP121 (5 μM) were collectedbetween 250 and 800 nm after sequential additions of 0.2 μLaliquots of DMSO-d6 stock solutions (0.15–83mM) of ligands. Difference spectra were obtained from the subtractionof the ligand-free CYP121 spectra from those obtained over the courseof the titration. The maximum change in absorbance from each differencespectrum was plotted against ligand concentration and data were fittedusing a modified version of the Morrison equation for tight bindingligands 24a and 25b(17) or a hyperbolic function for 19a.
Mentions: All subsequent Ar2 analogues containing a3-aminophenyl substituent(19b,c, 24a, 25a,b) caused type II shifts in the CYP121 optical spectrum,allowing their binding affinity to be determined by optical titrationin addition to ITC (Table 2 and Figure 5). The magnitude of the type II shift decreasedwith increasing steric bulk ortho to the 3-aminophenylsubstituent. These results supported the hypothesis that torsionalstrain in the biphenyl system, or the steric bulk of the benzyl ormethyl hydroxy protecting groups, in analogues 19b and 19c, respectively, prevented the ligands approaching the cofactorfor heme coordination.

Bottom Line: Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM).Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands.Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, U.K.

ABSTRACT
The essential enzyme CYP121 is a target for drug development against antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to bind to CYP121 using a cascade of biophysical assays. Synthetic merging and optimization of 1 produced a 100-fold improvement in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of structural motifs to be assessed, the identification of more LE scaffolds for optimization and highlighted binding affinity hotspots. Structure-guided addition of a metal-binding pharmacophore onto LE retrofragment scaffolds produced low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV-vis spectroscopy, and native mass spectrometry provides insight for subsequent drug development.

No MeSH data available.


Related in: MedlinePlus