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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

Representative ITC binding isotherms andintegrated enthalpy plotsfor (a) fused Ar1–Ar3 fragment 6 (500 μM);(b) non-heme binding Ar3 analogue 26a (1 mM); (c) hemebinding analogue 25a (500 μM). The isotherm ofeach compound titrated into buffer alone was subtracted from thatcontaining CYP121 (50 μM). The integrated enthalpy change foreach injection plotted as a function of molar ratio of ligand-to-CYP121was fitted using a one-site equilibrium binding model to calculatebinding affinity (KD). The binding stoichiometry(N) was allowed to vary for analogues 6, 26a, and 25a.
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fig6: Representative ITC binding isotherms andintegrated enthalpy plotsfor (a) fused Ar1–Ar3 fragment 6 (500 μM);(b) non-heme binding Ar3 analogue 26a (1 mM); (c) hemebinding analogue 25a (500 μM). The isotherm ofeach compound titrated into buffer alone was subtracted from thatcontaining CYP121 (50 μM). The integrated enthalpy change foreach injection plotted as a function of molar ratio of ligand-to-CYP121was fitted using a one-site equilibrium binding model to calculatebinding affinity (KD). The binding stoichiometry(N) was allowed to vary for analogues 6, 26a, and 25a.

Mentions: The bindingaffinity (KD) of analogues for CYP121was determined by ITC (Table 2, Figure 6a–c). The KDs of heme binding Ar2 analogues were also determined by UV–visoptical titration (Table 2, Figure 5).43−45 Comparison of the KDs obtained for theAr2 series by ITC and optical titration enabled robust comparisonsto be made with the affinity of the non-heme binding Ar1 and Ar3 seriesof analogues, as well as with previously reported type II inhibitorsin the literature.12,27,28 In general, KD values obtained by opticaltitration were 10–100-fold lower than those measured in ITC,consistent with previous reports in the literature.45 Variations in affinity data generated using different biophysicaltechniques have been previously attributed to the kinetically significantbut spectrally silent, multistep binding interactions of P450s withligands, the potential presence of heterogeneous enzyme populationsin solution, requirement for conformational change, and/or enzymecooperativity.45,46 High resolution (1.6–2.2Å) X-ray crystal structures of CYP121 in complex with a numberof analogues were obtained, allowing inhibitor binding modes to beconfirmed and enabling robust comparison of the SAR contributing tobinding affinity (Figure 7,8).


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)

Representative ITC binding isotherms andintegrated enthalpy plotsfor (a) fused Ar1–Ar3 fragment 6 (500 μM);(b) non-heme binding Ar3 analogue 26a (1 mM); (c) hemebinding analogue 25a (500 μM). The isotherm ofeach compound titrated into buffer alone was subtracted from thatcontaining CYP121 (50 μM). The integrated enthalpy change foreach injection plotted as a function of molar ratio of ligand-to-CYP121was fitted using a one-site equilibrium binding model to calculatebinding affinity (KD). The binding stoichiometry(N) was allowed to vary for analogues 6, 26a, and 25a.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835159&req=5

fig6: Representative ITC binding isotherms andintegrated enthalpy plotsfor (a) fused Ar1–Ar3 fragment 6 (500 μM);(b) non-heme binding Ar3 analogue 26a (1 mM); (c) hemebinding analogue 25a (500 μM). The isotherm ofeach compound titrated into buffer alone was subtracted from thatcontaining CYP121 (50 μM). The integrated enthalpy change foreach injection plotted as a function of molar ratio of ligand-to-CYP121was fitted using a one-site equilibrium binding model to calculatebinding affinity (KD). The binding stoichiometry(N) was allowed to vary for analogues 6, 26a, and 25a.
Mentions: The bindingaffinity (KD) of analogues for CYP121was determined by ITC (Table 2, Figure 6a–c). The KDs of heme binding Ar2 analogues were also determined by UV–visoptical titration (Table 2, Figure 5).43−45 Comparison of the KDs obtained for theAr2 series by ITC and optical titration enabled robust comparisonsto be made with the affinity of the non-heme binding Ar1 and Ar3 seriesof analogues, as well as with previously reported type II inhibitorsin the literature.12,27,28 In general, KD values obtained by opticaltitration were 10–100-fold lower than those measured in ITC,consistent with previous reports in the literature.45 Variations in affinity data generated using different biophysicaltechniques have been previously attributed to the kinetically significantbut spectrally silent, multistep binding interactions of P450s withligands, the potential presence of heterogeneous enzyme populationsin solution, requirement for conformational change, and/or enzymecooperativity.45,46 High resolution (1.6–2.2Å) X-ray crystal structures of CYP121 in complex with a numberof analogues were obtained, allowing inhibitor binding modes to beconfirmed and enabling robust comparison of the SAR contributing tobinding affinity (Figure 7,8).

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