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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

X-ray crystal structuresof heme binding Ar2 analogues 25b (PDB 5IBJ), 25a (PDB 5IBE),and 19a (PDB 5IBF) in complex with CYP121. (a) 25b (cyan)closely recapitulated the binding of lead 2,forming polar contacts (yellow dashes) with amino acids (blue sticks)in binding hotspots as well as additional binding contacts to Ser237,Val228, and the heme iron. (b) 25a (orange) was rotatedaway from the I-helix (yellow cartoon), placing the 5-aminopyrazolering in the Ar3 pocket. Polar contacts to the heme iron and Ser237are indicated (yellow dashes). (c) Two molecules of 19a bound per active site, each ligand fulfilling a subset of the interactionsidentified as binding hotspots. Inset: Heme binding 19a recapitulated the binding mode of 25a while the non-hemebinding 19a ligand satisfied aromatic interactions inthe Ar1 pocket, as well as the hydrogen bonding interactions of fragment 7. (d) Extracted structures of 25b, 25a, and 19a annotated to show metal coordination (yellowdashes) distance, angle of approach to the porphyrin ring (yellowangle), and dihedral torsion in the biphenyl-aniline motif. Removalof the Ar2 4-hydroxy group reduced the dihedral angle of the biphenylaniline system in 19a and 25a by 30°compared to 25b and allowed the analogues to approachthe plane of the porphyrin at a sharper angle (31–33°).The omit Fo – Fc electron density maps of ligands 19a, 25a, and 25b contoured to 3σ have beenprovided in Supporting Information Figure S5. Figures prepared using PyMOL v1.7.4 (Schrödinger, LLC).
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fig7: X-ray crystal structuresof heme binding Ar2 analogues 25b (PDB 5IBJ), 25a (PDB 5IBE),and 19a (PDB 5IBF) in complex with CYP121. (a) 25b (cyan)closely recapitulated the binding of lead 2,forming polar contacts (yellow dashes) with amino acids (blue sticks)in binding hotspots as well as additional binding contacts to Ser237,Val228, and the heme iron. (b) 25a (orange) was rotatedaway from the I-helix (yellow cartoon), placing the 5-aminopyrazolering in the Ar3 pocket. Polar contacts to the heme iron and Ser237are indicated (yellow dashes). (c) Two molecules of 19a bound per active site, each ligand fulfilling a subset of the interactionsidentified as binding hotspots. Inset: Heme binding 19a recapitulated the binding mode of 25a while the non-hemebinding 19a ligand satisfied aromatic interactions inthe Ar1 pocket, as well as the hydrogen bonding interactions of fragment 7. (d) Extracted structures of 25b, 25a, and 19a annotated to show metal coordination (yellowdashes) distance, angle of approach to the porphyrin ring (yellowangle), and dihedral torsion in the biphenyl-aniline motif. Removalof the Ar2 4-hydroxy group reduced the dihedral angle of the biphenylaniline system in 19a and 25a by 30°compared to 25b and allowed the analogues to approachthe plane of the porphyrin at a sharper angle (31–33°).The omit Fo – Fc electron density maps of ligands 19a, 25a, and 25b contoured to 3σ have beenprovided in Supporting Information Figure S5. Figures prepared using PyMOL v1.7.4 (Schrödinger, LLC).

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)

X-ray crystal structuresof heme binding Ar2 analogues 25b (PDB 5IBJ), 25a (PDB 5IBE),and 19a (PDB 5IBF) in complex with CYP121. (a) 25b (cyan)closely recapitulated the binding of lead 2,forming polar contacts (yellow dashes) with amino acids (blue sticks)in binding hotspots as well as additional binding contacts to Ser237,Val228, and the heme iron. (b) 25a (orange) was rotatedaway from the I-helix (yellow cartoon), placing the 5-aminopyrazolering in the Ar3 pocket. Polar contacts to the heme iron and Ser237are indicated (yellow dashes). (c) Two molecules of 19a bound per active site, each ligand fulfilling a subset of the interactionsidentified as binding hotspots. Inset: Heme binding 19a recapitulated the binding mode of 25a while the non-hemebinding 19a ligand satisfied aromatic interactions inthe Ar1 pocket, as well as the hydrogen bonding interactions of fragment 7. (d) Extracted structures of 25b, 25a, and 19a annotated to show metal coordination (yellowdashes) distance, angle of approach to the porphyrin ring (yellowangle), and dihedral torsion in the biphenyl-aniline motif. Removalof the Ar2 4-hydroxy group reduced the dihedral angle of the biphenylaniline system in 19a and 25a by 30°compared to 25b and allowed the analogues to approachthe plane of the porphyrin at a sharper angle (31–33°).The omit Fo – Fc electron density maps of ligands 19a, 25a, and 25b contoured to 3σ have beenprovided in Supporting Information Figure S5. Figures prepared using PyMOL v1.7.4 (Schrödinger, LLC).
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Related In: Results  -  Collection

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fig7: X-ray crystal structuresof heme binding Ar2 analogues 25b (PDB 5IBJ), 25a (PDB 5IBE),and 19a (PDB 5IBF) in complex with CYP121. (a) 25b (cyan)closely recapitulated the binding of lead 2,forming polar contacts (yellow dashes) with amino acids (blue sticks)in binding hotspots as well as additional binding contacts to Ser237,Val228, and the heme iron. (b) 25a (orange) was rotatedaway from the I-helix (yellow cartoon), placing the 5-aminopyrazolering in the Ar3 pocket. Polar contacts to the heme iron and Ser237are indicated (yellow dashes). (c) Two molecules of 19a bound per active site, each ligand fulfilling a subset of the interactionsidentified as binding hotspots. Inset: Heme binding 19a recapitulated the binding mode of 25a while the non-hemebinding 19a ligand satisfied aromatic interactions inthe Ar1 pocket, as well as the hydrogen bonding interactions of fragment 7. (d) Extracted structures of 25b, 25a, and 19a annotated to show metal coordination (yellowdashes) distance, angle of approach to the porphyrin ring (yellowangle), and dihedral torsion in the biphenyl-aniline motif. Removalof the Ar2 4-hydroxy group reduced the dihedral angle of the biphenylaniline system in 19a and 25a by 30°compared to 25b and allowed the analogues to approachthe plane of the porphyrin at a sharper angle (31–33°).The omit Fo – Fc electron density maps of ligands 19a, 25a, and 25b contoured to 3σ have beenprovided in Supporting Information Figure S5. Figures prepared using PyMOL v1.7.4 (Schrödinger, LLC).
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