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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 structures of lead compound 2 and componentretrofragments 4–6 in complex withCYP121. The I- (yellow), and F-, and G- (blue) helices are shown incartoon representation. (a) Overlaid structures of retrofragments 4 (purple) (PBD 4KTJ), 5 (yellow) (PDB 4KTF), and 6 (cyan) (PDB 5IBJ) with lead compound 2 (salmon) (PDB 4KTL), illustrating theconserved binding mode distal to the heme cofactor (magenta).28 (b) Structures, binding affinity, and ligandefficiency (LE) of compound 2 and retrofragments 4, 5, and 6. (c–e) X-raycrystal structures of CYP121 in complex with retrofragments 4, 5, and 6, respectively, indicatingpolar interactions (yellow dashes) with active site residues (blue)and solvent (red spheres). Co-crystallized sulfate (yellow lines)is also indicated in (d). The associated omit FoFc electron density map of fragment 6 contoured to 3σ has been provided in the SupportingInformation, Figure S5. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
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fig2: X-ray crystal structures of lead compound 2 and componentretrofragments 4–6 in complex withCYP121. The I- (yellow), and F-, and G- (blue) helices are shown incartoon representation. (a) Overlaid structures of retrofragments 4 (purple) (PBD 4KTJ), 5 (yellow) (PDB 4KTF), and 6 (cyan) (PDB 5IBJ) with lead compound 2 (salmon) (PDB 4KTL), illustrating theconserved binding mode distal to the heme cofactor (magenta).28 (b) Structures, binding affinity, and ligandefficiency (LE) of compound 2 and retrofragments 4, 5, and 6. (c–e) X-raycrystal structures of CYP121 in complex with retrofragments 4, 5, and 6, respectively, indicatingpolar interactions (yellow dashes) with active site residues (blue)and solvent (red spheres). Co-crystallized sulfate (yellow lines)is also indicated in (d). The associated omit FoFc electron density map of fragment 6 contoured to 3σ has been provided in the SupportingInformation, Figure S5. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).

Mentions: Retrosynthetic fragmentation oflead compound 2 indicated that the relative binding contributionsof Ar1, Ar2, and Ar3 could be determined from a combination of monophenoland biphenol fragments 3, 4, 5, and 6 (Figure 3).32 The sequential reconstructionof 2 from these fragments enabled the GE of each aromaticring to be calculated from the difference in the free energy of bindingper heavy atom added at each stage of reconstruction (GE = −ΔΔG/ΔHA).31 Retrofragments 3 and 5 were synthesizedaccording to the previously reported literature procedure,28,33 which was also used to access fragment 4. Biphenolfragment 6 was synthesized according to the general routefor accessing 3,5-disubstituted aminopyrazoles described by Johnsonet al.34 X-ray crystal structures of fragments 3–628 in complexwith CYP121 (Figure 2a–c) were obtained in order to aidthe interpretation of binding affinity and GE trends. Retrofragments 3–6 each reproduced the privileged non-hemebinding mode of lead compound 2, which is unusual forP450 ligands containing azole motifs. Analysis of the conserved andvariable binding interactions made by the retrofragments highlightedstructural features likely to be important for driving the bindingaffinity of 2.32,35 Monophenol 4 bound in an orientation that overlapped with that of Ar2 of thelead compound 2 (Figure 2a). The water-bridged hydrogen bonding network betweenAsn85, Thr229, and the heme propionate group with the 4-hydroxy substituentof 4 was conserved, while the 5-aminopyrazole ring wasshifted toward the I-helix (highlighted yellow in Figure 2) to enable hydrogen bondinginteractions with Val228 and aromatic interactions with Phe168 andTrp182, mimicking the function originally fulfilled by Ar1 of leadcompound 2. The small shift in position of the aminopyrazolering of 4 indicated that interactions made at the Ar1site might contribute more significantly to the affinity of lead compound 2 than do the hydrogen-bonding interactions made by 2 with Gln385.


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 structures of lead compound 2 and componentretrofragments 4–6 in complex withCYP121. The I- (yellow), and F-, and G- (blue) helices are shown incartoon representation. (a) Overlaid structures of retrofragments 4 (purple) (PBD 4KTJ), 5 (yellow) (PDB 4KTF), and 6 (cyan) (PDB 5IBJ) with lead compound 2 (salmon) (PDB 4KTL), illustrating theconserved binding mode distal to the heme cofactor (magenta).28 (b) Structures, binding affinity, and ligandefficiency (LE) of compound 2 and retrofragments 4, 5, and 6. (c–e) X-raycrystal structures of CYP121 in complex with retrofragments 4, 5, and 6, respectively, indicatingpolar interactions (yellow dashes) with active site residues (blue)and solvent (red spheres). Co-crystallized sulfate (yellow lines)is also indicated in (d). The associated omit FoFc electron density map of fragment 6 contoured to 3σ has been provided in the SupportingInformation, Figure S5. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4835159&req=5

fig2: X-ray crystal structures of lead compound 2 and componentretrofragments 4–6 in complex withCYP121. The I- (yellow), and F-, and G- (blue) helices are shown incartoon representation. (a) Overlaid structures of retrofragments 4 (purple) (PBD 4KTJ), 5 (yellow) (PDB 4KTF), and 6 (cyan) (PDB 5IBJ) with lead compound 2 (salmon) (PDB 4KTL), illustrating theconserved binding mode distal to the heme cofactor (magenta).28 (b) Structures, binding affinity, and ligandefficiency (LE) of compound 2 and retrofragments 4, 5, and 6. (c–e) X-raycrystal structures of CYP121 in complex with retrofragments 4, 5, and 6, respectively, indicatingpolar interactions (yellow dashes) with active site residues (blue)and solvent (red spheres). Co-crystallized sulfate (yellow lines)is also indicated in (d). The associated omit FoFc electron density map of fragment 6 contoured to 3σ has been provided in the SupportingInformation, Figure S5. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
Mentions: Retrosynthetic fragmentation oflead compound 2 indicated that the relative binding contributionsof Ar1, Ar2, and Ar3 could be determined from a combination of monophenoland biphenol fragments 3, 4, 5, and 6 (Figure 3).32 The sequential reconstructionof 2 from these fragments enabled the GE of each aromaticring to be calculated from the difference in the free energy of bindingper heavy atom added at each stage of reconstruction (GE = −ΔΔG/ΔHA).31 Retrofragments 3 and 5 were synthesizedaccording to the previously reported literature procedure,28,33 which was also used to access fragment 4. Biphenolfragment 6 was synthesized according to the general routefor accessing 3,5-disubstituted aminopyrazoles described by Johnsonet al.34 X-ray crystal structures of fragments 3–628 in complexwith CYP121 (Figure 2a–c) were obtained in order to aidthe interpretation of binding affinity and GE trends. Retrofragments 3–6 each reproduced the privileged non-hemebinding mode of lead compound 2, which is unusual forP450 ligands containing azole motifs. Analysis of the conserved andvariable binding interactions made by the retrofragments highlightedstructural features likely to be important for driving the bindingaffinity of 2.32,35 Monophenol 4 bound in an orientation that overlapped with that of Ar2 of thelead compound 2 (Figure 2a). The water-bridged hydrogen bonding network betweenAsn85, Thr229, and the heme propionate group with the 4-hydroxy substituentof 4 was conserved, while the 5-aminopyrazole ring wasshifted toward the I-helix (highlighted yellow in Figure 2) to enable hydrogen bondinginteractions with Val228 and aromatic interactions with Phe168 andTrp182, mimicking the function originally fulfilled by Ar1 of leadcompound 2. The small shift in position of the aminopyrazolering of 4 indicated that interactions made at the Ar1site might contribute more significantly to the affinity of lead compound 2 than do the hydrogen-bonding interactions made by 2 with Gln385.

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