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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

Fragment merging and in silico directed optimization of fragment 1 (green, PDB 4G47) led to the discovery of low micromolar CYP121 inhibitor 2 (salmon, PDB 4KTL). Fragment 1 and lead 2 retaineda conserved binding mode distal to the heme cofactor (magenta). Hydrogenbonding interactions between the 5-aminopyrazole ring and CYP121 residuesGln385 and Ala167, and the phenol group with Asn85 and active sitewater molecules are shown as yellow dashed lines. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
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fig1: Fragment merging and in silico directed optimization of fragment 1 (green, PDB 4G47) led to the discovery of low micromolar CYP121 inhibitor 2 (salmon, PDB 4KTL). Fragment 1 and lead 2 retaineda conserved binding mode distal to the heme cofactor (magenta). Hydrogenbonding interactions between the 5-aminopyrazole ring and CYP121 residuesGln385 and Ala167, and the phenol group with Asn85 and active sitewater molecules are shown as yellow dashed lines. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).

Mentions: Fragment-based drug discovery (FBDD) is an established techniquein both academia and industry that has been applied to a wide rangeof molecular targets.20−25 The availability of high resolution crystal structures of CYP121,in both the substrate-free and substrate/ligand-bound forms, makeit amenable to a fragment-based approach.26 We have previously reported the development of low micromolar affinityCYP121 inhibitors, developed from fragments that were identified ina biophysical screen of our fragment library.27,28 Six fragment hits were crystallized with CYP121, one of which was4-(1H-1,2,4-triazol-1-yl)phenol 1 (KD = 1.7 mM) (Figure 1). Fragment 1 bound in two differentoverlapping orientations in the CYP121 active site, suggesting anapproach based on fragment merging. Synthesis of a range of analogues,guided by in silico screening to optimize the binding interactionsof the heterocyclic fragment core, resulted in the identificationof the triphenol pyrazole-amine 2 (KD = 15 μM). An X-ray crystal structure of 2 bound to CYP121 revealed that 2 maintainedthe binding mode of the original fragment 1. The optimizedhydrogen bonding interactions between the 5-aminopyrazole core of 2 and distal pocket residues Gln385, Ala167, and Thr77 representeda privileged binding mode for an azole-containing compound, whichgenerally bind directly to the heme cofactor of P450s using a heterocyclicnitrogen atom. The combination of the low micromolar affinity of 2 and its unique binding mode, which was conserved with fragment 1, led us to pursue further optimization of this compoundseries.


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)

Fragment merging and in silico directed optimization of fragment 1 (green, PDB 4G47) led to the discovery of low micromolar CYP121 inhibitor 2 (salmon, PDB 4KTL). Fragment 1 and lead 2 retaineda conserved binding mode distal to the heme cofactor (magenta). Hydrogenbonding interactions between the 5-aminopyrazole ring and CYP121 residuesGln385 and Ala167, and the phenol group with Asn85 and active sitewater molecules are shown as yellow dashed lines. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Fragment merging and in silico directed optimization of fragment 1 (green, PDB 4G47) led to the discovery of low micromolar CYP121 inhibitor 2 (salmon, PDB 4KTL). Fragment 1 and lead 2 retaineda conserved binding mode distal to the heme cofactor (magenta). Hydrogenbonding interactions between the 5-aminopyrazole ring and CYP121 residuesGln385 and Ala167, and the phenol group with Asn85 and active sitewater molecules are shown as yellow dashed lines. Figures preparedusing PyMOL v1.7.4 (Schrödinger, LLC).
Mentions: Fragment-based drug discovery (FBDD) is an established techniquein both academia and industry that has been applied to a wide rangeof molecular targets.20−25 The availability of high resolution crystal structures of CYP121,in both the substrate-free and substrate/ligand-bound forms, makeit amenable to a fragment-based approach.26 We have previously reported the development of low micromolar affinityCYP121 inhibitors, developed from fragments that were identified ina biophysical screen of our fragment library.27,28 Six fragment hits were crystallized with CYP121, one of which was4-(1H-1,2,4-triazol-1-yl)phenol 1 (KD = 1.7 mM) (Figure 1). Fragment 1 bound in two differentoverlapping orientations in the CYP121 active site, suggesting anapproach based on fragment merging. Synthesis of a range of analogues,guided by in silico screening to optimize the binding interactionsof the heterocyclic fragment core, resulted in the identificationof the triphenol pyrazole-amine 2 (KD = 15 μM). An X-ray crystal structure of 2 bound to CYP121 revealed that 2 maintainedthe binding mode of the original fragment 1. The optimizedhydrogen bonding interactions between the 5-aminopyrazole core of 2 and distal pocket residues Gln385, Ala167, and Thr77 representeda privileged binding mode for an azole-containing compound, whichgenerally bind directly to the heme cofactor of P450s using a heterocyclicnitrogen atom. The combination of the low micromolar affinity of 2 and its unique binding mode, which was conserved with fragment 1, led us to pursue further optimization of this compoundseries.

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