Limits...
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

Representativenative mass spectra of CYP121 and selected compounds 26a, 25a, and 19a demonstrated thatcompounds formed stable complexes with both monomeric (m/z 5250–6750) and dimeric (m/z 5250–6750) CYP121. The number of boundligands (orange spheres) increased in a concentration-dependent mannerand was proportionate to the binding affinity (KD) of the compounds. Spectra were collected for CYP121 (8.7or 5 μM) at three different ligand-to-protein ratios (0.5:1,1:1, and 25:1) and in the presence of DMSO-d6 (2.2% or 5% v/v) alone. The concentration of protein or DMSO-d6 did not affect the quality of the spectraor ligand binding interactions, but higher concentrations of DMSO-d6 reduced the charge state of the protein. Ligandbinding stoichiometry was calculated from the difference in mass ofligand-bound and unbound (orange dashed lines) protein peaks dividedby the molecular weight of ligands (Table S1, Supporting Information).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835159&req=5

fig9: Representativenative mass spectra of CYP121 and selected compounds 26a, 25a, and 19a demonstrated thatcompounds formed stable complexes with both monomeric (m/z 5250–6750) and dimeric (m/z 5250–6750) CYP121. The number of boundligands (orange spheres) increased in a concentration-dependent mannerand was proportionate to the binding affinity (KD) of the compounds. Spectra were collected for CYP121 (8.7or 5 μM) at three different ligand-to-protein ratios (0.5:1,1:1, and 25:1) and in the presence of DMSO-d6 (2.2% or 5% v/v) alone. The concentration of protein or DMSO-d6 did not affect the quality of the spectraor ligand binding interactions, but higher concentrations of DMSO-d6 reduced the charge state of the protein. Ligandbinding stoichiometry was calculated from the difference in mass ofligand-bound and unbound (orange dashed lines) protein peaks dividedby the molecular weight of ligands (Table S1, Supporting Information).

Mentions: Native mass spectra were collected for CYP121 with the mostpotent Ar3 analogue 26a, Ar2 analogues 19a, 24a, 25a, and 25b and thebiaryl retrofragment 6 (Figure 9a–c). Allanalogues formed stable complexes with the CYP121 monomer and dimer.The proportion of ligand-bound CYP121 increased in a concentration-dependentmanner as the ratio of ligand-to-protein was increased from sub- (0.5:1)to superstoichiometric (25:1) values. The relative occupancy of monomericand dimeric CYP121 and binding stoichiometry observed at three differentligand concentrations allowed analogues to be ranked in terms of theirrelative binding affinities (Table S1,Supporting Information). The rank order of affinities correlated wellwith KD values calculated previously byITC and UV–vis optical titration (Table 2). Both the CYP121 monomer and dimer wereentirely in the bound state at stoichiometric ligand-to-protein concentrationsof Ar2 analogues 25a and 25b, consistentwith the low nanomolar affinities of these compounds. In contrast,complete occupancy of the CYP121 dimer by the more weakly bindinganalogues 19a and 26a was not achieved untilligands were present in 25-fold excess ligand-to-protein concentrations.Despite the comparable KD of retrofragment 6 to Ar2 analogue 19a, the CYP121 dimer remainedsingly bound even at 25-fold excess concentration of 6. This apparent difference in binding strength between 6 and 19a observed by native mass spectrometry mightreflect the different binding mode of the two analogues. Heme bindinginteractions made by 19a would likely be strengthenedin the gas phase of the mass spectrometer, while the closer proximityof 6 to the entrance of the active site could allow theligand to be displaced more easily during ionization. The double-occupancyof the CYP121 monomer observed for analogue 19a in X-raycrystallography was not detected within the concentration ranges testedin native mass spectrometry experiments (2.5–218 μM ligand).


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)

Representativenative mass spectra of CYP121 and selected compounds 26a, 25a, and 19a demonstrated thatcompounds formed stable complexes with both monomeric (m/z 5250–6750) and dimeric (m/z 5250–6750) CYP121. The number of boundligands (orange spheres) increased in a concentration-dependent mannerand was proportionate to the binding affinity (KD) of the compounds. Spectra were collected for CYP121 (8.7or 5 μM) at three different ligand-to-protein ratios (0.5:1,1:1, and 25:1) and in the presence of DMSO-d6 (2.2% or 5% v/v) alone. The concentration of protein or DMSO-d6 did not affect the quality of the spectraor ligand binding interactions, but higher concentrations of DMSO-d6 reduced the charge state of the protein. Ligandbinding stoichiometry was calculated from the difference in mass ofligand-bound and unbound (orange dashed lines) protein peaks dividedby the molecular weight of ligands (Table S1, Supporting Information).
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Representativenative mass spectra of CYP121 and selected compounds 26a, 25a, and 19a demonstrated thatcompounds formed stable complexes with both monomeric (m/z 5250–6750) and dimeric (m/z 5250–6750) CYP121. The number of boundligands (orange spheres) increased in a concentration-dependent mannerand was proportionate to the binding affinity (KD) of the compounds. Spectra were collected for CYP121 (8.7or 5 μM) at three different ligand-to-protein ratios (0.5:1,1:1, and 25:1) and in the presence of DMSO-d6 (2.2% or 5% v/v) alone. The concentration of protein or DMSO-d6 did not affect the quality of the spectraor ligand binding interactions, but higher concentrations of DMSO-d6 reduced the charge state of the protein. Ligandbinding stoichiometry was calculated from the difference in mass ofligand-bound and unbound (orange dashed lines) protein peaks dividedby the molecular weight of ligands (Table S1, Supporting Information).
Mentions: Native mass spectra were collected for CYP121 with the mostpotent Ar3 analogue 26a, Ar2 analogues 19a, 24a, 25a, and 25b and thebiaryl retrofragment 6 (Figure 9a–c). Allanalogues formed stable complexes with the CYP121 monomer and dimer.The proportion of ligand-bound CYP121 increased in a concentration-dependentmanner as the ratio of ligand-to-protein was increased from sub- (0.5:1)to superstoichiometric (25:1) values. The relative occupancy of monomericand dimeric CYP121 and binding stoichiometry observed at three differentligand concentrations allowed analogues to be ranked in terms of theirrelative binding affinities (Table S1,Supporting Information). The rank order of affinities correlated wellwith KD values calculated previously byITC and UV–vis optical titration (Table 2). Both the CYP121 monomer and dimer wereentirely in the bound state at stoichiometric ligand-to-protein concentrationsof Ar2 analogues 25a and 25b, consistentwith the low nanomolar affinities of these compounds. In contrast,complete occupancy of the CYP121 dimer by the more weakly bindinganalogues 19a and 26a was not achieved untilligands were present in 25-fold excess ligand-to-protein concentrations.Despite the comparable KD of retrofragment 6 to Ar2 analogue 19a, the CYP121 dimer remainedsingly bound even at 25-fold excess concentration of 6. This apparent difference in binding strength between 6 and 19a observed by native mass spectrometry mightreflect the different binding mode of the two analogues. Heme bindinginteractions made by 19a would likely be strengthenedin the gas phase of the mass spectrometer, while the closer proximityof 6 to the entrance of the active site could allow theligand to be displaced more easily during ionization. The double-occupancyof the CYP121 monomer observed for analogue 19a in X-raycrystallography was not detected within the concentration ranges testedin native mass spectrometry experiments (2.5–218 μM ligand).

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