A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen)

Frederickson, Martyn; Selvam, Irwin; Evangelopoulos, Dimitrios; McLean, Kirsty J.; Katariya, Mona M.; Tunnicliffe, Richard B.; Kavanagh, Madeline E.; Charoensutthivarakul, Sitthivut; Blankley, Richard T.; Levy, Colin; Carvalho, Luiz Pedro S. de; Leys, D.; Munro, Andrew W.; Coyne, Anthony G.; Abell, Chris

doi:10.1016/j.ejmech.2022.114105

Cited by 5 publications

(8 citation statements)

References 43 publications

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“…This enzyme is, by all indicators, remarkably rigid. An overlay of ten representative CYP121 structures; its ligand-free structure (3G5F), its cYY-bound structure (3G5H), and the 8 most recently deposited crystal structures in the Protein Data Bank (7S0O, 7RUK, 7NQO, 7NQN, 7NQM, 6TEV, 6TET, and 6TE7), , reflects an α carbon root-mean-square deviation of a mere 0.12–0.29 Å. Despite the high degree of conformational uniformity in the crystallographic data, our previous solution NMR studies confirmed that conformational heterogeneity does occur at least at the ligand-sensitive FG-loop of the distal (substrate-binding) surface .…”

Section: Discussionmentioning

confidence: 99%

Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis

Campomizzi,

Uttamrao,

Stallone

et al. 2024

Biochemistry

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The cytochrome P450 enzyme CYP121A1 endogenously catalyzes the formation of a carbon−carbon bond between the two phenol groups of dicyclotyrosine (cYY) in Mycobacterium tuberculosis (Mtb). One of 20 CYP enzymes in Mtb, CYP121A1 continues to garner significant interest as a potential drug target. The accompanying reports the use of 19 F NMR spectroscopy, reconstituted activity assays, and molecular dynamics simulations to investigate the significance of hydrogen bonding interactions that were theorized to stabilize a static active site water network. The active site residue Asn-85, whose hydrogen bonds with the diketopiperazine ring of cYY contributes to a contiguous active site water network in the absence of cYY, was mutated to a serine (N85S) and to a glutamine (N85Q). These conservative changes in the hydrogen bond donor side chain result in inactivation of the enzyme. Moreover, the N85S mutation induces reverse type-I binding as measured by absorbance difference spectra. NMR spectra monitoring the ligand-adaptive FG-loop and the active site Trp-182 side chain confirm that disruption of the active site water network also significantly alters the structure of the active site. These data were consistent with dynamics simulations of N85S and N85Q that demonstrate that a compromised water network is responsible for remodeling of the active site B-helix and a repositioning of cYY toward the heme. These findings implicate a slowly exchanging water network as a critical factor in CYP121A1 function and a likely contributor to the unusual rigidity of the structure.

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Section: Discussionmentioning

confidence: 99%

Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis

Campomizzi,

Uttamrao,

Stallone

et al. 2024

Biochemistry

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Section: Compounds In the Distal Region Of The Heme May Disrupt The H...mentioning

confidence: 99%

“…Further chemical modifications were performed on this series of compounds as a route to developing a specific CYP121A1 inhibitor. The triazole ring was replaced with a pyrimidine ring to improve water solubility [36]. The lipophilicity of the compounds was reduced by replacing unsaturated aromatic sidechains with achiral saturated rings containing solubilizing or heavier heteroatoms such as sulfur [36].…”

Section: Compounds In the Distal Region Of The Heme May Disrupt The H...mentioning

confidence: 99%

Structure–Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1

Padayachee,

Lamb,

Nelson

et al. 2024

IJMS

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Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1’s overall activity were identified and highlighted this enzyme’s rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug–P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure–function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

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“…5,11−19 both Phe-168 and Trp-182 side chains. 20 Notably, these two residues are located one-half helical-turn away from the outward facing nonpolar side chains of Ile-166 and Ile-180; two residues that are known to affect the monomer−dimer equilibrium. 21 Alanine substitutions at either Ile-166 or Ile-180 induce observable monomer formation of CYP121.…”

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confidence: 99%

“…In most published structures, overlapping ligand-binding modes cluster into a region of the active site located between the reactive iron center of the heme and the expected access channel near the distal-facing F and G α-helices. ,− A consistent theme in CYP121 active site–ligand interactions is a hypothetical contribution from π-stacking interactions between aromatic ligands and the active site residues Phe-168 and Trp-182. For example, morpholine derivatives coordinate their indole moiety within 4–5 Å of both Phe-168 and Trp-182 side chains . Notably, these two residues are located one-half helical-turn away from the outward facing nonpolar side chains of Ile-166 and Ile-180; two residues that are known to affect the monomer–dimer equilibrium .…”

mentioning

confidence: 99%

Active Site Aromatic Residues Play a Dual Role in the Substrate Interaction and Protein Structure in Functional Dimers of CYP121A1 of Mycobacterium tuberculosis

et al. 2023

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The essential enzyme CYP121A1 of Mycobacterium tuberculosis forms a functional dimer, which when disrupted results in a decrease of activity and substrate specificity. The crystal structure of CYP121A1 in complex with its substrate dicyclotyrosine (cYY) indicates that the aromatic side chains of Phe-168 and Trp-182 form stabilizing π−π interactions with a tyrosyl ring of cYY. In the enclosed study, we utilize targeted 19 F labeling of aromatic residues to label CYP121A1 for detection by nuclear magnetic resonance (NMR) spectroscopy. 19 F-NMR spectra and functional characterization of mutations to Phe-168 and Trp-182 are combined with all-atom molecular dynamics simulations of substrate-bound and substrate-free CYP121A1. This study shows that these aromatic residues interact with cYY predominantly through π−π stacking. In addition to playing an essential role in substrate binding, these active site residues also stabilize the tertiary and quaternary structures of CYP121A1. An additional unexpected finding was the presence of cYY-induced long-range allostery that affects residues located near the homodimer interface. Taken together, this study highlights a structural relationship between the active site environment of this essential enzyme with its global structure that was previously unknown.

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A new strategy for hit generation: Novel in cellulo active inhibitors of CYP121A1 from Mycobacterium tuberculosis via a combined X-ray crystallographic and phenotypic screening approach (XP screen)

Cited by 5 publications

References 43 publications

Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis

Asparagine-85 Stabilizes a Structural Active Site Water Network in CYP121A1 of Mycobacterium tuberculosis

Structure–Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1

Active Site Aromatic Residues Play a Dual Role in the Substrate Interaction and Protein Structure in Functional Dimers of CYP121A1 of Mycobacterium tuberculosis

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