Domain-Swap Dimerization of Acanthamoeba castellanii CYP51 and a Unique Mechanism of Inactivation by Isavuconazole

Sharma, Vandna; Shing, Brian; Hernández-Álvarez, Lilian; Debnath, Anjan; Podust, L.M.

doi:10.1124/molpharm.120.000092

Cited by 4 publications

(21 citation statements)

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“…The oligomeric state of target enzymes is an important interactomic factor. It was shown that a dimeric form is characteristic for cholestenol delta-isomerase (EBP) [ 40 ], sterol 14-alpha-demethylase (CYP51A1) [ 41 ] and MVK [ 42 ]. Squalene epoxidase (SQLE) exists in a monomeric form [ 43 ], while 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) forms tight tetramers that implies the influence of the enzyme’s oligomeric state on the activity and mechanisms for allosteric modulation by ligands [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

et al. 2021

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A global protein interactome ensures the maintenance of regulatory, signaling and structural processes in cells, but at the same time, aberrations in the repertoire of protein–protein interactions usually cause a disease onset. Many metabolic enzymes catalyze multistage transformation of cholesterol precursors in the cholesterol biosynthesis pathway. Cancer-associated deregulation of these enzymes through various molecular mechanisms results in pathological cholesterol accumulation (its precursors) which can be disease risk factors. This work is aimed at systematization and bioinformatic analysis of the available interactomics data on seventeen enzymes in the cholesterol pathway, encoded by HMGCR, MVK, PMVK, MVD, FDPS, FDFT1, SQLE, LSS, DHCR24, CYP51A1, TM7SF2, MSMO1, NSDHL, HSD17B7, EBP, SC5D, DHCR7 genes. The spectrum of 165 unique and 21 common protein partners that physically interact with target enzymes was selected from several interatomic resources. Among them there were 47 modifying proteins from different protein kinases/phosphatases and ubiquitin-protein ligases/deubiquitinases families. A literature search, enrichment and gene co-expression analysis showed that about a quarter of the identified protein partners was associated with cancer hallmarks and over-represented in cancer pathways. Our results allow to update the current fundamental view on protein–protein interactions and regulatory aspects of the cholesterol synthesis enzymes and annotate of their sub-interactomes in term of possible involvement in cancers that will contribute to prioritization of protein targets for future drug development.

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Section: Resultsmentioning

confidence: 99%

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

et al. 2021

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“…AcCYP51 (UniProtKB L8GJB3) and NfCYP51 (UniProtKB A0A2H4A2U9_NAEFO) proteins were expressed in Escherichia coli and purified according to the published protocols. , Both proteins were expressed with a 6xHis tag attached to the C-terminus and modified N-terminal membrane-anchoring residues. Specifically, AcCYP51 and NfCYP51 had 42 N-terminal residues replaced by MAKKTSSKGK and 34 N-terminal residues replaced by MAKKTSSKGKL, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Accordingly, CYP51s are popular drug targets in pathogenic fungi and protozoa, including kinetoplastids and amoebae. − Acanthamoeba castellanii CYP51 (AcCYP51) is a pharmacologically validated drug target in A. castellanii; ,, however, the effectiveness of azole antifungal agents has been found to vary greatly …”

Section: Introductionmentioning

confidence: 99%

“…Although CYPs characterized in vitro are catalytically competent as monomers, biophysical and biochemical studies revealed oligomer formation in microsomal membranes, which may play regulatory or stabilizing roles. − In the absence of a bilayer membrane, homodimerization has been demonstrated by size-exclusion chromatography and X-ray crystallography only for the recombinant CYP51 of the free-living amoeba and opportunistic human pathogen A. castellanii (AcCYP51) …”

Section: Introductionmentioning

confidence: 99%

“…In the presence of inhibitory drugs (clotrimazole and isavuconazole), AcCYP51 was monomeric in the crystals. The monomeric state had a more flexible polypeptide chain, as indicated by greater mean B-factors and 74 disordered N-terminal residues in the AcCYP51–isavuconazole complex . In the AcCYP51–clotrimazole complex, the 74 N-terminal residues were resolved and folded into the typical CYP monomer structure.…”

Section: Introductionmentioning

confidence: 99%

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Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51

et al. 2022

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Acanthamoeba castellanii is a free-living amoeba that can cause severe eye and brain infections in humans. At present, there is no uniformly effective treatment for any of these infections. However, sterol 14α-demethylases (CYP51s), heme-containing cytochrome P450 enzymes, are known to be validated drug targets in pathogenic fungi and protozoa. The catalytically active P450 form of CYP51 from A. castellanii (AcCYP51) is stabilized against conversion to the inactive P420 form by dimerization. In contrast, Naegleria fowleri CYP51 (NfCYP51) is monomeric in its active P450 and inactive P420 forms. For these two CYP51 enzymes, we have investigated the interplay between the enzyme activity and oligomerization state using steady-state and time-resolved UV–visible absorption spectroscopy. In both enzymes, the P450 → P420 transition is favored under reducing conditions. The transition is accelerated at higher pH, which excludes a protonated thiol as the proximal ligand in P420. Displacement of the proximal thiolate ligand is also promoted by adding exogenous nitrogenous ligands (N-ligands) such as imidazole, isavuconazole, and clotrimazole that bind at the opposite, distal heme side. In AcCYP51, the P450 → P420 transition is faster in the monomer than in the dimer, indicating that the dimeric assembly is critical for stabilizing thiolate coordination to the heme and thus for sustaining AcCYP51 activity. The spectroscopic experiments were complemented with size-exclusion chromatography and X-ray crystallography studies. Collectively, our results indicate that effective inactivation of the AcCYP51 function by azole drugs is due to synergistic interference with AcCYP51 dimerization and promoting irreversible displacement of the proximal heme–thiolate ligand.

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Combating the causative agent of amoebic keratitis, Acanthamoeba castellanii, using Padina pavonica alcoholic extract: toxicokinetic and molecular docking approaches

Abdel-Hakeem,

Hassan,

Hifney

et al. 2024

Sci Rep

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Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography–mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.

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Domain-Swap Dimerization of Acanthamoeba castellanii CYP51 and a Unique Mechanism of Inactivation by Isavuconazole

Cited by 4 publications

References 68 publications

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer Context

Homodimerization Counteracts the Detrimental Effect of Nitrogenous Heme Ligands on the Enzymatic Activity of Acanthamoeba castellanii CYP51

Combating the causative agent of amoebic keratitis, Acanthamoeba castellanii, using Padina pavonica alcoholic extract: toxicokinetic and molecular docking approaches

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