Allosteric Activation of Cytochrome P450 3A4 via Progesterone Bioconjugation

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of the majority of drugs. As such, it is implicated in many adverse drug–drug and food–drug interactions, and is of significant interest to the pharmaceutical industry. This enzyme is known to simultaneously bind multiple ligands and display atypical enzyme kinetics, suggestive of allostery and cooperativity. As well, evidence of a postulated peripheral allosteric binding site has provoked debate around its significance and location. We report the use of bioconjugation to study the significance of substrate binding at the proposed allosteric site and its effect on CYP3A4 activity. CYP3A4 mutants were created and covalently modified with various small molecules including progesterone. The labeled mutants displayed enhanced kinetic stability and improved activity in testosterone and 7-benzyloxy-(4-trifluoromethyl)­coumarin oxidation assays. Our work applies a new strategy to study cytochrome P450 allostery and supports the hypothesis that substrate binding at the postulated allosteric site of CYP3A4 may induce functional cooperativity

Inhibitors of Aminoglycoside Resistance Activated in Cells

The most common mechanism of resistance to aminoglycoside antibiotics entails bacterial expression of drug-metabolizing enzymes, such as the clinically widespread aminoglycoside <i>N</i>-6′-acetyltransferase (AAC­(6′)). Aminoglycoside-CoA bisubstrates are highly potent AAC(6′) inhibitors; however, their inability to penetrate cells precludes <i>in vivo</i> studies. Some truncated bisubstrates are known to cross cell membranes, yet their activities against AAC(6′) are in the micromolar range at best. We report here the synthesis and biological activity of aminoglycoside-pantetheine derivatives that, although devoid of AAC(6′) inhibitory activity, can potentiate the antibacterial activity of kanamycin A against an aminoglycoside-resistant strain of <i>Enterococcus faecium</i>. Biological studies demonstrate that these molecules are potentially extended to their corresponding full-length bisubstrates by enzymes of the coenzyme A biosynthetic pathway. This work provides a proof-of-concept for the utility of prodrug compounds activated by enzymes of the coenzyme A biosynthetic pathway, to resensitize resistant strains of bacteria to aminoglycoside antibiotics

Site-Specific Fluorescent Labeling and Oriented Immobilization of a Triple Mutant of CYP3A4 via C64

The generation of site-specific bioconjugates of proteins is highly desired for a number of biophysical and nanotechnological applications. To this end, many strategies have been developed that allow the specific modification of certain canonical amino acids and, more recently, noncanonical functional groups. P450 enzymes are heme-dependent monooxygenases involved in xenobiotic metabolism and in the biosynthesis of a variety of secondary metabolites. We became interested in the site-specific modification of these enzymes, CYP3A4 in particular, through our studies of their <i>in vitro</i> biocatalytic properties and our desire to exploit their remarkable ability to oxidize unactivated C–H bonds in a regio- and stereospecific manner. Obtained via a partial cysteine-depletion approach, a functional triple mutant of CYP3A4 (C98S/C239S/C468G) is reported here which is singly modified at C64 by maleimide-containing groups. While cysteine-labeling of the wild-type enzyme abolished >90% of its enzymatic activity, this mutant retained ≥75% of the activity of the unmodified wild-type enzyme with 9 of the 18 maleimides that were tested. These included both fluorescent and solid-supported maleimides. The loss of activity observed after labeling with some maleimides is attributed to direct enzyme inhibition rather than to steric effects. We also demonstrate the functional immobilization of this mutant on maleimide-functionalized agarose resin and silica microspheres

3‑Oxo-hexahydro‑1<i>H</i>‑isoindole-4-carboxylic Acid as a Drug Chiral Bicyclic Scaffold: Structure-Based Design and Preparation of Conformationally Constrained Covalent and Noncovalent Prolyl Oligopeptidase Inhibitors

Bicyclic chiral scaffolds are privileged motifs in medicinal chemistry. Over the years, we have reported covalent bicyclic prolyl oligopeptidase inhibitors that were highly selective for POP over a number of homologous proteins. Herein, we wish to report the structure-based design and synthesis of a novel class of POP inhibitors based on hexahydroisoindoles. A docking study guided the selection of structures for synthesis. The stereochemistry, decoration, and position within the molecule of the bicyclic scaffolds were assessed virtually. Following the synthesis of the best candidates, <i>in vitro</i> assays revealed that one member of this chemical series was more active than any of our previous inhibitors with a <i>K</i>_<i>i</i> of 1.0 nM. Additional assays also showed that the scaffold of this potent inhibitor, in contrast to one of our previously reported chemical series, is highly metabolically stable, despite the foreseen potential sites of metabolism. Interestingly, computer docking calculations accurately predicted the optimal features of the inhibitors

3‑Oxo-hexahydro‑1<i>H</i>‑isoindole-4-carboxylic Acid as a Drug Chiral Bicyclic Scaffold: Structure-Based Design and Preparation of Conformationally Constrained Covalent and Noncovalent Prolyl Oligopeptidase Inhibitors

Bicyclic chiral scaffolds are privileged motifs in medicinal chemistry. Over the years, we have reported covalent bicyclic prolyl oligopeptidase inhibitors that were highly selective for POP over a number of homologous proteins. Herein, we wish to report the structure-based design and synthesis of a novel class of POP inhibitors based on hexahydroisoindoles. A docking study guided the selection of structures for synthesis. The stereochemistry, decoration, and position within the molecule of the bicyclic scaffolds were assessed virtually. Following the synthesis of the best candidates, <i>in vitro</i> assays revealed that one member of this chemical series was more active than any of our previous inhibitors with a <i>K</i>_<i>i</i> of 1.0 nM. Additional assays also showed that the scaffold of this potent inhibitor, in contrast to one of our previously reported chemical series, is highly metabolically stable, despite the foreseen potential sites of metabolism. Interestingly, computer docking calculations accurately predicted the optimal features of the inhibitors

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 10

<p><b>Percentage parasite proliferation of the different lines in the presence of the pantothenate analogues (a) N5-trz-C1-Pan and (b) <i>N</i>-PE-αMe-PanAm, and inhibition of [¹⁴C]pantothenate phosphorylation in parasite lysates by various pantothenate analogues: (c) PanOH, (d) CJ-15,801, (e) N5-trz-C1-Pan and (f) <i>N</i>-PE-αMe-PanAm.</b> Symbols represent Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasite lines. The Y-axes of c−f indicate the percentage of total pantothenate phosphorylation. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate for the parasite proliferation assays and duplicate for the phosphorylation assays. Error bars represent SEM and are not visible if smaller than the symbols.</p

The pantothenate requirement of the different parasite lines observed in this study.

<p>(a) Percentage proliferation of parasites grown in different pantothenate concentrations. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate. Error bars represent SEM and are not visible if smaller than the symbols. For clarity, only data from the Parent (white circles), CJ-A (black diamonds), and CJ-A^+WTPfPanK1 (grey diamonds) lines are shown. (b) The pantothenate stimulatory concentration 50 (SC₅₀) values obtained for Parent, PanOH-A, PanOH-B, CJ-A, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 line parasites, which is the concentration of pantothenate required in the medium to support parasite proliferation by 50% (with 100% set to parasites grown in complete medium containing 1 <i>μ</i>M pantothenate). Errors represent SEM (n ≥ 3). An asterisk indicates that the value is significantly different from that obtained for the Parent line (CJ-A SC₅₀ value 95% CI compared to Parent: 2.7 to 30.9). No significant difference was observed between the SC₅₀ value of the Parent and those of PanOH-A, PanOH-B, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 (95% CI compared to Parent: PanOH-A = -6.8 to 7.1, PanOH-B = -3.8 to 9.3, Parent^+WTPfPanK1 = -9.3 to 3.3 & CJ-A^+WTPfPanK1 = -7.8 to 6.6).</p

Mutations in <i>Pfpank1</i> and the affected residues within the <i>Pf</i>PanK1 protein.

<p>(a) The single nucleotide polymorphisms detected in the <i>Pfpank1</i> gene (accession number: PF3D7_1420600) of PanOH-A, PanOH-B and CJ-A, and the corresponding amino acid changes in the <i>Pf</i>PanK1 protein. (b) A three-dimensional homology model of the <i>Pf</i>PanK1 protein (pink) based on the solved structure of human PanK3 (PDB ID: 5KPR), overlaid on the human PanK3 structure in its active conformation (blue), with an ATP analogue (AMPPNP; carbon atoms coloured green) and pantothenate (carbon atoms coloured yellow) bound. <i>Pf</i>PanK1 shares 28% sequence identity with human PanK3 over the parts of the protein that have been modeled. Red spheres indicate the residues (G95 and D507) affected by the mutations in the parasite proteins. Human PanK3 has been shown to exist as a dimer. Here, individual monomers are shown in different shades of pink and blue.</p

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 2

<p><b>Percentage proliferation of parasites from the Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) lines in the presence of (a) PanOH, (b) CJ-15,801 or (c) chloroquine.</b> Drug-pressured lines were generated by exposing Parent line parasites to 11 − 13 weeks of continuous drug-pressuring with either PanOH (for PanOH-A and PanOH-B) or CJ-15,801 (for CJ-A), followed by limiting dilution cloning. Values are averaged from ≥ 4 independent experiments, each carried out in triplicate. All error bars represent SEM. Error bars are not visible if smaller than the symbols.</p

The fitness of the different mutant lines generated in this study relative to the Parent line as determined from parasite competition assays.

<p>(a) A flow chart illustrating how the competition assay was performed. For each competition culture, an equal number of parasites from the Parent and a mutant line were combined into a single flask. These mixed cultures were maintained for a period of 6 weeks. The fitness cost associated with the <i>Pfpank1</i> mutations was assessed by determining the PanOH sensitivity of the mixed cultures: (b) Parent vs PanOH-A (grey triangles), (c) Parent vs PanOH-B (grey squares) and (d) Parent vs CJ-A (grey diamonds), at Week 0 (W0; dashed lines) and Week 6 (W6; solid lines). It was expected that the greater the fitness cost to the mutant, the greater the shift of its mixed culture PanOH dose-response curve toward the Parent line curve after 6 weeks. Arrows indicate this shift between W0 and W6. The parasite proliferation curves (dotted lines) of the respective mutant clones (black symbols) and Parent line (white circles) are also shown for comparison. Values for the mixed cultures are averaged from 2 independent experiments, each carried out in triplicate. Error bars represent SEM (n ≥ 4) for the individual cultures and range/2 for the mixed cultures, and are not visible if smaller than the symbols.</p