Design and synthesis of aryl-piperidine derivatives as potent and selective PET tracers for cholesterol 24-hydroxylase (CH24H).

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC50 = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a18F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC50 = 8.8 nM) and 3g (IC50 = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [18F]3f and [18F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers.

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

Potent mechanism-based inactivation of cytochrome P450 2B4 by 9-ethynylphenanthrene: implications for allosteric modulation of cytochrome P450 catalysis.

The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 µM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, ß1-ß2 loop, and ß4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.

Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme.

Residues located outside the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability and drug metabolism. However, contributions of non-active-site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over 11 sites were constructed, expressed in Escherichia coli and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D and E474Q) were chosen for detailed functional analysis. Among these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for wild-type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin, 7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M) for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yielded distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.

Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity.

A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.

Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR.

Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

Structures of cytochrome P450 2B4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .

Prior X-ray crystal structures of rabbit cytochrome P450 2B4 (2B4) in complexes with various imidazoles have demonstrated markedly different enzyme conformations depending on the size of the inhibitor occupying the active site. In this study, structures of 2B4 were determined with the antiplatelet drugs clopidogrel and ticlopidine, which were expected to have greater freedom of movement in the binding pocket. Ticlopidine could be modeled into the electron density maps in two distinct orientations, both of which are consistent with metabolic data gathered with other mammalian P450 enzymes. Results of ligand docking and heme-induced NMR relaxation of drug protons showed that ticlopidine was preferentially oriented with the chlorophenyl group closest to the heme. Because of its stereocenter, clopidogrel was easier to fit in the electron density and exhibited a single orientation, which points the chlorophenyl ring toward the heme. The C(α) traces of both complexes aligned very well with each other and revealed a compact, closed structure that resembles the conformation observed in two previously determined 2B4 structures with the small molecule inhibitors 4-(4-chlorophenyl)imidazole and 1-(4-chlorophenyl)imidazole. The 2B4 active site is able to accommodate small ligands by moving only a small number of side chains, suggesting that ligand reorientation is energetically favored over protein conformational changes for binding of these similarly sized molecules. Adjusting both protein conformation and ligand orientation in the active site gives 2B4 the flexibility to bind to the widest range of molecules, while also being energetically favorable.

Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity.

The Tbd_0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 A resolution X-ray crystal structure reported here revealed a hexameric assembly with D(3) symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

Kinetic properties of ATP sulfurylase and APS kinase from Thiobacillus denitrificans.

The Thiobacillus denitrificans genome contains two sequences corresponding to ATP sulfurylase (Tbd_0210 and Tbd_0874). Both genes were cloned and expressed protein characterized. The larger protein (Tbd_0210; 544 residues) possesses an N-terminal ATP sulfurylase domain and a C-terminal APS kinase domain and was therefore annotated as a bifunctional enzyme. But, the protein was not bifunctional because it lacked ATP sulfurylase activity. However, the enzyme did possess APS kinase activity and displayed substrate inhibition by APS. Truncated protein missing the N-terminal domain had <2% APS kinase activity suggesting the function of the inactive sulfurylase domain is to promote the oligomerization of the APS kinase domains. The smaller gene product (Tbd_0874; 402 residues) possessed strong ATP sulfurylase activity with kinetic properties that appear to be kinetically optimized for the direction of APS utilization and ATP+sulfate production, which is consistent with an enzyme that functions physiologically to produce inorganic sulfate.

Crystal structures of cytochrome P450 2B4 in complex with the inhibitor 1-biphenyl-4-methyl-1H-imidazole: ligand-induced structural response through alpha-helical repositioning.

Two different ligand occupancy structures of cytochrome P450 2B4 (CYP2B4) in complex with 1-biphenyl-4-methyl-1H-imidazole (1-PBI) have been determined by X-ray crystallography. 1-PBI belongs to a series of tight binding, imidazole-based CYP2B4 inhibitors. 1-PBI binding to CYP2B4 yields a type II spectrum with a K(s) value of 0.23 microM and inhibits enzyme activity with an IC(50) value of 0.035 microM. Previous CYP2B4 structures have shown a large degree of structural movement in response to ligand size. With two phenyl rings, 1-PBI is larger than 1-(4-chlorophenyl)imidazole (1-CPI) and 4-(4-chlorophenyl)imidazole (4-CPI) but smaller than bifonazole, which is branched and contains three phenyl rings. The CYP2B4-1-PBI complex is a structural intermediate to the closed CPI and the open bifonazole structures. The B/C-loop reorganizes itself to include two short partial helices while closing one side of the active site. The F-G-helix cassette pivots over the I-helix in direct response to the size of the ligand in the active site. A cluster of Phe residues at the fulcrum of this pivot point allows for dramatic repositioning of the cassette with only a relatively small amount of secondary structure rearrangement. Comparisons of ligand-bound CYP2B4 structures reveal trends in plastic region mobility that could allow for predictions of their position in future structures based on ligand shape and size.