Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound.

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes.

The study of drug metabolism is fundamental to drug discovery and development (DDD) since by mediating the clearance of most drugs, metabolic enzymes influence their bioavailability and duration of action. Biotransformation can also produce pharmacologically active or toxic products, which complicates the evaluation of the therapeutic benefit versus liability of potential drugs but also provides opportunities to explore the chemical space around a lead. The structures and relative abundance of metabolites are determined by the substrate and reaction specificity of biotransformation enzymes and their catalytic efficiency.

Future of Biotransformation Science in the Pharmaceutical Industry.

Over the past decades, the number of scientists trained in departments dedicated to traditional medicinal chemistry, biotransformation and/or chemical toxicology have seemingly declined. Yet, there remains a strong demand for such specialized skills in the pharmaceutical industry, particularly within drug metabolism/pharmacokinetics (DMPK) departments. In this position paper, the members of the Biotransformation, Mechanisms, and Pathways Focus Group (BMPFG) steering committee reflect on the diverse roles and responsibilities of scientists trained in the biotransformation field in pharmaceutical companies and contract research organizations.

Case Study 10: A Case to Investigate Acetyl Transferase Kinetics.

Major routes of metabolism for marketed drugs are predominately driven by enzyme families such as cytochromes P450 and UDP-glucuronosyltransferases. Less studied conjugative enzymes, like N-acetyltransferases (NATs), are commonly associated with detoxification pathways. However, in the clinic, the high occurrence of NAT polymorphism that leads to slow and fast acetylator phenotypes in patient populations has been linked to toxicity for a multitude of drugs.

Investigation of Ocular Bioactivation Potential and the Role of Cytochrome P450 2D Enzymes in Rat.

Background: Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification.

Do We Need to Study Metabolism and Distribution in the Eye: Why, When, and Are We There Yet?

The liver is known to be the principal site of drug metabolism. Depending on the route of administration, especially in cases of topical and local delivery, evaluation of local drug metabolism in extrahepatic tissues is vital to assess fraction of the drug metabolized. This parameter becomes important from the point of view of drug availability or the contribution to overall clearance.

Human cytochrome P450 27C1 catalyzes 3,4-desaturation of retinoids.

In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction.

Cyp27c1 Red-Shifts the Spectral Sensitivity of Photoreceptors by Converting Vitamin A1 into A2.

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown.

Oxidation of pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene by human cytochrome P450 2A13.

1. The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2.

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine, a Key Step in Endogenous Mammalian Morphine Biosynthesis.

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated.

Structural and kinetic basis of steroid 17α,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5.

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol.

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

The "GyrA-box" is required for the ability of DNA gyrase to wrap DNA and catalyze the supercoiling reaction.

DNA gyrase is the only topoisomerase that can introduce negative supercoils into DNA. It is thought that the binding of conventional type II topoisomerases, including topoisomerase IV, to DNA takes place at the catalytic domain across the DNA gate, whereas DNA gyrase binds to DNA not only at the amino-terminal catalytic domain but also at the carboxyl-terminal domain (CTD) of the GyrA subunit. The binding of the GyrA CTD to DNA allows gyrase to wrap DNA around itself and catalyze the supercoiling reaction.