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. Preclinical drug biotransformation studies are done to quantify in vitro intrinsic clearance to estimate likely in vivo pharmacokinetic parameters, to predict an appropriate dose, and to anticipate interindividual variability in response, including from drug-drug interactions. Such studies need to be done rapidly and cheaply, but native enzymes, especially in microsomes or hepatocytes, do not always produce the full complement of metabolites seen in extrahepatic tissues or preclinical test species. Furthermore, yields of metabolites are usually limiting. Engineered recombinant enzymes can make DDD more comprehensive and systematic. Additionally, as renewable, sustainable, and scalable resources, they can also be used for elegant chemoenzymatic, synthetic approaches to optimize or synthesize candidates as well as metabolites. Here, we will explore how these new tools can be used to enhance the speed and efficiency of DDD pipelines and provide a perspective on what will be possible in the future. The focus will be on cytochrome P450 enzymes to illustrate paradigms that can be extended in due course to other drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Protein engineering can generate enhanced versions of drug-metabolizing enzymes that are more stable, better suited to industrial conditions, and have altered catalytic activities, including catalyzing non-natural reactions on structurally complex lead candidates. When applied to drugs in development, libraries of engineered cytochrome P450 enzymes can accelerate the identification of active or toxic metabolites, help elucidate structure activity relationships, and, when combined with other synthetic approaches, provide access to novel structures by regio- and stereoselective functionalization of lead compounds.

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. The BMPFG is affiliated with the International Society for the Study of Xenobiotics (ISSX) and was specifically created to promote the exchange of ideas pertaining to topics of current and future interest involving the metabolism of xenobiotics (including drugs). The authors also delve into the relevant education and diverse training skills required to successfully nurture the future cohort of industry biotransformation scientists and guide them toward a rewarding career path. The ability of scientists with a background in biotransformation and organic chemistry to creatively solve complex drug metabolism problems encountered during research and development efforts on both small and large molecular modalities is exemplified in five relevant case studies. Finally, the authors stress the importance and continued commitment to training the next generation of biotransformation scientists who are not only experienced in the metabolism of conventional small molecule therapeutics, but are also equipped to tackle emerging challenges associated with new drug discovery modalities including peptides, protein degraders, and antibodies. SIGNIFICANCE STATEMENT: Biotransformation and mechanistic drug metabolism scientists are critical to advancing chemical entities through discovery and development, yet the number of scientists academically trained for this role is on the decline. This position paper highlights the continuing demand for biotransformation scientists and the necessity of nurturing creative ways to train them and guarantee the future growth of this field.

Novel advances in biotransformation and bioactivation research - 2020 year in review.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

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. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid L-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O(3)- and the O(6)-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O(3)-demethylation and the O(6)-demethylation are members of the Fe(II)/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O(3)-demethylation. We report that demethylation of thebaine at the O(6)-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O(6)-demethylation of thebaine by an Fe(II)/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O(6)-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O(6)-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.

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. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.

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. Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3. 1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4. Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5. These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans.

Computer-Aided Design and Biological Evaluation of Diazaspirocyclic D4R Antagonists.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra, resulting in motor dysfunction. Current treatments are primarily centered around enhancing dopamine signaling or providing dopamine replacement therapy and face limitations such as reduced efficacy over time and adverse side effects. To address these challenges, we identified selective dopamine receptor subtype 4 (D4R) antagonists not previously reported as potential adjuvants for PD management. In this study, a library screening and artificial neural network quantitative structure-activity relationship (QSAR) modeling with experimentally driven library design resulted in a class of spirocyclic compounds to identify candidate D4R antagonists. However, developing selective D4R antagonists suitable for clinical translation remains a challenge.

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. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.

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. A key example of this is the observed clinical toxicity in patients who exhibit the slow acetylator phenotype and were treated with isoniazid. Toxicity in patients has led to detailed characterization of the two NAT isoforms and their polymorphic genotypes. Investigation in recombinant enzymes, genotyped hepatocytes, and in vivo transgenic models coupled with acetylator status-driven clinical studies have helped understand the role of NATs in drug development, clinical study design and outcomes, and potential roles in human disease models. The selected case studies herein document NAT enzyme kinetics to explore substrate overlap from two human isoforms, preclinical species considerations, and clinical genotype population concerns.

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. An understanding of drug metabolism in the eye is important for clinical ocular therapeutics and potential drug candidates. METHODS: We aimed to investigate the metabolism of timolol in rat ocular and liver S9 fractions, as well as rat ocular tissue and plasma following a 0.5% topical (ocular) dose of timolol. We explored the potential in vitro metabolic bioactivation in the eye/liver by conducting trapping studies for putative aldehyde and iminium ion intermediates that may arise from the morpholine functionality. RESULTS: Oxidative metabolism of timolol to its major metabolite (M4) in ocular S9 and recombinant rat cytochrome P450 (CYP) isoforms supports the possible role of rat ocular CYP2D2, 2D4, and/or 2D18. Observation of N-acetyl-timolol (M5) is suggestive that the ocular N-acetyltransferases may also play a larger role in ocular disposition of timolol, a previously unreported finding. This research is the first comprehensive report of in vitro ocular metabolism of timolol in rat. CONCLUSION: This study also indicates that in vitro hepatic metabolism is over-predictive of ocular metabolism following topically ocular dosed timolol. The research, herein, highlights the eye as an organ capable of first pass metabolism for topical drugs. Thus, new ophthalmologic considerations for studying and designing long term topical therapies in preclinical species are needed in drug discovery.

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. Examples include fraction metabolized in the gut for oral drugs and contribution of pulmonary or renal clearance to total clearance of a drug. Diseases of the eye represent a rising unmet medical need and a number of therapeutics are currently being developed in the form of small molecules and biologics. Treatment of ocular diseases has expanded to explore various topical formulations and local short- and long-term therapies by ocular routes of administration. Until recently, metabolism in the eye for any species, including human, was not well documented, but this topic is gaining wide interest. Many in vitro-ex vivo models, each with separate pros and cons, are being used for studying ocular metabolism. This review is aimed at providing a perspective on the relevance and application of ocular metabolism, melanin binding, and the use of tissue- and cell-derived ocular models in discovery and preclinical development.

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. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis-retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation.

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. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, ß-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). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (ß-nicotyrine) and (-)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 µM and 110 µM, respectively). ß-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 µM). Neither menthol nor ß-nicotyrine was an inactivator of CYP2A13. Whereas, ß-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 µM, kinact was 0.61 min(-1)). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 µM). The inactivation of CYP2A6 by either ß-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that ß-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.

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. Recent structural studies, however, have revealed striking similarities between the GyrA CTD and the ParC CTD, as well as the ability of the ParC CTD to bind and bend DNA. Thus, the molecular basis of gyrase-mediated wrapping of DNA needs to be reexamined. Here, we have conducted a mutational analysis to determine the role of the "GyrA-box," a 7-amino acid-long motif unique to the GyrA CTD, in determining the DNA binding mode of gyrase. Either a deletion of the entire GyrA-box or substitution of the GyrA-box with 7 Ala residues abolishes the ability of gyrase to wrap DNA around itself and catalyze the supercoiling reaction. However, these mutations do not affect the relaxation and decatenation activities of gyrase. Thus, the presence of a GyrA-box allows gyrase to wrap DNA and catalyze the supercoiling reaction. The consequence of the loss of the GyrA-box during evolution of bacterial type II topoisomerases is discussed.