Week 1 - Phase 1 Metabolism: Detailed Study of Esterases and Epoxide Hydrolases

Phase 1 metabolism involves more than just the Cytochrome P450 (CYP450) system; it includes other critical enzymes such as Epoxide Hydrolase and Various Esterases.
These enzymes catalyze the addition of water (hydrolysis or hydration) to specific functional groups, such as esters, amides, and epoxides.
Key Goals of these enzymes in metabolism include:
- Deactivation of active drug molecules.
- Activation of prodrugs into their active forms.
- Increasing the water solubility of compounds to facilitate renal elimination.

Esterase enzymes are responsible for the hydrolysis of drugs containing ester and amide functional groups.
Genetic Basis: Human carboxyl esterases are primarily encoded by two genes:
- $CES1$ (encoding for HCE1).
- $CES2$ (encoding for HCE2).
Structural Characteristics: The human carboxyl esterase enzyme is a trimeric enzyme, meaning it consists of three associated amino acid chains.
Reaction Mechanism (Serine Esterase):
- The active site contains a serine residue whose hydroxyl ( $OH$ ) group is positioned to interact with the carbonyl group ( $C=O$ ) of the substrate.
- A catalytic triad, involving a histidine residue, facilitates the reaction. The histidine deprotonates the serine, making the oxygen a strong nucleophile.
- Step 1: The activated oxygen of serine attacks the carbonyl carbon of the ester, forming a tetrahedral intermediate.
- Step 2: The alcohol component of the original ester is released, and the carboxylic acid portion remains covalently bonded to the enzyme as an ester.
- Step 3: A molecule of water is activated by the histidine (intermolecular interaction) to become a good nucleophile.
- Step 4: The water molecule attacks the enzyme-bound intermediate, hydrolyzing the bond and releasing the final carboxylic acid product.
- Outcome: The ester is successfully split into an alcohol and a carboxylic acid ( $\text{Ester} + H_2O \rightarrow \text{Alcohol} + \text{Carboxylic Acid}$ ).

Enzymes function by lowering the activation energy ( $E_{a}$ ) of a chemical reaction.
Uncatalyzed Reaction Example: Hydrolyzing isopropyl acetate in plain water at room temperature might take several years or even decades because the activation energy is too high.
Reaction Equation: $\text{Isopropyl Acetate} + H_2O \rightarrow \text{Acetic Acid} + \text{Isopropanol}$ .
Chemical Catalysis in a Lab: To speed up this reaction, chemists use two methods:
- Adding a catalyst (e.g., a few drops of $H_2SO_4$ to provide hydronium ions, $H_3O^+$ ) to protonate the carbonyl and provide an alternative, lower-energy pathway.
- Increasing the temperature ( $T$ ). For example, heating the reaction to $100^\text{o}C$ ( $373.15\,K$ ) increases the number of molecules with enough energy to overcome the barrier.
Biological Catalysis: In the human body, temperature is constant (approx. $310.15\,K$ ), so carboxyl esterases lower the $E_a$ significantly through hydrogen bonding and the catalytic triad, allowing the reaction to occur in minutes rather than decades.
Arrhenius Equation: The rate constant ( $k$ ) is defined by $k = A e^{-\frac{E_a}{RT}}$ :
- $A$ is the pre-exponential factor.
- $E_a$ is the activation energy in Joules.
- $R$ is the gas constant.
- $T$ is the temperature in Kelvin.

Aspirin is hydrolyzed by Human Carboxyl Esterase 2 (HCE2) into salicylic acid.
Mixed Clinical Outcome:
- Activation: This reaction activates the anti-inflammatory activity of the molecule.
- Deactivation: This reaction deactivates the antiplatelet activity of aspirin.

Oseltamivir contains an ethyl ester group that is hydrolyzed by Human Carboxyl Esterase 1 (HCE1).
Outcome: Activation: Oseltamivir is a prodrug; the hydrolysis converts it into a carboxylic acid, which is the active species.
The resulting carboxylic acid has high affinity for the neuraminidase enzyme due to interactions with hydrogen bond donors in the enzyme active site.

Contains a methyl ester ( $CH_3$ group) that undergoes hydrolysis by carboxyl esterases.
Outcome: Deactivation: The parent methyl ester is the active species; the resulting carboxylic acid is inactive and highly water-soluble, allowing it to be eliminated renally.

Prasugrel: Hydrolyzed by esterases at an O-acetate group. A subsequent step involves hydration, though complexly mediated by Cytochrome P450.
Clopidogrel: Undergoes deactivation via esterase hydrolysis of its methyl ester into an inactive carboxylic acid metabolite.
Mechanism Note: In cases like clopidogrel, the active metabolite is formed through a different pathway (oxidation by CYP450), while the esterase pathway is a competing deactivation route.

Cocaine is subject to hydrolysis by multiple enzymes at two different sites:
- Butyrylcholinesterase (BChE): Hydrolyzes the benzoyl ester.
- Human Carboxyl Esterase (HCE): Hydrolyzes the methyl ester to form benzoylecgonine.
Outcome: Deactivation: Both hydrolysis reactions lead to the loss of cocaine's pharmacological activity.

Epoxide hydrolases ( $EPHX1$ and $EPHX2$ ) catalyze the addition of water to highly strained, three-membered epoxide rings (also known as arene oxides when part of an aromatic system).
Substrates: Epoxides are often formed as reactive intermediates during the metabolism of alkenes or aromatic groups by Cytochrome P450.
Chemical Reactivity: Epoxides are highly electrophilic and reactive due to ring strain. While normal ethers are stable, the weak carbon-oxygen bonds in the strained epoxide ring allow nucleophiles to attack and open the ring.
Mechanism:
- The epoxide binds to the enzyme.
- A carboxylate group in the enzyme's catalytic triad acts as a nucleophile to open the epoxide ring, forming an ester intermediate.
- A water molecule then hydrolyzes the ester intermediate, resulting in a 1,2-diol (a molecule with two adjacent hydroxyl groups).
Biological Examples:
- Dietary Lipids: Unsaturated fatty acids like oleic acid and linoleic acid can form epoxides via CYP450 or superoxide (reactive oxygen species).
- Detoxification: Epoxide hydrolase serves as a detoxification mechanism to prevent electrophilic epoxides from reacting with cellular proteins.

Soluble epoxide hydrolase inhibitors are currently being developed as pharmaceutical treatments for neuropathic pain.

The transcript highlights several errors found in published academic articles regarding chemical structures and metabolic pathways:
- Prasugrel: An article on "Impact of Genetic Polymorphisms… on Clopidogrel and Prasugrel Response" (2013) incorrectly illustrated the structure with an inverted methyl ester (methyl ester vs. O-acetate ester).
- Cocaine: An article on "Experimental treatments for cocaine toxicity" (2013) incorrectly attributed N-demethylation of cocaine to Butyrylcholinesterase. N-demethylation is actually performed by Cytochrome P450 (specifically $CYP3A4$ ), as established in earlier literature (e.g., 1994).
- These discrepancies underscore the importance of verifying chemical structures and mechanisms against established chemistry principles.