MedChem Module 4 Lecture 4.6

Introduction to Phase I Enzymes

Presenter: Wenjun Xie, Assistant Professor in the Department of Medicinal Chemistry at UF.
This lecture discusses Phase I enzymes, following a previous discussion on cytochrome P450 (CYP450).
Importance of Phase I Enzymes: Catalyze essential chemical reactions for drug metabolism.

Definition: FMO is a mono oxygenase enzyme that catalyzes the introduction of an oxygen atom to substrates.
Comparison to P450: FMO is similar to P450 in function (it is also a mono oxygenase) and is membrane-bound, allowing closer proximity to lipophilic drugs.
Key Characteristics:
- FMO mainly contributes to drug metabolism but has fewer isoforms (only five: FMO1, FMO2, FMO3, FMO4, FMO5).
- It catalyzes oxidation reactions involving heteroatoms like oxygen, nitrogen, sulfur, and phosphorus in organic molecules.
Flavin Group's Role:
- The flavin group facilitates electron transfer from the cofactor (NADPH).
- Differs from P450 enzymes which require coupling with a reductase.

Example with Chlorpyrifos:
- Substrates: FMO can oxidize sulfur and nitrogen heteroatoms in chlorpyrifos, especially adding oxygen atoms from molecular oxygen to reactions involving these atoms (marked in red).

Definition: Dehydrogenases are another class of enzymes involved in Phase I reactions.
Types of Dehydrogenases:
- Alcohol dehydrogenase (ADH): Acts on alcohols.
- Aldehyde dehydrogenase (ALDH): Acts on aldehydes.
Cofactors Required: Can utilize either NADH or NADPH (difference is a phosphate group).
Differences in Reaction:
- Alcohol Dehydrogenase: No introduction of new oxygen.
- Aldehyde Dehydrogenase: Introduces a new oxygen atom, transforming aldehyde to carboxylic acid.
Location of Enzymes: Typically found in cell plasma, as substrates are more hydrophilic (e.g., alcohols and aldehydes). No need for being membrane-bound.
Impact of Inefficient ALDH in Certain Populations:
- Some individuals, particularly in Asian populations, have inefficient ALDH leading to accumulation of acetaldehyde (a toxic byproduct causing flushing and headaches).

Definition: Enzyme that catalyzes oxidizing reactions for nitrogen-containing drugs (amines).
Function: MAO found in mitochondria and brain tissues; regulates neurotransmitter activity.
Cofactors: Requires NADH for its reactions.
Example Reaction: Converts amine groups to ketones and ammonia.
Negative Implications:
- MAO can catalyze the formation of toxic metabolites leading to neurological disorders, such as in Parkinson's disease (e.g., conversion of MTTP to harmful byproducts).

Cytochrome P450 (CYP450): Most versatile enzyme, with over 50 members involved in various oxidation reactions (hydroxylation, epoxylation, desaturation, dehalogenation).
FMO: Adds oxygen to heteroatoms; fewer isoforms compared to P450, representing a small fraction of overall metabolism.
Dehydrogenases:
- ADH for alcohol, ALDH for aldehyde, both important for alcohol digestion and metabolism.
MAO: Modulates neurotransmitter levels, potential to generate toxic compounds.
Statistics on Reactions:
- CYP450 catalyzes 75-80% of Phase I reactions, with CYP3A4 alone accounting for 40% and others (CYP2D6, CYP2C9, CYP2C19) contributing about 10% each.
- Esterases and epoxide hydrolases contribute 5-10%.
- Though fewer in number, other enzymes are crucial for maintaining metabolic balance.
Role in Drug Development: Enzymes in drug metabolism enhance water solubility which is vital for subsequent phases of biotransformation (Phase II and Phase III).
Following lectures will cover Phase II and Phase III metabolism.

Key Concept: Hydrolysis is another major category of Phase I drug metabolism reactions involving various substrates.
- Ester Hydrolysis:
- Definition: An ester bond (formed from carboxylic acid and a hydroxyl group) can easily be hydrolyzed by esterase.
- Example: Procaine (ester) can be broken down by esterase into acid and hydroxyl components.
- Amide Hydrolysis:
- Stronger and more resistant than esters; requires specific enzymes for hydrolysis.
- Example: Acetaminophen is hydrolyzed into acetic acid and an amine group by amidases.
- Peptide Hydrolysis:
- Peptide bonds (special amid bonds) form proteins comprising amino acids.
- Importance: Peptidases are crucial for protein digestion; they need to be highly specific to prevent excess degradation of essential proteins.
- Example: Hydrolysis of aspartame (a sweetener) into its amino acid components.
- Epoxide Hydrolysis:
- Epoxides (3-membered oxygen-containing rings) can be hydrolyzed by epoxide hydrolase enzymes.
- Two forms exist: microsomal and cytosolic; both catalyze the addition of water to open the epoxide ring.

Context: Reduction reactions in drug metabolism are less common due to the higher presence of molecular oxygen in cells.
Low Oxygen Environments: Reduction mainly occurs in low oxygen environments like the intestinal tract where bacteria can facilitate these reactions.
**Example Types of Reduction:
- Nitrile to Amino Group:** Catalyzed by nitrile reductase using NADPH.
- Azo to Amino Group: Azo compounds can convert to amino groups through similar reducing reactions.

Functional Role: Phase I drug metabolism primarily serves to functionalize molecules to prepare them for the next metabolism stages.
Prevalence of Oxidation: Most reactions involve oxidation, with hydrolysis as a significant follow-up reaction.
Commonality of Reduction: Reduction is much rarer and localized to specific areas within the body.
Cohesive Role of Enzymes: All Phase I enzymes are critical for processing drugs and preventing toxic accumulation.
Acknowledgment of Future Lectures: Further exploration into Phase II and III drug metabolism will follow.
Conclusion: Understanding enzyme roles in Phase I drug metabolism lays the foundation for comprehending broader pharmacokinetics and drug action.