Enzymes as Drug Targets: Antiviral Drug Discovery

Introduction to Chemical Biology, Drug Discovery, and Medicinal Chemistry

Definition: A pharmaceutical agent with a desired biological effect or a compound that interacts with biological systems to produce a biological response.
The term "drugs" encompasses a wide range of substances, including those with both therapeutic and addictive properties.
Examples: Caffeine, nicotine, ethanol, THC.
The perception of drugs can change over time (e.g., nicotine, THC).
Classification of drugs:
- By pharmacological effect (e.g., antibiotics, antivirals).
- By chemical structure (e.g., penicillins, steroids, opioids).
- By target system (e.g., protease inhibitors, antiviral drugs, anticancer drugs).

Organisms:
- Human (e.g., cancer drugs).
- Bacterial (e.g., antibiotics).
- Viral (e.g., antivirals).
Types of drug targets:
- Receptors (e.g., in cancer).
- Nucleic Acids (e.g., in cancer).
- Enzymes (major class of drug targets).

Human enzymes: Targeting overexpressed enzymes in diseases like cancer.
Bacterial enzymes: Example: Penicillins inhibit D-Alanyl transpeptidase, preventing bacterial cell wall formation. Bacteria can develop resistance through enzymes like beta-lactamase, leading to a continuous cycle of drug development and adaptation.
Viral enzymes: Viruses encode enzymes necessary for replication within host cells. These enzymes are selective drug targets.

HIV protease: Inhibitors like Saquinavir target this enzyme to suppress viral replication.
Flavivirus proteases (e.g., Zika, West Nile, Dengue, Yellow Fever): These viruses utilize proteases for replication, making them attractive targets for broad-spectrum inhibitors. Developing inhibitors that act against multiple Flaviviruses due to the similarity of their proteases.

Enzymes are proteins made up of amino acids.
Central dogma of molecular biology: DNA → RNA → Proteins
20 amino acids encoded by DNA.
Three nucleotides encode one amino acid (genetic code).
Amino acid categories:
- Hydrophobic (collapse into the protein core).
- Charged (Arginine, Lysine, Aspartate, Glutamate).
- Polar (engage in hydrogen bonds; Serine, Threonine).
- Special (Cysteine with Sulphur for nucleophilic interactions).
Enzymes accelerate reactions by arranging building blocks in the right orientation.

Primary Structure:
- Defined by translation.
- Amino acids linked by amide bonds (peptide bonds).
- Backbone: Repeating amide linkages.
- Side chains: Variable groups defining each amino acid.
- N-terminus (amine end) and C-terminus (carboxylic acid end).
Secondary Structure:
- Hydrogen bonds between backbone atoms.
- Alpha Helix: Hydrogen bonds within a single strand.
- Beta Sheet: Hydrogen bonds between two strands (parallel or anti-parallel).
Tertiary Structure:
- Driven by forces: van der Waals, hydrogen bonds, ionic interactions.
- Hydrophobic Collapse: Hydrophobic amino acids move to the protein's interior, while hydrophilic amino acids interact with water on the exterior. Folding of the protein leads to lower energy state.
Quaternary Structure:
- Multiple protein subunits combine to form a functional complex.
- Example: PCNA forms a trimer that functions as a DNA clamp.

Involves enthalpy and entropy considerations.
Hydrophobic effect: Minimizing interactions between oil and water molecules, similar to protein folding.
Side chain interactions: Ionic interactions (salt bridges), hydrogen bonds, van der Waals forces.
Cysteine: Can form disulfide bonds (covalent) for extra stability.

Enzymes catalyze reactions with high speed and enantioselectivity.
Example: Conversion of Purific Acid to Lactic Acid, where a new Stereocenter is created.
Catalysis is driven by:
- Collision of molecules in the correct orientation.
- Reduction of activation energy by stabilizing the transition state.
- Enzymes do not alter thermodynamics ($\Delta G remains the same).

Substrate binds and is converted to product.
Michaelis-Menten kinetics: Enzyme (E) + Substrate (S) ⇌ Enzyme-Substrate complex (ES) → Enzyme (E) + Product (P).
Intermolecular forces (van der Waals, hydrogen bonds, ionic interactions) govern substrate recognition in the active site.
Fisher's Lock and Key hypothesis (static active site) is incorrect.
Koshland's induced fit theory: Proteins (and active sites) are dynamic; substrates induce conformational changes upon binding.

Enzyme and substrate are in equilibrium with the enzyme substrate complex.
Two constants:
- V_{max}, which is the maximum speed
- K_m$$, which is the substrate concentration at half maximum speed. The Michelles Matten constant
The velocity increases linearly vs substrate amount until the enzymes are saturated with substrate.

Drugs can mimic substrates or products.
Product inhibition: Product binds to the active site, preventing further substrate binding (natural regulation mechanism).
Competitive Inhibition: Drug competes with substrate for the active site.
Irreversible Inhibition:
- Drug binds to the active site and forms a covalent bond with a nucleophilic residue.
- More efficient because the enzyme is deactivated forever.
Allosteric Inhibition: Inhibitor binds to a site other than the active site, causing a conformational change that prevents substrate binding.

Competitive Inhibition:
- Reversible.
- Can be outcompeted by high substrate concentrations.
Irreversible Inhibition:
- Covalent bond formation between drug and enzyme.
- Increased efficiency and longer-lasting effects.
- Potential selectivity problems due to off-target effects.
Benefits of irreversible inhibition: increased efficiency (independent of substrate concentration) and lower doses required.
Problem: potential for off-target effects.