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Drug-Target Interactions
and Enzyme Inhibitors

Explore how drugs bind to proteins, the kinetics of competitive and non-competitive inhibition, and how \( K_i \) and \( IC_{50} \) shape modern pharmacology.

Biochemistry

What Are Drug-Target Interactions?

Most drugs work by binding to a specific biological target: a protein, enzyme, receptor, or nucleic acid. This binding event alters the target's function, producing a therapeutic effect. The interaction is governed by the same forces that drive all molecular recognition: hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic effects.

Essential Vocabulary

Ligand
Any molecule that binds to a target: substrates, drugs, hormones, neurotransmitters.
Affinity
How tightly a drug binds. Measured by \( K_d \): a lower \( K_d \) means higher affinity.
Selectivity
A drug's preference for one target over others: reduces off-target side effects.

Binding Models: Lock-and-Key vs Induced Fit

Lock-and-Key Enzyme Drug Bound Induced Fit Enzyme Drug Bound Shape change

Lock-and-Key: the active site is pre-shaped to fit the ligand exactly. Induced Fit: the enzyme changes conformation upon ligand binding, wrapping around the drug for a tighter interaction.

Enzymes as Drug Targets

Enzymes catalyze biochemical reactions by lowering activation energy. When an enzyme drives a disease process (e.g., viral replication, inflammation), blocking that enzyme with an inhibitor is a powerful therapeutic strategy.

Reversible
Drug binds and unbinds: equilibrium between free and bound states. Effect is dose-dependent and temporary.
Irreversible
Drug forms a covalent bond with the enzyme, permanently inactivating it. New enzyme must be synthesized.

Michaelis-Menten Kinetics Review

\([S]\) (substrate concentration) Reaction rate (\(v\)) \(V_{\max}\) \(V_{\max}/2\) \(K_m\)

The \( K_m \) value tells us enzyme-substrate affinity: a low \( K_m \) means the enzyme reaches half-max velocity at low substrate: it binds substrate tightly.

Competitive Inhibition

Active Site S I ✕ Competitive Inhibitor Binds the active site Competes with substrate \(K_m\) (apparent) Increases \(V_{\max}\) Unchanged Overcome by excess \([S]\)

Non-Competitive Inhibition

Active Site S I Allosteric site Non-Competitive Inhibitor Binds an allosteric site Distorts enzyme shape \(K_m\) (apparent) Unchanged \(V_{\max}\) Decreases NOT overcome by excess \([S]\)

Competitive vs Non-Competitive

Competitive Non-Competitive
Binding Site Active site Allosteric site
Effect on \( K_m \) Increases (apparent) Unchanged
Effect on \( V_{\max} \) Unchanged Decreases
Overcome by [S]? Yes No
Lineweaver-Burk Same y-intercept, different x-intercept Same x-intercept, different y-intercept

Uncompetitive and Mixed Inhibition

Uncompetitive
Binds only the ES complex (not free enzyme). Both \( K_m \) and \( V_{\max} \) decrease. Lineweaver-Burk: parallel lines.
Mixed
Binds both free enzyme and ES complex with different affinities. Both \( K_m \) and \( V_{\max} \) change. Lineweaver-Burk lines intersect left of y-axis.

Lineweaver-Burk Plots

\(1/[S]\) \(1/v\) 0 No I Comp. Non-C. \(1/V_{\max}\) Uninhibited Competitive Non-Comp.

Measuring Potency: \( K_i \) and \( IC_{50} \)

\( K_i \) (Inhibition Constant)
The dissociation constant for the enzyme-inhibitor complex. Lower \( K_i \) = tighter binding = more potent inhibitor.
\( IC_{50} \)
The inhibitor concentration that reduces enzyme activity by 50%. Depends on assay conditions (substrate conc.).

How to Identify an Inhibitor Type

1
Run kinetic assays at multiple \([S]\) with and without inhibitor to get \( v \) vs \([S]\) data.
2
Construct a Lineweaver-Burk plot (\( 1/v \) vs \( 1/[S] \)). Fit lines for each inhibitor concentration.
3
Analyze the intersection. Same y-intercept = competitive. Same x-intercept = non-competitive. Parallel lines = uncompetitive.
4
Confirm with \( K_m^{\text{app}} \) and \( V_{\max}^{\text{app}} \). Compare apparent values with the uninhibited enzyme.
5
Calculate \( K_i \) from the slope change or use the Cheng-Prusoff equation from \( IC_{50} \) data.

Example 1: Competitive Inhibitor Effect

An enzyme has \( K_m = 5 \text{ mM} \) and \( V_{\max} = 100 \text{ } \mu\text{mol/min} \). A competitive inhibitor is added at \([I] = 10 \text{ mM}\) with \( K_i = 5 \text{ mM} \). Find the apparent \( K_m \) and predict what happens to \( V_{\max} \).

Example 2: Non-Competitive Inhibitor Effect

Same enzyme (\( K_m = 5 \text{ mM} \), \( V_{\max} = 100 \text{ } \mu\text{mol/min} \)). Now a non-competitive inhibitor is added at \([I] = 10 \text{ mM}\) with \( K_i = 10 \text{ mM} \).

Example 3: \( IC_{50} \) to \( K_i \) Conversion

A competitive inhibitor of HIV protease has \( IC_{50} = 30 \text{ nM} \) measured at \([S] = 2K_m\). Find the intrinsic \( K_i \).

Common Mistakes

✕ Confusing \( K_m \) and \( V_{\max} \) changes
Competitive changes \( K_m \) (not \( V_{\max} \)). Non-competitive changes \( V_{\max} \) (not \( K_m \)). Mix these up and every inhibition analysis fails.
✕ Treating \( IC_{50} \) as \( K_i \)
\( IC_{50} \) depends on substrate concentration and assay conditions. Only \( K_i \) is an intrinsic measure of inhibitor potency.
✕ Forgetting "overcome by \([S]\)"
Only competitive inhibition can be overcome by adding more substrate. Non-competitive and uncompetitive cannot.

Summary of Inhibitor Types

Property Competitive Non-Comp. Uncompetitive Mixed
Binds E only E and ES ES only E and ES
\( K_m^{\text{app}} \) ↑ Same ↓ Varies
\( V_{\max}^{\text{app}} \) Same ↓ ↓ ↓
LB Plot Intersect y-axis Intersect x-axis Parallel Left of y-axis
Overcome by [S]? Yes No No No

Real-World Drug Examples

Statins
Competitive inhibitors of HMG-CoA reductase. Lower cholesterol by blocking the rate-limiting step of its synthesis.
Methotrexate
Competitive inhibitor of dihydrofolate reductase (DHFR). Blocks DNA synthesis: used in cancer and autoimmune disease.
HIV Protease Inhibitors
Competitive inhibitors that mimic the transition state of the protease substrate, preventing viral maturation.

Question 1 of 5

A competitive inhibitor is added to an enzyme-catalyzed reaction. Which of the following is true?

Question 2 of 5

On a Lineweaver-Burk plot, a non-competitive inhibitor produces lines that:

Question 3 of 5

An enzyme has \( K_m = 4 \text{ mM} \). A competitive inhibitor (\( K_i = 2 \text{ mM} \)) is added at \([I] = 6 \text{ mM}\). What is the apparent \( K_m \)?

Question 4 of 5

Which type of inhibition cannot be overcome by increasing substrate concentration?

Question 5 of 5

A competitive inhibitor has \( IC_{50} = 60 \text{ nM} \) measured at \([S] = 2K_m\). Using the Cheng-Prusoff equation, what is \( K_i \)?