Biomolecule Structure, Drug Design, and Cellular Processes

Posted on Mar 18, 2025 in Biotechnology

Biomolecule Structure Determination

Several methods exist for determining the structure of biomolecules. Here are two prominent examples:

X-Ray Crystallography

X-ray crystallography is a high-resolution technique analogous to microscopy. It allows visualization of protein structures at the atomic level, enhancing our understanding of protein function. We can study protein interactions with other molecules, conformational changes, and catalysis (in enzymes). This knowledge aids in designing novel drugs targeting specific proteins or engineering enzymes for industrial processes.

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Advantages: High resolution.

Disadvantages: Requires a crystalline enzyme/inhibitor complex; provides a static snapshot; potential for crystal packing artifacts.

To visualize proteins in atomic detail, electromagnetic radiation with a wavelength of approximately 0.1 nm (1 Å), i.e., X-rays, is required.

NMR (Nuclear Magnetic Resonance) Spectroscopy

NMR spectroscopy uses multidimensional experiments to obtain information about proteins in solution.

  1. Each distinct nucleus in the molecule experiences a unique chemical environment, resulting in a distinct chemical shift.
  2. Proteins have thousands of resonances; multidimensional experiments correlate frequencies of distinct nuclei.
  3. Additional dimensions reduce overlap and provide more information by correlating signals from specific molecular regions.
  4. Magnetization is transferred using radiofrequency pulses and delays; this is described by pulse sequences.

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Advantages: Measurements are taken in solution, providing a more dynamic and physiologically relevant picture compared to crystals.

Disadvantages: Traditionally limited to soluble proteins that don’t aggregate (newer methods are addressing membrane proteins); requires N15 or C13 labeled proteins; historically, resolution was lower than X-ray crystallography (modern NMR achieves resolutions of 10 angstroms or less).

Ultimately, the NMR data is used to calculate the structure.

Influenza Virus Infection Pathway

Influenza viruses are classified based on their surface proteins:

  • H (Hemagglutinin): Attaches to cell surface proteins, enabling viral entry.
  • N (Neuraminidase): Cleaves the connections between the virus and the cell surface, releasing new viral particles to infect more cells.

The infection process proceeds as follows:

  1. The virus attaches to the host cell via Hemagglutinin (H).
  2. The virus enters the cell.
  3. Viral genes are released into the cell.
  4. The host cell is tricked into producing new viral particles.
  5. Neuraminidase (N) releases the newly formed viruses.

Ligand Binding Modes

Optimizing binding affinity is an iterative process. Modifications to a lead structure are made, and their effects are analyzed. Rational drug design relies on predicting the consequences of these structural changes.

Analogue Binding Mode

Example: Tiorphan & retro-Tiorphan

This involves using groups with equivalent molecular recognition properties. These groups:

  • Occupy comparable volumes in the binding pocket.
  • Interact with the same surface of the protein.
  • Interact with the same amino acid side chains.

Alternative Binding Mode

Example: N-Cbz-Phenylalanine & N-β-Phenylpropionyl-Phe

This describes structurally similar ligands exhibiting different binding modes. Ligands with similar functional groups may interact with *different* amino acids. “Multiple binding modes” refers to a single ligand adopting different positions within the protein’s binding pocket.

Pain Transmission in Nerve Cells

Key Facts:

  • Calcium, sodium, and potassium ions regulate crucial intracellular functions. For instance, calcium regulates muscle contraction.
  • Ion channels control ion flow into and out of cells.
  • Some conotoxins act as analgesics by interacting with ion channel receptors, preventing the channel from opening. This blocks ion entry into neighboring nerve fibers, preventing electrical impulse generation and thus blocking the “pain” message.

The Nerve Impulse

  1. An electrical impulse travels along the axon: sodium ions (red) rush in, and potassium ions (green) rush out.
  2. Sodium ion accumulation triggers the opening of calcium ion channels.
  3. Calcium influx causes the release of acetylcholine into the synaptic junction.
  4. Acetylcholine binds to receptor proteins, altering the shape of the ion channel.
  5. This opens the sodium ion channel, allowing sodium ions to enter.
  6. These sodium ions initiate an electrical impulse in the next nerve cell, propagating the pain message.

To block pain, we can target these ion channels.

Quantitative Methods for Describing Drug Efficiency

Hammett Equation

This describes the relationship between the electronic properties of substituents and the reactivity of aromatic compounds, quantified by the Hammett constant (σ).

  • Electron-accepting substituents (e.g., nitro, cyano) have positive σ values.
  • Electron-donating substituents (e.g., hydroxy, amino) have negative σ values.

Each reaction type has a reaction constant (ρ). The general Hammett equation is:

ρσ = logKR-X – logKR-H

Where KR-X is the equilibrium constant for the substituted compound, and KR-H is the equilibrium constant for the unsubstituted compound.

Hansch Analysis

Later, a lipophilic parameter, P, was defined analogously to σ. Different parameters were combined into a model, including a model for non-linear lipophilic interactions. The Hansch equation is:

P = logPR-X – logPR-H

(P = distribution coefficient in a mixture of n-octanol and water)

Knowing П values and PR-H allows for the calculation of values for new compounds.

Hansch Analysis correlates physicochemical properties with biological effects:

log 1/C = -k1(log P)2 + k2logP + k3σ + …

Where:

  • C = molar concentration causing a biological effect
  • P = distribution coefficient (n-octanol/water)
  • σ = Hammett constant
  • k = coefficients determined by regression analysis

Hansch analysis uses regression analysis to establish a quantitative relationship between biological activities and physicochemical parameters, based on a hypothetical model.

Designing a Specific Inhibitor for a Viral Infection

Neuraminidase (NA) inhibitors reduce viral infection. To improve their clinical efficacy, non-carbohydrate inhibitors are designed based on the NA active site structure. This is an iterative process involving:

  1. Ligand modeling and electrostatic calculations.
  2. Chemical synthesis of compounds.
  3. Biological testing.
  4. NA-inhibitor complex structure determination via X-ray crystallography.

QSAR of Adrenergic Antagonists

QSAR (Quantitative Structure-Activity Relationships) is a discipline that relates chemical structures to biological effects. For example, the adrenergic effect of N,N-dimethyl-α-bromophenylamines is a well-studied QSAR example. These substances can abolish the agonistic effects of adrenaline, depending on their structure. The value C represents the dose of an antagonist that inhibits 50% of the adrenaline effect.

Statistical parameters in QSAR analysis:

  • r (correlation coefficient): Should be close to 1.
  • s (standard deviation): Should be as small as possible.
  • F: Should be as large as possible.

Examples of Rational Drug Design

  • Hormones: Insulin binds to receptors on cell membranes, signaling cells to take up glucose.
  • Protein Channels: Regulate the movement of substances across membranes (e.g., the CFTR protein pumps ions).
  • Transport: Hemoglobin in red blood cells transports oxygen.

Enzyme Efficiency

Enzymes have specific active sites where reactions occur. Substrates enter the active site, and the enzyme’s specific shape ensures it performs only a specific job.

  • Example: Catalase breaks down hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O).
  • Example: Amylase breaks down starch into maltose. Maltase then breaks down maltose into glucose, which is absorbed into the blood for energy.