Enzymes as Drug Targets

Proteases, Reverse Transcriptases, and Antiviral Drug Discovery

Enzymes like proteases and reverse transcriptases are useful in antiviral drug discovery.
HIV's mechanism:
- It goes RNA -> DNA -> RNA -> protein, against the central dogma.
- This is a weakness, allowing the development of reverse transcriptase inhibitors.
Integrase integrates viral DNA into host DNA, making infection chronic and hard to cure.

Current HIV Treatment

HIV is suppressed using Reverse Transcriptase Inhibitors, Protease Inhibitors, and Integrase Inhibitors.
These suppress viral protein production.
Eradication is difficult as the virus is integrated into the host cells.

Enzymes: Structure, Function, and Drug Targets

Enzymes have active sites that recognize substrates.
Drugs that bind to the active site compete with the substrate, called competitive inhibition.
Irreversible inhibition involves a covalent modifier that forms a covalent bond with the enzyme.

Other Enzyme Inhibition Mechanisms

Non-competitive or allosteric inhibition: Inhibitor and substrate bind to different sites and do not directly compete.
Uncompetitive inhibition: Inhibitor binds only to the enzyme-substrate complex, preventing processing.
Mixed inhibition: A combination of different inhibition mechanisms.

Michaelis Menten Kinetics and Lineweaver-Burk Plot

Michaelis Menten kinetics and Lineweaver Burk plots help identify inhibition mechanisms through kinetics.
Competitive mode: Intersection with the y-axis.
Non-competitive mode: Intersection with the x-axis.
Uncompetitive mode: Parallel lines on the plot, requiring accurate data.
$K<em>m$ and $V</em>{max}$ values help in identifying the mechanism of inhibition.

Serine Proteases: Catalytic Triad

Serine proteases have a catalytic triad: serine, histidine, and aspartate.
This triad is crucial for catalysis.
The aspartate abstracts a proton from the histidine, which then abstracts a proton from the serine, making the serine nucleophilic.
This is a conserved motif in many proteins.

Zika Virus Protease

Viral proteases are highly conserved, especially at the active site.
Superimposition of Dengue, West Nile, and Zika virus proteases shows structural similarity.
Enzymes bring residues close in 3D space to speed up reactions, such as proteases (10^10 times) and nucleases (10^15 times).

Protease Mechanism

Substrate binding pockets are labeled S1, S2, S3, etc., with corresponding side chains P1, P2, P3, etc.
Interactions with side chains orient the peptide in the active site.
Proteases cleave specifically; for example, some cleave after arginine, while the COVID protease cleaves after glutamine.

Enzyme Catalysis

Aspartate and histidine make serine nucleophilic, attacking the amide bond.
A tetrahedral intermediate with high energy is formed.
Enzymes lower this transition state energy.
The oxyanion is stabilized by the enzyme via NH bonds, decreasing the transition state energy.
The peptide cleaves, serine remains bound, forming an ester.
The catalytic triad uses a water molecule to hydrolyze the ester.

Enzyme Function

Enzymes accelerate reactions by bringing residues close to each other and increasing the effective concentration of nucleophiles.
Nature splits the reaction into two steps, lowering the activation energy.

Enzyme Assays

An understanding of the enzyme and its substrate is required to develop an assay.
For proteases, one needs to know the cleavage preference.
A fluorescent dye can be placed next to the cleavage site.
For instance, Amino Methyl Coumarin (AMC) is coupled via an amide bond.
Cleavage releases fluorescent amine.
Increase in fluorescence over time indicates enzyme activity.
Inhibitors will reduce or eliminate fluorescence over time.
Academic labs use cuvettes, whereas industry uses 96 or 384 well plates with robotic pipetting for high-throughput screening.
Assay limitations: Unspecific binding, fluorescent compounds, quenchers.

SARS CoV-2 and Drug Targets

SARS CoV-2 is similar to SARS CoV, so major drug targets were known.
Entry inhibitors target ACE2 receptor interaction and TMPRSS2 protease cleavage of the spike protein.
The virus injects RNA genome into the cell, hijacking the cell for replication.

Viral Genome Replication

The SARS CoV-2 genome is long and encodes two polyproteins.
Ribosome shifts create two different proteins.
Polyproteins are cleaved by viral protease: Main protease and papain-like protease. The Main protease is a good drug target.
Cleavages release components necessary for RNA genome replication.
The replication complex produces mRNAs that are translated into viral proteins, such as spike, envelope, and membrane proteins.

Main Protease Structure

The main protease is a cysteine protease with histidine and cysteine in the active site.
Histidine activates cysteine via proton abstraction to enhance its nucleophilicity.
Similar to HIV protease, it functions as a dimer.

Drug Discovery Approaches

Active site targeting: Mimicking the substrate and modifying it to act as a reversible or irreversible inhibitor.
Dimerization interface interruption: Targeting the dimerization to prevent it, as only the dimer is catalytically active.
Protein-protein interaction inhibitors (PPIs): Disrupting the dimerization of the protease.
Dissociation constant (Kd) of the dimerization is 10 micromolar.

Schecter and Berger: Protease Specificity

Protease cleavage depends on residue nature (1967).
Residues on the N-terminus of the cleavage site are P1, P2, P3, etc.
Residues on the C-terminus of the cleavage site are P1', P2', P3', etc.
Corresponding pockets in the enzyme are S1, S2, S1', S2', etc.
Glutamine in P1 indicates cleavage after glutamine; Arginine in P1 and Serine in P1' indicate cleavage between arginine and serine.

SARS CoV-2 Protease Specificity and Assay Design

Substrate preference resembles SARS CoV-1, cleaving after glutamine in P1.
Only glutamine is tolerated in P1; small residues like serine or alanine are preferred in P1'.
Residue conservation decreases further from the cleavage site.
Assay uses the peptide substrate with two dyes at either end.
Fluorescent dye and quencher system: FRET (Förster Resonance Energy Transfer) is used.
FRET: Non-radiating mechanism from the excited electron to come down, causing fluorescence quenching.
Cleavage increases the distance between dyes, increasing fluorescence.
Linear range measures activity and initial velocity (V0).

Early Inhibitors and Covalent Interactions

Early inhibitors were based on substrates with a glutamine mimetic in the P1 side.
Covalent interactions were also incorporated.
Alpha Keto Amide and Michael acceptors were used for irreversible inhibition with the active cysteine in the active site.

Paxlovid (Nirmatrelvir)

Paxlovid, developed by Pfizer, entered the market via emergency approvals.
It contains a glutamine mimetic in P1 with valine mimetics with a covalent modifier.
Nitrile group covalently binds to active site cysteine, forming a thio imidate.

Variants and Mutations

Early variants emerged due to random fitness increases; later, vaccines influenced mutations.
Spike protein mutations were common.
Little mutation was observed in the protease; Delta was like the wild type, Omicron had P132H.
Mutations had little impact on catalytic activity, hence random.

Paxlovid Resistance

Mutations had no impact on the activity of Paxlovid.
IC50 for Paxlovid was around 10 nanomolar for all variants.
A screen of 96 potential mutations revealed that half were inactive, half had low activity.
Five mutations (N40, N142L, E166M, etc.) had wild-type activity.
E166M mutation caused resistance to Paxlovid: 25-fold decrease in activity and 100-fold decrease in activity for other inhibitors.
Resistance is evolving, but not at the same pace as with HIV protease inhibitors.

Non-Peptidic Inhibitors

Later drugs are less peptidic and may not need covalent modifiers.
Ensitrelvir is a small molecule with equal activity compared to Paxlovid.
It is a reversible competitive inhibitor with similar S1 pocket interactions.
The activity is similar across mutants.

Chemical Biology Curriculum Overview

Covers biomolecules (DNA, carbohydrates, lipids) on a chemical basis.
Includes protein and enzyme topics: Enzyme kinetics, inhibition, drug development.
Examines protein folding, protein-protein and protein-ligand interactions (CHEM Bio I).
CHEM Bio II focuses on protein synthesis, expression, analysis, and modification.