🧪Synthetic Biology Unit 12 – Protein Synthesis for Drug Discovery

Key Concepts in Protein Synthesis

• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Transcription process where DNA is used as a template to synthesize complementary RNA strands catalyzed by RNA polymerase enzymes
• Translation process where the genetic code in mRNA is decoded by ribosomes to synthesize polypeptide chains that fold into functional proteins
  • Occurs in the cytoplasm of cells and involves tRNA molecules that carry specific amino acids to the ribosome
  • Ribosomes read the codons in mRNA and link the corresponding amino acids together to form polypeptide chains
• Post-translational modifications can alter the structure and function of proteins after synthesis (phosphorylation, glycosylation)
• Protein folding determines the three-dimensional structure of proteins critical for their biological functions
  • Misfolded proteins can lead to various diseases (Alzheimer's, Parkinson's)
• Protein degradation pathways break down and recycle proteins no longer needed by the cell (ubiquitin-proteasome system)

Molecular Biology Fundamentals

• DNA double helix structure consists of two complementary strands held together by hydrogen bonds between base pairs (A-T, G-C)
• DNA replication occurs during cell division to ensure genetic information is passed on to daughter cells
  • Involves DNA polymerase enzymes that synthesize new strands using the original DNA as a template
• RNA molecules are single-stranded and contain uracil (U) instead of thymine (T) found in DNA
  • Three main types of RNA: mRNA (messenger), tRNA (transfer), and rRNA (ribosomal)
• Genetic code determines how the sequence of nucleotides in mRNA is translated into amino acids
  • Consists of 64 codons, each specifying a particular amino acid or stop signal
• Mutations can alter the genetic code and lead to changes in the amino acid sequence of proteins
  • Point mutations involve single nucleotide changes (substitutions, insertions, deletions)
• Gene expression regulation controls which genes are transcribed and translated in different cell types and conditions
  • Transcription factors bind to specific DNA sequences to activate or repress gene expression

Protein Structure and Function

• Primary structure refers to the linear sequence of amino acids in a polypeptide chain determined by the genetic code
• Secondary structure describes the local folding patterns of polypeptide chains stabilized by hydrogen bonds (α-helices, β-sheets)
• Tertiary structure represents the three-dimensional shape of a single polypeptide chain resulting from interactions between amino acid side chains
  • Stabilized by various forces (hydrogen bonds, disulfide bridges, hydrophobic interactions)
• Quaternary structure involves the assembly of multiple polypeptide chains into a functional protein complex (hemoglobin)
• Protein domains are distinct structural and functional units within a protein that can evolve and function independently
• Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy
  • Active site is the region where substrates bind and the reaction occurs
  • Specificity depends on the complementary shape and chemical properties of the active site
• Protein-protein interactions mediate many cellular processes (signal transduction, cell adhesion)
  • Interaction interfaces involve complementary surfaces and specific amino acid contacts

Techniques for Protein Engineering

• Recombinant DNA technology allows the manipulation and expression of genes in different host organisms
  • Restriction enzymes cut DNA at specific sequences enabling the insertion of genes into plasmid vectors
  • Plasmids are circular DNA molecules that can replicate independently and carry foreign genes
• Polymerase chain reaction (PCR) amplifies specific DNA sequences using primers and DNA polymerase
  • Enables the rapid generation of large quantities of DNA for cloning and mutagenesis
• Site-directed mutagenesis introduces specific changes to the DNA sequence of a gene to alter the amino acid sequence of the encoded protein
  • Oligonucleotide primers containing the desired mutation are used in PCR to amplify the modified gene
• Protein expression systems produce recombinant proteins in various host organisms (bacteria, yeast, mammalian cells)
  • Choice of expression system depends on factors such as post-translational modifications and yield
• Protein purification techniques isolate the protein of interest from the host cell lysate
  • Affinity chromatography uses tags (His-tag) that bind specifically to a matrix
  • Size-exclusion chromatography separates proteins based on their molecular weight
• Directed evolution mimics natural selection to evolve proteins with desired properties
  • Involves rounds of mutagenesis, screening, and amplification to identify improved variants

Drug Discovery Process Overview

• Target identification involves selecting a protein or pathway implicated in a disease that could be modulated by a drug
  • Genomics, proteomics, and computational methods aid in identifying potential targets
• Target validation confirms the role of the target in the disease and its suitability for drug development
  • Uses techniques such as gene knockouts, RNA interference, and small molecule probes
• High-throughput screening (HTS) rapidly tests large libraries of compounds against the target to identify hits that show desired activity
  • Automated robotic systems and miniaturized assays enable screening of millions of compounds
• Hit-to-lead optimization improves the potency, selectivity, and pharmacokinetic properties of the initial hits
  • Medicinal chemistry modifies the chemical structure to enhance drug-like properties
• Lead optimization further refines the lead compounds to improve their efficacy and safety
  • In vitro and in vivo studies assess the pharmacology, toxicology, and pharmacokinetics
• Preclinical development involves extensive testing of the optimized lead in animal models to establish safety and efficacy
  • Investigational New Drug (IND) application filed with regulatory agencies to begin clinical trials
• Clinical trials test the drug candidate in humans to determine its safety, dosage, and effectiveness
  • Phase 1 assesses safety in healthy volunteers, Phase 2 evaluates efficacy in a small patient group, Phase 3 confirms safety and efficacy in a larger patient population
• FDA approval is required before the drug can be marketed and prescribed to patients
  • New Drug Application (NDA) submitted with data from preclinical and clinical studies

Protein-Based Drug Design

• Structure-based drug design (SBDD) uses the three-dimensional structure of the target protein to guide the design of small molecule inhibitors
  • X-ray crystallography and NMR spectroscopy determine the atomic structure of proteins
  • Computational methods (molecular docking) predict the binding of ligands to the target
• Ligand-based drug design (LBDD) uses the structure of known ligands that bind to the target to design new compounds with similar properties
  • Pharmacophore modeling identifies the essential features of ligands responsible for their activity
  • Quantitative structure-activity relationship (QSAR) models correlate the chemical structure of ligands with their biological activity
• Protein-protein interaction inhibitors disrupt the formation of protein complexes involved in disease pathways
  • Peptidomimetics are small molecules that mimic the structure of peptides and can bind to protein interfaces
• Antibody-based therapeutics use monoclonal antibodies that specifically bind to target proteins and modulate their function
  • Antibody engineering optimizes the affinity, specificity, and effector functions of antibodies
• Targeted protein degradation induces the degradation of disease-causing proteins using small molecules
  • Proteolysis-targeting chimeras (PROTACs) recruit the ubiquitin-proteasome system to degrade the target protein

Synthetic Biology Applications

• Metabolic engineering modifies the metabolic pathways of organisms to produce desired compounds (drugs, biofuels)
  • Uses enzymes and regulatory elements to optimize the flux through the pathway
  • Examples include the production of artemisinin (antimalarial drug) and isobutanol (biofuel) in engineered microbes
• Biosensors are engineered proteins that detect specific molecules and generate a measurable output signal
  • Transcription factor-based biosensors activate gene expression in response to the target molecule
  • Fluorescent protein-based biosensors change their fluorescence properties upon binding the target
• Genome editing tools (CRISPR-Cas9) enable precise modification of DNA sequences in living cells
  • Guide RNA directs the Cas9 nuclease to cut the target DNA, allowing for gene knockouts or insertions
  • Potential applications in gene therapy, disease modeling, and agricultural biotechnology
• Synthetic gene circuits are engineered genetic networks that perform complex functions in living cells
  • Toggle switches and oscillators create bistable and oscillatory gene expression patterns
  • Logic gates (AND, OR) process multiple input signals to control gene expression
• Cell-free systems use purified components (enzymes, ribosomes) to carry out biochemical reactions outside of living cells
  • Enable rapid prototyping and optimization of metabolic pathways and genetic circuits
  • Applications in point-of-care diagnostics, vaccine production, and biomanufacturing

Challenges and Future Directions

• Protein misfolding and aggregation pose challenges for the production and stability of recombinant proteins
  • Chaperone engineering and directed evolution strategies can improve protein folding and solubility
• Off-target effects of drugs can lead to adverse side effects and limit their therapeutic window
  • Improving the specificity of drug-target interactions and targeted delivery methods can mitigate off-target effects
• Drug resistance can emerge due to mutations in the target protein or activation of alternative pathways
  • Combination therapies and targeting multiple nodes in a pathway can reduce the risk of resistance
• Immunogenicity of protein-based drugs can elicit an immune response and reduce their efficacy
  • Humanization of antibodies and PEGylation can reduce immunogenicity
• Scalability and cost-effectiveness of synthetic biology applications remain challenges for industrial implementation
  • Advances in bioprocess engineering and metabolic optimization can improve the economics of biomanufacturing
• Ethical and societal considerations surrounding the use of synthetic biology and genome editing technologies
  • Public engagement, regulatory oversight, and responsible innovation frameworks are needed to address these concerns
• Integration of artificial intelligence and machine learning methods can accelerate the drug discovery process
  • Deep learning can aid in target identification, compound screening, and de novo drug design
• Personalized medicine approaches tailor drugs to an individual's genetic makeup and disease profile
  • Pharmacogenomics and companion diagnostics can guide the selection of optimal therapies for each patient