Fragment based drug design Flashcards
What is fragment based screening?
Fragment-based screening is a drug discovery approach that involves the screening of small, low molecular weight compounds known as fragments to identify potential lead compounds for further development into drug candidates. These fragments typically consist of 10-20 heavy atoms and possess a range of chemical functionalities.
It is based on the principle that small fragments can bind to target proteins with high affinity and can serve as starting points for the design and optimization of larger, more potent molecules.
Explain the process of fragment-based drug design.
Fragment Library Generation: A diverse library of small, low molecular weight compounds (fragments) is assembled. These fragments typically consist of 10-20 heavy atoms and possess a range of chemical functionalities.
Biophysical Screening: Fragments are screened against the target protein using biophysical techniques to detect binding interactions. These techniques include nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and others. The goal is to identify fragments that bind to the target protein with reasonable affinity.
Hit Validation and Expansion: Fragments that show promising binding to the target protein are further characterized and validated using additional biophysical and biochemical assays. This helps confirm their binding affinity and selectivity. Hits can be further expanded by synthesizing analogs or by exploring fragment linking strategies to improve binding potency.
Fragment Growing and Optimization: Once validated, the identified fragments are used as starting points for the development of larger molecules through fragment growing or linking strategies. This involves adding functional groups or extending the fragments to increase binding affinity and optimize properties such as selectivity, solubility, and bioavailability.
Lead Optimization: The optimized fragments or fragment-derived molecules are further optimized through iterative cycles of medicinal chemistry, structural biology, and biological testing to improve their drug-like properties and efficacy. This includes modifying the structure, optimizing pharmacokinetic properties, and minimizing off-target effects.
Define Lipinski rule of 5 with mention of the fragment rule of 3.
Molecular Weight (MW): The molecular weight should be less than or equal to 500 daltons (Da). Higher molecular weight compounds may have difficulties crossing biological barriers.
Lipophilicity (LogP): The octanol-water partition coefficient (LogP) should be less than or equal to 5. LogP measures the compound’s hydrophobicity. High lipophilicity can lead to poor aqueous solubility and absorption issues.
Hydrogen Bond Donors (HBD): The number of hydrogen bond donors should be less than or equal to 5. Hydrogen bond donors are functional groups, such as hydroxyl or amino groups, capable of donating hydrogen bonds.
Hydrogen Bond Acceptors (HBA): The number of hydrogen bond acceptors should be less than or equal to 10. Hydrogen bond acceptors are functional groups, such as oxygen or nitrogen atoms, capable of accepting hydrogen bonds.
Fragment rule of 3 is identical however 300mw, 3 logp, 3 acceptors, 3 doners.
What makes something a small molecule vs a fragment ?
Size: Small molecules generally have a molecular weight of up to a few hundred daltons (Da), typically ranging from 150 to 900 Da. They are larger and more structurally complex than fragments.
Complexity: Small molecules exhibit greater structural complexity, typically consisting of multiple functional groups and a well-defined three-dimensional structure. They often possess a complete pharmacophore, which is a set of structural features responsible for the molecule’s biological activity.
Functional Groups: Small molecules commonly contain multiple functional groups, such as hydroxyl (-OH), amino (-NH2), carbonyl (-C=O), or aromatic rings. These functional groups contribute to their interactions with target proteins and biological activity.
Pharmacological Activity: Small molecules are typically designed or selected based on their specific pharmacological activity against a target, such as binding to an enzyme, receptor, or protein target, and exerting a desired biological effect.
What is the main goal of the fragment-based drug discovery approach?
The main goal of the fragment-based drug discovery (FBDD) approach is to identify small, low molecular weight compounds called fragments that bind to a target protein with high affinity. These fragments serve as starting points for the development of more potent and selective drug candidates
Binding Affinity: The primary aim is to identify fragments that bind to the target protein with reasonable affinity. Fragments are typically designed to have weak binding affinity to explore a larger chemical space and identify potential binding sites on the target.
Structural Information: Fragments provide valuable structural information about the target-ligand interactions. They offer insights into the binding modes, hotspots, and potential drug-target interactions, guiding the design of optimized compounds.
Scaffold Exploration: Fragment-based approaches allow for the exploration of diverse chemical scaffolds. By screening a library of fragments, a wide range of chemical functionalities and structural motifs can be assessed, enabling the identification of unique starting points for drug development.
Hit-to-Lead Optimization: Fragments that show promising binding characteristics are further optimized through a process called fragment growing or fragment linking. This involves expanding or linking fragments to enhance their binding affinity and develop lead compounds with improved potency, selectivity, and drug-like properties.
Efficiency and Speed: Fragment-based drug discovery is known for its efficiency and speed. By focusing on small, manageable compounds, the screening process can be streamlined, and the synthesis and evaluation of a fragment library can be accomplished relatively quickly.
Why are alternative screening methods needed for fragment-based drug design and list these methods?
Due to their smaller size fragments interact more weakly than standard small molecules hence more sensitive detection methods are required. Low affinity makes false positives likely so use multiple techniques to verify.
Commonly used methods include
Thermal shift assays e.g. DSF.
Surface plasmon resonance (SPR)
NMR
X-ray crystallography required to verify binding and binding mode.
Explain the principle of Differential Scanning Fluorimetry (DSF)?
When heated proteins unfold at a temperature (Tm) determined by buffer composition and sequence.
Ligands that bind the protein stabilise the structure and increase Tm
Dyes bind hydrophobic portions of the protein and fluorescence increases allowing us to measure Tm.
Explain the principle of Surface Plasmon Resonance (SPR)?
SPR measures the binding of ligand to a protein immobilised onto the surface of a chip.
Changes in the intensity of light reflected from a gold surface allow us to monitor binding.
Provides KD values.
Less high throughput than DSF.
Does not prove fragments inhibit activity.
Explain the process of protein NMR?
Protein NMR spectroscopy is a technique used to study proteins in solution. It involves placing the protein sample in a strong magnetic field and applying radiofrequency pulses to excite the atomic nuclei. When the excited nuclei relax, they emit weak radiofrequency signals that are detected and transformed into a spectrum. This spectrum provides information about the structure and dynamics of the protein.
Allows screening of fragment mixtures and can determine KD.
Explain the principle of Isothermal Calorimetry (ITC)
Gold standard method to measure ligand affinity.
Measures small changes in temperature resulting from ligand binding.
Main disadvantage is it is low throughput
Also does not directly measure inhibition.
What is fragment-based drug design targeted or ligand-based drug design?
Targeted as All FBDD projects rely upon knowledge of the structure of the target.
Most commonly used method is X-ray crystallography.
Has advantage of high resolution (<3 Å)
Advances in Cryo-EM may soon change this.
Two main uses:
Hit identification
Identification of binding mode for fragment linking/growth.
What strategies are used to improve fragment hits ?
Fragment Growing: Fragments with initial binding affinity can be grown by adding functional groups or extending the molecule’s size. This strategy involves systematically adding atoms or chemical moieties to the fragment scaffold to enhance interactions with the target protein and improve binding affinity.
Fragment Linking: Fragments that bind to adjacent regions on the target protein can be linked together to create larger molecules with improved binding affinity. By connecting two or more fragments with a linker, the orientation and distance between the fragments can be optimized to enhance their interactions with the target.
Fragment Merging: Fragments with distinct binding sites on the target protein can be merged into a single molecule, creating a multi-fragment compound. This strategy leverages the binding interactions of multiple fragments simultaneously, potentially leading to synergistic effects and improved affinity.
Structure-Guided Optimization: High-resolution structural information, such as X-ray crystallography or NMR, can provide insights into the fragment-target interactions. This information guides the design of optimized compounds by identifying key binding interactions, hotspots, and potential areas for molecular modifications.
Fragment-to-Lead Evolution: Fragments with improved binding affinity undergo iterative cycles of structural modifications, synthesis, and testing to optimize their potency, selectivity, pharmacokinetic properties, and drug-like characteristics. This process involves studying the structure-activity relationship (SAR) to guide the design of lead compounds.
Explain the principle of X-ray crystallography in FBDD?
Mix highly concentrated protein (~10 mg/mL) with precipitant
Seal and leave at RT or 4 for several days.
Pick up microscopic (<100 mm) crystals
Flash freeze in cryoprotectant
High intensity X-rays are used to image the crystals
Provides binding location
Also provides information on key interactions.
Provides information on how to grow the hit.
Can provide information on how to combine fragments.
What are the advantages of FBDD over high-throughput screening approaches ?
Coverage of Chemical Space: FBDD allows for the exploration of a larger chemical space compared to HTS. Fragments are smaller, simpler molecules that can sample a wide range of chemical functionalities and structural motifs. This diversity of fragments increases the chances of identifying novel and diverse lead compounds.
Efficient Target Sampling: FBDD is particularly effective for challenging drug targets, such as protein-protein interactions or allosteric sites, where traditional HTS methods may struggle. Fragments are more likely to bind weakly to these challenging targets, making them ideal starting points for optimization.
Higher Hit Rates: FBDD often achieves higher hit rates compared to HTS. Fragments, by design, have a higher probability of binding to their target proteins due to their smaller size and simpler structure. This higher hit rate streamlines the optimization process, as a larger proportion of identified fragments have the potential for further development.
Better Understanding of Binding Interactions: FBDD provides detailed structural information about fragment-protein interactions. By using techniques such as X-ray crystallography or NMR spectroscopy, the binding modes and key interactions between fragments and the target protein can be elucidated. This structural insight guides the optimization process and helps in the design of more potent compounds.
Rational Drug Design: FBDD is well-suited for rational drug design. Fragments can be systematically grown or linked to optimize their binding affinity and selectivity based on the structural information obtained from fragment-protein complexes. This rational approach accelerates the drug discovery process and minimizes the need for extensive screening.
Reduced Compound Libraries: FBDD requires smaller libraries of compounds compared to HTS. Instead of screening large libraries with millions of compounds, FBDD focuses on a smaller, well-curated library of fragments. This reduces costs and resources required for screening and synthesis.
Lower False Positives: FBDD has a lower rate of false positive hits compared to HTS. Fragments typically exhibit weak binding affinities, reducing the likelihood of non-specific interactions and false positives. This improves the reliability of identified hits, making the optimization process more efficient.
What makes a good fragment in drug discovery ?
Size and Molecular Weight: Fragments are typically small molecules with low molecular weight (less than 300-350 Daltons). This small size allows fragments to explore a larger portion of chemical space and increases the probability of finding binding interactions with the target protein.
Rigidity: Fragments with a certain degree of rigidity are preferred. Rigidity increases the likelihood of the fragment adopting a defined conformation and making specific interactions with the target protein. Flexible fragments can pose challenges in optimizing their binding affinity and selectivity.
Functional Group Diversity: Fragments should possess a range of functional groups, such as amines, carboxylic acids, hydroxyls, and heterocycles. This diversity allows for the exploration of various chemical interactions with the target protein and increases the chances of identifying promising binding interactions.
Low Ligand Efficiency: Fragments with low ligand efficiency, defined as the binding affinity divided by the number of non-hydrogen atoms, are preferred in the initial screening phase. This is because weaker binding fragments have a higher chance of revealing new binding sites on the target protein, which can be further optimized.
Solubility: Good fragments should have reasonable solubility in the biological and assay conditions. Solubility is an important consideration for the development and optimization of fragments into drug candidates.
Novelty: Fragments that are structurally distinct and differ from known drugs or common scaffolds are highly desirable. Novel fragments offer opportunities for exploring new chemical space and potentially discovering unique binding sites or interactions.
Synthetic Feasibility: Fragments should be amenable to efficient synthesis and chemical modifications. Synthetic accessibility is crucial for further optimization and the generation of more potent lead compounds.
Ligand Efficiency: Fragments with high ligand efficiency, defined as the binding affinity divided by the number of non-hydrogen atoms, are favorable during the optimization phase. Ligand efficiency measures the potency of the compound per unit of molecular weight and helps in the selection of fragments with better drug-like properties.
