lecture 3 notes

Lecture 3: 

Sample sources  

• Difficulties and time needed to obtain visas and permits restrict access to international sources of biota making research on local sources much easier for academic and industry researchers and samples are obtains from citizen scientists through crowdsourcing 

Natural product libraries  

• Original libraries were composed of crude extracts that were cheap and easy to produce  

• Partially-purified, pre-fractionated, natural product libraries are now produced using chromatographic techniques at greater cost, resources and time (e.g. library created using size exclusion chromatography etc) 

Natural product-inspired libraries  

• Combinatorial chemistry enabled the production of large libraries of synthetic compounds (combinational chemistry are series of functional groups and combining them together in different ways to create synthetic compounds --> however a lot of the synthesized drugs has poor drug-like properties meaning they are molecules that won't get into cells in the human body) 

• Synthetic libraries are now being pre-screened for similarity to natural compounds and structural scaffolds using in silico and AI tools 

Cell-based screens 

• Can utilize human cells --> can culture them and test different drugs and find a drug that works  

• Phenotypic screens (identify change of phenotype/function of the cell) can identify compounds that effect cell function, potentially by unknown mechanisms  

• Many different assay formats, but common approach is screening for cytotoxicity (cell death) --> can also monitor cell metabolism, migration/invasion, apoptosis, senescence, stress or changes in gene expression  

• Require down-stream mechanistic studies to determine which specific molecule or pathway is affected  

• Cell-based screens adds compounds to live active cells to measure the loss of activeness  

Cytotoxicity screens 

• All compounds and extracts will be toxic for human cells depending on its concentration  

• For cancer treatments or treatments where killing of the cell is the goal, compounds will be used at toxic levels  

• For other uses, must use compounds below the maximum non-toxic concentration/safe dose  

• First screened in cultured cells  

Cell viability assays  

• Cell health determined by measuring metabolic activity, DNA and/or protein content, or loss of membrane integrity  

• Relationship between loss of cell viability (death) and concentration determined  

Cell-free screens 

• Cell-free screens are done by targets that are already identified so it's less investigation because we are already working to find a molecule that interacts pharmacodynamically with the target  

• Allows for measurement of kinetic, thermodynamic or structural basis of target interactions  

• More prone to non-specific activity/false positives  

Antioxidants  

• Neutralise and stabilise free radicals produced during tissue damage and inflammation  

Antioxidant assays  

• Free radicals within the substrate are activated by removal of electrons; substrate is incubated with extracts, fractions or compounds; reduction of the substrate is determined by measuring the change in colour using a spectrophotometer; rate and extent of substrate reduction is determined 

Identifying active compounds  

• Once an active extract has been found, the compound(s) responsible for the activity are identified  

• Chromatography may be used to purify and increase the concentration of the desired compound with compound activity validated using phenotypic- or target-based screening assays (cell-based or cell-free screenings) 

• Then the compound structure will be determined using spectroscopy techniques