L5- Novel drugs & Bio-prospecting

Question 1

What is bioprospecting?

Answer 1

• Discovery/ search of new sources of chemical compounds, genes, enzymes, micro-organisms, and other valuable products from nature
• It involves natural resources, but also genetic engineering techniques

Question 2

What are the natural products found through bioprospecting?

Answer 2

• Bioproduct- a naturally- occuring, biologically-produced product
• Antiinfectives- compounds that kills or inhibits the growth of pathogens or parasites
  (Antimicrobial agent- compound that kills or inhibits the growth of microorganisms)
• Secondary metabolite- a product excreted by a microorganism near the end of exponential phase or in stationary phase
• Bioactive compound- a compound that has an effect on the biology of another organism
• Novel enzymes- new protease/ lipases/ amylases/ cellulases etc with indistrial applications

Question 3

What are antibiotics?

Answer 3

• Antibiotics or antibacterials are a type of antimicrobial used in the treatment and prevention of bacterial infection
• They may either kill or inhibit the growth of bacteria
  (several antibiotics are also effective against fungi and protozoans)
• Antibiotics are not effective against viruses such as the common cold or influenza, and may be harmful when taken inappropriately
• Widely used in medicine
• Penicillin antibiotics were among the first drugs to be effective against many previously serious diseases, such as bacterial infections caused by staphylococci and streptococci

Question 4

Why are new antibiotics required?

Answer 4

ISSUES RELATED TO THE DISCOVERY OF NEW ANTIBODIES:

• Many pharmaceutical companies have either left antibiotic development or reduced research (can take 8-10 yrs)
• Pharmaceutical companies have limited research into new antibodies
• Only 3 new classes of antibodies have made it to market since the 1960’s

ANTIBIOTIC RESISTANCE:
- the lack of new drugs + over-prescribing and misuse of antibiotics -> bacteria becoming increasingly resistant to modern medicines
- Presence of antibiotics is a strong selective pressure -> misuse forces adaptation
- Inappropriate antibiotic treatment and overuse of antibiotics have contributed to the emergence of antibiotic-resistant bacteria eg. self prescription of antibiotics
- Over-use of antibiotics in Agriculture
SUPERBUGS- antibacterial-resistant strains and species
- Superbugs now contribute to the emergence of diseases that were for a while well-controlled

Question 5

How bioprospecting can be used to identify new enzymes?

Answer 5

1. Isolate large numbers of strains of microorganisms that may produce bioactive compounds
2. Grow each up and make extracts (aqueous and organic solvents)
3. Test a number of different screening assays to detect an effect (eg. inhibition of growth, formation of an easily-detectable product, etc)
4. Chemically characterise bioactive compounds (is it new?)= lead compound
5. Study mode of action
6. Set up animal trials, clinical trials, improved productivity, regulatory approval, marketing

Question 6

How new bioprospecting techniques such as metagenomics can lead to the discovery of new enzymes?

Answer 6

NEW METHODS OF BIOPROSPECTING:
SYNTHETIC CHEMICAL LIBRARIES
- Structural/ biochemical information -> design of thousands of chemicals which may bind to active sites of vital enzymes involved in metabolic pathways
- Issue: Synthetic chemicals may lack the ability to cross cell membranes

METAGENOMICS
- Function-based or sequence-based cultivation-independent analysis of the microbes from a given environment

HIGH THROUGHPUT SCREENING (HTS):

• HTS involves screening a large chemical library for compounds that bind to the target in the hope of finding relatively potent drug leads
• Although a large number (millions) of compounds can be screened, it is expensive and activity seen in vitro may be different to in vivo

Question 7

How new bioprospecting techniques such as metagenomics can lead to the discovery of new enzymes? Meta-genome

Answer 7

SCREENING STRATEGIES: SOIL META-GENOMICS

1. FUNCTION-BASED (EXPRESSION-DEPENDENT) SCREENING
   - DNA extracted from a source
   - DNA fragmented and ligated into a vector (plasmid, etc)
   - Vector transformed into expression host (eg. E.coli)
   - Host grown and desired trait identified using HTS (look for phenotype)
2. SEQUENCE-BASED (HOMOLOGY-DEPENDENT) SCREENING
   - Detect gene variants with the conserved motif from the meta-genome
   - Generates a large dataset, which is then analysed by appropriate bioinformatic tools (HTS)
   - Prior knowledge of the gene of interest required (eg. common motifs for enzymes)

Question 8

How new culturing techniques are making it possible to isolate new soil bacteria

Answer 8

NEW METHOD FOR CULTURING SOIL BACTERIA
iChip
- Trap bacteria between two semi-permeable membranes (allow for nutrients/ growth factors to be supplied to bacteria)
- iChip placed back into soil
- Bacteria grow in the soil as pure cultures
- Bacteria assessed for antibiotic production