Lecture 5.4 (TCM)

Question 1

What is TCM?

Answer 1

Traditional Chinese Medicine

Question 2

What are some examples of alternate medicine?

Answer 2

chiropractic, herbal therapy, traditional Chinese medicine (TCM), meditation, yoga, acupuncture, and diet- based therapies.

Question 3

TCM has a long history of use in China with a proven record of high efficacy and low toxicity. Therefore, the use of empirical knowledge is an advantage over other screening strategies.

True or False

Answer 3

True

Question 4

Why use ‘knowledge based approach’ for TCMs?

Answer 4

TCM can be considered as a collection of “screened” natural products

Question 5

Describe construction of TCM-based sample libraries.

Answer 5

1. Select TCM based on empirical knowledge and available scientific data
2. construct a TCM-based sample library composed of formulae, crude extracts, active fractions, and pure compounds.
3. Develop and explore combinatorial libraries

Question 6

In how many categories does NMPA classify TCM into?

Answer 6

9

Question 7

What does category 1 of NMPA’s TCM classification refer to?

Answer 7

active component isolated and extracted from plants, animals, or minerals and its preparation (content of active component in the product > 90%) that have not been marketed in the domestic market previously.

Question 8

What does category 9 of NMPA’s TCM classification refer to?

Answer 8

generic drugs

Question 9

What do the categories of NMPA’s TCM classification (except for cat 1 and 9) entail?

Answer 9

technology innovation (e.g., optimization of dosage form and drug delivery methods) or

new discoveries (e.g., new active constituents, new plant parts for medicinal application, etc.)

Question 10

Previously, how were botanical products marketed?

Answer 10

as conventional food and dietary supplements with health-related claims.

Question 11

Under what circumstance is a botanical product considered a drug?

Answer 11

if it bears a disease claim.

Question 12

For a botanical product to be marketed as a drug, what requirements must the botanical drug meet?

Answer 12

the regulatory requirements for safety and effectiveness and comply with the manufacturing requirements to ensure product quality.

Question 13

What are the top 3 therapeutic categories for botanical drug products in the US?

Answer 13

1. oncology
2. endocrine metabolic
3. rheumatological analgesic

Question 14

What is a typical workflow for the generation of NP sample library?

Answer 14

solvent extraction of raw material
—-> organic extract + aqueous extract
——> fractionation
———> parellel multiple channel preparative HPLC

*activity screening can be performed after solvent extraction, at any of the steps

Question 15

What is the chemical process for NP-based drug discovery?

Answer 15

1. The NP is extracted, concentrated, fractionated and purified to yield biologically active compounds
2. identification of known compounds to avoid replication of previous effort
3. unknown structures determination thru high resolution NMR, MS, UV, IR
4. preliminary SAR studies
5. scale of of purification process
6. synthetic modification –> potential lead from ‘hit’
7. development of track candidate thru additional optimisation

Question 16

What is bio-assay guided fractionation?

Answer 16

starting with the extraction of natural materials of interest using organic solvents such as alcohol. –>
tested as crude extracts –>
isolation and identification of active compounds

Question 17

How to check purity of compound?

Answer 17

LC or LCMS

Question 18

What techniques are used to determine the structure of an unknown active compound?

Answer 18

NMR, MS, UV, IR, X-ray diffraction, optical rotatory dispersion (ORD), circular dichroism (CD)

Question 19

What are the key technologies in NP research?

Answer 19

1. Natural product databases + structure determination technologies –> identification of known compounds
2. extraction/isolation technologies –> bioassay guided fractionation
3. structure determination technologies

Question 20

Give some examples of extraction/isolation technologies

Answer 20

1. Supercritical Fluid Extraction (SFE)
2. Accelerated Solvent Extraction (ASE)
3. Pressurized Liquid Extraction (PLE)
4. Microwave-assisted Extraction (MAE)
5. Liquid – solid Isolation Technologies
   —> LC with solid absorbent as stationary phase
6. Liquid-liquid Isolation Technologies:
   —> Counter-current Chromatography (CCC)
7. gas chromatography

Question 21

What systems can HPLC coupled to for structure determination?

Answer 21

MS, NMR, CD

Question 22

Describe a HPLC-based identification system.

Answer 22

affinity selection –> second column for resolving into single components –> structure info by online UV, MS and NMR

Question 23

What kind of info does MS and NMR provide?

Answer 23

MS –> fragment ions

NMR –> substructure i.e. more extensive structural info

Question 24

What is directed biosynthesis?

Answer 24

Manipulate the genes associated with biosynthetic pathways to allow the engineering of compounds of interest.

Question 25

What is the genomic mining approach?

Answer 25

rapid ID of gene clusters that encode bioactive natural products --> allows computer predictions of chemical structure based on the sequence info

Question 26

Apart from conventional approaches in identifying the structure of NP or biological products, what technique could be used?

Answer 26

Genomic mining approach

Question 27

What is combinatorial biosynthesis (CB) and how can it complement the traditional chemical modification methodologies?

Answer 27

- introduce new functional groups onto natural product scaffolds - uses ‘tailoring’ enzymes in combinatorial approaches to expand natural product diversity HOWEVER, may not be applicable to synthetic compounds

Question 28

Combinatorial Biosynthesis (CB) is unique to natural products and may not be applicable to the modification of synthetic compounds. True or False

Answer 28

True

Question 29

Combinatorial Biosynthesis (CB) uses 'tailoring' enzymes, give some examples of tailoring reactions.

Answer 29

glycosylation, oxidation and reduction, methylation, halogenation, acylation and sulfation.

Question 30

A powerful approach to discover novel metabolites is to access sources of organisms that have not been explored previously. True or False

Answer 30

True

Question 31

What are some unexplored sources that could be used to discover novel metabolites?

Answer 31

1. microbes 2. marine environment microorganisms

Question 32

What are some microorganisms from the marine environment that will provide a high chance of finding novel drugs from natural sources?

Answer 32

actinomycetes fungi mycobacteria

Question 33

What are some obstacles in NP-based drug screening?

Answer 33

1. not possible to culture organism out of habitat 2. relevant NP not produced when organism out of habitat 3. presence of NPs that are known 4. presence of NPs that do not have drug-like property 5. bioactive NPs present in insufficient amounts 6. unknown molecular targets for NPS IDed with phenotypic assays

Question 34

What could be done to maximise the diversity of extracted NPs?

Answer 34

The biological material can be subjected to extraction with several solvents of different polarity.

Question 35

What are some solutions for overcoming obstacles in NP-based drug screening?

Answer 35

new methods for dereliction, extraction and pre-fractionation of extracts to eliminate false positives/interfering agents

Question 36

How to overcome the problem of the lack of possibility to culture the organism out of the natural habitat?

Answer 36

new methods for culturing and in situ analysis

Question 37

How to overcome the problem of 'relevant NP not produced when organism out of habitat'?

Answer 37

1. new methods for culturing and in situ analysis 2. new methods for NP synthesis induction and heterologous expression of biosynthetic genes

Question 38

How to overcome the problem of 'presence of NPs that are known'?

Answer 38

new methods for dereplication

Question 39

How to overcome the problem of 'presence of NPs that do not have drug-like property' and 'bioactive NPs present in insufficient amounts'?

Answer 39

New methods for extraction and pre-fractionation of extracts

Question 40

What does genome mining- based approaches to explore the biosynthetic capacity of microorganisms rely on?

Answer 40

DNA extraction, sequencing and bioinformatics analysis

Question 41

What is heterologous expression?

Answer 41

Heterologous expression is the introduction of either complementary DNA (cDNA) or RNA (cRNA) encoding for a protein of interest from one species into the cell of another species, such that the hosts' cellular machinery expresses the foreign protein

Question 42

What are the steps in genome mining-driven discovery of NPs?

Answer 42

1. DNA isolation from specific micorganism 2. sequencing 3. bioinformatic analysis 4a. heterologous expression --> NP isolation/characterization OR 4b. NP chemical synthesis OR 4c genetic manipulation of the native host --> NP isolation /characterisation

Question 43

What is the strength of 'synthetic-bioinformatic' approach?

Answer 43

it is entirely independent of microbial culture and gene expression

Question 44

What are the limitations of 'synthetic-bioinformatic' approach?

Answer 44

1. accuracy of computational chemical structure predictions 2. feasibility of total chemical synthesis

Question 45

Unmodified NPs often possess suboptimal properties, and superior analogues need to be obtained in order to yield valuable new drugs. True or false

Answer 45

True

Question 46

Unmodified NPs often possess suboptimal properties, and superior analogues need to be obtained in order to yield valuable new drugs. How can NP analogues be obtained?

Answer 46

1. Total chemical synthesis followed by chemical derivatization 2. Semisynthesis using a NP as a starting point 3. Biosynthetic engineering using manipulations of biosynthetic pathways of the producing organism to generate NP analogues

Question 47

Natural Product – based Drug Discovery is :

Answer 47

A multidisciplinary approach, involving the generation of truly novel molecular diversity from natural product sources, combined with total and combinatorial synthetic methodologies, and including the manipulation of biosynthetic pathways, provides the best solution to the current productivity crisis facing the industry and scientific community engaged in drug discovery and development.