PHC 502 Exam 1 midterm (Miller + ch1-3)

List sources of natural products for drug discovery

• Plants

• Fungi

• Bacteria

• Marine invertebrates

• Marine microbes

List 4 ways a source may be chosen for drug development

• Ethnobotany

• Natural observations

• Biodiversity

• Genetic sequencing

How are solid materials extracted?

Solvents

• Hydrophilic

• Lipophilic

can pull-out chemicals from a source (solid material)

How are bacterial cultures extracted?

1. Bacteria is grown

2. Transfer to centrifuge tube

3. Placed in centrifuge

4. Centrifuge it

   • Solids separate from liquids

What are natural products? (Definition Dr Miller)

Chemicals made by living organisms that are NOT required for immediate survival, but useful in defense, communication, and interaction with the environment

• “Secondary metabolites”

What are the five building blocks of primary metabolites

• Terpenoids

  • Isoprene

• Polyketides

  • Acetate

• Peptides

  • Amino acids

• Phenylpropanoids

  • Shikimate-derived molecules

• Alkaloids

  • N-containing primary metabolites

Why should NPs be used as drugs

•Natural products are chemically complex and contain many stereocenters

•Drug targets are 3D, not flat. Natural products evolved to bind to complex structures.

What technique is used to separate chemicals based on their polarity?

Chromatography

• Separates chemicals into different groups of compounds

• Sorted by polarity

Describe bio-assay guided drug discovery

A test done on a biological organism to check for an activity

• Determine active fractions

  • Narrow active fraction group down via chromatography

• Continue to do so until a molecule is pure and active

• One pure, determine how potent it is

  • How diluted can the concentration be while still retaining activity?

How are chemical extracts and purified compounds analyzed?

• High pressure liquid chromatography (HPLC)

  • Fancy chromatography machine that separates chemicals into individual compounds instead of fractions

    • Also see if molecules absorb light

• Mass spectrometry (MS)

  • “weighs” molecules, identifies molecular mass

• Nuclear magnetic resonance (NMR)

  • Used to determine purity and molecular structure of a sample

List famous natural products, what plants they derive from, and what they are used for

1. Morphine

   • Opium poppy

   • Painkiller

2. Mescaline

   • Peyote cactus

   • Hallucinogen

3. Caffeine

   • Coffea plant

   • Stimulant (considered the world’s most consumed psychoactive drug)

4. Ephedrine (and isomer pseudoephedrine)

   • Ephedra sinica

   • Nasal decongestant, stimulant

List the four marine invertebrates mentioned in lecture. What is unique about marine invertabrates as a whole?

1. Sponges

2. Corals

3. Mollusks

4. Tunicates

Unique because

• Extremely diverse

  • Classified into over 30 phyla

• Do not have a backbone

  • Often have soft bodies

Describe sponge anatomy

Choanocytes move water through sponges for feeding and respiration

Sclerocytes produce “spicules”; serve as skeletal structure.

list traits of sponges

• Most ancient animals

• Have unique cell types

• Skeletons are made of silica or calcium carbonate

List traits of tunicates

• the most “advanced” invertebrate

• has a notochord

  • “rudimentary spinal cord”

• filter feeders

How do you extract samples that are liquidated?

Separatory funnel

• Like dissolves like

What is LCMS-based metabolomics

Liquid chromatography–mass spectrometry

• Ultra high pressure chromatography + mass spec combined

• Learn molecular mass and all the compounds in a complex sample in one experiment

• Good way to “dereplicate” molecules/figure out what parts of the molecule are present that we already know about

What is the top down approach to natural products discovery

Traditional approach

• Culture condition screening

• diverse sampling

• comparative metabolomic profiling

  • = natural product discovery

What is the bottom up approach to natural products discovery?

Newer/modern approach

• Bioinformatics tools (antiSMASH)

• Native host

• Heterologous host

  • = Natural product discovery

Why are the effects of NP considered “hidden”? What modern tools are being used to work around this?

They’re not expressed under standard laboratory conditions

• Must be unlocked or elicited (especially in fungi)

Bioinformatics tools and genomics are currently being used to understand the biosynthetic capacity of microbes/plants

• Bioinformatics tools: antiSMASH

• Experimental tools” KOs, heterologous expression

What is antiSMASH

A software platform that analyzes a genome

• Spits out an inventory of predicted secondary metabolite gene clusters

• Sequences of enzymes can be compared for evidence of their potential functions

Describe heterologous expression. Pros?

expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment

• Expressing genes/gene clusters in heterologous host

• Isolate metabolites or show they are detected by LC-MS

Pro: Good system when the producing organism is hard to grow or doesn’t produce much

What is a gene cluster? What makes up a gene cluster?

A group of functionally related genes that are located close together in the genome, often sharing a single regulatory sequence / all the genes required to confer onto the host some advantage a secondary metabolite might provide

1. Biosynthetic enzymes

2. Regulatory genes

3. Transport systems

   • Efflux

   • Uptake

4. Mobile genetic elements

5. Additional “unknown” components that haven’t been recognized as necessary

Describe the role of biosynthetic enzymes

Produce the molecule

Describe the role of regulatory genes

Control the timing of production and maintenance of metabolite pools

Describe the role of efflux transport systems

Directs the molecule to where it needs to be

Describe the role of uptake transport systems

Brings in resources to the molecule, provides signals for molecule

Describe the role of mobile genetic elements

Play major roles in shaping gene function, genome evolution, and adaptation

• i.e. transposon fragments, etc

Of the FDA-approved small molecule drugs, how many are natural products and related molecules? (1981-2019)

Aprox. 67%

Briefly describe the golden age of antibiotic discovery

• 1940s-1960s

• People grew microbes in the lab and found out what chemicals they produced

• ~2/3 of all antibiotic drug classes currently in use came from this era

• Most drugs from this era are from soil-dwelling bacteria and fungi

Why did antibiotic discovery slow so rapidly? (1980s-present)

• “Low hanging fruit” had already been picked

  • Terrestrial, soil-dwelling actinomycetes

  • Regular rediscovery of known antibiotic classes that act through the same MOA

• Big pharma companies shifted focus to chronic illness

  • More profitable than acute illnesses that require antibiotics

Briefly describe modern US patent laws in relation to natural products

• “Products of nature”, “natural phenomena”, or “laws of nature” are merely discovered, not invented. Cannot patent a natural product because it’s not something you came up with.

• A molecule must be “markedly different” in structure or function from a natural substance to be patentable

  • You could discover the best anti-cancer drug ever, but cannot get (the best kind of) patent protection on the natural product itself – you must make changes to the chemistry

Describe taxol. What type of drug is it? Describe the scale-up process. What is it’s MOA?

• True natural product

• Discovered and developed as a drug without changing the molecular structure

• Natural source is limiting

  • Semi-synthesis of taxol allowed for large scale production

• MOA: Blocks cell division by binding to tubulin during mitosis

Why was Halichondrin B not practical as a drug despite it’s potency? What solution was identified?

• Natural isolation was inefficient: kilograms of sponge resulted in milligrams of compound

• Dozens of complex stereocenters were difficult to control for

• Not scalable due to chemical/structure complexity

• Couldn’t build molecule from scratch. Too complex, tedious, and had low yield

SOLUTION: Simplify the molecule, identify the pharmacophore

What is Eribulin? What type of drug is it/where was it derived from? What is it used to treat, MOA?

Halichondrin B pharmacophore

• Fully accessible to synthesis

• MOA: microtubule inhibition. Anti-cancer drug

• Approved for metastatic breast cancer and liposarcoma

Describe dolastatin. What type of drug is it? MOA? Where is it derived from?

• Cytotoxic agent. VERY VERY TOXIC. LOW IC50

  • Inhibits tubulin

  • Inhibits microtubule polymerization

    • Spindle cannot form, cell cannot divide

• Derived from sea hare (1mg isolated for each 100kg of sea hare. Very inefficent)

• Also harvested from cyanobacteria. ~10,000x more isolated compound extracted from cyanobacteria than a sea hare

Terpenoids are polymers of what?

Isoprene units (C₅)

Polyketides are polymers of what?

Acetate units (C2)

Peptides are polymers of what?

Amino acids

Where do phenylpropanoids derive from?

Phe/Tyr metabolism (Shikimic acid pathway)

Alkaloids contain what?

Metabolites with nitrogen

What is the FDA definition of a drug?

• A substance recognized by an official pharmacopeia or formulary

• A substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease

• A substance (other than food) intended to affect the structure or any function of the body

• A substance intended for use as a component of a medicine but not a device or component, part, or accessory of a device

How are approaches to drug discovery/development classified?

• Irrational approach

• Rational approach

• Antisense approach

• RNA interference approach

• Biopharmaceuticals/biologics

• Gene therapy

• Stem cell therapy

  • Somatic cell

  • Germ cell

Describe the irrational approach in drug discovery/development

Historical, traditional method

1. Uses empirical observations of pharmacological effects from multiple compounds

2. The active component that causes the observed effect is isolated

3. Chemical formula is determined

4. Modifications are made to improve properties

Describe the rational approach in drug discovery/development

• Requires 3D knowledge of target structure involved in a disease

• Drugs are designed to interact with target structure to create a response

• More modern, past 3 decades

Describe the antisense approach in drug discovery/development

• Modern approach

• Requires modifications to oligonucleotides that can bind to RNA and DNA

• Antisense drugs are used to stop transcriptional or translational pathways → interfere with process of disease

Describe the RNAi approach in drug discovery/development

Uses short interfering RNA (siRNA) to interfere with expression of a gene

Describe biopharmaceuticals/biologics drug discovery/development

Protein-based drugs

• Antibodies

• Vaccines

• Cytokines

Discovery starts from an understanding of the biological pathways that cause a specific disease. Manufacturing is based mainly on rDNA technologies using living organisms (bacteria, yeast, mammalian/insect cells)

Describe gene therapy drug discovery/development

Based on remedying a diseased gene

• Insert a missing gene into cells

• Insert a modified gene into cells

Lots of ethical considerations. Aim is for the inserted gene to influence the disease pathway, or to initiate synthesis of missing proteins/enzymes

Describe stem cell therapy drug discovery/development

• Aim is to grow body parts to replace defective human organs and nerves

• Stem cells are harvested from early embryos or umbilical cord blood

  • Due to young age, can be directed to grow into organs to replace diseased tissue

• The use of germ cells involves cloning → strict regulatory guidelines on research

Give a brief description of the drug development process from drug discovery to approval

Discovery → Preclinical → Clinical → Marketing application → Approval

Define FDA and EMA. What are their roles?

FDA= Food and Drug Administration (U.S.)

EMA = European Medicines Agency (E.U.)

Regulatory bodies that oversee the entire drug development process.

What does GCP stand for

Good clinical practice

Give a brief description of the drug discovery process

1. Identify and define medical needs

2. Research disease mechanisms

   • ID/validate targets involved in disease process

3. Search for lead compounds

4. Optimize the properties of the lead compounds

5. Perform drug development & pre clinical studies

   • in vitro

   • in vivo