Why medicinal chemists Why medicinal chemists
need molecular biologists
to discover
biodefense therapeutics
Lecture Presented to the University of
Hawaii, November 29th, 2018
Why a medicinal chemist needs molecular biology to
discover biodefense therapeutics
Lecture includes two example cases:
1. Anthrax and 2. Botulinum Neurotoxin
– History
– Important Questions to consider for a Biodefense– Important Questions to consider for a Biodefense
Therapeutic
– Help From Molecular Biology:
• Developing the Necessary Tools
• Applying the Tools
AMERITHRAX
Timeline of events:
https://www.cnn.com/2013/08/23/health/anthrax-fast-facts/index.html
https://www.fbi.gov/history/famous-cases/amerithrax-or-anthrax-investigation
Anthrax History
“Plague of Boils” in the Old Testament may have been referring to Anthrax in 15th century B.C. Egypt
Use in biowarfare and bioterrorism
• 1932-1945, WWII. Anthrax (and other agents) used by Japanese against Chinese
• 1932-1945, WWII. Anthrax (and other agents) used by Japanese against Chinese
• 1951-1953, Korean War. Aerial delivery of Anthrax possibly used by U.S. against North Koreans (?)
• 1979, Sverdlovsk, USSR. At least 68 deaths follow an accidental release of Anthrax spores from a biowarfarefacility
• 2001, AMERITHRAX. Unsolved (?) attacks with Anthrax spores sent through the U.S. Postal system kill 5
Bacillus anthracis
• Anthrax Pathogen
• Gram-Positive
• Rod-shaped
• Spores in Soil– World-wide– World-wide
– Hardy
• Herbivore Disease– Cattle, Sheep, Horses,
Mules, Goats
– Incidental Human Infection
Anthrax – forms of human disease
• Inhalational: – Infection via inhaled spores
– Long and variable dose-dependent incubation period (2-60 days)
– Onset looks like flu: aches, cough, fever
– Brief symptomatic improvement at day 3-4 is followed by rapid decline and death within 5-7 days (lungs � lymph � immunosuppression by toxins allows accelerated growth & dissemination)accelerated growth & dissemination)
– Fatality 80 - 100% even with treatment (sepsis, shock)
• Gastrointestinal:– From eating contaminated meat
– Severe GI symptoms, bloody vomit
– < 60% fatality with treatment
• Cutaneous:– Skin lesions which ulcerate and turn black (anthrakitis, Gr. “coal”)
– Rarely fatal with treatment
Anthrax Treatment: the key questions
• What does the disease actually do?– Pathogenesis (physiological and molecular mechanisms)
– How is it spread?Not person-to-person
– How can it be used as a weapon?SporesSpores
– How can it be cured? Prevention (vaccine)
or early postexposure intervention with antibiotics (<48 h)
• What tools do we need to answer these questions and develop a therapeutic?
The Molecular Mechanism of Anthrax Toxemia
1. Bacteria releases Protective
Antigen (PA), Edema Factor
(EF), and Lethal Factor (LF)
2. PA binds to cell surface
receptor (“Anthrax Toxin Receptor”
ATR)
3. Furin converts PA83 to PA63
4. Seven PA proteins combine
PA LF EF
B. anthracis
LF
7x
+ +
EF
- 20-kDa
1
23 4
5
4. Seven PA proteins combine
to provide a pore into cell
5. PA Heptamer binds either:
EF, forming Edema Toxin (ET); or
LF, forming Lethal Toxin (LT)
6. Endocytosis of LT or ET
7. EF or LF released into cytosol
8a. EF increases cAMP levels, leading to
edema
8b. LF cleaves Mitogen-Activated Protein
Kinase Kinases (MAPKKs) shutting
down cell machinery resulting
in cell death
Necrosis
Hypoxia
ATR
Endosome
EF
LF
cAMP
MAPKKs
Edema
Endocytosis
Adapted from Prince, A.S. J. Clin.Invest. 2003, 112, 656
6
7
8a
8b
Anthrax: Intervention Strategies?
PA LF EF
B. anthracis
LF
7x
+ +
EF
- 20-kDa
Necrosis
Hypoxia
ATR
Endosome
EF
LF
cAMP
MAPKKs
Edema
Endocytosis
Virulence Factors
• Determination of virulence factors
– Genetic modifications to organism establish that
LF deficient B. anthracis strains diminish lethality,
EF deficient bacteria still lethalEF deficient bacteria still lethal
– Molecular Biology: combine purified recombinant
toxin components to demonstrate bacteria-free
toxicity in cell culture and in vivo
Pezard C, Infect Immun 1991, 59, 3472-7.
Antibiotic Therapy
• Fluoroquinolines (“Cipro”), penicillins, and tetracyclines
• Recommended treatment course: 60 days• Recommended treatment course: 60 days
• Effective only if given in first 48 h of symptomatic inhalational disease, and works best if given before symptoms occur (Post-exposure prophylaxis)
Vaccine, Antitoxins,
Immunotherapeutics
• Biothrax Vaccine: (AVA, Anthrax Vaccine Adsorbed) is the only currently approved vaccine– Poor vaccination schedule (6 shots over 18 months for full protection +
annual boosters)– Poor reactogenicity profile– Not available to the general public; used for researchers working with
Anthrax, some veterinarians, selected military personnel
• Antitoxins: anti-PA antibody post-exposure therapeutics– Raxibacumab (Glaxo-SmithKline, FDA approval 2012); and – Obiltoxaximab, (Anthim, Elusys Therapeutics, FDA approval 2016)
• Human Immune Globulin, Anthrasil (Cangene) to be used with antibiotics; clears toxins from the circulation to improve morbidity and mortality (FDA approval 2015).
The bottom three therapies were not tested in humans, but are intended to improve survival based on animal studies (under the “Animal Rule”).
THE NEXT GENERATION:
Intracellular Postexposure Small Molecule Therapeutics
• Small Molecule (<500 MW) Inhibitors of LF
– Can act inside the cell, providing a longer window of opportunity than systemic clearance based of opportunity than systemic clearance based therapies
– Cheaper to prepare and stockpile compared to biologic drugs
Shoop WL, Proc Natl Acad Sci USA 2005 (Merck)
Jiao, G.-J. Bioorg. Med. Chem. Lett. 2012 (HBI)
Discovery and Development Needs
How can molecular biology help?
By providing purified proteins that can be:
1. Produced in sufficient quantity for study
and
2. Fine-tuned for experimental use.
3-D X-ray crystallographic analyses of LF
• Structural information about substrate binding – (Molecular Biology: mutations required at active site to inhibit
substrate cleavage; OR mutations required in protein substrate
sequence for the same purpose)
Turk, B.E., Nat. Struct. Mol.
Biol. 2004, 11, 60-66.
3-D X-ray crystallographic analyses of LF
Structural information about inhibitor binding
for design optimization – (Molecular Biology: truncated protein for stability and optimal
crystallization properties)
Shoop WL, Proc Natl
Acad Sci USA 2005.
Therapeutics discovery assays
– High throughput assay of the toxin activity you desire to
inhibit is essential (HTS to rapidly test tens of thousands of compounds)
• Reproducible
• Inexpensive
• Enzyme in vitro assays can tolerate a wide variety of possible • Enzyme in vitro assays can tolerate a wide variety of possible
inhibitor types (including those that are poorly soluble,
fluorescent), and allow large dynamic range
• Assay format must be amenable to high throughput, even
automation (very short assay run times are desired)
– Recombinant Proteins are superior for this purpose
• Purity, reproducibility, SAFETY!
The Dark Side
Molecular Biology in the Wrong Hands:
the Possible Development of Advanced
Biological WeaponsBiological Weapons
PART TWO: Botulinum Neurotoxins
Clostridium botulinum
• Botulinum
Pathogen
• Anaerobic
BacteriumBacterium
• Spores in Soil
World-wide
Botulism – forms of human disease
• Intoxication (no infection, active agent is the toxin):
– The U.S. has 150-200 naturally occurring cases per year, usually
from ingestion of improperly stored food (home canning,
fermented foods) or i.v. drug use
– Overdose of medical use toxin (Florida case)– Overdose of medical use toxin (Florida case)
– Intentional poisoning, bioterrorism & warfare
• Active Infections:
– Infant Botulism (rare)
– Gastrointestinal (very rare)
– Wound (i.v. drug users)
Botulism intoxication – symptoms and progression
• Initial symptoms include blurry vision, dry mouth,
difficulty swallowing
• Symptoms progress with descending paralysis
leading to respiratory failure and death
• Often accompanied by nonspecific gastrointestinal • Often accompanied by nonspecific gastrointestinal
distress
• Onset is dose-dependent; symptoms may continue to worsen up to 7 days
• Paralysis may persist up to several months, some symptoms may last for years (possibly PTSD or atrophy rather than toxin-related)
Botulism and Botulinum Toxin in History
“Sausage Poison”? from Latin botulus.
In 1895, Le Rustic restaurant, 3 musicians died, 50 fell ill after eating uncooked salted ham at a funeral in Ellezelles, Belgium.
Samples allowed first isolation of the bacterium by Emile Pierre van Ermengem, Professor of bacteriology at the University of Ghent. Named it Bacillus
botulinus (now Clostridium botulinum).
Use in biowarfare and bioterrorismUse in biowarfare and bioterrorism
• 1932-1945 WWII. Possible use by Japanese against Chinese
• 1989-91. Iraq military weaponizes Botulinum toxin but does not use Botulinum-containing bombs & missiles
• 1991-1995. Aum Shinrikyo cult in Japan cultures Clostridium and sprays culture supernatant in public streets and onto a U.S. Navy base; fortunately, their cultured strain did not produce toxin!
• Weaponization for military uses in programs in the U.S., Great Britain, and the Soviet Union (20th century)
Arnon, SS, JAMA, 2001, 285(8), 1059-1070.
Botulinum treatment:
the key questions
• What does the disease actually do?– Pathogenesis (physiological and molecular mechanisms)
– How is it spread?Not person-to-person, generally noninfectious intoxication (poisoning)
– How can it be used as a weapon?Aerosolized toxin, or poisoned food or drinkAerosolized toxin, or poisoned food or drink
– How can it be cured? Time. Supportive care only (respirator, ICU) – equine antitoxin (hBAT)
reduces hospital time if given quickly
• What tools do we need to answer these questions and develop a therapeutic?
Molecular mechanism of BoNT
intoxicationThe toxin’s enzymatic cleavage of
synaptic vesicle proteins inside the
nerve cells at nerve-muscle
junctions prevents acetylcholine
neurotransmitter release, resulting
in flaccid paralysis.in flaccid paralysis.
There are 7 serotypes A-H with
unique targets and varying
potencies.
Type A is the most potent:
Human LD50s
1 ng/kg i.v.,
14 ng/kg oral,
100 ng/kg inhalational.
Symptoms persist up to 3 months.
Arnon, SS, JAMA, 2001, 285(8), 1059-1070.
Botulinum: Intervention Strategies?
Vaccine Approach
• Postexposure vaccination is of no benefit.
• Preventive vaccination has a big drawback:
New medical uses for BoNTNew medical uses for BoNT
More than just Botox
• New medical uses for therapeutic botulinum
neurotoxin A include treatments for:
– Wrinkles ($ Botox $)
– Muscle spasms related to several disorders including
cerebral palsy, debilitating tics, rectal spasm, cerebral palsy, debilitating tics, rectal spasm,
overactive bladder, dystonia (musician’s dystonia)
and others
– Hyperhidrosis (excessive sweating)
– Chronic pain, including migraines
Individuals vaccinated against BoNT/A
could not receive these therapies
Passive Immunotherapy
• hBAT heptavalent despeciated equine antiserum (FDA approved, but supply expires after 2025) (doesn’t include new serotype H)
– Effective within 24-48 h window
Immunotherapeutic components of antiserum (antibodies) cannot enter
cells, and therefore will not affect toxin already inside the neuron.
– Effective within 24-48 h window
– Binds but may not clear circulating toxin
– Does not reverse symptoms
– ~10% adverse reactions
• Human antiserum (BabyBIG) (FDA approved)– Effective for infant botulism (shortens ICU time) with
accompanying antibiotic therapy
– Very limited supply (serum from immunized volunteers)
Extracellular postexposure antibody-based therapeutic
• Multicomponent humanized monoclonal antibodies clear extracellular toxin
Immunotherapeutic proteins cannot enter cells,
and therefore will not affect toxin already
inside the neuron.
antibodies clear extracellular toxin
– Narrow therapeutic window (before respiratory failure)
– In development (DoD 2024)
– These are recombinant proteins
THE NEXT GENERATION:
Intracellular postexposure small molecule therapeutics
• Small Molecule (<500 MW) Inhibitors of
botulinum toxin zinc metalloprotease
– Can act inside the cell, providing a longer window of – Can act inside the cell, providing a longer window of
opportunity than systemic clearance based therapies
– Cheaper to prepare and stockpile compared to
biologic drugs
Discovery and Development Needs
How can molecular biology help?
By providing purified proteins that can be:
1. Produced in sufficient quantity for study
and
2. Fine-tuned for experimental use.
3-D X-ray crystallography of BoNT/A LC
• Structural information about substrate binding – (Molecular Biology: mutations required at active site to inhibit substrate
cleavage to obtain crystal with bound substrate)
Breidenbach, MA,
Nature 2004, 432,
925-929.
SNAP-25
substrate
(pink) bound
to BoNT/A
LC
Structural information
about inhibitor binding
for design optimization (Molecular Biology provides truncated
protein for stability and improved
crystallization properties)
3-D X-ray crystallography of BoNT/A LC
Thompson, et al., Biochem., 2011, 50(19), 4019-4028.
crystallization properties)
Assays for Therapeutics discovery
– High throughput assay of the toxin activity you wish to
inhibit are essential (HTS to rapidly test tens of thousands of
compounds)
• Reproducible
• Inexpensive• Inexpensive
• Enzyme in vitro assays can tolerate a wide variety of possible
inhibitor types (including those that are poorly soluble,
fluorescent), and allow large dynamic range
• Assay format must be suitable for high throughput, even
automation (very short assay run times are desired)
– Recombinant Proteins are superior for this purpose
• Purity, reproducibility, SAFETY!
The Dark Side
Molecular Biology in the Wrong Hands:
the Possible Development of Advanced
Biological WeaponsBiological Weapons
(are the issues different for Botulinum than
they are for Anthrax?)
Review: helpful uses of molecular biology
in drug discovery for biodefense
Generally: molecular biology provides purified proteins that can
be:
– Produced in sufficient quantity for study, and
– Fine-tuned for experimental use, such as:
1. Elucidation of molecular mechanisms of disease and pathogenesis (testing of genetically altered organisms, isolated protein (testing of genetically altered organisms, isolated protein components and subunits)
2. Structural analysis (X-ray crystallography and mutant analysis of toxins with and without substrates and inhibitors)
3. Assay development for screening, discovery, and optimization of inhibitors (ideally using nontoxic subunit targets)
4. Production of protein-based therapeutics and vaccines