Why medicinal chemists need molecular biologists to discover biodefense therapeutics

Lecture Presented to the University of Hawaii, November 29 th , 2018 Why a medicinal chemist needs molecular biology to discover biodefense therapeutics

Lecture includes two example cases: 1. and 2. Botulinum Neurotoxin – History – 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 15 th century B.C. Egypt

Use in biowarfare and • 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 biowarfare facility • 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 – Hardy • Herbivore Disease – Cattle, Sheep, Horses, Mules, Goats – Incidental Human 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) – 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? Spores – How can it be cured? Prevention (vaccine) or early postexposure intervention with (<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 1 Antigen (PA), Edema Factor PA + LF + EF B. anthracis (EF), and Lethal Factor (LF) 2. PA binds to cell surface 2 receptor (“Anthrax Toxin Receptor” 3 4 EF ATR) - 20-kDa 7x 5 3. Furin converts PA 83 to PA 63 LF 4. Seven PA proteins combine to provide a pore into cell ATR 5. PA Heptamer binds either: Endocytosis EF, forming Edema Toxin (ET); or Edema cAMP EF 6 LF, forming Lethal Toxin (LT) 8a 7 6. Endocytosis of LT or ET 7. EF or LF released into cytosol Necrosis 8a. EF increases cAMP levels, leading to Hypoxia MAPKKs LF Endosome 8b edema 8b. LF cleaves Mitogen-Activated Protein Kinase Kinases (MAPKKs) shutting down cell machinery resulting Adapted from Prince, A.S. J. Clin.Invest. 2003 , 112 , 656 in cell death Anthrax: Intervention Strategies?

PA + LF + EF B. anthracis

EF

- 20-kDa 7x LF

ATR

Endocytosis

Edema cAMP EF

Necrosis Hypoxia MAPKKs LF Endosome Virulence Factors

• Determination of virulence factors – Genetic modifications to organism establish that LF deficient B. anthracis strains diminish lethality, EF 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. Therapy

• Fluoroquinolines (“Cipro”), penicillins, and tetracyclines

• 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 – , (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 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 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 Weapons PART TWO: Botulinum Neurotoxins Clostridium botulinum

• Botulinum Pathogen • Anaerobic Bacterium • 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) – Intentional poisoning, bioterrorism & warfare

• Active : – 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 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 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 (20 th 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 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 intoxication The toxin’s enzymatic cleavage of synaptic vesicle proteins inside the nerve cells at nerve-muscle junctions prevents acetylcholine neurotransmitter release, resulting in flaccid paralysis.

There are 7 serotypes A-H with unique targets and varying potencies. Type A is the most potent:

Human LD 50 s 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 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, 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

Immunotherapeutic components of antiserum (antibodies) cannot enter cells, and therefore will not affect toxin already inside the neuron.

• hBAT heptavalent despeciated equine antiserum (FDA approved, but supply expires after 2025 ) (doesn’t include new serotype H) – 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

Immunotherapeutic proteins cannot enter cells, and therefore will not affect toxin already inside the neuron.

• Multicomponent humanized monoclonal 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 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 )

SNAP-25 substrate (pink) bound Breidenbach, MA, to BoNT/A Nature 2004 , 432 , LC 925-929. 3-D X-ray crystallography of BoNT/A LC

Structural information about inhibitor binding for design optimization (Molecular Biology provides truncated protein for stability and improved crystallization properties)

Thompson, et al., Biochem., 2011 , 50(19), 4019-4028. 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 • 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 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 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