Lecture 1: History of antibiotic therapy






Russell E. Lewis
Associate Professor of Infectious Diseases
Department of Molecular Medicine
University of Padua


russelledward.lewis@unipd.it
https://github.com/Russlewisbo

Agenda

Appropriate perspective



Antimicrobial use: bacteria vs. humans

Bacteria

  • A millionth of a meter or less

  • Small amounts targeted to special circumstances

  • Minimal environmental exposure, minimized selective pressure

Humans

  • Considerable environmental exposure

  • Considerable selective pressure for resistant organisms

Chemical warfare in nature



Ancient antibiotic therapy



  • China: Moldy tofu to treat inflammation and infection of the skin 
  • Egypt: Moldy bread (Aish baladi) to treat skin lesions 
  • Greeks: Myrrh, wine, honey or caustic substances to treat wound infections 

Egyptians made tetracycline beer

(Nelson et al., 2010)

Paul Erlich (1854-1917) and the birth
of “modern” antibiotics



  • Developed methods for staining tissue made it possible to distinguish between different types of blood cells, which led to the ability to diagnose numerous blood diseases → “magic bullet hypothesis”

  • First screening of chemical libraries to identify active antibacterial compounds (synthetic compounds)

  • First effective treatment for syphilis- arsphenamine (Salvarsan) 1909

  • Developed methods for staining tissue made it possible to distinguish between different types of blood cells, which led to the ability to diagnose numerous blood diseases

  • First screening of chemical libraries to identify active antibacterial compounds (synthetic compounds)

  • First effective treatment for syphilis- arsphenamine (Salvarsan) 1909

Salvarsan is toxic



Side effects attributed to Salvarsan, included rashes, liver damage, and risks of life and limb, were thought to be caused by improper handling and administration of the relatively insoluble compound

Alexander Fleming (1881-1955): The serendipitous discovery






  • Fleming was skilled at administering Salvarsan to patients with syphilis, which led to a small but profitable practice
  • During World War I, Fleming worked in a wound-research laboratory and discovered that chemical antiseptics do not sterilize jagged wounds, and pus has its own antibacterial powers
  • After the war, Fleming focused on studying leukocytes and antisepsis, and in 1921 he discovered lysozyme, a substance that causes bacteria to disintegrate, but its effectiveness was limited against disease-causing bacteria

Discovery of penicillin





Gerhard Domagk (1895-1964):
Discovery of sulfonamides

  • Prontosil metabolized to sulfanilamide in vivo, initially found to be inactive in microbiological assays
  • Among the early patients was Domagk’s own 6-year-old daughter, Hildegard, who had contracted a severe streptococcal cellulitis from an accident with a sewing needle
  • Utterly desperate when the doctor recommended amputation to save his daughter’s life, Domagk treated Hildegard with Prontosil.
  • Hildegard recovered, but suffered a permanent reddish discolouration of her skin owing to the drug

Florey, Chain and Heatley:
Rediscovery and purification of penicillin


First hints of efficacy

Challenge: How to produce enough penicillin?



  • It was difficult to ferment enough crude peniciilin and to gain samples with few impurities
  • Heatley’s inventiveness shown both in this device and in his design of a dish to grow the mould at an experimental scale was of great importance to the development of penicillin

First trials in patients


Clinical response to penicillin


Clinical response to penicillin


Winston Churchill’s life saved with sulfapyridine in 1943



Industrial scale production of penicillin
in the United States



Structure of penicillin


  • Dorothy Hodgkin, a crystallography expert at Oxford University, used X-rays to analyse the structure of various natural products 

  • In 1946, she determined the structure of penicillin, earning her the Nobel Prize in Chemistry in 1964

  • Knowledge of the penicillin structure allowed scientists to modify penicillin, leading to the development of semisynthetic versions with broadened spectrum of activity, incrased stability, reduced toxicity

First reports of penicillin resistance


Repeating history

Discovery of cephalosporins



  • Bronzu performed epidemiological studies of typhoid infection in Cagliari, noted Salmonella typhi was not cultured once sewer water was discharged into the sea
  • Isolated the mold Cephalosporium (now known as Acremonium) in 1948 from seawater
  • Noticed that these cultures produced substances (he named mycetin) that were effective against Salmonella typhi, the cause of typhoid fever, which had beta-lactamase.
  • After he failed to find support from Italian government, he sent the fungus to Howard Florey at Oxford
  • 1962: Isolation of cephalosporin C

First effective therapies for tuberculosis

  • Selman Waksman and his team discovered multiple antimicrobials in the 1940s, including actinomycin, streptomycin, and neomycin

  • Their research focused on studying fungi and Actinobacteria, with a particular emphasis on soil bacteria belonging to the Streptomyces genus

  • Streptomyces bacteria were found to naturally produce a wide range of antimicrobials, leading to the discovery of these important compounds

  • Wakesman is credited with introduction of the term “antibiotic”

TB sanatoriums and impact of effective therapy

Antibiotic discovery: “The Golden Age”

Crises in new antibiotic development



Why has new antibiotic discovery faltered?

Empirical screening of fermentation broth was remarkably successful during the early phases of antibiotic discovery

Rediscovery of the old antibiotics through empiric screens becomes the biggest challenge

By the 1970’s antibiotic discovery began to shift from empirical to more “rationale” targeted approaches


  • Attempts to overcome the increasing work of dereplication with fewer possibilities for novel compound discovery

  • Initially based on the recognition that most useful antibiotics discovered to date targeted the cell wall or protein synthesis

  • Leveraged a growing ability to clone genes and manipulate bacterial strains to enhance whole cell screening (or specific purified proteins) for specific bacterial targets

1980s-1990s



As novel antibiotic discovery began to falter, modification of existing antibiotic scaffolds (pharmacophore) to overcome resistance became a dominant strategy for bringing “new” antibiotics to market


β-lactamases

Macrolides and tetracyclines

Fluoroquinolones

Sequencing of Haemophilus influenzae
genome in 1995

…but the crises still got worse

Why has biotechnology failed to deliver new antibiotics?


Problems with targeted antibiotic screening
in the 1990s and early 2000s



  • Bias to towards identification of single target inhibitors (high risk of rapid resistance development)

  • Biased towards human (eukaryotic) targets

    • Off target effects, higher toxicity potential

  • Chemical screening libraries- Mostly lipophilic compounds with limited structural diversity

    • Most drugs in libraries were highly flexible pharmacophores with few features found in antibacterial groups such as polar functional groups, complex ring systems, and chiral centers

  • Typical outcomes of whole cell screening: identification of detergent-like compounds or surface-active agents that affect integrity of bacterial cell membrane

  • Typical outcome of cell-free screens: discovery of compounds that would inhibit target protein but no activity against whole cells 

  • Screens rarely identified compounds with Gram-negative activity (because of excessive lipophilicity of screening library)

  • Biggest problem was probably the use of compound screening libraries that did not reflect the unique physicochemical property space occupied by known antibacterial agents

It is difficult to improve on nature


Densely deployed functional groups, allowing for maximal number of interactions with molecular targets, leads to exquisite selectivity for pathogen targets versus the host

Searching the wrong physiochemical space
for antibiotics


Physiochemical space varies by pathogen

An especially tricky problem for gram-negatives

How should we move forward? [.smaller]

Targeted screening

  • Specific inhibitors = rationale approach to develop still holds promise

  • Massive advances in synthetic biology (combinatorial biosynthesis, pathways engineering)

  • Development of new “antibiotic-like” chemical scaffolds for screening

  • Experience gained from 30 years of failure:

    • Knowledge of factors influencing bacterial cell penetration, transport, cellular efflux and protein binding is increasing rapidly- new rules similar to Lipinski 5 to help guide drug design

    • Use of more “druggable” libraries and combinatorial libraries that mimic natural products

    • Genetic modification of biosynthetic gene clusters in natural sources (i.e. actinomycetes)

Natural product screening

  • Billions of natural selection have optimized structures for protein interactions and antimicrobial activity

  • Estimated that less than 1% of prokaryotic and 7% of fungal strains have been isolated and cultured

  • Density and functionality in many natural products enables inhibition of multiple protein targets simultaneously

  • Complex structures make them poor substrates for metabolizing enzymes (superior pharmacokinetics and pharmacodynamics compared to small molecules)

  • More efficient and powerful methods for isolation and de-replication have been developed

Alternative strategies




  • Antimicrobial peptides (AMPs)

  • Monoclonal antibodies (MAbs)

  • Antibody-antibiotic conjugates

  • Bacteriophages

  • Antisense-based antimicrobials (ASOs)

  • Vaccination/immunotherapy

Remaining problem: Economics


References



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