- The golden era of antibiotic discovery from soil bacteria ended in the 1960s as actinomycetes species were overmined. Synthetic chemistry led to modifications of existing antibiotics but faced challenges penetrating gram-negative bacteria. - New antibiotic discovery requires exploring new sources like uncultured bacteria and targeting immutable aspects of bacterial cells. Recent promising compounds target the outer membrane proteins of gram-negatives. Combining natural and synthetic approaches may be most effective, like modifying natural products through medicinal chemistry. Chronic infections pose a challenge due to antibiotic tolerant persister cells, requiring multi-target approaches.

1. HAWASSA UNIVERSITY CM&HSC
School of pharmacy
Course: Pharmacology of natural products
Seminar on :The science of antibiotics discovery
by : Georgebush Diriba(Pharmacology Msc.
Candidate)
instructor : Dr. Serawit Deyno(Phd)
APRI,2023

2. The science of antibiotics
discovery

3. Introduction
• In all other therapeutic areas, there
is a positive correlation between accumulated
knowledge and the ability to discover new drugs.
But Discovering new antibiotics is uniquely
challenging and is contrary to this- (paradoxical)

4. • 1940s-Early 1960s : A golden era of antibiotic
discovery.
- In 1940s, Selman Wakman discovered
spectinomycin from soil actinomycetes
- The main classes of antibiotics were
discovered : —aminoglycosides, tetracyclines, b-
lactams, chloramphenicols, and macrolides
• Early 1960s, The golden era ended.
- Actinomycetes Species —was overmined.
- Things were looking up for synthetic compounds
.

5. • Synthetic compounds: (isoniazid, pyrazinamide,
and ethambutol)
• A broad-spectrum compound, metronidazole-
-Anaerobic or microaerophilic
• Finally, 1960s, Nalidixic acid(Less potent),
- Flourinated analogs (flouroquinolones-
ciprofloxacin

7. • Synthetic chemistry: introduce effective
analogs that converted (narrow-spectrum
G+ species into broadspectrum) antibiotics
- Penicillin-ampicillin;
- Erythromycin-azithromycin) and
- Analogs active against resistant pathogens.

8. • The industry combined several approaches to
produce a high-tech discovery platform
• However failed to discover new antimicrobials
• because Synthetic compounds were running into a
barrier
-Gram-negative bacteria – Has additional outer membrane.
-Outer membrane: lipopolysaccharide (LPS),hydrophilic
.Not permeable to large and hydrophobic compound
-The inner membrane, has a phospholipid bilayer and
membrane proteins. (hydrophobic)
-Extruded by Transenvelope multidrug resistance (MDR) pumps

9. Figure 2. Cell Envelope of Gram-Negative Bacteria and Its Drug Targets
The outer membrane contains a layer of negatively charged LPS that restricts the passage of large and hydrophobic compounds. LPS is the target of polymyxin.
The two essential proteins of the outer membrane are LptD, the transporter of LPS and the target of murepavadin, and BamA, which inserts b-barrel proteins
into
the membrane and is the target of darobactin and a polymyxin/murepavadin chimeric peptide. Penicillin binding proteins (PBPs), the transpeptidases that
assemble peptidoglycan, are the periplasmic targets of b-lactams. LepB is the periplasmic protease that cleaves signal peptides from proteins transported
across the inner membrane by the sec machinery, and is the target of modified arylomycin, GO775. Lipid II is the precursor of peptidoglycan and the target of
tridecaptin (and the target of teixobactin in Gram-positive species). Nutrients and many drugs pass through b-barrel porins in the outer membrane. Drugs that
pass through porins or the outer membrane are extruded by transenvelope MDR pumps, mainly of the resistance-nodulation-division (RND) family, such as

10. -
The AMR Crisis
• The World Health Organization (WHO) recently introduced
a list of priority pathogens (Tacconelli et al., 2018) and
‘‘critical priority’’— drug-resistant
- Enterobacteriaceae (E. coli, Salmonella typhimurium,
Klebsiella pneumoniae, Enterobacter),
- Pseudomonas aeruginosa, and
- Acinetobacter baumannii,
. All of the critical priority pathogens are Gram-negative
bacteria

11. • K. pneumoniae is of particular concern infection with
carbapenem-resistant strains --
-has a mortality rate of 30%–40% (USA)
- and 40%–50% in Europe
Antibiotic resistance comes in many forms
– penetration,
– efflux,
– target modification,
– destruction/modification of the antibiotic,
– target switching, and
– target sequestration
Antibiotic tolerance are primarily responsible for recalcitrance of
chronic infections.

12. Novel Lead Compounds
• NOVEL lead Compounds
• is not inhibitors of
-b-lactamases:- Clavulanic acid or
-Serine b-lactamases (SBLs)–tazobactam
- K. pneumoniae carbapenemase (KPC).
Avibactam, relebactamand vaborbactam
These compounds inhibit PBPs that are transpeptidases that
help build the peptidoglycan cell wall)
Mutations in the PBPs, or variants of these proteins borrowed
by plasmid acquisition, can provide resistance to b-lactams.
Novel compound a works in different way

13. NOVEL COMPOUND
TEIXOBACTIN,
• produced by from uncultured bacteria( Eleftheria
Terrae) and
- activite against all tested Grampositive bacteria,: - (MRSA) ,
Bacillus anthracis. It is highly efficacious in
• No detectable resistance
- hits two related targets—lipid II, precursor of
peptidoglycan, and
- lipid III, precursor of wall teichoicacid
• The targets are not mutable—not proteins and not
directly coded by DNA.

14. • MUREPAVADIN
• Murepavadin, a peptidomimetic
- exceptional activity against G-negative- (P. aeruginosa )
• Target outer memebrane
-LptD - translocator of LPS
• kidney damage in over half of the patients.
-Murepavadin is a polycation with 7 positive charges.
- Nephrotoxicity is a known liability of polycations such as
polymyxin

15. • Murepavadin is unusually potent, and it is
possible that a different regiment of
administration will result in a better
therapeutic window that will lead to its
development into a drug.

16. • Murepavadin/Polymyxin chimers
- outer membrane dual-targeting- to LPS and
BamA.
-broad activity against Gram-negative
- Hurdles Being a polycation.

17. DAROBACTIN A,
• A natural product discovered from screening P.
khanii (Photorhabdus symbionts)
- the outer membrane targets -BamA.
-It is active against a variety of Gram-negative
bacteria including MDR strains and

18. G0775
• An inhibitor of LepB: -a periplasmic protease
that cleaves signal peptides from proteins
exported across the cytoplasmic
membrane

19. Debio-1452-NH3,
• a version of afabicin modified for penetration
into Gram-negative bacteria.
• Of potential interest are inhibitors of LpxC
catalyzing the second step in the biosynthesis
of LPS. The protein is a Zn metalloenzyme.

20. From Ad Hoc Discovery to Platforms
Natural Products
. wakmans discover streptomycin From small hundred
strong collection, and is present in 1% of streptomycetes
• To find the next broad-spectrum antibiotic, one would
need to sieve through a library of at least 107 isolates.
• This makes discovery impractical; the typical size of a
strain collection in industry is around 105
.

21. • Actinomycetes are overmined
• A search of silent operons in actinomycetes is
unlikely to produce new drugs against Gram
negative pathogens, and
• One would need to look outside of
actinomycetes for novel antibiotics

22. New Sources of Natural Products
• Looking outside of actinomycetes for prolific
secondary metabolite producers,
• It makes sense to look for novel compounds among
groups of microorganisms that have been
underexploited.
• Most abundant soil taxa, more common than
actinomycetes—acidobacteria, verrucomicrobia,
rokubacteria, and gemmatimonadetes—devote a
considerable part of their genomes to BGCs

23. The challenges
• only 1% of cells from natural environments such as soil will
grow on a petri dish.
• 99% of species are uncultured
• uncultured bacteria seem like a good choice for novel lead
discovery
• Certain uncultured bacteria needs growth factors produced by
their neighbors
-iron-chelating siderophores and
- quinone components of the respiratory chain
- accounts oly 10%
- Scientist discovered iChip- to trick bacteria and bacteria
perceive as a natural habitat

24. Dereplication
• Dereplication, Identifying a Promising
Compound.
• by liquid chromatography-tandem mass
spectrometry (LC-MS/MS)
Membrane-Acting Compounds
• polymyxin and daptomycin. Polymyxin is a
polycationic peptide
• Daptomycin inhibits synthesis of lipid II,
precursor of peptidoglycan,

25. Synthetic Compounds and Rules of Permeation
• Rules of permeation into Gram-negative bacteria
• It is made of MDR pumps that extrude amphipathic compounds
across both membranes of the envelope.
To come up with rule of permeation Some of the parameters have
been established
- - a molecular weight cut-off of 600 Da and
- -low hydrophobicity
• - a low number of rotatable bonds (rigid structure)
• -a positive charge in the form of an amino group, -NH3+ are all
good for
permeation

26. Combining the Natural and Synthetic Chemistry
Platform
• Adding a positively charged amino group to penicillin(a
natural product) facilitated permeation of ampicillin through
porins of the outer membrane,.
• Modification of the narrow-spectrum macrolide antibiotic
erythromycin produced azithromycin, which is active against
Gram-negative bacteria with a relatively weak penetration
barrier, such as Neisseria and Moraxella.
There are good reasons to use natural products as a starting
point for a synthetic chemistry program
• Synthetic chemistry is good at producing small molecules
that inhibit enzymes or bind to receptors.
• Natural products do not seem to be limited by target or
mode of action constrains.

27. Chronic Infections and Antibiotic Tolerance
Drug tolerance
• additional threat posed by our pathogens
that is not common knowledge, but arguably causes greater
morbidity and mortality—
 by the presence of dormant persister cells produced stochastically
by bacterial populations
 Persisters are phenotypic variants of regular cells and are largely
responsible for recalcitrance of chronic infections
e.g cicystic fibrosis

28. Rational Approaches to Combat
Resistance
• To hit multiple targets.
• To hit immutable targets
• A strategy of bacteria attacking their
neighbors with a combination of compounds

29. THANK YOU!