Drug Development

Question 1

Chemical considerations

Answer 1

Electronic effect
Solubility effect
Steric effect

Question 2

Electronic effect

Answer 2

Resonance

Electronegativity - ability to donate or withdraw electrons

Question 3

Solubility effect

Answer 3

Water soluble groups
Ionizable groups
Hydrogen bonding
Lipid soluble groups

Question 4

Steric effect

Answer 4

Bulk and position
Modification close to functional group undergoing metabolic transformation can cause dramatic effect
Acetylcholine rapidly hydrolysed by acetylcholinesterase
Bethanechol is not hydrolysed by acetylcholinesterase as methyl group cannot be accommodated in active site of enzyme

Question 5

What does the functional group of a drug effect

Answer 5

ADME
Solubility
Shelf life
Target organisms

Question 6

Other factors affecting drug development

Answer 6

Scientific difficulty - screening of huge libraries of small molecules are not providing new antibiotics
Financial reward
Scale down of research in (big) pharma companies
Loss of “experts”
Socio-economic factors
Toxicology
Pharmacogenetics

Question 7

ADME

Answer 7

Absorbed
Distributed
Metabolised
Excreted

Question 9

Average time to market

Answer 9

15 years

Question 10

Drivers of research

Answer 10

Initial grant funding (BBSRC, EU and EPSRC)

Industrial funding
Can you patent a natural product ?

Question 12

ED50

Answer 12

Median effective dose
The dose of a medication that produces a specific effect in 50% of the population that takes the dose

Question 13

Median toxic dose

Answer 13

The dose required to produce a defined toxic effect in 50% of subjects

Question 14

Median lethal dose

Answer 14

The dose required to kill

Question 15

Process of development

Answer 15

The initial discovery hit on screen

Characterization purification and Identification of active compounds (HPLC / mass spec)

Measurement of Until microbial activity (MIC)

Measurement of in vitro toxicity (cell culture)(if low toxicity proceed to Galleria)

Measure of in vivo toxicity (Galleria mellonella)if MIC lower that ~30 ug/ml and low toxicity then test in mice)

Measurement of efficacy (Galleria mellonella) (if MIC lower that ~30 ug/ml and low toxicity then test in mice)

Measurement of efficacy mouse thigh model

Question 16

Identification of a drug molecule - the early antibiotics

Answer 16

In 1909, Paul Ehrlich (also worked with Robert Koch)
He discovered the very first antibiotics “a magic Bullet” , synthetic arsenic-based drugs (Salvarsan 606), in a large screen of hundreds of organoarsenic compounds for use in the treatment of syphili
It targeted the Syphilis bacteria
without damaging the rest of the body

The Idea of the therapeutic window

Question 17

Identification of penicillin

Answer 17

Alexander Fleming discovered penicillin, the first natural product antibiotic, in 1928.
Penicillin was also very difficult to isolate and purify, until the late 1930s. In 1939 by Florey and Chain
Dorothy Crowfoot Hodgkin elucidated the structure of penicillin in the early 1940’s
Following this discovery, penicillin started to be used systemically as an antibiotic, which then ushered in the golden age of antibiotics.
In 1945, Fleming, Chain, and Florey were awarded the Nobel Prize in Physiology or Medicine for their discovery of penicillin.

Question 18

Identification of sulfonamides

Answer 18

Gerard Domagk discovered Prontosil, a red dye, while experimenting with azo dyes against bacterial infections in mice in 1935

This led to the synthesis of more than 5000 sulfa drugs between 1935 and 1945

Sulfa drugs are broad-spectrum and work by inhibiting the dihydropteroate synthase (DHPS) enzyme in folate biosynthesis

Question 19

Waksman platform

Answer 19

Selman Waksman received the Nobel prize in 1952 for his “discovery of streptomycin, the first antibiotic effective against tuberculosis.” Waksman began systematically screening soil microorganisms for antibiotic production, He discovered that soil actinomycetes (streptomycetes) are prodigious producers

The screening method, often called the Waksman platform, involved preparing culture extracts from soil actinomycetes and overlaying filter paper discs infused with these extracts over a test organism on an agar plate, and then looking for zones of growth inhibition

Question 20

Anti microbial development step one identify in vitro antibacterial activity A simple Primary Screen

Answer 20

Collection of environmental sample
Plate out to culture diverse community
Isolation of pier microorganisms for screening
Isolation of microorganisms
Liquid culture
Extracts on disks

Question 21

Spheroplast screen

Answer 21

From the 1960s into the 1990s, spheroplast screens were used as a primary method for discovery of antibiotics that inhibit cell wall biosynthesis.
Growing bacteria were exposed to test substances under hypertonic conditions (concentration is higher than that inside the cell).

Inhibitors of cell wall synthesis caused growing bacteria to form spheroplasts. This screen enabled the discovery of fosfomycin, cephamycin C, thienamycin and several carbapenems.

Question 22

Semi-synthetic antibiotics

Answer 22

First response to bacterial resistance
Highly successful strategy where the natural antibiotic scaffold was modified chemically to produce new antibiotics with higher activity than the original
Express silent or cryptic biosynthetic genes

They are not routinely expressed under standard cultivation conditions, and there are several ongoing efforts to express these silent pathways and examine their products. Many of the following methods can also be used to increase antibiotic yields.

Question 23

New strategies and techniques for natural products

Answer 23

Less than 1% of the microbes present in the environment can be cultivated in the laboratory.
Traditional cultivation methods allow faster growing species to dominate, and rich medium supplies excess nutrients that may be toxic to oligophilic bacteria.
Many environmental bacteria only make microcolonies that require microscopic identification
A major reason why many species are uncultivatable is that synthetic medium lacks essential nutrients or growth factors that are present in their natural environment
In situ cultivation of previously uncultivable microorganisms using the ichip

Question 24

Ichip

Answer 24

introduced the diffusion chamber in 2002 for in situ cultivation by Epstein
The chamber allowed growth in pure culture while permitting the free diffusion of nutrients from the native environment.
The ichip consists of hundreds of miniature diffusion chambers that allow parallel in situ cultivation of hundreds of isolates.
Each chamber is inoculated with a single environmental cell
The chip is incubated in situ in the original environment. The method has high recovery rates (close to 50%) but also has recovered novel species.

Question 25

Genome mining

Answer 25

With advances in next-generation sequencing, several actinomycete genomes have been sequenced. Genomes of known antibiotic producers were found to have many other biosynthetic clusters of unknown functions. Streptomyces species, have 20-30 biosynthetic clusters. They are not routinely expressed under standard cultivation conditions There are several ongoing efforts to express these silent pathways and examine their products. Secondary metabolite biosynthetic gene clusters (smBGCs)

Question 26

Synthetic antibiotics

Answer 26

Following the boom in natural product antibiotic discovery in the 1940s and 1950s, New classes of antibiotics and several approved drugs resulted from fully synthetic approaches. Many successful attempts were also made towards fully synthetic routes to derivatives of natural product antibiotics since natural products were often difficult to isolate. eg,. The prototypical quinolone, nalidixic acid was discovered in the 1960s as a by-product during the synthesis of anti-malarial quinine compounds. 3 It was soon found to act by inhibiting the activity of bacterial topoisomerase type II enzymes

Question 27

Heterologous expression - genome mining

Answer 27

Specific strains of Streptomyces coelicolor, S. lividans, S. albus, and S. avermitilis have been developed as host strains for heterologous expression of biosynthetic gene clusters. These strains have been deleted of their native biosynthetic gene clusters, which leaves resources free for the expression of the heterologous product and also allows easier detection of the product without interfering background from the native products Tetarimycin A was discovered this way

Question 28

Ribosome engineering

Answer 28

This method was discovered when scientists observed that actinorhodin production by the non-producing Streptomyces lividans was stimulated by mutation of the ribosomal S12 protein. When more than 1000 actinomycetes from soil were tested, more than half of the non-producing strains could be made to produce antibiotics after they were made resistant to rifampicin or streptomycin. A new class of antibacterials named piperidamycin was discovered this way.

Question 29

High-throughput screening

Answer 29

High-Throughput Screening for Antimicrobial Compounds such as using a 96-Well Format Bacterial Motility Absorbance Assay. Bioprospecting for Antibacterial Drugs, plant extracts, soil, marine environments

Question 30

Conclusions

Answer 30

The discovery of the first antibiotics The golden era of natural product screening 1930 to 1970 with the discovery of many of the clinically used antibiotics. This was followed by a 30-year slump with a sharp drop in the number of new natural product antibiotics discovered. Synthetic antibiotics, and pharmaceutical companies dove into target-based antibiotic discovery campaigns. Return to natural products with new microbial cultivation and screening techniques increasing knowledge on the expression of cryptic pathways and genome mining

Question 31

Bacteriocins

Answer 31

ribosomally synthesized peptides with antibacterial activity. Bacteriocins can be post-translationally modified (Class I) or non-modified (Class II) They have different modes of action They often have a narrow spectrum of activity Eg. Sactibiotic Thuricin Cd is a two-component bacteriocin, consisting of the peptides Trnα and Trnβ These are membrane-acting and cause a collapse of the membrane potential

Question 32

Action of bacteriocins in gram-positive bacteria

Answer 32

Inhibition of peptidoglycan synthesis- pore formation Binding to protein channels- fore formation

Question 33

Action of bacteriocins in gram-negative bacteria

Answer 33

Inhibition of DNA gyrase Inhibition of RNA polymerase Inhibition of Asp-tRNA synthase

Question 34

Enzybiotics

Answer 34

Enzymes that break open bacterial cells by cleaving different bonds within the peptidoglycan layer Confusion of lysozyme, lysins, endolysins, lytic enzymes, murein hydrolase and autolysin. Many of these enzymes have complex substrates and they are highly specific. “Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases produced in phage-infected bacterial cells toward the end of the lytic cycle.

Question 35

Holin molecules

Answer 35

tunnel” through the membrane allowing the lysin access to the peptidoglycan.

Question 36

Bacteriophage lysis

Answer 36

involves at least two fundamentally different strategies. Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin in this review. The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer. This is necessary because phage-encoded lysins never have secretory signal sequences and are thus incapable of unassisted escape from the cytoplasm.

Question 37

Peptidoglycan bonds cleaved by lysins

Answer 37

N-acetyl-b-D-glucosamidase N-acetyl-(beta)-D-muramidase N-acetylmyramoyl-L-Ala-amidase L-alanoyl-D-Glu endopeptidase D-glutamyl-L-Lys endopeptidase

Question 38

Lytic cycle

Answer 38

The phage DNA circularizes. The virus uses the bacterial machinery to translate and transcribe the phage genome, resulting in new phage progeny and ultimate release of new phage partials.

Question 39

Lysogenic cycle

Answer 39

The phage DNA integrates into the bacterial chromosome. It may remain “dormant” for many generations (called a prophage), until an induction event normally brought about by bacterial stress. The phage DNA is excised and adopts a lytic cycle. This permits phage outbreaks to be separated in time.

Question 40

Endolysins

Answer 40

can be used to target specific bacteria. These are generally active on strains similar to those they are derived from. This offers the ability to selectively kill problematic pathogens with out destroying the natural flora. specificity is governed by the substrate specificity of the catalytic domains as well as the affinity of the targeting domains.

Question 41

Targeting and multi-domain enzymes

Answer 41

For example, the sf370.1 phage endolysin has a three domain architecture consisting of an N-terminal peptidoglycan hydrolase (“lysozyme”) catalytic domain, linked to an amidase domain which in turn is linked to a C-terminal CBD. In nature it is believed these interactions govern host specificity whilst also leaving the lysins tethered to the host cell-wall fragments post-lysis preventing them from wreaking further damage to the bacterial population (which would deplete the number of host cells available for further phage infection

Question 42

Problems with lysins

Answer 42

Gram –ve bacteria have an outer membrane which is hard to penetrate, most studies have been limited to Gram +ve’s.

Question 43

CBD

Answer 43

linked to a detection mechanism (GFP) may offer a rapid identification tool this occurs via interactions with specific cell wall carbohydrates. The CBD interaction is monovalent as so perhaps better suited to rapid photometric methods of detection (rather than antibodies based methods).