Present of drug discovery AMC exam 2

all done

2 limitations on synthetic libraries

limited by synthetic pathways, limited knowledge on the requirements for an antibacterial compound

what have natural abx proven to be (4)

more structurally diverse, more functionally complex, unique molecular architecture, higher antibacterial activity

what are the best sources to find new abx from natural products? (3)

actinomycetes/fungi/other soil bacteria, plant extracts, deep sea organisms (sponges, algae)

problem w/ soil bacteria

many do not grow in the lab

why have not all abx produced by soil bacteria been identified? (4 + additional points)

concept of “low hanging fruit”: only a small fraction of existing microbial diversity has been systematically sampled (limited to microorganisms that can be grown in the lab); only the most active abx have been identified (detection limited to the sensitivity of the assay); finding abx is a numbers game - screening of many strains is required (eg only 1 in 100 streptomyces produce streptomycin, only 1 in 100,000 produce daptomycin); organisms might not constantly produce abx compound - find the right growth conditions that triggers expression of new abx

what does the image show + descr

how soil bacteria are cultured to get natural abx out

eg of very simple abx

pyrazinamide

mechanisms of teixobactin (3)

produced by a gram- bacterium, kills gram+ bacteria, targets the lipid tails (lipid II and III) of precursors of teichoic acid and peptidoglycan, (also they though resistance would not be developed against it)

target based screening steps

from target (identification and validation of a potential drug target), enzyme inhibition (enzyme expression and purification, enzyme based screening (IC50), 3D structure determination, fragment based screening), to drug (validation against pathogen and MIC/MBC determination, preclinical development - lead optimization, med chem, in vivo studies)

pathogen based screening steps

from drug (potential anti bacterial molecules, compound libraries), in vitro activity (lead identification, MIC/MBC determination, cytotoxicity), to target (target identification and validation, preclinical development - lead optimization, medchem, in vivo studies)

major bottleneck in target based screening

validation against pathogen and MIC/MBC determination

ways of improving screens

a more sensitive and quantitative method to measure cell death - eg w/ resazurin reduction assay, easier readout esp for automation (add indicators like GFP and luciferase)

RRA long

resuzarin reducation assay - resazurin is a blue dye present in all living forms then turns into resorufin which is pink when dead - produced a quantifiable change, also fluorescent so can be detected by a machine

the present of abx selection - what ways are we doing better than the mistakes in the past (3)

diversity of synthetic compound library was too limited - trend towards finding abx from natural sources
effective compounds identified by TBDD failed to be effective against whole cells - trend towards screens using whole bacteria
target selection too strict - more focus on less conventional protein targets

ways to focus on less conventional targets general

do not focus only on essential targets w/ enzymatic activity localized in the cytosol

ways to focus on less conventional targets 4 specific points

select targets outside the cytosol - membrane/surface/secreted proteins can more easily be reacher by a compound
not only essential targets but also proteins important for virulence as potential target
not only enzymes but protein-protein interactions as a potential target
do not discard targets that are present in multiple copies - identification of inhibitors that block multiple targets, an effective way of reducing resistance development towards the new drug

eg for design a whole cell screen for a less conventional target

inhibition of secretion of virulence factors - eg toxins, extracellular degradative enzymes

how is it helpful if there are multiple copies of a gene in bacteria that is the drug target

more mutations needed before resistance can occur

eg of inhibition of protein secretion system

a baumannii depends on type II secretion systems for colonization - screen 6500 synthetic compounds for inhibiting extracellular lipase activity - substrate cleaved when lipase has high fluoresence
screening results that most were lipase inhibitors, no type II secretion inhibitors

BAM complex as drug target - descr, advantages

BamA (needed for folding outer membrane channel proteins) - is surface exposed, extracellular loop - abx does not have to overcome OM or deal w/ efflux pumps, essential, conserved in gram-, potential synergy

BAM inhibition assay for screening

between pathogen and drug based discovery

what part of an enzymes is the ideal target for an abx

active site of an essential enzyme

other less conventional target

efflux pumps - blocking them will efficiently decrease (multi-) drug resistance - get many drugs back!! blocking them witll inhibit virulence

how to screen for inhibitors of efflux pumps

bacterium overexpressing RND efflux pumps, screen compound library in presence of abx, abx kills the bacteria when efflux pumps are inhibited

other drug target + descr

quorum sensing - replace target genes w/ reporter gene, screen for compounds that induce, will affect virulence of bacteria

major bottleneck in pathogen based screening

target identification and validation - target should be known to be able to optimize the compound - also so thats its approved

what is special ab pyrazinamide

widely used abx to treat TB, its a vit B3 analogue, identified by pathogen based screening in the 1950s - BUT target still not known - would not be accepted for new abx identified today

how to find the target of pyrazinamide?

genetic approach - selection of resistant bacteria - all resistant colonies have mutations in pncA or in genes that code for transcription regulators of pncA - pncA is a nicotinamidase - required for modification of pyrazinamide into pyracinoic acid

why are there no mutations in the target gene of pyrazinamide (3)

mutations of target are lethal, pyanizoid acid has multiple targets, pyranizoid acid has no protein target - may work on lowering pH of cytosol or pathway toxification (NAD)

other approach to determine target of pyrazinamide

biochemical approach - binding of compound to protein extracts, pyranizoic acid does not bind to mycobacterial proteins w/ high affinity

likely target of pyrazinamide

it is acidifying the cytosol

what step now done earlier after hit found

go straight to animal studies to not do too many steps with a compound that would fail at animal studies anyways

how do mass spectrometry based thermal stability assays work

remove denatured proteins - centrifugation - identify non denatured protein (in solution) by mass spectrometry

hypomorphs espl

make bacteria more susceptible to compounds, knowing what target depletion causes sensitivity allows identification of target/MOA of compound

hypomorphic allele descr

gene mutation that results in a partial loss of normal gene function rather than a complete loss of function - “leaky” mutations, cannot delete essential genes but can knock them down

eg of hypomorphs

CRISPR-interference - CRISPR-I libararies, 1000s of guide RNAs per organism, one knock down per bacterium, inducible systems, identification of targets by looking at susceptibility profiles of hypomorphs, could be scaled up too - high throughput BUT does not work for every compound

advs and disadv of bacterial screens

adv: cell permeability tackled, unexpected targets can be identified, prodrugs can be selected, multi target inhibitors can be found
disadv: target identification can be difficult