Antibiotic Discovery Approaches and Targets

Approaches to Antibiotic Discovery

There are two primary approaches to discovering new antibiotics:

1. Target-First Approach:

   • Select a target: Identify a gene product that is essential for bacterial growth or survival. This target should ideally be unique to bacteria or significantly different from any homologous proteins in humans to minimize off-target effects.

   • Inhibit target function: Find a chemical compound that inhibits the function of the chosen target. This involves screening compound libraries for molecules that bind to and inhibit the activity of the essential bacterial protein.

2. Chemical-First Approach (Phenotypic Screening):

   • Select a chemical: Identify a chemical compound that inhibits bacterial growth. This is typically done through high-throughput screening of chemical libraries against bacterial cultures.

   • Identify the target and mode of action: Determine the specific target of the chemical and how it works to inhibit bacterial growth. This step often involves techniques like genetic screens, biochemical assays, and structural biology to understand the drug's mechanism.

Desirable Features of an Ideal Antibiotic Target

An ideal antibiotic target should possess the following characteristics:

• Essentiality: The target should be essential for bacterial growth or survival, meaning it is the product of an essential gene. Knocking out or inhibiting this gene should lead to bacterial death or growth arrest.

• Broad Spectrum: The target should be present in multiple pathogens, allowing the antibiotic to have a broad spectrum of activity. This makes the antibiotic useful against a wide range of bacterial infections.

• Absence in Humans: The target should be absent in humans to minimize the risk of toxicity. If the target is present in humans, the antibiotic should have a much higher affinity for the bacterial target than the human homolog.

• Druggability: The target should contain a site where an inhibitor can bind and inhibit its function. This means the target should have a structure conducive to small molecule binding and inhibition.

• Specificity: The interaction between the inhibitor and the target should be specific to minimize off-target effects and toxicity to humans. Off-target effects can lead to adverse side effects and the development of resistance.

• Partial Inhibition Sufficiency: Partial inhibition of the target should be sufficient to stop bacterial growth. This means that the antibiotic does not need to completely shut down the target to be effective.

• Low Resistance Potential: The target should have a low frequency of resistance development, or resistance should not arise easily. High rates of resistance development can quickly render an antibiotic useless.

• Limited Resistance Mechanisms: Resistance mechanisms should either not exist in other bacteria, or have a low probability of horizontal gene transfer (HGT). HGT can spread resistance genes rapidly among bacterial populations.

Identifying Essential Bacterial Genes

• Transposon Inactivation (Tn-seq):- Essential genes can be identified by transposon inactivation, where inactivation is lethal to the bacteria.

  • Tn-seq involves mapping transposon insertions in the target bacterial genome to identify genes where insertions are not tolerated. No Tn insertions are found in essential genes. Tn-seq libraries are generated by introducing transposons randomly into the bacterial genome, and then sequencing the insertion junctions to determine which genes can be disrupted without killing the bacteria.

• Validation of Essential Genes:- Expression knockdown: Use antisense RNA to reduce gene expression.

  • CRISPR interference: Employ CRISPR interference to reduce gene expression.

  • Expected outcome: Reduced expression of an essential gene should result in a reduced growth rate. This is because essential genes are required for bacterial survival, so reducing their expression impairs growth.

Tn-Seq Output and Interpretation

• (a) Saturated Transposon Insertion Profile: A saturated and evenly distributed profile of insertions is generated in a bacterial population with each cell containing a single insertion. Open reading frames are shown as rectangles colored by function (red is essential, green is nonessential, and purple is important). The saturation ensures that nearly every non-essential gene has been disrupted by a transposon insertion.

• (b) Growth and Selection: After insertions are produced, the cells are grown (in rich medium). Genes important for growth retain fewer insertions relative to their fitness contribution. This is because cells with insertions in important genes will grow more slowly and be outcompeted by cells with insertions in non-essential genes.

• (c) Quantification by Sequencing: The number of reads for each insertion in cultures is quantified with high-throughput sequencing. Sequence reads are measured before and after cells are grown. Comparing the number of reads before and after growth allows researchers to identify genes where insertions lead to a decrease in representation, indicating that the gene is important for growth.

Essential Genes and Antibiotic Targets

• Typically, a few hundred 'essential' genes are found in each bacterial genome. These are the genes that, if disrupted, lead to bacterial death or growth arrest.

• For example, E. coli has approximately 300 essential genes out of about 4500 total protein-coding genes. This means that about 7% of E. coli's genes are essential for survival under standard lab conditions.

• Essential gene products are potential targets for antibiotics. Inhibiting these gene products can disrupt bacterial growth and lead to cell death.

Complications and Solutions

• Operon Organization: Many essential genes are located within operons, which can lead to polar effects.- Polar effects: A gene knockout can have downstream polar effects on the expression of an intact essential gene. Polar effects occur when the disruption of one gene in an operon affects the expression of downstream genes.

  • Example from E. coli: The eno (essential) gene is located downstream of the pyrG (non-essential) gene. A Tn-knockout in pyrG reduces the expression of eno (polar effect). This can lead to misinterpretation of Tn-seq data. If pyrG appears essential in Tn-seq data due to the polar effect on eno, it might be incorrectly identified as an antibiotic target.

• Solution: Validate Essentiality:- Validate essentiality of individual genes by CRISPR interference (down-regulation) of expression. This helps to rule out false positives caused by polar effects.

CRISPR Interference for Gene Validation

• Mechanism: CRISPR-mediated interference of gene expression is caused by nuclease-deficient dCas9.- dCas9: Nuclease-deficient dCas9 contains two substitutions in the nuclease domains (D10A and H840A), rendering it without endonuclease activity. The dCas9 protein is guided to a specific DNA sequence by a guide RNA (sgRNA).

  • Transcription Inhibition: If the target DNA sequence is inside an open reading frame, the dCas9–sgRNA–DNA complex blocks the movement of RNAP, resulting in transcription inhibition of the target gene. This effectively reduces the expression of the targeted gene.

• Essentiality Confirmation: If the gene product is essential, induction of CRISPRi causes a reduced growth rate.- Tunable induction: Tunable induction of CRISPR-interference will reduce bacterial growth rate if a gene is essential. This allows researchers to control the level of gene expression and observe the resulting effect on bacterial growth.

Further Validation for CRISPR Interference

• Potential for Polar Effects: CRISPR interference still has the potential for polar effects on other genes in the operon if applied in the native gene location. Thus, further validation is often necessary.

• Engineered Gene Organization:- Delete original gene: Delete the original gene.

  • Express elsewhere: Express the gene elsewhere in the genome under a different promoter. This involves cloning the gene of interest into a plasmid and introducing it into the bacterial cell.

  • Induce CRISPRi: Induce CRISPRi on the expressed gene.

  • Normal growth: Normal growth indicates that the initial gene is not essential. This is because the gene is still being expressed from the new location in the genome.

  • No/poor growth: No/poor growth proves that the gene product is essential for normal growth. This confirms that the gene is essential for bacterial survival.

Comparative Genomics for Target Selection

• Broad-Spectrum Coverage: Use comparative genomics to identify targets that are conserved across multiple bacterial species to achieve broad-spectrum coverage. This involves comparing the genomes of different bacterial species to identify genes that are present in all or most of them.

• Unique or Conserved Proteins: Look for unique or conserved proteins using Venn diagrams of essential genes present in different species (P. aeruginosa, E. coli, K. pneumoniae, A. baumannii, S. aureus, S. pneumoniae).- Typical data:-

  • Each species has 2500 - 7000 genes.

  • Less than 100 are absent in humans. These are potential antibiotic targets.

  • Less than 10 are druggable targets. These are the most promising antibiotic targets.

  • About 300 are conserved genes. These are good targets for broad-spectrum antibiotics.

• Narrow Spectrum: Narrow spectrum = more potential targets (but smaller market). Narrow-spectrum antibiotics can be useful for treating specific infections and minimizing the disruption of the normal microbiota.

Target Validation and Assay Development

• Assay Development: Once a target (product of an essential gene) is chosen, an assay needs to be developed to screen for chemicals (Hits) that inhibit the gene product. The assay should be designed to be sensitive, specific, and reproducible.

• High-Throughput Screening (HTS): Develop a biochemical assay for high-throughput screening of chemicals. HTS allows for the rapid screening of large chemical libraries.

• Target Inhibition Assay: Focus on assays that measure target inhibition. This ensures that the identified hits are directly affecting the target of interest.

High-Throughput Screening and Hit Identification

• Industry Compound Collections: Industry compound collections typically contain 1–2 million compounds. These compounds are often proprietary and have been synthesized or isolated by pharmaceutical companies.

• Hit Rate: Less than 1% of compounds are confirmed hits. This means that only a small fraction of the screened compounds show activity against the target.

• Active Series: 2–3 active series are identified. An active series is a group of structurally related compounds that show activity against the target.

• Candidate Drugs: 1–2 candidate drugs are identified. These are the compounds that are most promising for further development.

• Process:- High throughput screening (biochemical assay). This involves using automated equipment to screen large numbers of compounds against the target.

  • Hit expansion/lead selection (chemistry). This involves synthesizing and testing additional compounds related to the initial hits to identify the most promising lead compounds.

  • Lead optimization (chemistry). This involves modifying the structure of the lead compounds to improve their potency, selectivity, and pharmacokinetic properties.

• Biochemical Assay: A biochemical assay must be developed for each chosen target (in vitro target inhibition). This assay should be designed to measure the activity of the target enzyme or protein in a test tube.

Examples of Biochemical Assays for Antibacterial Targets

Fluorescence Resonance Energy Transfer (FRET) Assays

• Principle: Fluorescence Resonance Energy Transfer (FRET) enables the study of molecular interactions by exciting a donor fluorophore, which transfers the energy to an acceptor fluorophore if they are in close proximity. The efficiency of energy transfer is highly dependent on the distance between the donor and acceptor fluorophores.

• Inhibition of FRET: An inhibitor (e.g., antibiotic) of Enzyme – Ligand interaction prevents FRET. This means that the inhibitor disrupts the close proximity of the donor and acceptor fluorophores, leading to a decrease in FRET signal.

Lipoprotein Signal Peptidase (Lsp, SPaseII)

• Function: An aspartic acid protease essential for bacterial lipoprotein maturation. Lipoproteins are important components of the bacterial cell envelope and play roles in various cellular processes.

• Validation: Lps is a validated antibacterial target. Inhibiting Lsp can disrupt lipoprotein maturation and lead to bacterial cell death.

• Known Inhibitors: Two natural product Lsp inhibitors, Globomycin and Myxovirescin, have antibacterial efficacy but limited stability and in vivo ineffectiveness, making them unsuitable for development. These inhibitors have served as leads for the development of more potent and stable Lsp inhibitors.

• New FRET Assay: A peptide-based Lsp FRET reporter was designed to mimic the canonical sequences of Lsp substrates for HTS biochemical assay. This assay allows for the rapid and sensitive screening of potential Lsp inhibitors.

FRET-Based HTS Assay for Lsp Inhibitors

• (B) Design for a Peptide-Based FRET Reporter Substrate:- N-terminal quencher (blue circle). The quencher absorbs the fluorescence emitted by the fluorophore, reducing the background signal.

  • C-terminal fluorophore (green circle). The fluorophore emits fluorescence when excited by light of a specific wavelength.

  • Rapidly quantitate Lsp activity in vitro. This assay allows for the measurement of Lsp activity in a test tube.

• (A) Lsp Cleavage of Lipobox Residues: Lsp recognizes a region of the signal sequence within the prolipoprotein termed the lipobox and removes all residues N-terminal to a diacylglyceryl (DAG)-cysteine residue.- Active Lps causes a Fluorescence signal. This is because the cleavage of the FRET substrate by Lsp separates the quencher and fluorophore, leading to an increase in fluorescence.

FRET Substrate to Monitor Lsp Activity

• (A) Structure of Lsp Peptide FRET Substrate:- N-terminal dabsyl quencher (blue). Dabsyl is a commonly used quencher in FRET assays.

  • C-terminal EDANS fluorophore (green). EDANS is a fluorescent dye that emits light at a specific wavelength.

  • Lsp from E. coli requires the DAG-modified cysteine residue (red) for activity. This modification is essential for Lsp to recognize and cleave the substrate.

• (B) Kinetic Assays with Lsp from E. coli: Michaelis-Menten kinetic constants of the optimized FRET substrate confirmed that the biochemical assay actually worked with Lps. This ensures that the assay is measuring the activity of Lsp and not some other enzyme.

Big Pharma’s Biochemical HTS Experience

• GSK: GSK screened 70 essential enzyme targets using 530,000 compounds (in-house library) over 6 years. 16 targets yielded Hits with chemically tractable low micromolar potency (on target enzyme activity) and 10× selectivity bacterial >mammalian enzymes. Only 5 were developed into leads, and none made it to the clinic. This highlights the challenges of translating hits from biochemical assays into effective drugs.

• Astra-Zeneca: Astra-Zeneca conducted 65 HTS on a library of 800,000 – 1,2 million compounds.

• Other Companies: Wyeth, Pfizer, Merck, etc. all did similar biochemical screens.

• Outcome: No new antibiotics reached the clinic. Problems included poor whole-cell activity, efflux, eukaryotic toxicity, resistance, etc. Each screen cost $300,000 – $600,000. This demonstrates the high cost and low success rate of traditional antibiotic discovery approaches.

Strategies for Target Identification

• Genomics & Bioinformatics

• Genetics: Identifies essential targets for growth/survival, confirm essentiality by gene knockout, confirm essentiality by reducing expression (RNAi, CRISPRi)

• Bioinformatics: Target is conserved in the ‘interesting’ bacterial species, target is absent in humans

• Structural biology: Target is ‘druggable’

• Biochemistry: HTS screening of compound libraries

Target-First vs Compound-First Approaches

• Biochemical pathway inhibition – target-first- Positive:-

  • Can choose novel target

  • Can set up high-throughput assay (HTS)

  • Can design drug to fit target

  • Negative:-

  • Drug may not enter bacteria

• Microbial growth inhibition – compound-first- Positive:-

  • Drug can enter relevant bacteria

  • Negative:-

    • Weak effects may not be detected

    • Slow throughput

    • Must locate target

    • Re-discover the same thing (natural products)

• Compromise: Hypomorphic whole cell- Positive:-

  • Drug can enter relevant bacteria

  • Focus on specific novel target

  • Negative:-

  • Slow initial throughput – must confirm targeting

• The major challenge is finding novel & suitable chemical starting points.

Multigene Targets vs Single Gene Targets

• Multigene targets:

• Many antibiotics targeting translation actually target ribosomal RNA: Aminoglycosides, Tetracyclines, Macrolides- 7 copies in E. coli (multicopy in most bacterial species)

• Antibiotics targeting cell wall synthesis usually bind multiple PBP’s: Beta-lactam antibiotics

• Antibiotics targeting DNA synthesis bind to Gyrase & Topoisomerase IV: Quinolones

• Antibiotics target two different steps in folic acid synthesis: Trimethoprim and Sulfonamides

• MULTIPLE GENES REDUCE THE POSSIBILITY OF TARGET MUTATIONS CAUSING RESISTANCE (PHENOTYPE NOT EXPRESSED)

• Single gene targets:

• RNA polymerase beta subunit: Rifampicin – spontaneous resistance is a problem

• Most novel targets are metabolic gene products: typically single gene products

• SINGLE GENE TARGET - MUTATIONS CAUSING RESISTANCE ARE LIKELY TO BE A PROBLEM

Exploiting Classic (Validated) Targets for Novel Antibiotics

• Novel targets in protein synthesis – example Odilorhabdins

• Novel targets in topoisomerases – example Gepotidicin – APPROVED!

• Novel targets in cell wall synthesis – example Dabocins

• Novel targets in the bacterial membrane - Daptomycin

• Re-using ‘old’ antibiotic targets

Novel Antibiotics Targeting Protein Synthesis

• Bacterial Protein Synthesis as a Target for Antibiotic Inhibition. Arentz & Wilson: Cold Spring Harb Pers Med, 2016;6:a025361

Odilorhabdins: Antibacterial Agents Binding at a New Ribosomal Site

• A new class of naturally produced, ribosome-targeting antibiotics (NOSO).

• Bind to the small ribosomal subunit at a site not exploited by known antibiotics.

• Induce miscoding (increase the affinity of aa-tRNAs to the ribosome).

• Promising antibacterial spectrum and efficacy in mouse infection models.

• Currently in Candidate to Phase 1 stage.

• ODL’s Bind in the Decoding Center on the Small Ribosomal Subunit

Gepotidacin: A Novel Topoisomerase Inhibitor

• Inhibits DNA topoisomerase II activity (DNA Gyrase & Topo IV).

• Gepotidacin had 0.12 and 0.25 mg/L MIC50 and MIC90 against gonococci, including ciprofloxacin-resistant strains.

• The combination of gepotidacin and moxifloxacin (a fluoroquinolone) had a synergistic effect.

• In Phase II trials, oral gepotidacin was 95% successful in treating uncomplicated genitourinary gonorrhea.

• Views of a gepotidacin complex formed with S. aureus gyrase and doubly nicked DNA at a resolution of 2.31 Å. ACS Infect. Dis. 2019, 5, 4, 570–581.

• Binds in the same DNA cleavage active site of gyrase as fluoroquinolones but displays distinct interactions.

• Gepotidicin retains activity against common gyrase mutations resistant to fluoroquinolones

• Successfully completed Phase III trials for UTI (2023). FDA has approved GSK’s gepotidacin (Blujepa) Uncomplicated urinary tract infections (uUTIs) in female adults and paediatric patients 12 years of age and older (2025).

Dabocins: Novel Antibacterials

• A novel class of antibacterials - derived from the l Diazabicyclooctane scaffold (as in Avibactam).

• Inhibit Penicillin-Binding Proteins (PBP) like beta-lactams and have excellent bactericidal properties.

• Designed to be a PBP binder – direct acting antibiotic (an alternative to traditional beta-lactam antibiotics)

• Unlike beta-lactams, Dabocins show an exceptional stability to class A, B, C, D beta-lactamases and are thus highly potent against problematic carbapenem-resistant isolates.

Daptomycin: Targeting the Bacterial Cell Membrane

• Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus.

• Indicated for skin and skin structure infections caused by Gram-positive infections, S. aureus bacteremia, and right-sided S. aureus endocarditis.

• Daptomycin inserts into the cell membrane in a phosphatidylglycerol-dependent fashion, where it aggregates.

• Aggregation of daptomycin alters the curvature of the membrane, which creates holes that leak ions.

• Causes rapid depolarization, resulting in a loss of membrane potential leading to inhibition of protein, DNA, and RNA synthesis, which results in bacterial cell death.

• Daptomycin is bactericidal against Gram-positive bacteria only!

• Polymyxins (colistin, polymyxin B) bind to LPS and phospholipids in the outer cell membrane of Gram-negative bacteria

Overall Summary of Antibacterial Targets

• The number of potential target enzymes (essential in bacteria, absent in humans, with a broad or useful spectrum) has been estimated to be ∼160.

• Only 16 enzyme classes are targets of clinical antibacterials, in addition to the non-enzyme targets: rRNA, lipid II, membranes, and DNA.