19. Comprehensive Study Notes on Antibiotic Discovery and Development
The Antibiotic Development Landscape: A Historical Perspective
The 'Golden Age' vs. The 'HTS Genomics Age'
A noticeable decline in the introduction of new antibiotic classes has been observed from 1980 to the present. While the emergence of entirely novel antibiotic classes has slowed, numerous individual antibiotics related to existing classes (e.g., new cephalosporins, fluoroquinolones, macrolides) have been introduced and refined.
Lynn L. Silver's review (2011) emphasizes the complexities and hurdles in antibacterial discovery, pointing out that the easily accessible targets have already been exploited, making new discoveries more challenging.
The Challenge of Limited Targets and Resistance
Limited Targets
Approved clinical antibiotics act on a restricted set of bacterial mechanisms, limiting the options for new drug development and increasing the likelihood of resistance.
Resistance Liabilities
All currently approved clinical antibiotics face the threat of resistance mechanisms, which evolve as bacteria adapt to the selective pressure exerted by these drugs.
WHO Priority-Ranked Antibiotic-Resistant Bacteria
The World Health Organization (WHO) has established a priority ranking system for antibiotic-resistant bacteria based on the severity of the threat they pose to public health. This ranking involved a comprehensive survey of 66 experts in the field. Key resistance designations include:
CR: Carbapenem-resistant
3GCR: 3rd Generation cephalosporin-resistant
VR: Vancomycin-resistant
MR: Methicillin-resistant
ClaR: Clarithromycin-resistant
FQR: Fluoroquinolone-resistant
PNS: Penicillin non-susceptible
AmpR: Ampicillin-resistant
WHO Priority Pathogens for New Antibiotic Development
The WHO has specified particular pathogens as critical targets for new antibiotic research and development, categorizing them by priority level:
Priority 1: Critical
Acinetobacter baumannii, carbapenem-resistant (Gram-negative): This bacterium is a major cause of hospital-acquired infections and is increasingly resistant to most antibiotics.
Pseudomonas aeruginosa, carbapenem-resistant (Gram-negative): Another significant cause of hospital-acquired infections, particularly in patients with compromised immune systems or cystic fibrosis.
Enterobacteriaceae, carbapenem-resistant, ESBL-producing (Gram-negative): These bacteria, including E. coli and Klebsiella pneumoniae, are becoming increasingly resistant to carbapenems and extended-spectrum beta-lactam antibiotics.
Priority 2: High
Enterococcus faecium, vancomycin-resistant (VRE): This bacterium is a common cause of hospital-acquired infections, especially in immunocompromised patients.
Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant (MRSA & VRE): A highly adaptable bacterium that can cause a variety of infections, from skin infections to life-threatening bloodstream infections.
Helicobacter pylori, clarithromycin-resistant: This bacterium infects the stomach and can cause ulcers and stomach cancer. Resistance to clarithromycin makes treatment more challenging.
Campylobacter spp., fluoroquinolone-resistant: A common cause of bacterial diarrhea worldwide.
Salmonellae, fluoroquinolone-resistant: These bacteria cause food poisoning and can lead to severe systemic infections.
Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant: This bacterium causes gonorrhea, a sexually transmitted infection, and resistance to multiple antibiotics is a major concern.
Priority 3: Medium
Streptococcus pneumoniae, penicillin-non-susceptible: This bacterium causes pneumonia, meningitis, and ear infections, and resistance to penicillin is increasingly common.
Haemophilus influenzae, ampicillin-resistant: A cause of respiratory infections, meningitis, and ear infections, particularly in children.
Shigella spp., fluoroquinolone-resistant: Another cause of bacterial diarrhea, particularly in developing countries.
Pre-Clinical Antibacterial Pipeline
Overview
Theuretzbacher et al. (2020) conducted an analysis of the pre-clinical antibacterial pipeline, identifying 407 projects in 2019. The analysis reveals a diverse array of approaches being pursued, but also highlights the high risks associated with translating these innovative approaches into clinical applications.
Institutions Involved
Small and medium-sized enterprises (SMEs) constitute the majority of institutions engaged in pre-clinical antibacterial development, accounting for 255 of the 314 institutions surveyed.
Academic institutions, large companies, non-profit institutions, and public-private partnerships are comparatively less represented in these efforts.
Geographically, more than half of the SMEs are located in North America, followed by Europe.
SMEs are particularly vulnerable to financial constraints, which can significantly impact their ability to sustain long-term research and development projects.
Spectrum of Direct-acting, Small-molecule Antibacterials
Approximately 20% of direct-acting small molecules in the preclinical antibiotic pipeline are derivatives of ‘old chemical classes’. This raises concerns about the potential for rapid development of resistance due to prior exposure to similar compounds.
The majority (>70%) of direct-acting antibacterials represent new chemical classes and/or have new targets. While this is promising, it also raises concerns about potential unknown toxicity liabilities, as these compounds have not been extensively studied.
Few compounds in development exhibit a broad spectrum of activity against both Gram-negative and Gram-positive bacteria. This narrow focus can be an economic liability for development, as it limits the potential market for these drugs.
Novel Approaches to Novel Antibiotics
Dual Targeting Approach
This strategy is designed to lower the risk of resistance by requiring a pathogen to simultaneously develop resistance to two different mechanisms of action. Ideally, a single compound should have dual targets within the bacterial cell.
Example: Martin et al. (2020) describe SCH-79797, a compound with two independent cellular targets: folate metabolism and bacterial membrane integrity. By hitting two essential pathways, the likelihood of resistance is significantly reduced.
They developed a lead compound, Irresistin-16, with increased potency. It demonstrated efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model, showing promise for treating drug-resistant gonorrhea.
The different parts of the compound target distinct bacterial mechanisms, enhancing its effectiveness and reducing resistance potential.
This research led to the establishment of an SME called ArrePath, which has received funding from CARB-X (Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator), highlighting the importance of public-private partnerships in antibiotic development.
Selective Targeting Approach
Selective targeting allows for the potential use of relatively toxic antibiotics as systemic agents by limiting their activity to the site of infection. This approach focuses the antibacterial effect on the pathogen while minimizing harm to the host.
The concept involves activating the antibiotic only in the presence of an infection, resulting in local toxicity that specifically kills the bacterial pathogen.
Example: Colistin, a known toxic antibiotic, was conjugated to a modified fragment of the human antimicrobial peptide, ubiquicidin. The release of colistin was controlled by a linker that is specifically cleaved by neutrophil elastase (NE), an enzyme secreted by neutrophil granulocytes at infection sites.
A proof-of-concept was achieved in vitro using co-cultures of primary human neutrophils and Escherichia coli. The setup induced the secretion of NE, leading to the release of free colistin and antibacterial efficacy comparable to that of free colistin.
While promising, this approach has not yet been tested in vivo, leaving its real-world effectiveness uncertain.
The diagram illustrates the infection-triggered release of antibiotics:
Structure of colistin B.
Overview of conjugate design.
Cellular mechanism of action:
Bloodstream infection with Gram-negative bacteria.
Binding of the peptide–colistin conjugate to a bacterium via the Ubi29–41 sequence.
Attraction of neutrophil granulocytes to the site of infection.
Secretion of neutrophil elastase (NE) from activated neutrophil granulocytes.
Cleavage of the linker and release of free colistin.
Killing of bacteria.
The Clinical Pipeline
Challenges and Needs
Most agents currently in clinical development are derivatives of established classes, which may limit their effectiveness against highly resistant strains.
Many new drugs aim to target the antibiotic-resistant priority pathogens identified by the WHO, addressing the most urgent threats in antibiotic resistance.
Pre-existing cross-resistance within antibacterial classes restricts the activity of many new agents against extensively drug-resistant (XDR) and pan-drug-resistant (PDR) Gram-negative pathogens, highlighting the need for novel mechanisms of action.
The development of new chemical classes is essential to provide effective therapeutic options against infections specifically caused by XDR and PDR Gram-negative bacteria.
Critical Analysis of Antibacterial Agents in Clinical Development
Theuretzbacher et al. (2020) offer a detailed assessment of antibacterial agents in clinical development, providing insights into their potential and limitations.
WHO Clinical Pipeline Analysis
While some new chemical classes are being developed, they often target classic bacterial mechanisms, raising concerns about the rapid emergence of resistance.
The WHO anticipates potential approval of a drug targeting Gonorrhea in 2025, reflecting the urgent need for new treatments for this increasingly resistant infection.
Novel Targets and Novel Chemicals – Higher Risks
Novel targets and novel chemicals generally carry a higher risk of unpredictable toxicity because the translatability of safety signals from preclinical models to humans is uncertain. Thorough preclinical testing is crucial to mitigate these risks.
Example 1: LpxC inhibitors target the first dedicated step in the synthesis of lipid A, a Gram-negative-specific molecule. Despite exploration since the mid-1990s, none have advanced beyond phase I clinical trials. The development of ACHN-975 was halted after a phase I trial due to local inflammation at the injection site and some toxicity signals in the mouse model.
Example 2: Murepavadin, a P. aeruginosa
specific LptD inhibitor (peptidomimetic), was terminated at Phase III clinical trials due to higher than expected rates of acute kidney injury. This demonstrates the challenges of unexpected toxicity of a new chemical not predicted from preclinical studies or studies in healthy individuals (Phase I).
New Directions: Non-Beta-Lactam Beta-Lactamase Inhibitors
The Importance of Beta-Lactams and Beta-Lactamase Inhibitors
Beta-lactam antibiotics (Penicillins, Cephalosporins, Monobactams, Carbapenems) remain the most crucial group of clinically used antibiotics, providing a broad spectrum of activity and generally good safety profiles.
The majority of resistance to beta-lactams is due to beta-lactamase enzymes, which hydrolyze the beta-lactam ring, rendering the antibiotic ineffective.
The primary strategy to combat this resistance has been to combine beta-lactam antibiotics with beta-lactamase inhibitors (BLIs), which protect the antibiotic from enzymatic degradation.
Traditionally, BLIs have been structurally related to beta-lactam antibiotics (e.g., Clavulanic acid + Amoxicillin, Sulbactam + Ampicillin, Tazobactam + Piperacillin).
Ceftazidime-Avibactam
New chemical classes of Non-beta-lactam betalactamase inhibitors, including diazabicyclooctanes (DBOs) and boronate classes, have been recently developed to overcome the limitations of traditional BLIs.
Avibactam–ceftazidime is the first DBO combination, offering a broader spectrum of activity against beta-lactamases.
Avibactam inhibits Klebsiella pneumoniae carbapenemases (KPCs) and AmpC-type β-lactamases, which are resistant to tazobactam and clavulanic acid, making it effective against many carbapenem-resistant Enterobacteriaceae.
However, Avibactam is not active against New Delhi metallo-β-lactamase 1 (NDM-1), a significant limitation in regions where NDM-1 is prevalent.
Avicaz is used to treat complicated intra-abdominal infections, urinary tract infections, and pneumonia caused by susceptible Gram-negative bacteria.
New DBO developments are in progress to create compounds with both BLI activity AND antibacterial activity through binding to PBP2, one of the target proteins of β-lactam antibiotics.
Boronate BLIs
Vaborbactam is a non-β-lactam β-lactamase inhibitor that broadens the spectrum of activity of carbapenems.
The combination drug meropenem/vaborbactam (Vabomere) is approved by the Food and Drug Administration for complicated urinary tract infections and pyelonephritis, and it is also used to treat complicated abdominal infections and hospital-acquired pneumonia.
Vaborbactam is a boronic acid β-lactamase inhibitor with a high affinity for serine β-lactamases, including Klebsiella pneumoniae carbapenemase (KPC) and the ESBL’s CTX-M-15 and SHV-12, enhancing the effectiveness of meropenem against resistant strains.
Boronate BLIs are being developed with a wider inhibitory spectrum to include some MBLs such as NDM and VIM (taniborbactam), addressing a critical gap in current treatment options.
The Trojan Horse Approach
Cefiderocol
Cefiderocol (Fetroja) represents the first successful application of the Trojan Horse approach, utilizing bacterial iron uptake mechanisms to facilitate antibiotic entry.
Bacteria are tricked into taking up the antibiotic by mimicking iron, which is essential for their growth. This allows the antibiotic to bypass resistance mechanisms that would otherwise prevent its entry into the cell.
Cefiderocol is a cephalosporin structurally related to ceftazidime and cefepime, but it is more stable to various β-lactamases, enhancing its resistance to enzymatic degradation.
The cephalosporin molecule is linked to a siderophore that can bind to iron, facilitating bacterial cell entry in addition to the usual entry via porin channels, ensuring effective penetration into the bacterial cell.
The main weakness of cefiderocol is its limited activity against NDM-producing E. coli as well as OXA-23- and OXA-24-producing Acinetobacter spp., restricting its use in certain infections.
Cefiderocol has been approved by the FDA for the treatment of complicated UTIs (including pyelonephritis) caused by susceptible Gram-negative bacteria in patients with limited or no alternative treatment options, providing a valuable tool against highly resistant infections.
However, the development of cefiderocol resistance during therapy is linked to poor outcomes, and there is little evidence to support the use of cefiderocol in patients with XDR Gram-negative bacteria, especially Acinetobacter spp. and Pseudomonas spp., indicating the need for careful monitoring and alternative strategies in these cases.
Gepotidacin (Blujepa)
Blujepa (gepotidacin) was approved by the US FDA (2025) for the treatment of uncomplicated urinary tract infections (uUTIs) in female adults and pediatric patients > 12 years of age
Blujepa is the first in a new class of oral antibiotics for uUTIs in nearly 30 years.
Over half of all women experience a uUTI in their lifetime, with approximately 30% suffering from a recurrent episode
The FDA approved the antibiotic on the basis of phase III EAGLE-2 and EAGLE-3 trials. These trials together enrolled over 3,100 female patients with uUTIs, who were randomized to gepotidacin or nitrofurantoin. The primary endpoint in both trials was the therapeutic response (success or failure) at test-of-cure in a subset of nearly 1,150 patients with nitrofurantoin-susceptible qualifying uropathogens who received at least one dose of study treatment.
In EAGLE-2, gepotidacin was non-inferior to nitrofurantoin, with respective therapeutic success rates of 50.6% and 47.0%. In EAGLE-3, gepotidacin was statistically superior to nitrofurantoin, with respective therapeutic success rates of 58.5% and 43.6%.
Gepotidacin targets bacterial DNA Gyrase and Topoisomerase IV
EAGLE-2 and 3 are similar trials and together provide substantial clinical evidence, with EAGLE-2 providing additional pharmacokinetic data and EAGLE-3 including an on- treatment ECG test.
Trial enrolment overlapped with the COVID-19 pandemic (the EAGLE-2 trial started in October 2019, and the EAGLE-3 trial began in May 2020), and trial sites are located across 12 countries, each with different infection and resistance patterns.
Potential Exam Questions on Antibiotic Discovery & Development
Describe the use of Tn-seq and CRISPR interference as methods to identify and validate a novel target for antibiotics
Describe the use of FRET as an assay to identify inhibitors of an antibiotic target
What is meant by the term ‘druggable’ target
What is an advantage of a multigene target? Give one example targeted by currently used clinical antibiotics
What is Gepotidicin and what is its target
What is LogD (or LogP) and what is its significance for antibiotics
Describe how the use of the iCHIP helps to identify novel natural product antibiotics
What are the main advantages of FBDD over classical chemical library screening
Describe the process of genome mining in terms of BCGs
Describe in outline the concept of making a DNA-encoded chemical library.
What advantages do DELs have over classical chemical libraries?
What is the general significance of pKa and Zwitterions in relation to antibiotic activity
What are the two most important primary assays used in evaluating novel antibacterial hit compounds
What are hERG, Nav 1.5 and Cav 1.2 and why are they important in antibiotic discovery
What is the purpose of a mouse PK study in antibiotic hit discovery
What is a CEREP assay and what is its purpose in antibiotic discovery
Describe what is meant by SAD in a phase 1 clinical trial
What is meant by MAD in a phase 1 clinical trial (give an outline of the study design)
Why are non-inferiority trials typically used to evaluate new antibiotics
Describe, with examples, what the term TPP means in an antibiotic development project
What are the primary and secondary objectives of a phase 1 clinical trial for an antibiotic
What are the main objectives of a phase 2 clinical trial for an antibiotic
Describe the differences between Push and Pull incentives in funding antibiotic development
Why was there a global shortage of PIP-TAZO in 2016
What are the species and resistance characteristics of the WHO Priority 1 pathogens
What is novel about avibactam
What is novel about varobactam
What is meant by the ‘trojan horse’ approach. Describe an example currently in clinical use
What is meant by ‘selective targeting’. What advantages could this approach have for therapy.