Leaks in the Pipeline: a Failure Analysis of Gram-Negative Antibiotic Development from 2010 to 2020.

The World Health Organization (WHO) has warned that our current arsenal of antibiotics is not innovative enough to face impending infectious diseases, especially those caused by multidrug-resistant Gram-negative pathogens. Although the current preclinical pipeline is well stocked with novel candidates, the last U.S. Food and Drug Administration (FDA)-approved antibiotic with a novel mechanism of action against Gram-negative bacteria was discovered nearly 60 years ago. Of all the antibiotic candidates that initiated investigational new drug (IND) applications in the 2000s, 17% earned FDA approval within 12 years, while an overwhelming 62% were discontinued in that time frame. These "leaks" in the clinical pipeline, where compounds with clinical potential are abandoned during clinical development, indicate that scientific innovations are not reaching the clinic and providing benefits to patients. This is true for not only novel candidates but also candidates from existing antibiotic classes with clinically validated targets. By identifying the sources of the leaks in the clinical pipeline, future developmental efforts can be directed toward strategies that are more likely to flow into clinical use. In this review, we conduct a detailed failure analysis of clinical candidates with Gram-negative activity that have fallen out of the clinical pipeline over the past decade. Although limited by incomplete data disclosure from companies engaging in antibiotic development, we attempt to distill the developmental challenges faced by each discontinued candidate. It is our hope that this insight can help de-risk antibiotic development and bring new, effective antibiotics to the clinic.

Modulating Pathogenesis with Mobile-CRISPRi.

Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

ß-Lactone formation during product release from a nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are multidomain modular biosynthetic assembly lines that polymerize amino acids into a myriad of biologically active nonribosomal peptides (NRPs). NRPS thioesterase (TE) domains employ diverse release strategies for off-loading thioester-tethered polymeric peptides from termination modules typically via hydrolysis, aminolysis, or cyclization to provide mature antibiotics as carboxylic acids/esters, amides, and lactams/lactones, respectively. Here we report the enzyme-catalyzed formation of a highly strained ß-lactone ring during TE-mediated cyclization of a ß-hydroxythioester to release the antibiotic obafluorin (Obi) from an NRPS assembly line. The Obi NRPS (ObiF) contains a type I TE domain with a rare catalytic cysteine residue that plays a direct role in ß-lactone ring formation. We present a detailed genetic and biochemical characterization of the entire Obi biosynthetic gene cluster in plant-associated Pseudomonas fluorescens ATCC 39502 that establishes a general strategy for ß-lactone biogenesis.