The purpose of this Notice of Funding Opportunity (NOFO) is to support milestone-driven projects focused on developing and utilizing novel predictive models, assays, tools, and/or platforms based on penetration and efflux of small molecules to facilitate therapeutic discovery for select Gram-negative bacterial pathogens: carbapenem-resistant Acinetobacter, carbapenem-resistant Enterobacteriaceae (CRE), and multidrug-resistant Pseudomonas aeruginosa. A number of Gram-negative bacterial pathogens are associated with the alarming increase in rates of drug resistance in healthcare and community settings. This group includes carbapenem-resistant Acinetobacter , carbapenem-resistant Enterobacterales (CRE), and MDR Pseudomonas aeruginosa that have been designated as “urgent” or “serious” threats in a recent report by the Centers for Disease Control and Prevention (CDC). A significant threat arises from the lack of effective therapeutic options available to treat some of these infections and is exacerbated by the scarcity of novel compounds effective against antimicrobial resistant (AR) and multidrug-resistant (MDR) Gram-negative bacteria in the discovery and development pipeline.

In recent years, several public forums (including NIAID-sponsored workshops) identified the lack of understanding of the principles that govern compound penetration into, and efflux out of, Gram-negative bacteria as a key bottleneck for the rational discovery of novel lead therapeutic compounds. The paucity of suitable assays/tools/models to inform structure-activity relationships and guide optimization of whole cell penetration (and efflux avoidance) is reflected in the failure of medicinal chemistry efforts to advance novel chemical classes of compounds with Gram-negative activity. As more Gram-negative bacteria become resistant to antimicrobials and therapeutic options become limited or nonexistent, it becomes imperative to understand and rationalize the principles that allow molecules to penetrate Gram-negative bacteria, while avoiding efflux and overt toxicity toward eukaryotic cells. Therefore, developing new assays, tools, and models is paramount for overcoming this key bottleneck and facilitating the development of novel compounds targeting Gram-negative pathogens.

Research Objectives and Scope
The objective of this NOFO is to support milestone-driven projects focused on developing and utilizing novel predictive models, assays, tools, and/or platforms aimed at gaining a better understanding of the rules and compound properties that govern the penetration and efflux of drug-like small molecules into Gram-negative bacterial pathogens. This NOFO also supports the preclinical development of novel Gram-negative antibacterial therapeutics based on the tools and models hereby developed.

Applications must focus on one or more of the following Gram-negative bacterial pathogens: carbapenem-resistant Acinetobacter, carbapenem-resistant Enterobacterales (CRE), and/or MDR Pseudomonas aeruginosa. Projects should complete assay/tool/model development prior to the end of the third year of the project period and initiate discovery activities to demonstrate its utility in supporting a corresponding medicinal chemistry program to generate a lead chemical series with demonstrated activity against one or more targeted Gram-negative bacteria. This NOFO will also support subsequent preclinical development of a promising lead antibacterial.

Given the complex challenges of this research, this initiative encourages applications from multi-disciplinary teams composed of relevant experts in areas such as bacterial physiology, microbiology, bacterial membrane biology, medicinal chemistry, pharmacology, computation, and specialized technologies (microscopy, spectroscopy, electrophysiology, machine learning, etc.), as appropriate. Collaboration with, and utilization of, the NIAID Chemistry Center for Combating Antibiotic-Resistant Bacteria (CC4CARB), is recommended for completion of project relevant medicinal chemistry tasks. Close collaboration between academic and industry partners is highly encouraged to optimally combine innovative basic science with drug discovery expertise and proper access to compound libraries more typically available from industry.

Examples of assay and model development activities include, but are not limited to:

Quantitative cellular (or model system) assays to measure drug penetration and efflux, independent from standard minimum inhibitory concentration (MIC) testing;
Innovative quantitative assays to measure drug concentrations in the bacterial cytoplasm and/or periplasmic space;
Innovative technologies for dissecting and assessment of the kinetics of drug penetration and efflux from bacteria; and
Computational algorithms for describing/predicting physical-chemical properties/guidelines needed by small molecules for optimal Gram-negative penetration and efflux avoidance.
Applicants should demonstrate the utility of the developed tools and/or assays to predict and measure potency of candidate therapeutics against Gram-negative targets through one or more approaches. For example, using the developed models and/or assays to guide a medicinal chemistry campaign aimed at producing a novel chemical series with Gram-negative activity; screening existing libraries using the computational algorithms developed as a tool to find compounds with Gram-negative activity; or profiling existing libraries of compounds with known Gram-negative activity.

Applications including the following will be considered non-responsive and will not be reviewed:

Projects that do not focus on at least one select Gram-negative pathogen (carbapenem-resistant Acinetobacter, carbapenem-resistant Enterobacterales, or MDR Pseudomonas aeruginosa);
Projects focused only on Gram-positive bacteria or Mycobacterium tuberculosis;
Projects that do not focus on penetration and efflux of small molecules;
Applications not containing a Milestone and Timeline attachment;
Applications proposing Clinical trials; and
Research on HIV/AIDS.