Courtney Aldrich, PhD

Antimicrobial resistance (AMR) is a natural phenomenon in response to—pervasive use of antibiotics, environmental pollution from pharmaceutical and agriculture antibiotic use, mass movement of people, loss of biodiversity, and lack of clean water and poor sanitation—that requires a multipronged approach beginning with addressing the underlying causes, followed by prevention, improved accessibility to treatment, and development of new therapeutic agents. The updated WHO Bacterial Priority Pathogen List released in 2024 includes 15 priority-pathogen drug-resistant phenotypes of concern with rifampicin-resistant Mycobacterium tuberculosis (the cause of tuberculosis or TB) catapulting from obscurity seven years ago to the critical group that has the highest threat and the largest unmet need along with Acinetobacter baumannii resistant to antibiotics of last resort.

Target-based approaches that seek to rationally design an antibiotic from first principles have failed to deliver a single new class of antibiotics, yet have been immensely successful in the antiviral arena and there are rationally-designed small-molecule inhibitors of nearly every open-reading frame of the HIV genome in clinical use. The divergence in outcomes between antibiotics and antivirals is astonishing and in part reflects the challenges of prioritizing antibiotic targets, caused by the amazing metabolic adaptability, functional redundancy—and metabolic buffering of bacteria. Integration of microbial physiology and functional genomics has begun to address many of the long-standing challenges in target identification and prioritization while significant progress has also been made to deconvolute the molecular rules predictive of antibiotic accumulation in bacteria. Nevertheless, the development of antibiotics against novel targets remains extremely challenging and requires a holistic approach balancing multiple factors, many of which are unknown a priori.

Antibiotics have evolved over millions of years to exploit metabolic vulnerabilities of competitors within their shared environmental microbiome, thus antibiotics effectively also provide target validation, which is guided by natural selection and may reveal other advantages given the selective pressure driving the evolution of their properties. Following this reasoning, identification of multiple natural products targeting a common biosynthetic pathway through convergent evolution should reinforce the importance of the pathway and its susceptibility to modulation by a small-molecule. Thus, it is not surprising that the ribosome, enzymes involved cell-wall, DNA and RNA synthesis are the most frequently observed targets of antibiotics. In addition to these established targets, there remains a wealth of overlooked antibiotics discovered during the 20th century, many of which have been long-forgotten, exemplified by the biotin antimetabolites acidomycin and amiclenomycin that were among the first reported microbial natural products and demonstrated excellent safety and intriguing activity against mycobacteria. While both antibiotics have proved intractable for development due to poor intrinsic stability and limited tolerance to modification, the discovery of multiple natural products targeting the same pathway reveals biotin biosynthesis is sufficiently vulnerable to inhibition and thereby provides strong pre-validation for targeting biotin biosynthesis. The historical antibiotics now can be easily found in the carefully curated Natural Products Atlas (www.npatlas.org) which includes nearly every reported microbial natural product ever described in the literature over the last 150 years while the Atlas of Biosynthetic Gene Clusters (BGCs) (https://img.jgi.doe.gov/) contains over a million BGCs encoding the historical antibiotics along with new ones awaiting discovery. Analysis of BCGs can sometimes provide further insight into mechanism and overall strategy used by the producing organism. We use historical data of antibiotics and genome mining to pre-validate targets.

Developing new antimicrobial agents with novel mechanisms has almost exclusively focused on core essential genes defined under nutrient-rich growth conditions that excludes potential targets and is unnecessarily restrictive. Advances in our understanding of microbial metabolism and physiology reveal numerous genes in cofactor, amino acid and nucleotide biosynthesis essential for infection in priority pathogens. The genetic background of the host and pathogen can profoundly impact gene essentiality in animal infection models, thus we integrate information from functional genomics studies to ensure potential targets are valid across a broad spectrum of host immune phenotypes and pathogen strains.

Biotin biosynthesis is essential for WHO critical group pathogens. We developed small-molecule inhibitors of various enzymes in the biotin pathway using structure-guided design. This project is representative and encompasses chemical synthesis and rigorous biochemical characterization to measure kinetic and thermodynamic parameters using steady-state kinetic analysis, isothermal titration calorimetry of the molecular target. Synthesis and compound design help build expertise in medicinal chemistry where novel methods are encouraged (chemoenzymatic synthesis, electrochemistry, photoredox, organocatalysis) and traditional methods emphasized (recrystallization, radial chromatography, sublimation, Schlenk technique, IR, UV). Microbiology characterization typically begins with measuring the minimum inhibitory concentration (MIC) followed by chemical complementation with biosynthetic pathway intermediates to pinpoint the blocked step. Further support for mechanism of action employs other traditional methods in microbiology as well as technologies including CRISPR and generation of conditional mutants to under and over-express the putative target, leading to predictable changes in drug susceptibility. Structural characterization and generation of resistant mutants helps confirm binding mode and molecular target(s), which provides important information to validate initial design and guidance for improving compounds. Studies to confirm the mechanism of action include a range of unbiased chemical genetic interaction approaches (Tn-Seq, RNA-Seq), targeted metabolomics of the pathway of interest, and quantitative proteomic analysis. Validation of the mechanism of action is crucial and provides outstanding foundational training in contemporary methods of chemical biology, biochemistry and genomics. We also concurrently evaluate and optimize physicochemical properties and measure solubility, lipophilicity (LogD), plasma protein binding, microsomal stability and pharmacokinetics (PK) to identify in vitro-in vitro correlates to optimize PK properties in order to evaluate compounds in vivo infection models. Learning elementary ADME assays (absorption, distribution, metabolism,
excretion) provides excellent training in bioanalytical chemistry.

In response to nutrient stress and many other stresses, the stringent response is generally activated leading to broad metabolic shutdown that allow bacteria to survive. This triggered persistence is responsible for the reduced rate of killing of many bactericidal antibiotics. Thus, we have also identified metabolically vulnerable targets, whose inhibition is bactericidal in priority pathogens through positive epistatic interactions through depletion of essential metabolite(s) and accumulation of a metabolite that is either toxic or dysregulates other pathways. Targets include coenzyme A, NAD, methionine, aspartate, and arginine.

Bacterial Metal Homeostasis. Iron is essential for bacteria and humans alike while excess iron is toxic, thus we tightly regulate the iron concentration in serum and mucosal surfaces using the high affinity iron transport proteins transferrin (Tf) and lactoferrin (Lf). These proteins bind two atoms of ferric (Fe 3+ ) iron and deliver it to host cells expressing cognate receptors, while withholding iron from bacteria as part of a general strategy known as nutritional immunity to limit trace minerals and prevent and or reduce infection. Pathogenic and commensal bacteria have evolved a multitude of ingenious mechanisms to overcome iron restriction imposed by the host, to meet their insatiable appetites for iron and commonly biosynthesize small-molecule multidentate iron chelators known as siderophores, that can pirate ferric iron from transferrin and lactoferrin. Mycobacterium tuberculosis, the causative agent of tuberculosis, has long served as an exemplary model system for studying the fundamental biology of iron homeostasis and produces a suite of siderophores known as the mycobactins and a water-soluble variant known as carboxymycobactin. Prior studies have revealed iron acquisition is essential for virulence and survival of virtually all pathogens, but there remain gaps in our knowledge regarding the relative contribution of the individual iron acquisition systems in vivo where heterogeneity of the bacterial population as well as host immune cells provide a rich diversity of host-pathogen interactions that are difficult to recapitulate in vitro. We are interested in bacterial metal homeostasis and are developing small-molecule probes to study virulence pathways involved in siderophore and metallophore biosynthesis to address long-standing gaps in the field.

Re-engineering Rifamycins. Finally, we seek to redesign classic antibiotics to overcome resistance and improve safety. We have focused on the rifamycins, which are antibiotics against some of the most difficult-to-treat bacterial infections including those caused by Mycobacterium tuberculosis (TB) and Mycobacterium abscessus because of their exceptional sterilizing activity against drug-tolerant extracellular and intracellular bacteria, outstanding pharmacokinetic properties, and superb penetration into lesion where mycobacteria reside. The potent bactericidal activity of rifamycins derives from inhibition of transcription through their tight-binding to the RpoB subunit of bacterial DNA-dependent RNA polymerase (RNAP) that leads to lethal double-stranded DNA breaks when the replisome collides with the stalled RNAP complex. Inhibition of transcription also prevents bacteria from rapidly mounting adaptive resistance. Hence, rifamycin resistance observed clinically through missense mutations at D435V, H445D and S450L residues in the RpoB subunit limits treatment options and is associated with significantly higher mortality. We have designed next-generation rifamycin analogs that overcome several resistance mechanisms and regain activity against the most challenging mutants. Our lead compound is progressing in advanced pre-clinical studies of M. abscessus.

PubMed

• Qu, D.; Ge, P.; Botella, L.; Park, S. W.; Lee, H. N.; Thornton, N.; Bean, J. M; Krieger, I. V.; Sacchettini, J. C.; Ehrt, S.; Aldrich, C. C.; Schnappinger, D. Mycobacterial Biotin Synthases Require an Auxiliary Protein to Convert Dethiobiotin into Biotin. Nature Commun. 2024, 15, 4161.
• Sullivan, M. R.; McGowen, K.; Liu, Q.; Akusobi, C.; Young, D. C.; Mayfield, J. A.; Raman, S.; Wolf, I. D.; Moody, D. B.; Aldrich, C. C.; Muir, A.; Rubin, E. J. Biotin-dependent cell envelope remodeling is required for Mycobacterium abscessus survival in lung infection. Nature Microbiol. 2023, 8, 481–497.
• Lan, T.; Ganapathy, U. S.; Sharma, S.; Ahn, Y-M.; Molodtsov, V.; Hegde, P.; Gengenbacher, M.; Ebright, R. H.; Dartois, V.; Freundlich, J. S.; Dick, T.; Aldrich, C. C. Redesign of Rifamycin Antibiotics to Overcome ADP-Ribosylation-Mediated Resistance. Angew. Chem. Int. Ed. 2022, 61, e202211498.
• Panda, S.; Poudel, T. N.; Hegde, P.; Aldrich, C. C. Innovative Strategies for the Construction of Diverse 1’-Modified C-Nucleoside Derivatives. J. Org. Chem. 2021, 86, 16625–16640.
• Alexander, E. M.; Kreitler, D. F.; Guidolin, V.; Hurben, A. K.; Drake, E.; Villalta, P. W.; Balbo, S.; Gulick, A. M.; Aldrich, C. C. Biosynthesis, Mechanism of Action, and Inhibition of the Enterotoxin Tilimycin Produced by the Opportunistic Pathogen Klebsiella oxytoca. ACS Infect. Dis. 2020, 6, 1976–1997.
• Buonomo, J. A.; Cole, M. S.; Eiden, C. G.; Aldrich, C. C. 1,3-Diphenyl-disiloxane Enables Additive-Free Redox Recycling Reactions and Catalysis with Triphenylphosphine. Synthesis, 2020, 52, 3583–3594.

• ​​​​​​Membrane-Active Anti-Bacterial Compounds and Uses Thereof,  Issued: September 9, 2022
• Next-Generation Remdesivir Antivirals, Issued: August 4, 2022