Discovery of Potent Isoindolinone Inhibitors that Target an Active Conformation of PARP1 Using DNA-Encoded Libraries.

Inhibition of poly (ADP-ribose) polymerase-1 (PARP1), a DNA repair enzyme, has proven to be a successful strategy for the treatment of various cancers. With the appropriate selection conditions and protein design, DNA-encoded library (DEL) technology provides a powerful avenue to identify small molecules with the desired mechanism of action towards a target of interest. However, DNA-binding proteins, such as PARP1, can be challenging targets for DEL screening due to non-specific protein-DNA interactions. To overcome this, we designed and screened a PARP1 catalytic domain construct without the autoinhibitory helical domain. This allowed us to interrogate an active, functionally-relevant form of the protein resulting in the discovery of novel isoindolinone PARP1 inhibitors with single-digit nanomolar potency. These inhibitors also demonstrated little to no PARP1-DNA trapping, a property that could be advantageous in the clinic.

Peptide Probes of Colistin Resistance Discovered via Chemically Enhanced Phage Display.

Colistin is an antibiotic of last resort used to treat infections caused by multidrug-resistant Gram-negative bacterial pathogens. The recent surge in reported cases of colistin-resistant infections urgently calls for fast and reliable diagnostic methods, which can be used for the facile detection and proper treatment of these challenging infections. A major mechanism of colistin resistance involves phosphoethanolamine (PE) modification of lipopolysaccharide (LPS), the molecular target of colistin. This LPS modification mechanism has been recently reported to be transferrable via a plasmid-carried mcr-1 gene, which is particularly concerning as it may readily confer colistin resistance to a wide array of bacterial pathogens. To develop molecular tools to allow facile detection of colistin resistance, we have herein enlisted a novel phage library that incorporates dynamic covalent warheads to recognize PE modifications on bacterial cells. Screening of this chemically modified phage library against colistin-resistant pathogens revealed a number of peptide probes that readily differentiate colistin-resistant bacterial strains from their colistin-susceptible counterparts. With a fluorophore label, these peptide probes selectively stain colistin-resistant bacteria at sub-to-low micromolar concentrations. The bacterial staining is minimally inhibited by the presence of serum proteins or even blood serum. Mechanistic studies indicate that our peptide probes bind colistin-resistant bacteria primarily by targeting PE-modified lipids. However, some species-specific features of the cell surface can also contribute to the peptides' association to bacterial cells. Further elucidation of such cell surface features may give molecular probes with improved species and strain specificity, which will enable bacterial infection diagnosis with high precision.

The Impact of Variable Selection Coverage on Detection of Ligands from a DNA-Encoded Library Screen.

DNA-encoded library (DEL) technology has become a prominent screening platform in drug discovery owing to the capacity to screen billions or trillions of compounds in a single experiment. Although numerous successes with DEL technology have been reported, we are unaware of a rigorous examination of the many different variables that can influence a screen's success. Herein, we explore the impact of variable sample sequencing depth on the detection of tool compounds with known affinities toward a given target while simultaneously probing the effect of initial compound input. Our sequencing data confirm reports that high-affinity compounds can be discovered directly from a DEL screen, but we demonstrate that a mismatch between selection output and sequencing quantity can obscure useful ligands. Our results highlight the importance of selection coverage in grasping the entire picture of a DEL screen where the signal of a weak or underrepresented ligand may be suppressed by the inherent noise of a selection. These potential missed ligands may be critical to the success or failure of a drug discovery program.

Radiolabeled Cationic Peptides for Targeted Imaging of Infection.

Molecular probes targeting bacteria provide opportunities to target bacterial infections in vivo for both imaging and therapy. In the current study, we report the development of positron emission tomography (PET) probes for imaging of live bacterial infection based on the small molecules HLys-DOTA, a polycationic peptide synthesized as the D-isomer (RYWVAWRNRG) conjugated to 1, 4, 7, 10-tetraazacyclododecane-N',N″,N‴,N-tetraacetic acid (DOTA) and AB1-HLys-DOTA, which includes an unnatural amino acid AB1 that preferentially binds to bacteria membrane lipids with amine groups via formation of iminoboronates. HLys-DOTA and AB1-HLys-DOTA peptides were radiolabeled with 64Cu and investigated as PET imaging agents to track bacterial infection in vitro and in intramuscularly infected (IM) mice models. Cell uptake studies at 37°C in Staphylococcus aureus (SA) show higher uptake of 64Cu-AB1-HLys-DOTA; 98.47 ± 3.54% vs 64Cu-HLys-DOTA; 39.12 ± 3.27% at 24 h. Standard uptake values (SUV) analysis of the PET images resulted in mean SUV of 0.70 ± 0.08, 0.49 ± 0.04, and 0.31 ± 0.01 for 64Cu-AB1-HLys-DOTA and 0.17 ± 0.06, 0.16 ± 0.02, and 0.13 ± 0.01 for 64Cu-HLys-DOTA at 1, 4, and 24 h post injection, respectively, in the infected muscles. Similarly, in the biodistribution studies, dose uptake in the infected muscles was 4 times higher in the targeted 64Cu-AB1-HLys-DOTA group than in the 64Cu-HLys-DOTA group and 2-3 times higher than in the PBS control group at 1, 4, and 24 h post injection. 64Cu-AB1-HLys-DOTA was able to distinguish between SA-infected muscle and Pseudomonas aeruginosa (PA) infected muscle with lower mean SUV of 0.28 ± 0.10 at 1 h post injection. This illustrates the utility of the AB1 covalently targeting group in synergy with the HLys peptide, which noncovalently binds to bacterial membranes. These results suggest that 64Cu-labeled AB1-HLys-DOTA peptide could be used as an imaging probe for detection of bacterial infection in vivo with specificity for Gram-positive bacteria.

Phage Display of Dynamic Covalent Binding Motifs Enables Facile Development of Targeted Antibiotics.

Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of our society and urgently calls for new strategies for infection diagnosis and antibiotic discovery. The antibiotic resistance problem at least partially arises from extensive use of broad-spectrum antibiotics. Ideally, for the treatment of infection, one would like to use a narrow-spectrum antibiotic that specifically targets and kills the disease-causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being. In this contribution, we describe a phage display platform that enables rapid identification of peptide probes for specific bacterial strains. The phage library described herein incorporates 2-acetylphenylboronic acid moieties to elicit dynamic covalent binding to the bacterial cell surface. Screening of the library against live bacterial cells yields submicromolar and highly specific binders for clinical strains of Staphylococcus aureus and Acinetobacter baumannii that display antibiotic resistance. We further show that the identified peptide probes can be readily converted to bactericidal agents that deliver generic toxins to kill the targeted bacterial strain with high specificity. The phage display platform described here is applicable to a wide array of bacterial strains, paving the way to facile diagnosis and development of strain-specific antibiotics.

Targeting bacteria via iminoboronate chemistry of amine-presenting lipids.

Synthetic molecules that target specific lipids serve as powerful tools for understanding membrane biology and may also enable new applications in biotechnology and medicine. For example, selective recognition of bacterial lipids may give rise to novel antibiotics, as well as diagnostic methods for bacterial infection. Currently known lipid-binding molecules primarily rely on noncovalent interactions to achieve lipid selectivity. Here we show that targeted recognition of lipids can be realized by selectively modifying the lipid of interest via covalent bond formation. Specifically, we report an unnatural amino acid that preferentially labels amine-presenting lipids via iminoboronate formation under physiological conditions. By targeting phosphatidylethanolamine and lysylphosphatidylglycerol, the two lipids enriched on bacterial cell surfaces, the iminoboronate chemistry allows potent labelling of Gram-positive bacteria even in the presence of 10% serum, while bypassing mammalian cells and Gram-negative bacteria. The covalent strategy for lipid recognition should be extendable to other important membrane lipids.