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Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis inBacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs inB. subtiliswere discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance inB. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies.

Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4-18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a β-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent β-lactone probes.

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b, or PBP4. Almost all penicillin scaffolds were coselective for all or combinations of PBP2a, 2b, and 4. Cephalosporins, on the other hand, possessed the lowest IC(50) values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin) and 2b (cefotaxime) or 2a, 2b, and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime, and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid), and various coselective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in β-lactam resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1’s active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.

Laponite is a clay-based material composed of synthetic disc-shaped crystalline nanoparticles with a large and highly ionic surface area. These characteristics enable the intercalation and dissolution of biomolecules in Laponite-based drug delivery systems. Furthermore, Laponite’s innate physicochemical properties and architecture enable the development of tunable pH-responsive drug delivery systems. Laponite’s coagulation capacity and cation exchangeability, determine its exchange capabilities, drug encapsulation efficiency, and release profile. These parameters have been exploited to design highly controlled and efficacious drug delivery platforms for sustained drug release. In this review, we give an overview of how the properties and specific interactions of various Laponite–polymer composite and drug moieties, have been leveraged to design efficient delivery of a number of therapeutics.

Peptidoglycan (PG) is a mesh-like heteropolymer made up of glycan chains cross-linked by short peptides and is the major scaffold of eubacterial cell walls, determining cell shape, size, and chaining. This structure, which is required for growth and survival, is located outside of the cytoplasmic membrane of bacterial cells, making it highly accessible to antibiotics. Penicillin-binding proteins (PBPs) are essential for construction of PG and perform transglycosylase activities to generate the glycan strands and transpeptidation to cross-link the appended peptides. The β-lactam antibiotics, which are among the most clinically effective antibiotics for the treatment of bacterial infections, inhibit PBP transpeptidation, ultimately leading to cell lysis. Despite this importance, the discrete functions of individual PBP homologues have been difficult to determine. These major gaps in understanding of PBP activation and macromolecular interactions largely result from a lack of tools to assess the functional state of specific PBPs in bacterial cells. We have identified β-lactones as a privileged scaffold for the generation of PBP-selective probes and utilized these compounds for imaging of the essential proteins, PBP2x and PBP2b, in Streptococcus pneumoniae. We demonstrated that while PBP2b activity is restricted to a ring surrounding the division sites, PBP2x activity is present both at the septal center and at the surrounding ring. These spatially separate regions of PBP2x activity could not be detected by previous activity-based approaches, which highlights a critical strength of our PBP-selective imaging strategy.

ABPP methods have been utilized for the last two decades as a means to investigate complex proteomes in all three domains of life. Extensive use in eukaryotes has provided a more fundamental understanding of the biological processes involved in numerous diseases and has driven drug discovery and treatment campaigns. However, the use of ABPP in prokaryotes has been less common, although it has gained more attention over the last decade. The urgent need for understanding bacteriophysiology and bacterial pathogenicity at a foundational level has never been more apparent, as the rise in antibiotic resistance has resulted in the inadequate and ineffective treatment of infections. This is not only a result of resistance to clinically used antibiotics, but also a lack of new drugs and equally as important, new drug targets. ABPP provides a means for which new, clinically relevant drug targets may be identified through gaining insight into biological processes. In this chapter, we place particular focus on the discussion of ABPP strategies that have been applied to study different classes of bacterial enzymes.

ABPP methods have been utilized for the last two decades as a means to investigate complex proteomes in all three domains of life. Extensive use in eukaryotes has provided a more fundamental understanding of the biological processes involved in numerous diseases and has driven drug discovery and treatment campaigns. However, the use of ABPP in prokaryotes has been less common, although it has gained more attention over the last decade. The urgent need for understanding bacteriophysiology and bacterial pathogenicity at a foundational level has never been more apparent, as the rise in antibiotic resistance has resulted in the inadequate and ineffective treatment of infections. This is not only a result of resistance to clinically used antibiotics, but also a lack of new drugs and equally as important, new drug targets. ABPP provides a means for which new, clinically relevant drug targets may be identified through gaining insight into biological processes. In this chapter, we place particular focus on the discussion of ABPP strategies that have been applied to study different classes of bacterial enzymes.